Application of dracocephalum moldavica extract in medicine for treating plateau polycythemia

Abstract

The invention discloses an application of dracocephalum moldavica extract in a medicine for treating plateau polycythemia, and belongs to the technical field of medicines. The dracocephalum moldavica extract disclosed by the invention can be used for inhibiting the excessive proliferation of red blood cells in altitude polycythemia (HAPC) and improving the physiological expression of hypoxia-induced hyperviscosity, and has a potential protection effect on relieving abnormal blood rheology caused by an altitude environment; the DML can effectively relieve the red blood cell excessive proliferation and multi-system pathological imbalance of the HAPC by adjusting iron homeostasis, relieving oxidative stress, inhibiting inflammatory response, improving metabolic reprogramming and intervening multiple target pathways such as an HIF-1 alpha-erythroid differentiation axis. Various flavonoid and phenolic acid components entering blood can form a core pharmacodynamic material basis, and an important scientific basis is provided for modern development of DML for plateau related diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the application of a *Cymbidium ensifolium* extract in a drug for high-altitude polycythemia. Background Technology

[0002] High-altitude polycythemia (HAPC) is a chronic mountain sickness caused by prolonged exposure to low-pressure, low-oxygen environments. Its main clinical features are abnormally elevated peripheral blood red blood cell count (RBC), hemoglobin (Hb), and hematocrit (HCT). Excessive erythrocyte proliferation increases blood viscosity, leading to microcirculatory disturbances, further exacerbating tissue hypoxia, and potentially causing thrombosis, pulmonary edema, right ventricular failure, and even death. Current research indicates that the pathogenesis of HAPC is not limited to compensatory proliferation of the erythroid lineage but also includes multiple systemic pathophysiological changes induced by prolonged low-pressure, low-oxygen exposure. These changes include multidimensional abnormalities such as oxidative stress imbalance, activation of the inflammatory cascade, iron metabolism disorders, bone marrow hematopoietic microenvironment remodeling, alterations in cellular energy metabolism, and vascular regulatory dysfunction. These factors interact to form a complex network regulatory system.

[0003] Under hypoxic conditions at high altitudes, HIF-2α exhibits enhanced stability, and the expression of erythropoietin (EPO) and its receptor (EPOR) is significantly upregulated. This, in turn, activates key erythroid transcription factors such as GATA1 through the JAK2 / STAT5 signaling pathway, accelerating erythropoiesis. This mechanism has been validated at the genetic and molecular levels in chronic mountain sickness (CMS) and high-altitude acute pancreatitis (HAPC). Notably, some HAPC patients exhibit persistent activation of the HIF pathway independent of EPO. Furthermore, the increased generation of reactive oxygen species (ROS) in hypoxic environments, coupled with decreased activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), leads to oxidative stress, activating the NF-κB / NLRP3 pathway and promoting the release of inflammatory factors such as IL-6 and TNF-α, thus creating a vicious positive feedback loop. In terms of metabolic reprogramming, HAPC is characterized by impaired lipid β-oxidation, disordered riboflavin metabolism, activation of the purine-uric acid pathway, and increased dependence of erythrocytes on glycolysis. These changes have been confirmed in animal models and metabolomics studies of patients.

[0004] Recent studies have revealed that HAPC can be viewed as a continuous process transitioning from physiological compensation to pathological transformation. Its main characteristics include: elevated thresholds in erythroid responses in individuals carrying EPAS1 / HIF-2α gene mutations; abnormalities in erythrocyte membrane proteins (such as Band 3), accompanied by weakened phagocytic function of splenic macrophages; and iron metabolism disorders leading to ineffective erythropoiesis. Despite significant progress in multi-omics research at the mechanistic level, no specific targeted therapy for HAPC has yet emerged. Existing clinical interventions, such as phlebotomy (which may exacerbate iron deficiency), angiotensin-converting enzyme inhibitors (ACEIs, with unstable efficacy), and HIF inhibitors (with off-target effects), have failed to fundamentally correct the core pathological link of the imbalance between erythroid proliferation and clearance.

[0005] Fragrant Blue Orchid ( Dracocephalum moldavica L., DML) is a commonly used traditional Chinese medicine. Its single-herb preparation, "Yixin Badiran Gibuya Granules", has been clinically proven to have significant efficacy in treating various cardiovascular diseases. However, there is no evidence that Xiangqinglan is used to treat high-altitude polycythemia. Summary of the Invention

[0006] This invention proposes the application of *Cymbidium ensifolium* extract in drugs for high-altitude polycythemia.

[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: Furthermore, *Xiangqinglan* refers to the dried above-ground part of *Xiangqinglan*.

[0008] Furthermore, the drug comprises orchid extract and pharmaceutically acceptable excipients, wherein orchid or orchid extract is the main component or active ingredient of the drug.

[0009] Furthermore, the *Cymbidium ensifolium* extract includes at least one or more of the following extracted from *Cymbidium ensifolium*: 6-{[2-(3,4-dihydroxyphenyl)-5-hydroxy-4-oxo-4H-chromogen-7-yl]oxy}-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid, N-maleicotryptophan, apigenin-7-O-β-D-glucuronide, juniper acetate, fulvic acid, daidzein, genistein, farnesin, tuberous acid, N-3-(benzoyloxy)-2-hydroxypropyl-β-D-glucuronide, acetyl-D-tryptophan, rosmarinic acid, glycyrrhizin, luteolin, and styracin.

[0010] Furthermore, the dosage form of the drug is selected from tablets, capsules, granules, powders, pills, or powders.

[0011] This invention also discloses a method for revealing the active ingredients and multi-target mechanism of action of *Cymbidium faberi* in combating high-altitude polycythemia. The method includes... (1) Based on serum pharmacology, a systematic analysis of the chemical components of DML and its blood-entry prototype components was conducted to clarify its potential pharmacodynamic material basis; (2) A rat model of high altitude polycythemia (HAPC) and a K562 cell erythroid differentiation system were constructed. The intervention effect of DML on HAPC and the multi-target mechanism of action were systematically evaluated by proteomics and non-targeted metabolomics.

[0012] The beneficial effects of the application of *Cymbidium ensifolium* extract in medications for high-altitude polycythemia are as follows: (1) The extract of the present invention can inhibit the excessive proliferation of erythrocytes in high-altitude polycythemia (HAPC), improve the physiological manifestations of hyperviscosity induced by hypoxia, and has a potential protective effect on alleviating blood rheological abnormalities caused by high-altitude environment. (2) The Chinese tallow tree and its extracts can significantly protect against the development of high altitude polycythemia (HAPC) by reducing the level of inflammatory factors and alleviating oxidative stress. They can effectively alleviate the spleen stress-induced hematopoietic abnormalities and kidney hypoxia damage induced by high altitude polycythemia (HAPC), showing that they have potential therapeutic value in maintaining blood homeostasis and protecting hypoxic target organs.

