Quality identification method and application of nettle mongolica for preparing anti-hyperuricemia medicine

Abstract

The invention provides a quality identification method and application of nettle mongolica for preparing an anti-hyperuricemia drug. The content of three anti-hyperuricemia active components, namely chlorogenic acid, New Zealand vitexin 2 and isovitexin, in nettle mongolica to be identified needs to be detected; the nettle at least meets one of the conditions that the contents of chlorogenic acid, New Zealand vitexin 2 and isovitexin are respectively greater than or equal to 2.0 mg / g, 1.5 mg / g and 1.5 mg / g, and when the total content of the three components is greater than or equal to 5.0 mg / g, the nettle can be used for preparing the anti-hyperuricemia medicine, the quality is high, and the application cost can be reduced. The three compound components have inhibition results on hyperuricemia key target proteins XOD and GLUT9, and the inhibition effect of the isovitexin on the two proteins is the best in the three compound components and is significant, so that the three compounds, especially the isovitexin, of the isovitexin, the New Zealand vitexin 2 and the chlorogenic acid, have the significant inhibition effect on the two proteins, and have the significant inhibition effect on the hyperuricemia key target proteins XOD and GLUT9 in the hyperuricemia key target proteins XOD and GLUT9 in the hyperuricemia key target proteins XOD and GLUT9 in the hyperuricemia key target proteins. The compound has a wide application prospect in preparation of anti-hyperuricemia drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a method and application for quality identification of Urticaria mongholica used in the preparation of drugs for treating hyperuricemia. Background Technology

[0002] Mongolian nettle (Urtica cannabina HERBA) is the dried aerial part of *Urtica cannabina* L., a perennial herbaceous plant belonging to the genus *Urtica* in the family Urticaceae. In Mongolian medicine, it is called *Hala Gai*. It tastes bitter and pungent, is warm in nature, and has strong effects. It is used to calm the stomach, regulate stomach temperature, detoxify, and relieve abdominal distension. It is used for dizziness, tinnitus, insomnia, palpitations, indigestion, belching, vomiting, diarrhea, joint pain, stomach distension, liver distension, rheumatic joint pain, itchy skin, and to detoxify snake venom. Currently, there is no national standard for Mongolian nettle. It is included in the *Inner Mongolia Mongolian Medicinal Herbs Standard (2021 Edition)* and the *Inner Mongolia Mongolian Medicinal Herbs Processing Specifications (2020 Edition)*, but the testing items are incomplete. This reflects a weak foundation in the research of the chemical composition and pharmacological activity of this medicinal herb, resulting in an incomplete quality standard system. According to literature reports, plants in this genus contain various chemical components such as flavonoids, alkaloids, lignans, coumarins, terpenes, steroids, organic acids, and volatile oils. They possess a wide range of pharmacological activities, including anti-inflammatory, analgesic, anti-prostate hyperplasia, antibacterial, antioxidant, hypoglycemic, and lipid-regulating effects. Therefore, it is necessary to improve their quality standards in order to comprehensively and effectively control the quality of medicinal materials.

[0003] Due to the diversification of dietary structures, the prevalence of metabolic diseases remains high and shows a trend towards affecting younger people, seriously impacting people's physical and mental health. Gout is a metabolic disease, and in recent years, its prevalence has been increasing year by year. Current clinical treatment mainly uses chemical drugs, primarily those that inhibit uric acid production and promote uricosuric excretion, such as allopurinol, benzbromarone, and febuxostat. However, long-term use of these drugs can lead to adverse reactions such as headaches, rashes, elevated liver transaminases, and gastrointestinal reactions. Furthermore, some drugs are contraindicated in patients with renal insufficiency. Mongolian medicine considers gout to fall under the category of joint edema, caused by improper diet and lifestyle leading to imbalances in the "three roots and seven elements," resulting in excessive edema that interacts with blood and accumulates in the joints, obstructing local blood and qi circulation. According to literature reports, ethyl acetate extract of Mongolian nettle has anti-inflammatory and analgesic effects, but its material basis and molecular mechanism remain unclear.

[0004] This invention collected 15 batches of *Urtica juncea* samples, established HPLC characteristic chromatograms reflecting the overall quality of *Urtica juncea*, and performed similarity evaluation and rapid identification of the chemical structures of characteristic components. Hierarchical cluster analysis (HCA), principal component analysis (PCA), and partial least squares-discriminant analysis (PLS-DA) were used to screen *Urtica juncea* Q-Markers. Molecular docking technology, in vitro XOD enzyme activity assays, and cell experiments were used to validate the characteristic and key components in *Urtica juncea* against anti-hyperuricemia-related target proteins in vitro, ensuring the effectiveness of the Q-Markers. Finally, using the Q-Markers and active ingredients as indicator components, content determination studies were conducted to provide a basis for improving the quality standards of *Urtica juncea*. Summary of the Invention

[0005] Therefore, one objective of this invention is to provide a method for quality identification of *Urtica montana* for preparing anti-hyperuricemia drugs, comprising the following steps:

[0006] 1) Detect the content of three active ingredients for treating hyperuricemia in the nettle plant to be identified: chlorogenic acid, vitexin 2, and isovitexin;

[0007] 2) Based on the content obtained in step 1), when the content of chlorogenic acid is greater than or equal to 2.0 mg / g and / or the content of vitexin 2 is greater than or equal to 1.5 mg / g and / or the content of isovitexin is greater than or equal to 1.5 mg / g, and the total content of chlorogenic acid, vitexin 2 and isovitexin is greater than or equal to 5.0 mg / g, the *Urtica montana* to be identified is *Urtica montana* that can be used to prepare anti-hyperuricemia drugs.

