Mechanistic insights into xanthomicrol as the active anti-HCC ingredient of Phytolacca acinosa Roxb.: A network pharmacology analysis and transcriptomics integrated experimental verification.

ETHNOPHARMACOLOGICAL RELEVANCE: Phytolacca acinosa Roxb. (PAR) is a Traditional Chinese Medicinal (TCM) plant with a broad global distribution encompassing 35 species, four of which are found in the People's Republic of China. It occupies a significant role in both Oriental and American traditional medicine, employed in treating a range of conditions such as edema, inflammation, dermatitis, and rheumatism. PAR is also used as a molluscicide and for addressing tumors and bronchitis. The plant is documented in the Chinese Pharmacopoeia and has a longstanding history in TCM, particularly for its diuretic properties and in treating ailments such as edema, swelling, and ulcers. Notably, PAR has demonstrated potent inhibitory effects against the A549 human lung cancer cell line, underscoring its potential in contributing to the development of novel cancer therapeutics. AIM OF THE STUDY: The research aims to elucidate the active components of PAR and their mechanisms in treating hepatocellular carcinoma (HCC). MATERIALS AND METHODS: Employing network pharmacology, this study predicted the principal active compounds and key targets of PAR. A holistic methodology incorporating biological network analysis, transcriptomics sequencing, molecular docking, and molecular dynamics (MD) simulations was utilized to forecast the effects of PAR on HCC, with empirical evidence supporting these findings. RESULTS: Network pharmacology identified xanthomicrol as the foremost active compound in PAR. The tumor-suppressive functions of PAR, as indicated by KEGG pathway analysis and transcriptomics sequencing, predominantly occur via the PI3K/AKT pathway. Molecular docking and dynamics simulations demonstrated the high affinity of xanthomicrol towards TNF, MMP9, PPARG, KDR, and MMP2. In vivo experiments verified the efficacy of xanthomicrol in curtailing HCC tumor growth, while in vitro assessments revealed its substantial impact on the proliferation and apoptosis of HepG2 and HCCLM3 cells. Moreover, the study indicates that xanthomicrol may modulate the expression of TNF, MMP9, PPARG, KDR, and MMP2 in HCC cells and inhibit the activation of the PI3K/AKT pathway. CONCLUSIONS: Xanthomicrol, a principal active component of PAR, has been identified to impede the growth of HCC by targeting the PI3K/Akt/MMP9 pathway. This insight could enhance therapeutic approaches for HCC.

Comparative Evaluation of Anticancer Activity of Natural Methoxylated Flavones Xanthomicrol and Eupatilin in A375 Skin Melanoma Cells.

Melanoma is a skin cancer caused by the malignant transformation of melanocytes and cutaneous melanoma represents the most aggressive and deadliest type of skin cancer with an increasing incidence worldwide. The main purpose of the present research was to evaluate the anticancer effects of the natural bioactive compounds xanthomicrol (XAN) and eupatilin (EUP) in human A375 malignant skin melanoma cells, a cell line widely used as an in vitro model of cutaneous melanoma. XAN and EUP are lipophilic methoxylated flavones with antioxidant, anti-inflammatory, and antitumor properties. The effects of XAN and EUP on cell viability, morphology, lipid profile, oxidative status, apoptosis, and mitochondrial membrane polarization were determined and compared in A375 cells. At 24 h-incubation (MTT assay), XAN significantly reduced viability at the dose range of 2.5-200 µM, while EUP showed a significant cytotoxicity from 25 µM. Moreover, both methoxylated flavones induced (at 10 and 25 µM, 24 h-incubation) marked cell morphological alterations (presence of rounded and multi-nucleated cells), signs of apoptosis (NucView 488 assay), and a noteworthy mitochondrial membrane depolarization (MitoView 633 assay), coupled to a marked lipid profile modulation, including variations in the ratio of phospholipid/cholesterol and a decrease in the oleic, palmitic, and palmitoleic acid amounts. Moreover, a remarkable time-dependent ROS generation (2',7'-dichlorodihydrofluorescein diacetate assay) was observed during 3 h-incubation of A375 cancer cells in the presence of XAN and EUP (10 and 25 µM). Our results confirm the potential antitumor effect of natural EUP and XAN in cutaneous melanoma by the activation of multiple anticancer mechanisms.

Xanthomicrol: Effective therapy for cancer treatment.

Cancer treatment is one of the main challenges of global health. For decades, researchers have been trying to find anti-cancer compounds with minimal side effects. In recent years, flavonoids, as a group of polyphenolic compounds, have attracted the attention of researchers due to their beneficial effects on health. Xanthomicrol is one of the flavonoids that has the ability to inhibit growth, proliferation, survival and cell invasion and ultimately tumor progression. Xanthomicrol, as active anti-cancer compounds, can be effective in the prevention and treatment of cancer. Therefore, the use of flavonoids can be suggested as a treatment along with other medicinal agents. It is obvious that additional investigations in cellular levels and animal models are still needed. In this review article, the effects of xanthomicrol on various cancers have been reviewed.