[0013] (3) DML can effectively reduce erythrocyte overproliferation and multi-system pathological imbalance in HAPC by regulating iron homeostasis, alleviating oxidative stress, inhibiting inflammatory response, improving metabolic reprogramming, and intervening in multiple target pathways such as the HIF-1α-erythroid differentiation axis. The various flavonoids and phenolic acids that enter the bloodstream may constitute the core pharmacodynamic material basis, providing important scientific basis for the modern development of DML for high-altitude related diseases. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 These are the BPC diagrams of each group of samples in Example 1. A is the BPC diagram of DML; B is the BPC diagram of HAPC model serum + DML; C is the BPC diagram of HAPC model group serum; D is the BPC diagram of DML administration group serum.

[0016] Figure 2 This is a BPC peak analysis chromatogram of DML (Dysphorus styracifolius) under positive and negative ion modes.

[0017] Figure 3 The effect of DML (Dysplasia spp.) on erythrocyte parameters in HAPC rats, where A represents RBC (10⁻¹⁰). 12 / L); B is HGB (g / L); C is HCT (%). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0018] Figure 4 The effects of DML (Dysplasia spp.) on serum oxidative stress and inflammatory factor levels in HAPC rats were investigated. A represents SOD; B represents GSH-Px; C represents MDA; D represents IL-6; E represents IFN-γ; F represents TNF-α; G represents VEGF; and H represents IL-1β. * P < 0.05, ** P <0.01, *** P < 0.001, **** P < 0.0001.

[0019] Figure 5 This describes the protective effect of DML on the spleen and kidneys of HAPC rats.

[0020] Figure 6 These are the results of rat proteomics analysis. A shows the PCA analysis results; B shows the differential gene volcano plot between MG and CG groups; C shows the differential gene volcano plot between HDML and MG groups; D shows the Venn analysis of differentially expressed proteins between groups; E shows the clustering heatmap analysis of differentially expressed proteins between groups; n=3.

[0021] Figure 7 Functional enrichment analysis of differentially expressed proteins GO and KEGG among groups (n=3). A represents MG. vs GO analysis of differentially expressed proteins between CG groups. B represents HDML. vs GO analysis of differentially expressed proteins between MG groups. C represents MG. vs Functional enrichment analysis of differentially expressed protein KEGG between CG groups. D represents HDML. vs Functional enrichment analysis of the differentially expressed protein KEGG between MG groups.

[0022] Figure 8This is an analysis of serum metabolites (n=6) from rats in each group, obtained using non-targeted metabolomics technology. A shows the PCA analysis under positive and negative ion modes; B shows the MG analysis. vs CG shows the intergroup OPLA-DA and S-Plot analysis; C represents LDML. vs MG group intergroup OPLA-DA and S-Plot analysis; D represents MDML. vs MG intergroup OPLA-DA and S-Plot analysis; E represents HDML. vs MG group intergroup OPLA-DA and S-Plot analysis plots; F is the Venn analysis plot.

[0023] Figure 9 Screening of differentially expressed metabolites among groups and KEGG functional enrichment analysis. A represents MG. vs CG differential metabolite volcano plot. B represents LDML. vs Volcano plot of MG differential metabolites. C represents MDML. vs Volcano plot of differential metabolites in MG. D is the volcano plot of differential metabolites between HDML and MG. E is the volcano plot of MG. vs Functional enrichment analysis of the differential metabolite KEGG in CG. F represents LDML. vs Functional enrichment analysis of the differential metabolite KEGG in MG. G represents MDML. vs Functional enrichment analysis of the differential metabolite KEGG in MG. H represents HDML. vs Functional enrichment analysis of the differential metabolite KEGG from MG.

[0024] Figure 10 The effects of DML intervention on key proteins in HAPC rats are shown, where A is DPP IV; B is Hepcidin; C is NPY; D is PON1; E is PRDX5; n=6; * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

[0025] Figure 11 The effects of DML on the expression of EPO, LDH, and HIF-1α in the kidneys of HAPC rats were investigated. In the figure, A represents DPPⅣ; B represents Hepcidin; C represents NPY; and D represents PON1, with N=6. * P < 0.05, ** P < 0.01, *** P< 0.001, **** P < 0.0001.

[0026] Figure 12 Effects of DML on erythroid differentiation of K562 cells. A represents the cell positivity rate detected by Hemin assay; B represents the screening of fetal bovine serum proportions using the CCK-8 assay; and C represents the concentration of serum containing the drug.

[0027] Figure 13 The mechanism by which DML inhibits erythroid differentiation of K562 cells is shown in the following graphs: A is a Western blotting graph of protein expression; B is a statistical graph of BAX protein expression; C is a statistical graph of BCL-2 protein expression; D is a statistical graph of EPO-R protein expression; E is a statistical graph of GATA-1 protein expression; F is a statistical graph of HIF-1α protein expression; and G is a statistical graph of PCNA protein expression. Detailed Implementation

[0028] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1

[0030] Application of *Cymbidium ensifolium* extract in drugs for high-altitude polycythemia.

[0031] The fragrant blue orchid described in this invention refers to the dried aerial part of the fragrant blue orchid. The dried aerial part of the fragrant blue orchid is extracted with water or organic solvents to obtain a fragrant blue orchid extract, wherein the organic solvents include organic alcohols, ethers, esters, or alkali extraction.

[0032] The *Cymbidium ensifolium* extract of this invention comprises at least one or more of the following extracted from *Cymbidium ensifolium*: 6-{[2-(3,4-dihydroxyphenyl)-5-hydroxy-4-oxo-4H-chromogen-7-yl]oxy}-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid, N-maleicotryptophan, apigenin-7-O-β-D-glucuronide, juniper acetate, fulvic acid, daidzein, genistein, farnesin, tuberous acid, N-3-(benzoyloxy)-2-hydroxypropyl-β-D-glucuronide, acetyl-D-tryptophan, rosmarinic acid, glycyrrhizin, luteolin, and styracin.

[0033] In this embodiment, the Dracocephalum moldavica extract at least includes all extracts of acacetin, tubulosin, oroxylin A-7-O-β-D-glucuronide, luteolin, 2',3,5,6',7-tetrahydroxyflavanone, apigenin-7-glucuronide, rosmarinic acid, N-acetyl-D-tryptophan, 3-(benzoic acid-2-hydroxypropyl-β-D-glucopyranosyluronide), daidzin, Tuberonic acid glucoside, N-malonyltryptophan extracted from Dracocephalum moldavica.

