OBJECTIVES: To assess the efficacy and safety of Tiaozhou Ziyin (TZZY) recipe for treatment of premature ovarian insufficiency (POI) and explore the possible mechanisms. METHODS: We used bioinformatics analyses and network pharmacology to identify the main active ingredients in TZZY recipe and their core targets, which were verified by Western blotting. We tested the efficacy and safety of the recipe in 60 POI patients, who were randomized into control group (n=30) with Femoston treatment and TZZY group (n=30) with additional TZZY recipe treatment for 3 menstrual cycles. RESULTS: The core active ingredients of TZZY recipe included kaempferol, β-sitosterol, luteolin, and quercetin. The core targets included SRC, TP53, STAT3, PIK3CA, and MAPK3, which were involved in positive regulation of cell movement and protein phosphorylation, the cancer pathways and the PI3K-Akt signaling pathway. Molecular docking showed that the core active ingredients had good binding ability with the core targets. In female rat models of POI, TZZY recipe treatment significantly up-regulated ovarian expressions of p-PI3K and p-Akt proteins. In the clinical trial, treatment with Femoston and Femoston plus TZZY recipe both significantly increased E₂ levels and reduced FSH and LH levels and Kupperman scores of the patients, and the combined treatment produced significantly stronger effects. Both treatments increased the number of antral follicles of the patients, but the combined treatment also significantly increased the levels of AMH. CONCLUSIONS The therapeutic mechanism of TZZY recipe for POI involves multiple active ingredients, multiple therapeutic targets and multiple pathways, and activating the PI3K /Akt pathway is one of its main mechanisms of action, to improve ovarian reserve function, alleviate clinical symptoms, and enhance clinical efficacy in POI patients.
Female ; Primary Ovarian Insufficiency/drug therapy* ; Humans ; Drugs, Chinese Herbal/therapeutic use* ; Animals ; Rats ; Molecular Docking Simulation ; Signal Transduction ; Sitosterols/therapeutic use* ; Kaempferols/therapeutic use*

This study investigated the mechanism by which kaempferol(KAE) affected intervertebral disc degeneration(IDD) through the p38 mitogen-activated protein kinase(p38 MAPK) signaling pathway. Rats were randomly divided into five groups: control group, model group, low-dose KAE group, medium-dose KAE group, and high-dose KAE group. An IDD model was established by needle puncture of the caudal intervertebral discs. Four weeks post-surgery, the rats were administered KAE via gavage for 8 consecutive weeks. Magnetic resonance imaging(MRI) was performed, and samples were collected. In vitro, an inflammation model of nucleus pulposus cells(NPCs) induced by tumor necrosis factor-alpha(TNF-α) was constructed. Anisomycin was used to activate the p38 MAPK signaling pathway. NPCs were divided into blank, model, KAE, agonist, and KAE + agonist groups. After 1 day of treatment, cell proliferation activity was assessed using the CCK-8. Protein expression levels were determined by Western blot, and mRNA expression was measured by real-time quantitative polymerase chain reaction. Cell apoptosis was detected by TUNEL staining, and immunofluorescence staining was used to detect type Ⅱ collagen and matrix metalloproteinase 3(MMP3). In vivo results indicated significant improvement in the degree of IDD in the treatment groups compared to the model group, with the medium-dose group showing more pronounced therapeutic effects than the low-and high-dose groups. In vitro results demonstrated that KAE treatment significantly enhanced NPC proliferation activity, down-regulated the expression levels of Bcl-2-associated X protein(Bax), interleukin-6(IL-6), interleukin-17A(IL-17A), MMP3, and a disintegrin and metalloproteinase with thrombospondin motifs 5, and inhibited the phosphorylation of p38 MAPK pathway-related proteins. Activation of the p38 MAPK signaling pathway by anisomycin reduced the therapeutic effects of KAE. The study concluded that KAE could improve the proliferation activity of degenerated NPCs, reduce inflammation levels, and slow the progression of IDD in rats, and the mechanism was likely related to the regulation of the p38 MAPK signaling pathway.
Animals ; p38 Mitogen-Activated Protein Kinases/genetics* ; Kaempferols/pharmacology* ; Intervertebral Disc Degeneration/genetics* ; Rats ; Rats, Sprague-Dawley ; Male ; Apoptosis/drug effects* ; Cell Proliferation/drug effects* ; Nucleus Pulposus/drug effects* ; Signal Transduction/drug effects* ; Humans ; MAP Kinase Signaling System/drug effects*