[0008] Furthermore, the method for detecting the content of three anti-hyperuricemia active ingredients—chlorogenic acid, vitexin 2, and isovitexin—in *Urtica juncea* includes the following steps:

[0009] A) Crush *Urtica montana*, pass through a 60-mesh sieve, accurately weigh 1.0 g, add 25 mL of 70% ethanol, weigh, extract ultrasonically for 30 min, make up the weight, centrifuge for 5 min at 3000 rpm / min, filter through a 0.22 μm microporous membrane to obtain the test solution, the volume of the test solution being 25 mL;

[0010] B) The test solution prepared in step A) was analyzed by high performance liquid chromatography (HPLC) to obtain the peak areas of three active ingredients: chlorogenic acid, vitexin 2, and isovitexin. The HPLC conditions were as follows: InertSustain C18, 5 μm, 4.6 mm × 250 mm; mobile phase: acetonitrile A - 0.4% formic acid water B; gradient elution program: 0–10 min, 10%–15% A; 10–25 min, 15%–20% A; 25–35 min, 20%–35% A; 35–37 min, 35%–90% A; 35–42 min, 90%–90% A; 42–44 min, 90%–10% A; 44–59 min, 10%–10% A; injection volume: 1–20 μL; flow rate: 1.0 mL / min; column temperature: 30 ℃; wavelength: 271 nm.

[0011] C) Substitute the peak area value obtained in step B) into the equation Y. 绿原酸 =46825X + 2475.8, Y 新西兰牡荆苷2 =108755X+22211 and Y 异牡荆苷 =171447X+2872.4, where Y is the peak area and X is the solute injection amount (μg), thus obtaining the solute injection amount X of the three active ingredients;

[0012] D) Substitute the solute injection amounts of the three active ingredients obtained in step C) into the formula to calculate the content (mg / g) = (solute injection amount (μg) / 1000) × (test solution volume (mL) × 1000 / solution injection amount (μL)) / 1 g, to obtain the content of the three active ingredients chlorogenic acid, vitexin 2 and isovitexin in the nettle to be identified.

[0013] Furthermore, the solution injection volume in step B) is 20 μL.

[0014] A second objective of this invention is to provide a method for detecting the content of anti-hyperuricemia active ingredients in *Urtica montana*, wherein the active ingredients are chlorogenic acid, vitexin 2, and isovitexin, and the method includes the following steps:

[0015] A) Crush *Urtica montana*, pass through a 60-mesh sieve, accurately weigh 1.0 g, add 25 mL of 70% ethanol, weigh, extract ultrasonically for 30 min, make up the weight, centrifuge for 5 min at 3000 rpm / min, filter through a 0.22 μm microporous membrane to obtain the test solution, the volume of the test solution being 25 mL;

[0016] B) The test solution prepared in step A) was analyzed by high performance liquid chromatography (HPLC) to obtain the peak areas of three active ingredients: chlorogenic acid, vitexin 2, and isovitexin. The HPLC conditions were as follows: InertSustain C18, 5 μm, 4.6 mm × 250 mm; mobile phase: acetonitrile A - 0.4% formic acid water B; gradient elution program: 0–10 min, 10%–15% A; 10–25 min, 15%–20% A; 25–35 min, 20%–35% A; 35–37 min, 35%–90% A; 35–42 min, 90%–90% A; 42–44 min, 90%–10% A; 44–59 min, 10%–10% A; injection volume: 1–20 μL; flow rate: 1.0 mL / min; column temperature: 30 ℃; wavelength: 271 nm.

[0017] C) Substitute the peak area values ​​obtained in step B) into the regression equation Y of the three active ingredients. 绿原酸 =46825X + 2475.8, Y 新西兰牡荆苷2 =108755X+22211 and Y 异牡荆苷 =171447X+2872.4, where Y is the peak area and X is the solute injection amount (μg), thus the solute injection amount X of the three active ingredients can be calculated;

[0018] D) Substitute the solute injection amounts of the three active ingredients obtained in step C) into the formula to calculate the content (mg / g) = (solute injection amount (μg) / 1000) × (test solution volume (mL) × 1000 / solution injection amount (μL)) / 1 g, to obtain the content of the three active ingredients in Urticaria mongholica: chlorogenic acid, vitexin 2, and isovitexin.

[0019] Furthermore, the solution injection volume in step B) is 20 μL.

[0020] The third objective of this invention is to provide an application of the above-described detection method in the separation and extraction of chlorogenic acid, vitexin 2, or isovitexin from nettle.

[0021] The fourth objective of this invention is to provide an application of *Urtica montana* in the preparation of drugs for treating hyperuricemia.

[0022] The fifth objective of this invention is to provide the application of isovitexin and / or daphnetin 2 and / or chlorogenic acid in the preparation of drugs for treating hyperuricemia.

[0023] Furthermore, the isovitilloside, nephrolepis 2, and chlorogenic acid were extracted from nettle.

[0024] This invention establishes a quality identification method for *Urtica montana* used in the preparation of anti-hyperuricemia drugs, which requires the detection of the content of three anti-hyperuricemia active ingredients in the *Urtica montana* to be identified: chlorogenic acid, vitexin 2, and isovitexin. This invention detected the content of three active ingredients in 15 batches of *Urtica juncea*, and found that the total content of the three active ingredients was 1.8904 mg / g, 1.4126 mg / g, 1.3768 mg / g, and 4.6798 mg / g, respectively. It also verified that the content of the three active ingredients was related to the inhibitory effect on hyperuricemia, and that there was a certain correlation between the amounts of the three active ingredients in the same *Urtica juncea*, i.e., a high content of one active ingredient likely indicates a relatively high content of the other two. Therefore, *Urtica juncea* was set to meet at least one of the following conditions: the content of chlorogenic acid, vitexin 2, and isovitexin is greater than or equal to 2.0 mg / g, 1.5 mg / g, and 1.5 mg / g, respectively; and the total content of the three ingredients is greater than or equal to 5.0 mg / g. This indicates that *Urtica juncea* with an active ingredient content exceeding 50% can be used as a *Urtica juncea* for preparing anti-hyperuricemia drugs. All three compounds showed inhibitory effects on the key target proteins of hyperuricemia, XOD and GLUT9. Among them, isovitexin showed the best and most significant inhibitory effect on both proteins. Therefore, isovitexin, vitexin-2, and chlorogenic acid, especially isovitexin, have broad application prospects in the preparation of anti-hyperuricemia drugs. Attached Figure Description

[0025] Figure 1 The HPLC characteristic chromatograms (S1-S15) of 15 batches of *Urtica montana* and their control characteristic chromatograms (R) are shown in this invention.