[0034] The extraction method of the Dracocephalum moldavica extract of the present invention is obtained by an existing conventional method.

[0035] The drug of the present invention includes a Dracocephalum moldavica extract and pharmaceutically acceptable excipients, and the drug takes Dracocephalum moldavica or the Dracocephalum moldavica extract as the main ingredient or active ingredient. The drug dosage form is selected from tablets, capsules, granules, infusion, pills or powders.

[0036] Example 2

[0037] Effect detection of Dracocephalum moldavica extract on high altitude polycythemia This embodiment mainly reveals the active ingredients of Dracocephalum moldavica against high altitude polycythemia, and systematically analyzes the chemical components of Dracocephalum moldavica (DML) and its prototype components in blood based on serum pharmacochemistry to clarify its potential pharmacodynamic substance basis; 2.1 Animals and cells 52 SPF-grade male SD rats (6-8 weeks old, body weight 180-220 g) were provided by the Animal Center of Xinjiang Medical University [License number: SYXK (Xin) 2023-0002]. The animals were housed in a standard environment (22±2°C, 40±10% humidity, 12h light-dark cycle), with free access to food and water, and the experiment began after 1 week of adaptation. K562 cells were kindly provided by the Central Laboratory of Xinjiang Medical University.

[0038] 2.2 Analysis of chemical components and serum prototype components of Dracocephalum moldavica 2.2.1 Preparation of serum samples containing drugs Twelve male SD rats (180-220 g) were selected and subjected to continuous low-pressure, low-oxygen exposure in a hypobaric chamber (simulating an altitude of 5000 m) for 30 days. They were then randomly divided into an HAPC group and a DML administration group (n=6). The administration group received 400 mg / kg of *Illicium verum* via gavage, while the HAPC group received an equal volume of solvent, for 7 consecutive days. Two hours after the last administration, the rats were anesthetized with sodium pentobarbital (40 mg / kg, ip), and 5 mL of blood was collected via the abdominal aorta. Serum was separated by centrifugation at 4 °C (3000 × g, 15 min) to obtain HAPC model serum and DML-administered rat serum; both were stored at -80 °C.

[0039] 2.2.2 Determination of chemical composition and serum prototype components of *Cymbidium faberi* After grinding the *Cymbidium faberi* sample into a fine powder, 100 mg was extracted with 1 mL of 70% methanol by sonication for 90 min, and the supernatant was collected by centrifugation. For the serum sample, 200 μL was added to 800 μL of methanol to precipitate the protein, and the supernatant was collected by centrifugation. All supernatants were filtered through a 0.22 μm filter membrane, lyophilized, and reconstituted with 40% methanol.

[0040] UHPLC-Q Exactive HF-X system, ACQUITY UPLC HSS T3 column (2.1 × 100 mm, 1.8 μm); mobile phase A: 0.1% formic acid in water, B: 0.1% formic acid in acetonitrile; flow rate: 0.3 mL / min, column temperature: 35 °C; injection volume: 5 μL. Full MS-dd-MS was used. 2 The mode is for simultaneous collection of positive and negative ions (m / z 90–1300), with a spray voltage of ±3.8 / 3.0 kV and an ion source temperature of 320 °C.

[0041] After the raw data was converted to mzXML by ProteoWizard, XCMS performed peak extraction, alignment, and baseline correction; using MS 1 Quality error <15 ppm, MS 2 A matching score >0.7 is used as the identification threshold, and confirmation is achieved by comparison with a standard spectral library. Each batch includes blank, QC, and sequence balancing injections; each sample group is repeated 3–5 times to evaluate method repeatability.

[0042] 2.3 Establishment and Drug Administration of HAPC Rat Model Forty male SD rats (200-220 g) were randomly selected, and 32 rats were placed in a hypobaric hypoxic chamber (simulating altitude 5000 m; atmospheric pressure 54.0 kPa, oxygen partial pressure 11.3 kPa; 18-24 °C; 40-60% humidity) for 30 consecutive days to establish a HAPC model. They were then randomly divided into four groups (n=8): a model group (4 mL / kg / d of physiological saline), a high / medium / low dose group of *Xiangqinglan* (400 / 200 / 100 mg / kg / d) treated by gavage for 28 days, and the remaining eight rats were fed normoxic conditions as a normal control group.

[0043] 2.4 Complete Blood Count (CBC) EDTA-K2 whole blood was tested for RBC, HGB, and HCT using a fully automated blood analyzer, and the testing was performed according to the manufacturer's quality control procedures.

[0044] 2.5 Pathological examination Spleen and kidney were removed after euthanasia and divided into two parts: ① Fixed in 4% paraformaldehyde for 24 h, routinely embedded in paraffin, sectioned (4 μm), and stained with hematoxylin and eosin (HE); ② The remaining tissue blocks were flash-frozen in liquid nitrogen and stored at -80°C for later use. The morphology of splenic follicles, red pulp, and glomeruli / tubules was observed under a light microscope.

[0045] 2.6 Serum ELISA detection After centrifugation (3000 rpm, 10 min), serum was stored at -80 °C, avoiding repeated freeze-thaw cycles. SOD, MDA, GSH-Px, IL-1β, and IL-6 were measured using the ELISA kit according to the manufacturer's instructions.

[0046] 2.7 Serum proteomics analysis Samples were diluted with PBS and protein was measured by BCA; DTT / IAM was used for reductive alkylation and acetone precipitation; trypsin (1:50 - 1:100, w / w) was used for digestion overnight at 37 °C; C18 was used for desalting and lyophilization, and the samples were reconstituted with 0.1% formic acid.

[0047] NanoElute separation, time-to-time Pro 2 Mass spectrometry was performed using DDA / DIA-PASEF (m / z 100-1700). Data were retrieved from spectral libraries using DIA-NN and then predicted using deep learning, with the free flow rate (FDR) controlled at 1%. Differentially expressed proteins were statistically screened and then enriched using GO and KEGG.

[0048] 2.8 Serum metabolomics analysis Proteins were precipitated from serum using methanol:acetonitrile (1:1, v / v), incubated at -20 °C for 30 min, and centrifuged (12,000 g, 10 min) to collect the supernatant. UHPLC-HRMS was used for acquisition in positive / negative ion mode (m / z 70-1000). XCMS was used for peak extraction, alignment, denoising, and batch effect correction. QC samples were used to monitor instrument drift and for signal correction based on QC-RLSC. PCA and OPLS-DA were then performed, and multiple comparisons were conducted using permutation tests (n=200) to assess the risk of model overfitting. Differential metabolites were screened using VIP thresholds and statistical tests.