OBJECTIVE: To investigate the mechanism of the effect of Astragalus membranaceus (A. membranaceus) on lung adenocarcinoma at the molecular level to elucidate the specific targets according to the network pharmacology approach. METHODS: The active components of A. membranaceus and their potential targets were collected from the Traditional Chinese Medicine Systems Pharmacology Database. Lung adenocarcinoma-associated genes were acquired based on GeneCards, Online Mendelian Inheritance in Man (OMIM), PharmGKB, and Therapeutic Targets databases. The PI3K/AKT signaling pathway-related genes were obtained using Reactome portal. Networks of "ingredient-target" and "ingredient-target-pathway-disease" were constructed using the Cytoscape3.6.0 software. The relationships among targets were analyzed according protein-protein interaction (PPI) network. Finally, molecular docking was applied to construct the binding conformation between active ingredients and core targets. Cell counting kit 8 (CCK8) and Western blot assays were performed to determine the mechanism of the key ingredient of A. membranaceus. RESULTS: A total of 20 active components and their 329 targets, and 7,501 lung adenocarcinoma-related genes and 130 PI3K/AKT signaling pathway-related genes were obtained. According to Venn diagram and PPI network analysis, 2 mainly active ingredients, including kaempferol and quercetin, and 6 core targets, including TP53, MAPK1, EGF, AKT1, ERBB2, and EGFR, were identified. The two important active ingredients of A. membranaceus, kaempferol and quercetin, exert the therapeutic effect in lung adenocarcinoma partly by acting on the 6 core targets (TP53, MAPK1, EGF, AKT1, ERBB2, and EGFR) of PI3K/AKT signaling pathway. Expressions of potential targets in lung adenocarcinoma and normal samples were analyzed by using UALCAN portal and found that ERBB2 was overexpressed in lung adenocarcinoma tissues and upregulation of it correlated with clinicopathological characteristics. Finally, quercetin repressed viabilities of lung adenocarcinoma cells by targeting ERBB2 on PI3K/AKT signaling confirmed by CCK8 and Western blot. CONCLUSION Our finding unraveled that an active ingredient of A. membranaceus, quercetin, significantly inhibited the lung adenocarcinoma cells proliferation by repressing ERBB2 level and inactivating the PI3K/AKT signaling pathway.
Humans ; Astragalus propinquus ; Kaempferols ; Network Pharmacology ; Phosphatidylinositol 3-Kinases ; Proto-Oncogene Proteins c-akt ; Epidermal Growth Factor ; Molecular Docking Simulation ; Quercetin ; Adenocarcinoma of Lung ; Lung Neoplasms ; Signal Transduction ; ErbB Receptors ; Drugs, Chinese Herbal

The flavonoids in Panax notoginseng were qualitatively analyzed by ultra-high performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q-TOF-MS), and the content of three main flavonoids in P. notoginseng of different specifications and grades collected from different habitats was determined by HPLC-DAD. Flavonoids and anthocyanins were analyzed by UPLC-Q-TOF-MS/MS in the positive and negative ion modes, respectively. Twelve flavonoid glycosides and one anthocyanin glycoside in P. notoginseng were identified, but no flavonoid aglycones were detected. Among them, 12 compounds were identified in the underground part of P. notoginseng for the first time and eight compounds were first reported in this plant. Moreover, six and four compounds were identified in the Panax genus and the Araliaceae family for the first time, respectively. A method for simultaneous determination of three flavonoids in P. notoginseng was established by HPLC-DAD. The content of flavonoids in 721 P. notoginseng samples of 124 specifications and grades collected from 20 different habitats was simultaneously determined. Among three flavonoids determined, the content of quercetin-3-O-(2″-β-D-xylosyl)-β-D-galactoside was the highest with the average content in the tested samples of 161.0 μg·g~(-1). The content of compounds quercetin-3-O-hexosyl-hexoside and kaempferol-3-O-pentosyl-hexoside was relatively low, with the average content of 18.5 μg·g~(-1)(calculated as quercetin-3-O-sophoroside) and 49.4 μg·g~(-1)(calculated as kaempferol-3-O-sangbu diglycoside). There were significant differences in flavonoids content of samples from different production area. The content of flavonoids in spring P. notoginseng was significantly lower than that in winter P. notoginseng when the other influencing factors such as production areas, germplasm resources, and cultivation conditions were fixed. As for P. notoginseng of different specifications, the flavonoid content in the part connecting the taproot and the aboveground stem was significantly higher than that in other parts. The results of large-scale data showed that the flavonoid content gradually increased with the increase in the number of heads. There were significant differences between the flavonoid content in most specifications and grades, especially the 20-head P. notoginseng and countless head P. notoginseng, whose content was significantly lower and significantly higher than that of other specifications and grades, respectively. This study provides a scientific basis for the study of the effective components and quality control of P. notoginseng from the perspective of flavonoids.
Flavonoids/analysis* ; Anthocyanins/analysis* ; Quercetin ; Chromatography, High Pressure Liquid/methods* ; Kaempferols ; Tandem Mass Spectrometry/methods* ; Glycosides