[0026] Figure 2 This is the characteristic chromatogram of the *Urtica montana* reference standard and the HPLC chromatogram of the reference solution (mixed reference standard) of the present invention;

[0027] Figure 3 This is an HPLC chromatogram of the reference solution (mixed reference standard) and the test solution of *Urtica montana*.

[0028] Figure 4 The HCA curves for 15 batches of *Urtica montana* from this invention are shown below.

[0029] Figure 5 shows the PLS-DA model (A) and VIP value (B) of 15 batches of *Urtica montana* in this invention.

[0030] Figure 6 This invention relates to the interaction between chlorogenic acid, vitexin 2, and isovitexin and the target protein XOD.

[0031] Figure 7 This invention relates to the interaction between chlorogenic acid, vitexin 2, and isovitexin and the target protein GLUT9.

[0032] Figure 8 The present invention utilizes chlorogenic acid, vitexin 2, and isovitexin to inhibit XOD activity;

[0033] Figure 9 This invention investigates the effects of chlorogenic acid, vitexin-2, and isovitexin on HK2 cell survival.

[0034] Figure 10 This invention investigates the effects of chlorogenic acid, vitexin 2, and isovitexin on GLUT9 protein expression in HK2 cells. Detailed Implementation

[0035] The present invention will be described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of the present invention. The present invention is not limited to the following embodiments or examples. Any modifications and variations made without departing from the spirit of the present invention should be included within the scope of the present invention. Unless otherwise specified, the experimental materials or reagents used in the following embodiments are commercially available, and the solutions are aqueous solutions prepared with purified water.

[0036] 1. Instruments and Materials

[0037] 1.1 Instruments

[0038] Inertsustain C18 analytical column (4.6×100 mm, 5 μm, Shimadzu); KQ-5200DB ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); Shimadzu LC-2030CN high-performance liquid chromatography; PTY-323 / 423 0.0001 g electronic balance (HZ Electronics Technology Co., Ltd., Connecticut, USA); PT-124 / 85S 0.0001 g electronic balance (HZ Electronics Technology Co., Ltd., Connecticut, USA); KQ-600DB CNC ultrasonic cleaner (Kunshan Ultrasonic Instrument Co., Ltd.); BUCHI rotary evaporator (BUCHI AG, Switzerland); FW177 traditional Chinese medicine pulverizer (Tianjin Test Instrument Co., Ltd.); BJ-2CD clean bench (Shanghai Boxun Medical Biological Instrument Co., Ltd.); 3951-CO2 cell culture incubator (Thermo Scientific, USA); Epoch fully automated enzyme-linked immunosorbent assay (ELISA) reader (Biotek, USA); ChemiScope Mini 3300 chemiluminescence imaging system (model ChemiScope 3300). The Mini was purchased from Shanghai Qinxiang Scientific Instruments Co., Ltd.

[0039] 1.2 Materials and Reagents

[0040] Acetonitrile (chromatographic grade, TEDIA, USA); formic acid (chromatographic grade, Shanghai Jingchun Biochemical Technology Co., Ltd.); anhydrous ethanol (analytical grade, Tianjin Damao Chemical Reagent Factory); Wahaha purified water. Reference standards: Chlorogenic acid (batch number MUST-20032310) and isovitexin (batch number MUST-20070110) were purchased from Chengdu Mansite Biotechnology Co., Ltd.; New Zealand vitexin-2 (batch number P01F9F54173), xanthine (batch number S18024), and xanthine oxidase (XOD) (batch number S10113) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.; sodium hydroxide (batch number 10019718) and potassium dihydrogen phosphate (batch number 10017628) were purchased from Sinopharm Chemical Reagent Co., Ltd. DMEM culture medium (batch number PM150210), MEM (containing NEAA) (PM150410), HK2 cells (model CL-0109), and HK2 cell-specific culture medium (batch number CM-0109) were purchased from Wuhan Pronosei Biotechnology Co., Ltd.; DMSO (Solarbio, batch number 1213C0321), CCK8 (batch number CA1210), and BCA kit (PC0020) were purchased from Beijing Solarbio Technology Co., Ltd.; allopurinol (batch number A8003) was purchased from Sigma-Aldrich (USA). Anti-GAPDH antibody (batch number T0004) was purchased from Affinity; Anti-GLUT9 antibody (primary antibody GLUT9, batch number Ab223470) was purchased from Abcam; Goat anti-Rabbit IgG (H+L) and HRP Conjugated (batch number S004F) were purchased from Tiandeyue (Beijing) Biotechnology Co., Ltd.; PMSF (batch number ST506) was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; Prestained Protein Ladder (batch number 26616) was purchased from Fermentas; PVDF membrane (batch number IPVH00010) and ECL chemiluminescence kit (batch number WBKLS0050) were purchased from Millipore.

[0041] 1.3 Medicinal Materials

[0042] The medicinal materials used in this embodiment are all the above-ground parts of the perennial herbaceous plant *U. cannabina* L., belonging to the genus *U.* of the family Urticaceae, and were collected from Inner Mongolia. The origin and batch of the collected medicinal materials are shown in Table 1.

[0043] Table 1. Origin and Batch of *Ulva montana*

[0044] serial number batch number Origin Part Origin S1 2020071902 U. cannabina above ground Yuzhoudi Town, Keshiketeng Banner, Chifeng City S2 2020071903 U. cannabina above ground G303, Linxi County, Chifeng City S3 2020071904 U. cannabina above ground Shangxin Gacha, Saihantala Sumu, Arukorqin Banner, Chifeng City S4 2020071905 U. cannabina above ground Bayinwendusumulaga Saihua Gacha, Arukorqin Banner, Chifeng City S5 2020071906 U. cannabina above ground Saihantala Sumu, Arukorqin Banner, Chifeng City S6 2020071907 U. cannabina above ground Bayinwendusumulaga Saihua Gacha, Arukorqin Banner, Chifeng City S7 2020071908 U. cannabina above ground Bayinhushu Gacha, Hansumu Sumu, Arukorqin Banner, Chifeng City S8 2020071909 U. cannabina above ground Saihantala Sumu, Arukorqin Banner, Chifeng City S9 2020081610 U. cannabina above ground Bagajigastai, Zhenglan Banner, Xilingol League, Inner Mongolia S10 2020072611 U. cannabina above ground Bayanhushuo Town, Dongwuzhumuqin Banner, Xilingol League, Inner Mongolia S11 2020071912 U. cannabina above ground Ulijimuren Sumu, Zhalute Banner, Tongliao City S12 2020062113 U. cannabina above ground Chaolutu, Houqi, Tongliao City S13 2020062115 U. cannabina above ground Xizhelimu, Xing'an League S14 2020071916 U. cannabina above ground Unknown S15 2020071918 U. cannabina above ground Hanshan Forest Farm, Tongliao City