[0049] 2.9 Screening and Validation of Key Proteins 2.9.1 Molecular docking 3D structures of key targets were obtained from the PDB database, and water molecules and ligands in the proteins were removed using Pymol software. The SDF structures of the active ingredients obtained from the PubChem database, along with the pretreated proteins, were imported into Autodock 1.5.7 software for modification, followed by molecular docking and calculation of docking binding energies. Finally, Pymol software was used for visualization.

[0050] 2.9.2 Validation of Key Proteins Key proteins such as DPP-IV, Hepcidin, PRDX5, NPY, and PON1 were selected for ELISA validation. Standard curves, blanks, and quality control samples were set for each batch, and the results were calculated according to the instructions and normalized appropriately.

[0051] 2.9.3 Measurement of EPO, LDH, and HIF-1α in renal tissue Kidney tissue was homogenized with lysis buffer containing protease inhibitors, centrifuged (12,000 rpm, 10 min, 4 °C), and the supernatant was collected. The supernatant was quantified using a rat EPO and LDH ELISA kit.

[0052] Kidney proteins were extracted and quantified using BCA. An equal volume of protein was loaded onto a membrane, separated by SDS-PAGE, and transferred to a PVDF membrane. After blocking (5% skim milk powder, 1 h, room temperature), HIF-1α (1:2000) primary antibody was added (4 °C, 18 h). The membrane was washed and incubated with goat anti-rabbit secondary antibody (1 h, room temperature). ECL staining was performed, and images were acquired using a gel imaging system. β-actin was used as an internal control, and band grayscale analysis and normalization were performed using ImageJ.

[0053] 2.10 Regulatory effect of DML on erythroid differentiation of K562 cells 2.10.1 Cell Culture K562 cells were routinely cultured in IMDM medium (containing 10% FBS and 1% penicillin and streptomycin), incubated at 37 °C with saturated humidity at 5% CO2, and the passage density was controlled at [missing information]. Cells in the logarithmic growth phase were used for experiments.

[0054] 2.10.2 Benzidine staining differentiation rate Hemin (6.25 mg / mL, dissolved in DMSO, stored at 4 °C protected from light) and 0.4% benzidine staining solution (12% acetic acid) should be prepared fresh before use. Resuspend K562 cells in... / mL, seeded in 6-well plates and induced with 40 μM Hemin. After washing with PBS according to the procedure, add benzidine staining solution for 3 min, add 1 μL of 30% H2O2 and react for 5 min. Observe and photograph the slides; count ≥200 cells, calculate the positive rate (%) = positive / total number × 100%, repeat 3 times and take the average.

[0055] 2.10.3 Screening of FBS Concentration Basal media containing 0%, 2%, 4%, 6%, 8%, and 10% FBS were prepared, corresponding to uninduced and Hemin-induced conditions, respectively. Each group had 6 replicates. After 48 h of hypoxic culture, CCK-8 was added for 2 h of incubation, and the absorbance at 450 nm was measured to select the FBS concentration that maintained the best activity.

[0056] 2.10.4 Screening of DML-containing serum concentrations Different treatments were set up, including V0 (200 μL FBS), V1 (200 μL drug-containing serum), V2 (200 μL drug-containing serum evaporated under nitrogen + 200 μL FBS reconstituted), V3 (400 μL drug-containing serum evaporated under nitrogen + 200 μL FBS reconstituted), V4 (600 μL drug-containing serum evaporated under nitrogen + 200 μL FBS reconstituted), and V5 (800 μL drug-containing serum evaporated under nitrogen + 200 μL FBS reconstituted). 8% final concentration of drug-containing serum was added to IMDM induction medium containing 2% FBS, and induction was performed with 40 μM Hemin. After 48 h of hypoxic culture, the absorbance at 450 nm was measured, and the relative survival rate was calculated.

[0057] Relative survival rate (%) = .

[0058] 2.10.5 Western blotting Cellular proteins were extracted and quantified using BCA. After protein denaturation, equal volumes of protein were loaded onto a PVDF membrane, separated by SDS-PAGE gel electrophoresis, and electroporated onto the membrane. The PVDF membrane was blocked (5% skim milk powder, 1 h, room temperature) and then incubated with primary antibodies (4℃, 18 h). Antibody dilutions were: anti-β-actin (1:5000), anti-EPO-R (1:1000), anti-HIF-1α (1:1000), anti-BCL-2 (1:2000), anti-GATA1 (1:1000), anti-PCNA (1:2000), and anti-BAX (1:5000). After primary antibody incubation and washing, the membrane was incubated with the corresponding horseradish peroxidase-conjugated secondary antibody (1 h, room temperature). The membrane was then developed using ECL and images were acquired using a gel imaging system. ImageJ was used for band grayscale analysis, and the relative protein expression level was calculated using β-actin as a reference.

[0059] 2.11 Statistical Analysis Data are expressed as mean ± standard error (Mean ± SEM). Normality was assessed using the Shapiro–Wilk test; homogeneity of variance was assessed using the Levene test. Independent samples t-tests were used for comparisons between two groups that met both normality and homogeneity of variance. One-way ANOVA was used for comparisons among multiple groups, with Tukey post-hoc tests performed when necessary. Non-parametric tests were used when normality and homogeneity of variance were not met. The statistical significance threshold was set to two-tailed. P <0.05.

[0060] Metabolite profiles of groups a, b, c, and d were compared and analyzed using UHPLC-HRMS to screen for potentially bioactive compounds. The groups are as follows: a) DML, a type of orchid extract; b. Serum from the HAPC model; c. HAPC serum + DML in vitro mixing system (HAPC model serum and DML of *Cymbidium goeringii* were mixed in vitro). d. Rat serum after DML administration.

[0061] The resulting BPC graph is as follows Figure 1 As shown. In the positive and negative ion BPC chromatograms of DML, the peaks with higher abundance were confirmed by chromatographic shape and analyzed by secondary mass spectrometry, and were numbered and labeled sequentially. Figure 2 The obtained components are shown in Table 1.