The growth environment of medicinal plants plays an important role in the formation of their medicinal quality. However, there is a lack of combined analysis studying the close relationship between the growth environment, chemical components, and related biological activities of medicinal plants. Therefore, this study investigated the effect of different soil moisture treatments on the efficacy to eliminate dampness and relieve jaundice and the flavonoid content of Sedum sarmentosum, and explored their correlation. The flavonoid content in the decoction of S. sarmentosum growing under field conditions with soil moisture levels of 35%-40%(T1), 55%-60%(T2), 75%-80%(T3), and 95%-100%(T4) was compared. The effects of these treatments on liver function parameters, liver inflammation, and oxidative damage in mice with dampness-heat jaundice were evaluated, and the correlation between pharmacological indicators and flavonoid content was analyzed. The results showed that the total flavonoid and total phenolic acid content in the decoction of S. sarmentosum were highest in the T1 treatment, followed by the T3 treatment. The content of quercetin, kaempferol, and isorhamnetin was highest in the T2, T1, and T3 treatments, respectively. Among the different moisture treatments, the T3 group of S. sarmentosum effectively reduced the levels of serum ALT, AKP, TBIL, DBIL, TBA, as well as hepatic TNF-α and IL-6 in mice with jaundice, followed by T2 treatment, especially in reducing AST level. The T4 treatment had the poorest effect. Correlation analysis showed a significant negative correlation between AST, ALT, AKP levels in mice and the total content of quercetin and the three flavonoids. MDA showed a significant negative correlation with the total flavonoid content and kaempferol. TNF-α exhibited a significant negative correlation with the content of isorhamnetin. In conclusion, S. sarmentosum growing under field conditions with a soil moisture level of 75%-80% exhibited the best efficacy to eliminate dampness and relieve jaundice. This study provides insights for optimizing the cultivation mode of medicinal plants guided by pharmacological experiments.
Mice ; Animals ; Flavonoids/chemistry* ; Plant Extracts/pharmacology* ; Quercetin ; Sedum/chemistry* ; Kaempferols ; Soil ; Tumor Necrosis Factor-alpha ; Plants, Medicinal/chemistry* ; Jaundice/drug therapy*

Simiao Yong'an Decoction is a classic prescription for treating gangrene. Modern medical evidence has proven that Si-miao Yong'an Decoction has therapeutic effects on atherosclerosis(AS), vascular occlusion angeitides, and hypertension, while its pharmacodynamic mechanism remains unclear. The evidence of network pharmacology, molecular docking, literature review, and our previous study suggests that luteolin and kaempferol are two major flavonoids in Simiao Yong'an Decoction and can inhibit macrophage inflammation and exert anti-AS effects. However, due to lack of the metabolism studies in vivo, little is known about the metabolic characteristics of luteolin and kaempferol. This study employed ultra-performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS/MS) and relevant software to identify the metabolites and metabolic pathways of luteolin and kaempferol in rat plasma, urine, and feces, after oral administration of luteolin and kaempferol, respectively. After the administration of luteolin, 10, 11, and 3 metabolites of luteolin were detected in the plasma, urine, and feces, respectively. After the administration of kaempferol, 9, 3, and 1 metabolites of kaempferol were detected in the plasma, urine, and feces, respectively. The metabolic pathways mainly involved methylation, glucuronidation, and sulfation. This study enriches the knowledge about the pharmacological mechanism of luteolin and kaempferol and supplies a reference for revealing the metabolic process of other flavonoids in Simiao Yong'an Decoction, which is of great significance for elucidating the pharmacological effects and effective substances of this decoction in vivo.
Rats ; Animals ; Tandem Mass Spectrometry/methods* ; Luteolin/analysis* ; Drugs, Chinese Herbal/chemistry* ; Kaempferols/analysis* ; Chromatography, High Pressure Liquid/methods* ; Molecular Docking Simulation