[0045] 2. Methods and Results

[0046] 2.1 HPLC Characteristic Chromatography of Mongolian Nettle (Urtica mongholicus)

[0047] 2.1.1 Chromatographic conditions

[0048] InertSustain C18 (5 μm, 4.6 mm × 250 mm); Mobile phase: acetonitrile (A) - 0.4% formic acid water (B); Gradient elution program: 0–10 min, 10%–15% A; 10–25 min, 15%–20% A; 25–35 min, 20%–35% A; 35–37 min, 35%–90% A; 35–42 min, 90%–90% A; 42–44 min, 90%–10% A; 44–59 min, 10%–10% A; Injection volume: 20 μL; Flow rate: 1.0 mL / min; Column temperature: 30 ℃; Wavelength: 271 nm.

[0049] 2.1.2 Preparation of the test solution

[0050] Fifteen batches of medicinal materials were pulverized, passed through a 60-mesh sieve, and 1.0 g was accurately weighed and placed in 100 mL Erlenmeyer flasks. 25 mL of 70% ethanol was added, and the samples were weighed. The samples were then extracted by ultrasonication for 30 min, the weight was adjusted, and the samples were centrifuged for 5 min at 3000 rpm / min and filtered through a 0.22 μm microporous membrane for later use.

[0051] 2.1.3 Preparation of reference solution

[0052] Accurately weigh appropriate amounts of chlorogenic acid, viicenin-2, and isovitexin reference standards, and prepare C using 70% ethanol solution. 绿原酸 =32.80 μg / mL, C 新西兰牡荆苷2 =45.12 μg / mL, C 异牡荆苷 A mixed solution with a concentration of 59.52 μg / mL was prepared to obtain the reference solution. Before analysis, the solution was filtered through a 0.22 μm microporous membrane and kept for later use.

[0053] 2.1.4 Precision Test

[0054] Take *Urtica macrantha* (batch number 2020071908) with the serial number S7, prepare it according to the method in section "2.1.2", and inject it continuously 6 times under the chromatographic conditions in section "2.1.1", and record the peak area. Using peak 2 as the reference peak, the relative peak area RSD of each common peak is less than 1.98%, indicating that the instrument precision is good.

[0055] 2.1.5 Repeatability Test

[0056] Six samples of *Urtica montana* (batch number 2020071908) with the serial number S7 were prepared according to the method described in section "2.1.2" and injected separately according to the chromatographic conditions described in section "2.1.1". The peak areas were recorded. Using peak 2 as the reference peak, the relative peak area RSD of each common peak was less than 2.03%, indicating good method repeatability.

[0057] 2.1.6 Stability Test

[0058] Take *Urtica montana* (batch number 2020071908) with batch number S7, prepare it according to the method in section "2.1.2", and inject it at 0 h, 6 h, 12 h, 14 h, 16 h, 18 h, 20 h, 22 h, and 24 h according to the chromatographic conditions in section "2.1.1", and record the peak area. Using peak 2 as the reference peak, the relative peak area RSD of each common peak is less than 2.07%, indicating that the test solution has good stability within 24 h.

[0059] 2.1.7 Generation and Similarity Evaluation of HPLC Characteristic Chromatography

[0060] Fifteen batches of *Urtica juncea* (S1–S15) were prepared according to the method described in section “2.1.2” and injected separately according to the chromatographic conditions described in section “2.1.1”. The AIA files of the recorded chromatograms were analyzed using the *Traditional Chinese Medicine Fingerprint Similarity Evaluation System (2012A)*. Using S1 as the reference chromatogram, with a time window width of 0.5 min, multi-point correction and full-peak matching of the chromatographic peaks were performed to generate characteristic chromatogram matching diagrams for the 15 batches of *Urtica juncea* and a control fingerprint chromatogram (R). See [link to relevant documentation]. Figure 1 Peak 4 showed good separation and a moderate retention time, and was selected as the reference peak. The similarity between the characteristic spectra of S1 to S15 and the control characteristic spectra was calculated, and the results were 0.754, 0.975, 0.954, 0.989, 0.97, 0.956, 0.983, 0.912, 0.946, 0.956, 0.853, 0.757, 0.967, 0.967, and 0.815, respectively. The similarity between S1 and S12 and other samples was low, but the chemical composition of each batch of samples was basically the same.

[0061] 2.1.8 Attribution and Identification of Common Peaks

[0062] Inject the sample according to the chromatographic conditions described in section "2.1.1". Compare the test solution with the reference solution. Three common peaks were identified, namely peaks 2-4, with the active ingredients being chlorogenic acid, vitexin 2, and isovitexin, respectively. The results are shown in the figure. Figure 2 , 3.

[0063] 2.2 Chemical Pattern Analysis

[0064] 2.2.1 HCA Analysis

[0065] SPSS 19.0 software was used for analysis. The between-groups linkage method was employed, with Euclidean distance as the measure, and the peak areas of the five characteristic peaks in the *Urtica montana* characteristic spectrum as the observations. Cluster analysis diagrams were plotted. (See attached diagram). Figure 4 The 15 batches of samples were divided into three categories: S1, S8, S11, S12, and S15 were in category 1; S2, S4, S5, S10, and S14 were in category 2; and S3, S6, S7, S9, S13, and S16 were in category 3, indicating that there are certain differences in the quality of the samples from each batch.