[0062]

[0063]

[0064] As shown in Table 1, 37 major chemical components were identified in *Cymbidium faberi*, including: carboxylic acids and their derivatives, flavonoids and their aglycones (acacetin, luteolin, oroxyloside), cinnamic acid and its derivatives, fatty acyl groups and glycerophospholipids, isoflavones, amino acid derivatives and organic oxides, etc. By comparing the qualitative and quantitative analysis results of three groups of samples—fragrant orchid, HAPC model serum, and model-treated serum—15 candidate components for blood entry were screened out from the 37 identified chromatographic peaks. These included: 6-{[2-(3,4-dihydroxyphenyl)-5-hydroxy-4-oxo-4H-chromogenen-7-yl]oxy}-3,4,5-trihydroxyoxacyclohexane-2-carboxylic acid, N-maleicotryptophan, apigenin-7-O-β-D-glucuronide, safflower acetate, fulvic acid, daidzein, genistein, farnesin, tuberous acid, N-3-(benzoyloxy)-2-hydroxypropyl-β-D-glucuronide, acetyl-D-tryptophan, rosmarinic acid, glycyrrhizin, luteolin, and citronellol. The peak areas of the above compounds in the DML administration group were all ≥3-fold higher than those in the model group, meeting the screening criteria for "blood-entering components" in serum medicinal chemistry. These components may constitute the key pharmacodynamic material basis for DML intervention in HAPC.

[0065] Example 3 The effect of *Cymbidium faberi* on high-altitude polycythemia (HAPC) This embodiment mainly constructs a rat model of high-altitude polycythemia (HAPC) and a K562 cell erythroid differentiation system. Through proteomics and non-targeted metabolomics technologies, it systematically evaluates the intervention effect of DML on HAPC and its multi-target mechanism of action.

[0066] 3.1 Effects of *Cymbidium ensifolium* intervention on erythrocyte parameters in HAPC rats Red blood cell parameters are the most crucial and direct hematological indicators for evaluating the occurrence and progression of HAPC. Therefore, detecting changes in RBC, Hb, and HCT can reflect whether the model has been successfully established and the intervention effect of DML on abnormal red blood cell proliferation. Results showed that compared with the normal control group (CG), the levels of RBC, Hb, and HCT in the model group (MG) rats were significantly increased (…). P The value <0.01 indicates that continuous hypoxia exposure successfully induced high-altitude erythrocyte proliferation characteristics, and the model establishment is reliable.

[0067] After intervention with different doses of DML, compared with the model group, the levels of RBC, Hb, and HCT in rats all decreased to varying degrees. P <0.05. Among them, the intervention effect was more significant in the medium and high dose groups, and showed a certain dose-dependent trend ( Figure 3In the HAPC rat model, DML significantly reversed excessive erythroid proliferation, especially in the medium and high dose groups. Specifically, after DML intervention, the increasing trends of hemoglobin (Hb), red blood cell count (RBC), and hematocrit (HCT) were significantly inhibited, improving the physiological manifestations of hypoxia-induced hyperviscosity syndrome and potentially protecting against hemorheological abnormalities caused by high-altitude environments.

[0068] 3.2 Effects of *Cymbidium ensifolium* on serum oxidative stress and inflammatory markers in HAPC rats Oxidative stress and inflammatory response are important driving factors for abnormal erythrocyte proliferation induced by high-altitude hypoxia. Therefore, analyzing serum antioxidant enzyme activity and inflammatory factor levels can further assess whether DML plays a role in regulating pathological mechanisms.

[0069] Regarding oxidative stress, compared with CG, the activities of SOD and GSH-Px in the serum of MG rats were significantly decreased ( P <0.01), while MDA levels were significantly elevated ( P <0.01 indicates significant oxidative stress damage in the HAPC model rats. After DML intervention, the activities of SOD and GSH-Px in the medium- and high-dose groups of rats were significantly increased ( P <0.05%, MDA content decreased significantly ( P The value of DML is <0.05, indicating that DML can effectively improve the oxidative stress state of HAPC rats.

[0070] Regarding inflammatory factors, the serum levels of IL-6, IFN-γ, TNF-α, VEGF, and IL-1β in the MG group rats were significantly increased. P <0.01, indicating a significant systemic inflammatory response. After DML intervention, the levels of the above-mentioned inflammatory factors in rats of all dose groups decreased to varying degrees, with the medium and high dose groups showing the most significant decrease. P <0.05). These results suggest that *Cymbidium faberi* can significantly protect against the development and progression of HAPC by reducing inflammatory cytokine levels and alleviating oxidative stress. Figure 4 ).

[0071] 3.3 Protective effect of DML on spleen and kidney tissues in HAPC rats Given that the spleen reflects the state of hematopoietic regulation and the kidneys are sensitive target organs for hypoxia damage, changes in the histological morphology of these two organs can be used to assess the pathological process of HAPC and the effectiveness of drug intervention.

[0072] In the spleen tissue, MG showed significant structural damage, manifested as blurred boundaries between the white and red pulp, marked dilation of the splenic sinuses, and aggregation of erythrocytes and granulocytes at different developmental stages, suggesting that the spleen was in a state of stress-induced hematopoiesis and blood retention. After DML intervention, the tissue structure gradually recovered, the white and red pulp became clearly distinguishable, and no abnormal pathological changes were observed. Figure 5 A) indicates that DML can effectively alleviate splenic hematopoietic disorders caused by hypoxia.

[0073] Renal pathology results showed significant damage in the MG group, including glomerular capillary congestion (green arrow), focal tubular atrophy and narrowing or disappearance of lumens, epithelial cell cytoplasmic vacuolation (black arrow), accompanied by brownish-yellow granular deposition (yellow arrow) and tubular dilation (red arrow), indicating severe hypoxic damage and oxidative stress. With increasing DML dosage, this damage gradually improved: the LDML group still showed focal congestion and mild edema, but the renal tissue structure remained basically intact; the pathological manifestations in the MDML group were significantly reduced; and the HDML group only had mild residual vascular congestion (…). Figure 5 B).

[0074] In summary, histological evidence suggests that *Cymbidium faberi* can dose-dependently alleviate HAPC-induced splenic stress-induced hematopoietic abnormalities and renal hypoxic injury, indicating its potential therapeutic value in maintaining blood homeostasis and protecting hypoxic target organs.

[0075] Venn analysis results are as follows Figure 6 As shown in Figure C, there are 135 common differentially expressed proteins among the comparison groups. Figure 6 (D) suggests that DML intervention has a wide-ranging impact on multiple protein regulatory pathways.

[0076] To reveal the biological significance of differentially expressed proteins, GO and KEGG functional enrichment analyses were performed. GO annotation results showed that differentially expressed proteins in the MG group were mainly enriched in core biological processes such as cellular process, biological regulation, and regulation of biological process; at the molecular functional level, they mainly involved functional categories such as binding, catalytic activity, and molecular function regulator activity. Figure 7 After DML intervention, the GO enrichment characteristics of differentially expressed proteins were consistent with those of the model group, and were still mainly concentrated in the aforementioned key biological processes and molecular functions.