OBJECTIVE: To investigate the effect of kaempferol on proliferation of acute myeloid leukemia (AML) KG1a cells and its mechanism. METHODS: Human AML KG1a cells in logarithmic growth stage were taken and set at 25, 50, 75 and 100 μg/ml kaempferol group, another normal control group (complete medium without drug) and solvent control group (add dimethyl sulfoxide) were also set. After 24 and 48 hours of intervention, the cell proliferation rate was detected by CCK-8 assay. In addition, interleukin-6 (IL-6) combined with kaempferol group (Plus 20 μg/l IL-6 and 75 μg/ml kaempferol) was set up, 48 hours after culture, the cell cycle and apoptosis of KG1a cells were detected by flow cytometry, the mitochondrial membrane potential (MMP) of KG1a cells was detected by MMP detection kit (JC-1 method), and the expression of Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway related proteins in KG1a cells were detected by Western blot. RESULTS: The cell proliferation rate of 25, 50, 75 and 100 μg/ml kaempferol group decreased significantly (P<0.05), and with the increase of kaempferol dose (r_{24 h}=-0.990, r_{48 h}= -0.999), the cell proliferation rate decreased gradually (P<0.05). The inhibitory effect of 75 μg/ml kaempferol on cell proliferation reached half of effective dose after 48 hours of intervention. Compared with normal control group, the G₀/G₁ phase cell proportion and apoptosis rate of cells in 25, 50 and 75 μg/ml kaempferol group increased, while the S phase cell proportion, MMP, phosphorylated JAK2 (p-JAK2)/JAK2 and phosphorylated STAT3 (p-STAT3)/STAT3 protein expression decreased in a dose-dependent manner (r=0.998, 0.994, -0.996, -0.981, -0.997, -0.930). Compared with 75 μg/ml kaempferol group, the G₀/G₁ phase cell proportion and apoptosis rate of cells in IL-6 combined with kaempferol group decreased, while the S phase cell proportion, MMP, p-JAK2/JAK2 and p-STAT3/STAT3 protein expression increased significantly (P<0.05). CONCLUSION Kaempferol can inhibit KG1a cell proliferation and induce KG1a cell apoptosis, its mechanism may be related to the inhibition of JAK2/STAT3 signal pathway.
Humans ; STAT3 Transcription Factor/metabolism* ; Interleukin-6/metabolism* ; Kaempferols/pharmacology* ; Signal Transduction ; Apoptosis ; Janus Kinase 2 ; Cell Proliferation ; Leukemia, Myeloid, Acute

OBJECTIVE: Although there have been improvements in targeted therapy and immunotherapy, the majority of lung adenocarcinoma (LUAD) patients still lack effective therapies. Consequently, it is urgent to screen for new diagnosis biomarkers and pharmacological targets. Junctional adhesion molecule-like protein (JAML) was considered to be an oncogenic protein and may be a novel therapeutic target in LUAD. Kaempferol is a natural flavonoid that exhibits antitumor activities in LUAD. However, the effect of kaempferol on JAML is still unknown. METHODS: Small interfering RNA was used to knockdown JAML expression. The cell viability was determined using the cell counting kit-8 assay. The proliferation of LUAD cells was evaluated using the 5-ethynyl-2'-deoxyuridine incorporation assay. The migration and invasion of LUAD cells were evaluated by transwell assays. Molecular mechanisms were explored by Western blotting. RESULTS: JAML knockdown suppressed proliferation, migration and invasion of LUAD cells, and JAML deficiency restrained epithelial-mesenchymal transition (EMT) via inactivating the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. Using a PI3K activator (740Y-P), rescue experiments showed that phenotypes to JAML knockdown in LUAD cells were dependent on the PI3K/AKT/mTOR pathway. Kaempferol also inhibited proliferation, migration and invasion of A549 and H1299 cells and partially suppressed EMT through the PI3K/AKT/mTOR pathway. Knockdown of JAML ameliorated the inhibitory effect of kaempferol on LUAD cells. Kaempferol exerted anticancer effects by targeting JAML. CONCLUSION JAML is a novel target for kaempferol against LUAD cells. Please cite this article as: Wu Q, Wang YB, Che XW, Wang H, Wang W. Junctional adhesion molecule-like protein as a novel target for kaempferol to ameliorate lung adenocarcinoma. J Integr Med. 2023; 21(3): 268-276.
Humans ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Junctional Adhesion Molecules/metabolism* ; Kaempferols/pharmacology* ; Cell Line, Tumor ; Cell Movement/genetics* ; Adenocarcinoma of Lung/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Lung Neoplasms/metabolism* ; Cell Proliferation ; Gene Expression Regulation, Neoplastic