Xanthomicrol Activity in Cancer HeLa Cells: Comparison with Other Natural Methoxylated Flavones.

The methoxylated flavone xanthomicrol represents an uncommon active phenolic compound identified in herbs/plants with a long application in traditional medicine. It was isolated from a sample of Achillea erba-rotta subsp. moschata (musk yar-row) flowering tops. Xanthomicrol promising biological properties include antioxidant, anti-inflammatory, antimicrobial, and anticancer activities. This study mainly focused on the evaluation of the xanthomicrol impact on lipid metabolism in cancer HeLa cells, together with the investigation of the treatment-induced changes in cell growth, morphology, and apoptosis. At the dose range of 5-100 µM, xanthomicrol (24 h of incubation) significantly reduced viability and modulated lipid profile in cancer Hela cells. It induced marked changes in the phospholipid/cholesterol ratio, significant decreases in the levels of oleic and palmitic acids, and a marked increase of stearic acid, involving an inhibitory effect on de novo lipogenesis and desaturation in cancer cells. Moreover, marked cell morphological alterations, signs of apoptosis, and cell cycle arrest at the G2/M phase were observed in cancer treated cells. The bioactivity profile of xanthomicrol was compared to that of the anticancer methoxylated flavones eupatilin and artemetin, and structure-activity relationships were underlined.

Non-Volatile Terpenoids and Lipophilic Flavonoids from Achillea erba-rotta Subsp. moschata (Wulfen) I. Richardson.

Musk yarrow (Achillea erba-rotta subsp. moschata (Wulfen) I. Richardson) is endemic to the Central Alps, and is used to flavour alcoholic beverages. Despite its popularity as aromatizing agent and its alleged beneficial effects on digestion, the phytochemical profile of the plant is still largely unknown and undiscovered. As a consequence, its authentication in aromatized products is impossible beyond sensory analysis allowing forgery. To address these issues, we phytochemically characterized a sample of musk yarrow from the Italian Eastern Alps, identifying, in addition to widespread phytochemicals (taraxasterol, apigenin), the guaianolides 3, 8, 9; the seco-caryophyllane 6; and the polymethoxylated lipophilic flavonoids 1, 4, and 5. The flavonoid xanthomicrol 1, a major constituent of the plant, was cytotoxic to HeLa cells, but only modestly affected primary 3T3 fibroblasts. On account of their stability, detectability by UV absorption, and concentration, the oxygenated flavonoids qualify as markers to validate the supply chain of the plant growers to consumers.

Xanthomicrol suppresses human hepatocellular carcinoma cells migration and invasion ability via Μu-opioid receptor.

BACKGROUND: Xanthomicrol is one of the methoxylated flavones and a promising cancer chemopreventive agent, but its anti-migration and anti-invasion ability on human hepatocellular carcinoma (HCC) remains unknown. OBJECTIVES: This study aims to explore Xanthomicrol's effects on migration and invasion ability of the human HCC Huh7 cell line. METHODS: Viability of Huh7 cells was measured by cell counting kit-8 (CCK8) assay. Cell apoptosis was assayed with flow cytometry analysis. The ability of migration and invasion of Huh7 cells was then detected through Transwell assays. Epithelial-mesenchymal transition (EMT)-related proteins were also detected through Western blot. KEY FINDINGS: Xanthomicrol inhibits the migration and invasion of Huh7 cells. The overexpression of Μu-opioid receptor (MOR) increases Huh7 cells' proliferation and enhances migration and invasion ability, while xanthomicrol treatment decreases the expression of MOR. Moreover, xanthomicrol can reverse migration, invasion and EMT-related protein expression by overexpressed MOR. CONCLUSIONS: These results suggest that xanthomicrol is a potential MOR antagonist, and it possesses potent anti-migration and anti-invasion ability on Huh7 cells.

Phenols from Origanum dictamnus L. and Thymus vulgaris L. and their activity against Malassezia globosa carbonic anhydrase.

Malassezia spp. are lipophilic fungi that are part of the normal flora of the human skin and are the etiological agents of dandruff and seborrheic dermatitis. ß-Carbonic Anhydrases (CAs; EC 4.2.1.1) expressed from the pathogenic fungi are an alternative/complementary drug target. Previous work by our groups demonstrated that flavonoids and depsides can effectively inhibit Malassezia globosa ß-CA (MgCA). In continuation of this study herein we report the inhibitory activity of a variety of phenols from Origanum dictamnus L. and Thymus vulgaris L. against ß-MgCA, among them I4-II7-di-carvacrol, a new natural product. Structure elucidation of the compounds was performed by 1 D, 2 D NMR and spectrometric analyses. Xanthomicrol and rosmarinic acid were active in the (sub)micromolar range (KIS 0.6 and 2.2 µM, respectively vs 40.0 µM of the standard inhibitor acetazolamide). Finally, the compounds were not cytotoxic, but showed in vitro no activity against Malassezia furfur.