[0066] 2.2.2 PCA and PLS-DA Analysis

[0067] To further analyze the chemical components causing the differences between different batches of samples, samples S1-S15 were used as the Y variable, and the peak areas of the five components were used as the X variable. PCA and PLS-DA were performed using SIMCA 14.1 software. The results are shown in [Figure number missing]. Figure 5A , 5B Fifteen batches of samples were divided into three categories. The significance projection (VIP) value > 1.0 in the PLS-DA model was used to screen for differentially expressed components among different batches. The magnitude of the VIP value indicates the degree of contribution to sample classification. The results showed that PCA and PLS-DA yielded two components, peaks 4 and 3, indicating that these two components are the characteristic components that significantly contribute to the quality differences between different batches of samples. (See...) Figure 5A 5B. This suggests that these two components should be monitored when establishing quality standards for *Urtica montana* to reduce batch-to-batch variability. They also serve as potential *Urtica montana* Q-Markers and provide a reference for their selection.

[0068] 2.3 In vitro experimental verification

[0069] 2.3.1 Molecular docking

[0070] This study selected XOD (1FIQ) and GLUT9 (4YGB), key target proteins for hyperuricemia, as docking proteins to dock with active components in *Urtica montana* samples and screened potential *Urtica montana* Q-Markers to verify their interaction activity. The 3D structure of the xanthine oxidase target protein XOD was downloaded from the PDB database (http: / / www.rcsb.org / ). The GLUT9 target protein was used as a molecular docking target using a homology model previously established by the research group. The ligands were downloaded in .sdf format from the PubChem database (https: / / pubchem.ncbi.nlm.nih.gov / ) to obtain their 2D structures. The prepared target proteins and compounds were imported into Discovery Studio 2016 Client software, and molecular docking was performed using the Libdock module. The docking results showed that the three compounds bound to the XOD (1FIQ) and GLUT9 (4YGB) target proteins through hydrogen bonds, pi-Alkyl conjugation hydrophobic interactions, van der Waals forces, and pi-pi conjugation interactions. (See attached table). Figure 6 The highest Libidock score for the interaction with the XOD (1FIQ) target protein was chlorogenic acid (126.7), while the highest Libidock score for the interaction with the GLUT9 (4YGB) target protein was davidin 2 (148.376). The results are shown in Table 2. All three compounds exhibited interaction activity with their target proteins. Therefore, chlorogenic acid, davidin 2, and isovitexin were used as indicator components to study their in vitro inhibitory activity against key target proteins of hyperuricemia.

[0071] Table 2. Libidock score for docking of active ingredients

[0072]

[0073] 2.3.2 XOD Inhibition Activity Test

[0074] The reaction system volume for this experiment was 200 μL. All solutions used in this experiment were dissolved and prepared using DMSO. The reaction was carried out in a 96-well plate. The required sample volumes of chlorogenic acid, vitexin-2, and isovitexin were all 40 μL. The specific concentrations were calculated and prepared according to different experimental groups, so that the final concentrations of the three compounds in the system included three groups: 25 μM, 50 μM, and 100 μM, respectively. The required volume of 20 U / L XOD solution was 80 μL. After adding the required reagents to the wells according to different groups, the plates were incubated at 40 ℃ for 30 min. Then, 80 μL of 500 μmol / L xanthine substrate solution was added, and the plates were incubated at 40 ℃ for 15 min. The absorbance value was then recorded at 292 nm.

[0075] The experiment included sample groups (three samples combined with XOD and substrate, i.e., each group contained one sample, XOD, and substrate), blank groups (substrate and three samples combined separately, none containing XOD), positive control groups (containing allopurinol, XOD, and substrate), and enzyme groups (containing XOD and substrate, without sample). Each group had three replicates depending on the conditions, and the experiment was repeated three times. The final concentration of each sample was 20 mmol / L. The XOD inhibition rate was calculated using the formula: Inhibition rate (%) = [1 - (OD value of sample group - OD value of blank group) / (OD value of enzyme group - OD value of blank group)] × 100%. Figure 8 (Note: ** vs 100 μM of chlorogenic acid, *P<0.01; ### vs 100 μM of Vicenin-2, P<0.001). The experimental results showed that the inhibitory activity of the positive control drug allopurinol increased with increasing drug concentration, and the inhibition rates of each concentration group were higher than those of chlorogenic acid, vitexin-2, and isovitexin. Compared with the 100 μM chlorogenic acid group, the inhibition rate of the 100 μM vitexin-2 group was lower than that of the 100 μM chlorogenic acid group (P<0.01). Compared with the 100 μM vitexin-2 group, the inhibition rate of the 100 μM isovitexin-2 group was higher than that of the 100 μM isovitexin-2 group. μM group (P<0.01); In addition, it can be seen that the inhibition rate of chlorogenic acid, vitexin 2 and isovitexin against in vitro XOD activity increases with increasing concentration, which is concentration-dependent. Among the three compounds at the three concentrations, isovitexin has the best inhibitory effect on XOD and the effect is significant.

[0076] 2.3.3 Effects on the expression of GLUT9 protein, the HK2 uric acid transporter, in human renal tubular epithelial cells in vitro.

[0077] 2.3.3.1 Cell Culture

[0078] HK2 cells were revived in a 37 ℃ water bath and 8 mL of MEM (containing NEAA) medium (10% FBS, 1% PS) was added. Cells were cultured in T-25 cell culture flasks at 37 ℃ under saturated humidity and 5% CO2. The medium was changed every other day, and the cells reached confluence after 3-4 days of growth. Cells were passaged using a 0.25% trypsin-EDTA digestion solution. Cells were observed under an inverted phase-contrast microscope, and those that were evenly distributed across the bottom of the flask and showed good growth were selected for experimental use.