[0077] In the MG model, KEGG enrichment analysis of differentially expressed proteins showed significant enrichment of pathways related to cell junction damage, cytoskeleton remodeling, immune activation, and inflammatory responses, including tight junction, Focal adhesion, regulation of actin cytoskeleton, phagosome, and T cell receptor signaling pathway. This suggests that significant cellular structural damage, enhanced immune response, and oxidative stress imbalance occur during HAPC. After DML intervention, the characteristics of KEGG-enriched pathways changed significantly, with differentially expressed proteins mainly concentrated in pathways related to lipid metabolism regulation, energy homeostasis maintenance, and immune regulation, such as the PPAR signaling pathway, adipocytokine signaling pathway, regulation of lipolysis in adipocytes, and multiple signaling pathways related to T cell regulation and infection response.

[0078] 3.4 Non-targeted metabolomics analysis reveals the regulatory role of DML in metabolic abnormalities. Considering the complementary nature of protein regulation and metabolic changes, we adopted a method using positive and negative ions ( Non-targeted metabolomics detection was performed on CG, MG, and the three-dose DML intervention group under the ) mode, and multidimensional statistical analysis was conducted. After data preprocessing and standardization, QC samples showed high clustering in the score plot, indicating that the overall detection process had good stability and repeatability.

[0079] PCA results in both positive and negative ion modes showed a significant separation between the CG and MG groups. Figure 8 A) suggests that high-altitude hypoxia led to widespread metabolic disturbances. After DML treatment, the metabolic characteristics of all three dose groups approached those of the CG group, reflecting a dose-dependent regulatory effect of DML on metabolic abnormalities. To further improve the inter-group discrimination, we constructed an OPLS-DA model based on the PCA results. The model's R... 2 Y and Q 2 The values ​​reached 0.994 and 0.978 respectively, and no overfitting was observed in 200 permutation tests. Figure 8 B) indicates that the model has reliable fitting and predictive capabilities. The OPLS-DA score plot further shows that each DML dose group is clearly separated from the MG group ( Figure 8 CE). Feature variables selected based on S-Plot plots (VIP>1, P <0.05) Far from the origin, suggesting that these metabolites may be key metabolic markers for distinguishing DML from MG and are involved in their main pharmacodynamic processes.

[0080] Volcano plots were drawn based on the difference screening criteria (Fold change ≥ 1.5, P < 0.05). Figure 9 The results showed significant metabolic changes between the MG and CG groups. In the MG group, 712 metabolites were significantly upregulated, while 565 were significantly downregulated in the CG group. KEGG enrichment analysis indicated that these differentially expressed metabolites were mainly concentrated in key metabolic pathways such as metabololic pathways, biosynthesis of cofactors, Bile secretion, Riboflavin metabolism, and ABC transporters, covering multiple biological processes including energy metabolism, bile acid metabolism, and transmembrane transport, suggesting that HAPC-related metabolic disorders involve a broad and multi-level metabolic network. After drug intervention, 1011, 1147, and 1167 differentially expressed metabolites were screened in the high, medium, and low dose DML groups, respectively. These metabolites were mainly enriched in pathways such as metabololic pathways, biosynthesis of cofactors, and Bile secretion, indicating that DML acts on HAPC-related metabolic abnormalities through a multi-target regulatory mechanism. Figure 9 EH).

[0081] To further identify the core metabolic targets of DML, Venn analysis was performed on the differential metabolites among the three dose groups. Figure 8F). The results showed that 416 metabolites underwent significant changes in all three groups, and these metabolites may represent key metabolic markers of DML's anti-HAPC effect. In the MG group, multiple lysophospholipids and membrane lipids (such as LPE (18:2 / 0:0), LPC (14:0 / 0:0), PC (16:0 / 2:0), and PS (20:4 / 20:4)) were significantly decreased, suggesting that cell membrane lipid metabolism was inhibited. Acylcarnitines related to fatty acid transport and β-oxidation (such as Stearoylcarnitine and Carnitine C14:0) were also significantly reduced, indicating restricted energy metabolism. At the same time, cholic acid and taurine were significantly increased, suggesting enhanced hepatobiliary metabolism and hypoxic stress response. After DML intervention, membrane lipid and acylcarnitine levels generally rebounded, while bile acid and taurine levels declined, indicating that DML can effectively correct the metabolic imbalance caused by hypoxia. The MG group exhibited typical metabolic remodeling characteristics, primarily manifested by a significant decrease in glycerophospholipids, lysophospholipids, and acylcarnitines, reflecting disturbances in lipid homeostasis and energy metabolism. Simultaneously, the increase in bile acids and taurine further suggests enhanced hepatobiliary metabolism and hypoxic stress. After DML intervention, these metabolite changes showed a trend towards normalization across different dose groups, particularly in the recovery of membrane lipids and acylcarnitines, indicating a significant regulatory role of DML in metabolic homeostasis. DML also demonstrated its potential in restoring lipid metabolism and energy homeostasis by reversing restricted fatty acid oxidation and inhibiting the abnormal increases in bile acids and taurine. These results provide solid metabolic evidence for the pharmacodynamic mechanism of DML, further supporting its ability to restore HAPC-induced metabolic abnormalities by regulating lipid metabolism, energy homeostasis, and bile acid metabolism.

[0082] 3.5 Screening and validation of key target proteins using combined proteomics and metabolomics analysis Based on the combined proteomic and metabolomic results, the molecular characteristics of the MG model can be summarized as follows: impaired cell junctions and cytoskeleton structure, activation of immune-inflammatory pathways, accompanied by dysregulation of membrane lipid homeostasis and restricted fatty acid energy metabolism. After DML intervention, the focus of pathway enrichment shifted to lipid metabolism and energy homeostasis maintenance (such as PPAR / lipolysis / adipokine signaling) and immune regulation, consistent with the trend of increased membrane lipids and acylcarnitine and decreased stress-related metabolites in the metabolome, suggesting that DML may improve HAPC-related pathological changes through a synergistic mechanism of "metabolic remodeling-immune regulation". Therefore, six key proteins were screened for further validation: Hepcidin (master regulator of iron homeostasis), PRDX5 (mitochondrial antioxidant protein), PON1 (antioxidant and anti-inflammatory enzyme), DPPⅣ (hematopoietic microenvironment regulatory enzyme), NPY (vasoactive neuropeptide), and HIF-1α (hypoxia transcription regulator).