Isomers are widely distributed in Chinese herbal medicines,and can be discriminated by energy-resolved mass spectrometry( ER-MS). However,ER-MS was performed through direct injection of reference compounds with syringe pump,which encountered a significant technical barrier for high-throughput and automated measurements. Herein,online ER-MS was conducted using LC-MS platform,and a pair of isomers,kaempferol vs luteolin,were employed as a case study to illustrate and assess the utility of online ER-MS for isomeric discrimination. High-resolution tandem mass spectrometry data of both flavonoids were acquired on LC-QE-Orbitrap-MS,and the fragmentation pathways responsible for the primary fragment ions were proposed. The primary signal in MS1 occurred at m/z 285( [M-H]-),and the primary signals of either compound generated by retro-Diels-Alder fragmentation were observed at m/z 151 and 133. The spectral information was subsequently transferred onto LC-Qtrap-MS platform to carry out online ER-MS. Two precursor-to-product ion transition candidates were constructed as m/z 285>151 and 285>133,and either afterward derived a set of pseudo-ion transitions( PITs) and so forth,exactly corresponding to a series of progressive collision energies( eg-5,-8,-11 e V,and so on). All PITs were typed into the monitoring list of multiple reaction monitoring program to generate the peak area datasets. Either dataset was normalized using the highest values in the set and imported into Graph Pad Prism software to plot the Gaus-sian-shaped curve that was termed as the break-down graph. The apex of the regressive curve was termed as optimal collision energy( OCE). The OCE values corresponding to m/z 285>151 were calculated as-29. 06 e V and-35. 71 e V for kaempferol and luteolin,respectively. In the case of m/z 285>133,the OCEs were yielded as-44. 15 e V for kaempferol and-49. 01 e V for luteolin. With re-ference to their chemical structures,the location of hydroxyl group was regarded to be responsible for the differences of either m/z 285>151 or 285>133 between the isomers,attributing to their different bond properties. Above all,online ER-MS offers an eligible tool for isomeric discrimination,and provides meaningful information for the accurate chemical composition characterization based on LC-MS,which is not limited to Chinese herbal medicines.
Chromatography, Liquid ; Flavonoids ; Kaempferols ; Luteolin ; Tandem Mass Spectrometry

Several chemical compounds can restore pigmentation in vitiligo through mechanisms that vary according to disease etiology. In the present study, we investigated the melanogenic activity of six structurally distinct compounds, namely, scopoletin, kaempferol, chrysin, vitamin D, piperine, and 6-benzylaminopurine. We determined their effectiveness, toxicity, and mechanism of action for stimulating pigmentation in B16F10 melanoma cells and in a zebrafish model. The melanogenic activity of 6-benzylaminopurine, the compound identified as the most potent, was further verified by measuring green fluorescent protein concentration in tyrp1 a: eGFP (tyrosinase-related protein 1) zebrafish and mitfa: eGFP (microphthalmia associated transcription factor) zebrafish and antioxidative activity. All the tested compounds were found to enhance melanogenesis responses both in vivo and in vitro at their respective optimal concentration by increasing melanin content and expression of TYR and MITF. 6-Benzyamino-purine showed the strongest re-pigmentation action at a concentration of 20 μmol·Lin vivo and 100 μmol·Lin vitro, and up-regulated the strong fluorescence expression of green fluorescent protein in tyrp1a: eGFP and mitfa: eGFP zebrafish in vitro. However, its relative anti-oxidative activity was found to be very low. Overall, our results indicated that 6-benzylaminopurine stimulated pigmentation through a direct mechanism, by increasing melanin content via positive regulation of tyrosinase activity in vitro, as well as up-regulating the expression of the green fluorescent protein in transgenic zebrafish in vivo.
Alkaloids ; chemistry ; pharmacology ; Animals ; Benzodioxoles ; chemistry ; pharmacology ; Benzyl Compounds ; chemistry ; pharmacology ; Cholecalciferol ; chemistry ; pharmacology ; Flavonoids ; chemistry ; pharmacology ; Humans ; Kaempferols ; chemistry ; pharmacology ; Melanins ; genetics ; metabolism ; Monophenol Monooxygenase ; genetics ; metabolism ; Pigmentation ; drug effects ; Piperidines ; chemistry ; pharmacology ; Polyunsaturated Alkamides ; chemistry ; pharmacology ; Purines ; chemistry ; pharmacology ; Scopoletin ; chemistry ; pharmacology ; Vitiligo ; drug therapy ; enzymology ; metabolism ; Zebrafish