[0079] 2.3.3.2 CCK-8 assay for cell viability

[0080] To select the optimal dosing time and concentration, cell culture medium with a density of 1×10⁻⁶ was used to prepare the cells. 5 Cell suspension at 100 μL / mL was added to a 96-well plate, resulting in a cell count of 1 × 10⁶ cells per well. 4 Cells / mL (blank wells without cells) were cultured for 12 h. After culturing, the old culture medium was discarded, and 90 μL of fresh culture medium and 10 μL of compound solution (chlorogenic acid, vitexin 2, and isovitexin solution) were added to the experimental wells. The stock solution of chlorogenic acid, vitexin 2, and isovitexin was prepared with cell culture medium to a concentration of 200 mmol / L, filtered through a 0.22 μm filter membrane for sterilization, and diluted according to different concentration requirements so that the final concentrations of the compounds in the experimental wells were 10, 20, 40, 60, 80, 100, 120, 160, and 200 μM, respectively. A control group (0 μM group) was also set up. 100 μL of fresh culture medium was added to both the control and blank wells. Each group was in triplicate. After culturing in a cell culture incubator for 12 h, 24 h, and 48 h, the cells were removed, 10 μL of CCK-8 solution was added to each well, and the cells were placed in the incubator for another 2.5 h. The absorbance A value was measured at 450 nm using a microplate reader. Cell viability (%) = (A 实验孔 -A 空白孔 ) / (A 对照孔 -A 空白孔 () × 100%. According to Figure 9 Experimental results showed (Note: * vs 0 μM, *P<0.05, **P<0.01) that, compared with the control group (0 μM group), chlorogenic acid at all concentrations had no effect on the survival rate of HK2 cells after 12 h, 24 h, and 48 h of administration (P>0.05). Figure 9 -ABC); Compared with the control group (0 μM group), with the extension of the dosing time (48 h), both New Zealand vitexin-2 and isovitexin 80-200 μM groups had an inhibitory effect on the survival rate of HK2 cells (P<0.01). Figure 9-EFG-HIJ). Therefore, based on the above experimental results, the following experiments were conducted with a dosing time of 12 h, chlorogenic acid concentrations of 50, 100, and 200 μM, vitexin-2 concentrations of 20, 40, and 80 μM, and isovitexin concentrations of 25, 50, and 100 μM.

[0081] 2.3.3.3 Grouping and Dosing

[0082] The experiment was divided into 10 groups, with 3 replicates per group and 1 × 10⁶ cells per well. 4 Cells were cultured at a concentration of 1 cell / mL, with one control group and nine experimental groups. The control group was cultured in serum-free DMEM medium. The experimental groups were chlorogenic acid, vitexin 2, and isovitexin. The compounds were dissolved in DMSO to prepare a stock solution of 20 mmol / L. The final cell concentrations for the chlorogenic acid group were 50, 100, and 200 μM, for the vitexin 2 group were 20, 40, and 80 μM, and for the isovitexin group were 25, 50, and 100 μM. The drug administration time was 12 h.

[0083] 2.3.3.4 Western blot analysis of GLUT9 protein expression in HK2 cells as a uric acid transporter

[0084] After cell grouping and intervention, the cells were carefully rinsed 2-3 times with pre-cooled physiological saline, discarding any residual liquid in the plate. Then, 100 μL of 2% SDS lysis buffer was added, and the lysis buffer was carefully collected into 1.5 mL pre-cooled EP tubes. The tubes were boiled at 100 °C for 15-20 min to ensure complete cell lysis, and total cell protein was extracted. Protein concentration was then determined according to Method 2 of the BCA kit. Based on the sample concentration, all samples were prepared into equal volumes and masses. After electrophoresis on a 10% SDS-PAGE gel, the protein was transferred to a PVDF membrane at 250 mA (60 min). After blocking with 3% skim milk powder for 1 h, the PVDF membrane was incubated overnight at 4 °C with primary antibody GLUT9 (1:1000) and internal control GAPDH (1:5000). The PVDF membrane was washed 5 times with TBST solution for 5 min each time. The PVDF membrane was then reacted with HRP-labeled secondary antibody (1:3000), gently shaken on a shaker at room temperature for 1 h, and then washed 5 times with TBST solution for 5 min each time. ECL development was performed, and the experimental results were analyzed using a gel imaging analyzer. Figure 10The results showed that, compared with the control group, all doses of chlorogenic acid, 2, 40 and 80 μM of vitexin, and all doses of isovitexin all downregulated the expression of the HK2 uric acid transporter GLUT9 protein. Among the three, isovitexin showed the best inhibitory effect, and the inhibitory effect of isovitexin was significant.

[0085] 2.4 Determination of the content of multiple components of *Urtica montana* in combating hyperuricemia

[0086] 2.4.1 Examination of Linear Relationships

[0087] Take the reference solution (C) 绿原酸 =32.80 μg / mL, C 新西兰牡荆苷2 =45.12 μg / mL, C 异牡荆苷 =59.52 μg / mL), the solution was injected into the HPLC system under the liquid chromatography conditions described in 2.1.1, with injection volumes of 1, 2, 4, 8, and 16 μL, respectively. The chromatographic peak areas were recorded. A graph was plotted with the solute injection amount (μg, which is the solute concentration in the reference solution multiplied by the injection volume) on the x-axis and the chromatographic peak area (determined by HPLC) on the y-axis, yielding the regression equation Y. 绿原酸 =46825X + 2475.8, R 2 =0.9996; Y 新西兰牡荆苷2 =108755X+22211,R 2 =0.9996, Y 异牡荆苷 =171447X + 2872.4, R 2 =1.

[0088] 2.4.2 Precision Test

[0089] Same as item 2.1.4.

[0090] 2.4.3 Repeatability Test

[0091] Same as item 2.1.5.

[0092] 2.4.4 Stability Test

[0093] Same as item 2.1.6.

[0094] 2.4.5 Spiking Recovery Test

[0095] Six portions of *Urtica montana* sample (batch number 2020071904), each 0.5 g, were taken. The reference standards for the three components to be added were calculated based on approximately 50% of the sample. The test solutions were prepared according to the method described in section "2.1.2," and then injected and determined under the chromatographic conditions described in section "2.1.1." The peak areas were recorded, and the recoveries of each component were calculated. The results showed that the average recoveries of chlorogenic acid, vitexin-2, and isovitexin were 109.61%, 92.07%, and 100.01%, respectively, with RSDs of 3.24%, 1.02%, and 2.21%, respectively, meeting the recovery limits and indicating good accuracy of the method.