[0083] To assess the potential interactions between DML-infused components and the aforementioned potential target proteins, we further performed molecular docking analysis. Lower binding energies indicate a more stable ligand-receptor complex conformation and a stronger binding likelihood. The results showed that the binding energies of all seven components to the six target proteins were below -5.0 kcal / mol, suggesting good binding potential. Specifically, DPP Ⅳ showed a higher overall binding affinity to PON1, while NPY showed a relatively weaker affinity; the optimal binding conformations were Glycitin-DPP Ⅳ, Oroxyloside-PON1, Glycitin-HIF-1α, and Rosmarinic acid-PRDX5, respectively. Considering the multi-target binding performance, Glycitin and Oroxyloside exhibited lower binding energies at multiple target sites, suggesting they may be important active components in DML.

[0084] ELISA validation results further support the above screening ( Figure 10 Compared with the CG group, the serum levels of DPPⅣ, Hepcidin, and NPY in MG rats were significantly increased, while PON1 and PRDX5 were significantly decreased, indicating that iron homeostasis, antioxidant activity, and hematopoietic regulation were all disrupted. After DML intervention, the above abnormalities were significantly corrected: the levels of DPPⅣ, Hepcidin, and NPY decreased to varying degrees, while PON1 and PRDX5 were upregulated in a dose-dependent manner. The overall trend was highly consistent with molecular docking and omics predictions.

[0085] Proteomics results further revealed the multi-target regulatory mechanism of DML. The occurrence of HAPC involves multiple pathological processes, including iron homeostasis disturbance, enhanced oxidative stress, activation of inflammatory responses, alteration of the hematopoietic microenvironment, and persistent activation of hypoxia signaling pathways. Related proteins and metabolites play key roles in these processes. Hepcidin, as a core regulator of iron metabolism, typically leads to iron retention and impaired iron utilization when elevated, further enhancing erythroid stress and erythropoietin-stimulating signals, and is a significant driver of excessive erythrocyte proliferation. PRDX5 and PON1 in the antioxidant system are responsible for scavenging peroxides and maintaining redox stability of lipids and proteins; decreased expression of these enzymes leads to the accumulation of reactive oxygen species and exacerbates oxidative damage. SOD and GSH-Px are major endogenous antioxidant enzymes; their decrease indicates impaired ROS scavenging capacity, while elevated MDA, as an end product of lipid peroxidation, reflects oxidative damage to the cell membrane. Elevated levels of inflammatory cytokines IL-6, TNF-α, IFN-γ, and IL-1β in HAPC promote erythropoietin signaling, angiogenesis, and stress responses in the hematopoietic microenvironment, while VEGF plays a crucial role in hypoxia-driven vascular remodeling. Abnormal expression of DPP IV and NPY, related to hematopoiesis and neurohumoral regulation, reflects disruption of the hematopoietic microenvironment and neurovascular regulatory network, further amplifying erythroid stress responses. In the hypoxia signaling pathway, HIF-1α, as a core oxygen-sensing factor, drives EPO release and enhanced glycolysis upon activation, while elevated LDH indicates increased metabolic stress and damage caused by tissue hypoxia. These proteins and metabolites constitute key nodes in the complex pathological network of HAPC.

[0086] 3.6 Regulation of hypoxia-related molecules in renal tissue of HAPC rats by DML To verify the key molecules and their regulatory directions obtained from multi-omics analysis, we focused on the hypoxia-response pathway, which is consistently pointed to by multi-omics, and examined the changes of HIF-1α and its downstream EPO and LDH in kidney tissue to confirm the regulatory trend of DML at the level of overall oxygen metabolism and tissue damage.

[0087] Western blot results showed that HIF-1α expression was significantly increased in the kidneys of MG rats compared with CG rats. After intervention with different doses of DML, HIF-1α expression decreased to varying degrees, with the medium and high dose groups showing more significant decreases, suggesting that DML can inhibit hypoxic response signals in the kidneys of model animals. Figure 11 A, Figure 11 B).

[0088] ELISA testing further showed that the levels of EPO and LDH in the kidney tissue of the MG group were significantly elevated. P<0.01). After DML intervention, the above indicators decreased to varying degrees, especially in the high-dose group, suggesting that DML has a corrective effect on hypoxia-induced erythema and damage markers. Figure 11 C, 11D).

[0089] 3.7 Regulatory effect of DML on erythroid differentiation of K562 cells 3.7.1 Results of Hemin concentration screening To obtain a stable and controllable in vitro erythroid differentiation model for subsequent mechanism validation using DML-containing serum, this study first evaluated the effects of Hemin concentration and induction time on the erythroid differentiation capacity of K562 cells. The results showed that Hemin-induced erythroid differentiation exhibited clear dose- and time-dependent effects. At 48 h, the positivity rates of benzidine in all concentration groups were significantly increased (20%, 19.67%, 22.17%, 35.17%, and 34.17%, respectively). By 72 h, the positivity rate further increased, indicating that extending the induction time also enhanced the erythroid differentiation effect.

[0090] However, when the Hemin concentration exceeded 40 μM, the cell condition deteriorated significantly, exhibiting adverse reactions such as decreased growth viability. Therefore, we ultimately selected a Hemin concentration of 40 μM and an induction time of 48 h as the optimal conditions for subsequent experiments. Figure 12 A) 3.7.2 Results of screening for fetal bovine serum concentration To ensure good survival of K562 cells in subsequent Hemin-induced erythroid differentiation experiments, we evaluated the effect of different fetal bovine serum (FBS) concentrations (0%, 2%, 4%, 6%, 8%, 10%) on cell viability. The results showed that the proliferation capacity of K562 cells gradually increased with increasing FBS concentration. 2% FBS was sufficient to maintain the basic growth status of the cells. Figure 12 (B) Therefore, 2% FBS was used as the culture medium supplement concentration in the subsequent drug-containing serum intervention experiment.

[0091] 3.7.3 Results of screening for drug-containing serum concentrations in DML To ensure that DML-containing serum could function effectively while maintaining K562 cell viability in in vitro experiments, we screened serums with different treatment concentrations. The experimental results are as follows: Figure 12 C showed that cell activity increased with increasing DML-containing serum concentration. Since V1 concentration significantly increased K562 cell activity, V1-containing serum was selected for subsequent studies.

[0092] 3.7.4 Western blot validation Western blot results showed that hypoxia stimulation significantly upregulated the pro-apoptotic protein BAX (Bax). P <0.05), while DML treatment significantly reduced its expression, suggesting that DML alleviates hypoxia-induced apoptosis stress. Correspondingly, the anti-apoptotic protein BCL-2 decreased under hypoxic conditions ( P <0.001), but significantly increased under hypoxia + DML conditions ( P The value <0.0001 indicates that DML has a survival-protective effect on K562 cells in a hypoxic environment.