[0096] 2.4.6 Sample Measurement Results

[0097] Take 15 batches (S1~S15) of *Urtica montana*, prepare test solutions according to section “2.1.2”, and inject 20 μL of each solution sequentially under the chromatographic conditions of “2.1.1”. Record the peak areas. Substitute the regression equations for the three components in “2.4.1” into the peak area Y to calculate the solute injection amount (μg) of the three components in the sample. Calculate the content of the three components in the sample based on the volume of the test solution prepared under section “2.1.2” and the injection amount. The content calculation formula is as follows:

[0098] Content (mg / g) = (Solute injection volume (μg) / 1000) × (Test solution volume (mL) × 1000 / Solution injection volume (μL)) / 1 g

[0099] Since the volume of the test solution is 25 mL and the injection volume is 20 μL, then:

[0100] Content (mg / g) = Solute injection volume (μg) × 1.25

[0101] The results are shown in Table 3. The contents of chlorogenic acid, vitexin-2, and isovitexin in 15 batches of *Urtica juncea* medicinal materials were 0.1434–4.2346 mg / g, 0.1186–2.5812 mg / g, and 0.0569–3.6700 mg / g, respectively. The contents of each component fluctuated greatly, but there was a correlation among the three components. That is, in the same batch, either all three components were at a high level or all three components were at a low level. This shows that the contents of each of the three components and the sum of the contents of the three components have an impact on the quality of *Urtica juncea*. The average content of the three components individually and in total was 1.8904 mg / g, 1.4126 mg / g, 1.3768 mg / g, and 4.6798 mg / g, respectively. When the content of chlorogenic acid, vitexin 2, and isovitexin was set to be greater than or equal to 2.0 mg / g, 1.5 mg / g, and 1.5 mg / g, respectively, and the total content of the three components was greater than or equal to 5.0 mg / g, the content of active ingredients was high, exceeding 50% of that in *Urtica juncea*. The cost-effectiveness was significantly improved, demonstrating good practical and application value.

[0102] Table 3. Contents of chlorogenic acid, vitexin 2 and isovitexin in batches of *Urtica juncea* ( )

[0103]

[0104] 3. Discussion

[0105] 3.1 Selection and Analysis of the Q-Marker for *Urtica montana*

[0106] This study identified three components from the characteristic spectrum of *Urtica montana* using a comparison with reference standards, ensuring the specificity, traceability, and measurability of the Q-Marker. Chemometric analysis suggested that vitexin-2 and isovitexin should be given special attention, providing a reference for Q-Marker screening. Previous studies have shown that vitexin-2 has anti-inflammatory, antispasmodic, and hepatoprotective effects. Isovitexin possesses anti-inflammatory, antioxidant, antitumor, and hepatoprotective biological activities, basically meeting the principles for Q-Marker screening.

[0107] This invention verifies the bioactivity of *Urtica montana* Q-Marker from three perspectives. First, potential Q-Markers were screened using molecular docking technology. The Q-Markers were docked with uric acid-lowering target proteins XOD and GLUT9. The results showed that chlorogenic acid, vitexin 2, and isovitexin were bound together through hydrogen bonds, hydrophobic bonds, and π-π bonds, exhibiting good activity. Second, in vitro enzyme inhibition experiments were conducted, binding the Q-Marker to xanthine oxidase (XOD) in vitro. The results showed that chlorogenic acid, vitexin 2, and isovitexin all exhibited in vitro inhibitory activity against XOD. Finally, cell experiments verified that *Urtica montana* Q-Markers had no effect on HK2 cell survival while downregulating the HK2 uric acid transporter GLUT9 protein.

[0108] 3.2 Selection of Extraction Method

[0109] This experiment investigated the effects of different solvents (methanol, 70% methanol, 50% methanol, 95% ethanol, 70% ethanol, 50% ethanol), extraction methods (ultrasound and reflux), solvent volume (25 mL, 50 mL, 100 mL), and extraction time (15 min, 30 min, 60 min) on the chromatograms. The extraction method of ultrasonic extraction with 25 mL of 70% ethanol for 30 min was determined to be the best method.

[0110] 3.3 Investigation of Chromatographic Conditions

[0111] The *Urtica montana* sample solution was compared between 254 nm and 271 nm using a UV detector. Considering the maximum absorption wavelength of each component, chromatographic baseline, peak shape, and resolution, 271 nm was selected as the detection wavelength for the three Q-Markers. This study investigated the effects of methanol-water and acetonitrile-0.4% formic acid aqueous solution as mobile phases on the separation performance and baseline of the three Q-Markers. Ultimately, acetonitrile-0.4% formic acid aqueous solution was determined to be the optimal mobile phase, providing the best separation performance and a stable baseline.

[0112] 4. Conclusion

[0113] This invention establishes HPLC characteristic chromatograms of the Mongolian medicine *Urtica juncea* and methods for determining the content of three chemical components. Combined with chemometric analysis, the results show significant differences in the content or proportion of components in *Urtica juncea* from different origins. Therefore, in formulating quality standards for *Urtica juncea* medicinal materials, the origin factors should be fully considered; appropriate specificity, integrity, and measurability indicators should be selected from characteristic chromatograms and indicative components. This study provides a more scientific and reasonable innovative approach and method for Q-Marker research in *Urtica juncea*. To improve the quality control level of *Urtica juncea*, the contents of chlorogenic acid, vitexin-2, and isovitexin in 15 batches of *Urtica juncea* medicinal materials were 0.1434–4.2346 mg / g, 0.1186–2.5812 mg / g, and 0.0569–3.6700 mg / g, respectively. The average contents of the three components individually and the total contents of the three components were 1.8904 mg / g, 1.4126 mg / g, 1.3768 mg / g, and 4.6798 mg / g, respectively. The limits for chlorogenic acid, vitexin-2, and isovitexin were set to be greater than or equal to 2.0 mg / g, 1.5 mg / g, and 1.5 mg / g, respectively, and the total contents of the three components were set to be greater than or equal to 5.0 mg / g. At a concentration of mg / g, the active ingredient content is high, exceeding 50% of that in *Urtica juncea*, significantly improving efficiency at the same cost and demonstrating good practical and application value. Based on the inhibitory effects of the three compounds on the key target proteins XOD and GLUT9 of hyperuricemia, with the inhibitory effect increasing with concentration, and the significant inhibitory effect of isovitilloside on both proteins, it is evident that isovitilloside, vitexin-2, and chlorogenic acid, especially isovitilloside, have broad application prospects in the preparation of anti-hyperuricemia drugs.