[0093] In the regulation related to erythroid differentiation ( Figure 13 Hypoxia significantly upregulated EPO-R and GATA1 ( P <0.01, consistent with the pattern that hypoxia promotes erythroid differentiation; while DML treatment inhibited the upregulation of these two proteins ( P <0.05), suggesting that DML can inhibit hypoxia-induced erythroid differentiation signals.

[0094] Regarding the hypoxia response pathway, HIF-1α is significantly elevated under hypoxic conditions. P <0.0001), while DML intervention significantly reduced its expression ( P <0.001 indicates that DML may weaken the hypoxic response of erythroid cells by inhibiting HIF-1α stability or expression. Furthermore, the proliferation-related protein PCNA significantly rebounded under hypoxia + DML conditions ( P <0.05), suggesting that DML can partially restore the proliferation capacity of K562 cells in a hypoxic environment.

[0095] In the rat model of this invention, significantly elevated Hepcidin levels indicated disruption of iron homeostasis and enhanced erythroid stress; downregulation of PRDX5 and PON1, along with decreased SOD and GSH-Px activities accompanied by increased MDA, indicated oxidative stress and oxidative damage; elevated levels of IL-6, TNF-α, IFN-γ, IL-1β, and VEGF indicated activation of systemic inflammation and vascular stress, while abnormally elevated DPP IV and NPY further pointed to disruptions in the hematopoietic microenvironment and neurovascular regulation. Simultaneously, high expression of HIF-1α, EPO, and LDH revealed persistent hypoxic response, excessive erythropoietin signaling, and tissue damage in the model animals. These changes are consistent with lipid, cofactor, and bile acid metabolism disorders in metabolomics, indicating that the pathological changes in HAPC are characterized by multi-system imbalances. Subsequent DML intervention reversed these pathological abnormalities at multiple levels. The decrease in hepcidin suggests the restoration of iron homeostasis; the increase in PRDX5 and PON1, along with the enhanced activities of SOD and GSH-Px and the decrease in MDA, collectively indicate the re-establishment of antioxidant capacity. The reduction in inflammatory factors and VEGF indicates the suppression of systemic inflammation and vascular stress, while the downregulation of DPP IV and NPY suggests a gradual return to stability in the hematopoietic microenvironment and neural regulation. Simultaneously, DML significantly inhibited the increase of HIF-1α and its downstream EPO and LDH, suggesting its ability to block hypoxia signaling pathways and alleviate tissue damage. Furthermore, the recovery trend of lipid and cofactor metabolism in metabolomics is highly consistent with the improvement in antioxidant and anti-inflammatory indicators, further supporting the systemic regulatory role of DML in the oxidation-inflammation-metabolic network.

[0096] Serum pharmacochemistry analysis provided direct evidence for the pharmacodynamic basis of DML. Using UHPLC-HRMS to identify 37 major chemical components in *Dalbergia latifolia*, we screened out 15 potentially active components. These components were significantly elevated in blood and are known to have antioxidant, anti-inflammatory, microcirculation-improving, and energy metabolism-regulating effects. These chemical components are consistent with changes in the proteome and metabolome of DML, further supporting the idea that DML exerts its comprehensive protective effect through the synergistic action of multiple components.

[0097] K562 cell experiments also validated the mechanism of action of DML at the cellular level. Under hypoxic conditions, the stabilization of HIF-1α leads to the upregulation of EPO-R and GATA1, thereby promoting erythroid differentiation. DML significantly inhibited HIF-1α and its downstream differentiation signals, suggesting that it may intervene in the erythroid maturation process by regulating the stability or activity of HIF-1α. Furthermore, DML promoted BCL-2 expression, reduced BAX, and restored PCNA levels under hypoxic conditions, indicating its protective role in alleviating hypoxic stress and maintaining cell proliferation. This is highly consistent with the effect of DML in inhibiting abnormal erythrocyte proliferation in vivo.

[0098] Based on multi-level evidence from both in vivo and in vitro studies, hypoxia induces erythroid hyperproliferation and multi-pathway imbalance by triggering oxidative stress, inflammatory responses, metabolic reprogramming, and HIF-1α stabilization. DML, through multi-target synergistic action, reverses these abnormalities, restores metabolic homeostasis, hematopoietic and immune balance, and cellular energy status, thereby inhibiting the progression of HAPC.

[0099] DML can effectively alleviate erythrocyte overproliferation and multi-system pathological imbalance in high-altitude clinical practice by regulating iron homeostasis, alleviating oxidative stress, inhibiting inflammatory responses, improving metabolic reprogramming, and intervening in multiple pathways such as the HIF-1α-erythroid differentiation axis. Its various flavonoids and phenolic acids that enter the bloodstream may constitute the core pharmacodynamic material basis, providing important scientific evidence for the modern development of DML for high-altitude-related diseases.

[0100] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0101] Finally, it should be noted that the embodiments disclosed in this invention are merely preferred embodiments of this invention and are only used to illustrate the technical solutions of this invention, not to limit it. Although this invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this invention.

Claims

1. Application of a Dracocephalum moldavica extract in a medicine for high altitude polycythemia.

2. Use according to claim 1, characterized in that, The Dracocephalum moldavica is a dry above-ground part of Dracocephalum moldavica.

3. Use according to claim 1, characterized in that, The medicine comprises the Dracocephalum moldavica extract and pharmaceutically acceptable adjuvants, and the medicine takes the Dracocephalum moldavica or the Dracocephalum moldavica extract as a main component or an effective component.

4. Use according to claim 1, characterized in that, The Dracocephalum moldavica extract at least comprises any one or several of 6-{[2-(3,4-dihydroxyphenyl)-5-hydroxy-4-oxo-4H-chromen-7-yl]oxy}-3,4,5-trihydroxyoxane-2-carboxylic acid, N-maleoyltryptophan, apigenin-7-O-β-D-glucuronide, thujyl acetate, furilic acid, daidzein glycoside, fargesin, acacetin, tuberostemonoside, N-3-(benzoyloxy)-2-hydroxypropyl-β-D-glucuronide, acetyl-D-tryptophan, rosmarinic acid, licochalcone A, luteolin, and baiyunoside extracted from the Dracocephalum moldavica.

5. The use according to claim 1, characterized in that, The dosage form of the medicine is selected from tablets, capsules, granules, powders, pills, or powders.

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