[0114] The conventional techniques and solutions not described in detail in the above embodiments are all well known in the art, and therefore will not be elaborated upon here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A method for quality identification of *Urtica montana* used in the preparation of drugs for treating hyperuricemia, characterized in that, Includes the following steps: 1) Detect the content of three active ingredients for treating hyperuricemia in the nettle plant to be identified: chlorogenic acid, vitexin 2, and isovitexin; 2) Based on the content obtained in step 1), when the content of chlorogenic acid is greater than or equal to 2.0 mg / g and / or the content of vitexin 2 is greater than or equal to 1.5 mg / g and / or the content of isovitexin is greater than or equal to 1.5 mg / g, and the total content of chlorogenic acid, vitexin 2 and isovitexin is greater than or equal to 5.0 mg / g, the *Urtica montana* to be identified is *Urtica montana* that can be used to prepare anti-hyperuricemia drugs.

2. The identification method as described in claim 1, characterized in that, The method for detecting the content of three anti-hyperuricemia active ingredients—chlorogenic acid, vitexin 2, and isovitexin—in *Urtica juncea* includes the following steps: A) Crush nettle, pass through a 60-mesh sieve, accurately weigh 1.0 g, add 25 mL of 70% ethanol, weigh, extract ultrasonically for 30 min, make up the weight, centrifuge for 5 min at 3000 rpm / min, filter through a 0.22 μm microporous membrane to obtain the test solution, the volume of the test solution is 25 mL; B) The test solution prepared in step A) was analyzed by high performance liquid chromatography (HPLC) to obtain the peak areas of three active ingredients: chlorogenic acid, vitexin 2, and isovitexin. The HPLC conditions were as follows: InertSustain C18, 5 μm, 4.6 mm × 250 mm; mobile phase: acetonitrile A - 0.4% formic acid water B; gradient elution program: 0–10 min, 10%–15% A; 10–25 min, 15%–20% A; 25–35 min, 20%–35% A; 35–37 min, 35%–90% A; 35–42 min, 90%–90% A; 42–44 min, 90%–10% A; 44–59 min, 10%–10% A; injection volume: 1–20 μL; flow rate: 1.0 mL / min; column temperature: 30 ℃; wavelength: 271 nm. C) Substitute the peak area value obtained in step B) into the equation Y. 绿原酸 =46825X + 2475.8, Y 新西兰牡荆苷2 =108755X+22211 and Y 异牡荆苷 =171447X+2872.4, where Y is the peak area and X is the solute injection amount (μg), thus obtaining the solute injection amount X of the three active ingredients; D) Substitute the solute injection amounts of the three active ingredients obtained in step C) into the formula to calculate the content (mg / g) = (solute injection amount (μg) / 1000) × (test solution volume (mL) × 1000 / solution injection amount (μL)) / 1 g, to obtain the content of the three active ingredients chlorogenic acid, vitexin 2 and isovitexin in the nettle to be identified.

3. The identification method as described in claim 2, characterized in that, The solution injection volume in step B) is 20 μL.

4. A method for detecting the content of an anti-hyperuricemia active ingredient in *Urtica juncea*, characterized in that, The active ingredients are chlorogenic acid, vitexin 2, and isovitexin, and the method includes the following steps: A) Crush nettle, pass through a 60-mesh sieve, accurately weigh 1.0 g, add 25 mL of 70% ethanol, weigh, extract ultrasonically for 30 min, make up the weight, centrifuge for 5 min at 3000 rpm / min, filter through a 0.22 μm microporous membrane to obtain the test solution, the volume of the test solution is 25 mL; B) The test solution prepared in step A) was analyzed by high performance liquid chromatography (HPLC) to obtain the peak areas of three active ingredients: chlorogenic acid, vitexin 2, and isovitexin. The HPLC conditions were as follows: InertSustain C18, 5 μm, 4.6 mm × 250 mm; mobile phase: acetonitrile A - 0.4% formic acid water B; gradient elution program: 0–10 min, 10%–15% A; 10–25 min, 15%–20% A; 25–35 min, 20%–35% A; 35–37 min, 35%–90% A; 35–42 min, 90%–90% A; 42–44 min, 90%–10% A; 44–59 min, 10%–10% A; injection volume: 1–20 μL; flow rate: 1.0 mL / min; column temperature: 30 ℃; wavelength: 271 nm. C) Substitute the peak area values ​​obtained in step B) into the regression equation Y of the three active ingredients. 绿原酸 =46825X + 2475.8, Y 新西兰牡荆苷2 =108755X+22211 and Y 异牡荆苷 =171447X+2872.4, where Y is the peak area and X is the solute injection amount (μg), thus the solute injection amount X of the three active ingredients can be calculated; D) Substitute the solute injection amounts of the three active ingredients obtained in step C) into the formula to calculate the content (mg / g) = (solute injection amount (μg) / 1000) × (test solution volume (mL) × 1000 / solution injection amount (μL)) / 1 g, to obtain the content of the three active ingredients in Urticaria mongholica: chlorogenic acid, vitexin 2, and isovitexin.

5. The detection method as described in claim 4, characterized in that, The solution injection volume in step B) is 20 μL.

6. The application of the detection method according to claim 4 or 5 in the separation and extraction of chlorogenic acid, vitexin 2 or isovitexin from nettle.

7. Application of *Urtica montana* in the preparation of drugs for treating hyperuricemia.

8. Application of isovitexin and / or vitexin-2 and / or chlorogenic acid in the preparation of antihyperuricemia drugs.

9. The application as described in claim 8, characterized in that, The isovitinoside, nephrolene 2, and chlorogenic acid were extracted from nettle.

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