Inductive effect of titanium dioxide nanoparticles on the anticancer compounds production and expression of rosmarinic acid biosynthesis genes in Dracocephalum kotschyi transformed roots.

Methoxylated flavonoids, mainly xanthomicrol and cirsimaritin that can be extracted from Dracocephalum kotschyi Boiss, have anticancer, antispasmodic and antiplatelet effects. The production of these valuable pharmaceutical compounds is one of the major goals of biotechnology studies. In this work, induced transformed roots were influenced by various concentrations of titanium dioxide nanoparticles (TiO2 NPs) at 24 or 48 h exposure time. The effects of TiO2 NPs were assessed on growth rate, activity of antioxidant enzymes, total phenol and flavonoid content (TPC and TFC) and rosmarinic acid (RA) and some flavonoids accumulation. The gene expression level of phenylalanine ammonia-lyase (pal) and rosmarinic acid synthase (ras) genes were assessed by real time PCR analysis. The transformed roots biomass was substantially increased in elicited roots in comparison with the control. The TPC, TFC and antioxidant enzymes activitywere affected by TiO2 NPs concentration and exposure time. Valuable flavonoids with anticancer characteristics along with xanthomicrol, cirsimaritin and isokaempferide exhibited an increase (70, 34.28 and 7.81-fold, respectively) versus the control. The maximum content of RA (530.5 µg g-1 FW), which was 4.30 times as great as that of control was detected in samples treated with TiO2 NPs (50 mg L-1) 24 h after elicitation. Real-time PCR analysis revealed a considerable increase in pal and ras expression rate engaged by TiO2 NPs levels and exposure time. Overall D. kotschyi transformed roots elicitation by TiO2 NPs led to a massive increment in the production of valuable anticancer flavonoids such as xanthomicrol, cirsimaritin and RA as polyphenol.

Inhibitory effect of flavonoid xanthomicrol on triple-negative breast tumor via regulation of cancer-associated microRNAs.

Breast cancer is the leading cause of cancer death in women worldwide. Due to the side effects of current chemo-reagents on healthy tissues, it is essential to search for alternative compounds with less toxicity and better efficacy. In the present study, we have investigated the anticancer effects of flavonoid xanthomicrol on the mice breast cancer model using MTT assay, cell cycle and Annexin/PI analysis, colony formation assay, H&E staining, immunohistochemistry, and miRNA analysis. Our results demonstrated that xanthomicrol decreased the cell viability and clonogenic capability, induced G1-arrest and apoptosis in the breast cancer cells in vitro, and caused a significant reduction in the volume and weight of mice tumors in vivo. In addition, xanthomicrol reduced the expression of TNFα, VEGF, MMP9, and Ki67, while upregulating the expression of apoptotic markers such as Bax, caspase3, and caspase9. Finally, the expression of miR21, miR27, and miR125, known as oncomirs, decreased significantly after xanthomicrol administration, while the expression of miR29 and miR34, functioning as tumor suppressors, increased significantly (p < .001). Our data demonstrated that xanthomicrol can induce apoptosis and suppress angiogenesis in breast cancer cells due to its inhibitory effect on oncomirs and its stimulatory effect on tumor suppressor miRNAs.

Identification of Xanthomicrol as a Major Metabolite of 5-Demethyltangeretin in Mouse Gastrointestinal Tract and Its Inhibitory Effects on Colon Cancer Cells.

5-Demethyltangeretin (5DT) is a unique polymethoxyflavone mainly found in the peel of citrus, and has shown potent suppressive effects on multiple human cancer cells. Biotransformation plays a critical role in the biological activities of dietary bioactive components because their metabolites may exert significant bioactivities. In the present study, the metabolic fate of 5DT in mouse gastrointestinal (GI) tract after long-term oral intake and the anti-cancer effects of its major metabolite were determined. It was found that 5DT underwent extensive biotransformation after oral ingestion in mice. A major demethylated metabolite was produced via phase I metabolism, while conjugates (glucuronide and sulfate) were generated via phase II metabolism. Specifically, 4'-position on the B ring of 5DT was the major site for demethylation reaction, which led to the production of xanthomicrol (XAN) as a major metabolite. More importantly, the level of XAN in the colon was significantly higher than that of 5DT in 5DT-fed mice. Thus, we further determined the suppressive effects of XAN on human colon cancer HCT116 cells. We found that XAN effectively inhibited the proliferation of HCT116 cells by arresting cell cycle and inducing cellular apoptosis, which was further evidenced by upregulated p53 and p21 and downregulated cyclin D and CDK4/6 level. In conclusion, this study identified XAN as a major metabolite of 5DT in mouse GI tract, and demonstrated its suppressive effects on HCT116 colon cancer cells.

Pharmacokinetics of calycopterin and xanthmicrol, two polymethoxylated hydroxyflavones with anti-angiogenic activities from Dracocephalum kotschyi Bioss.

BACKGROUND: Recently flavonoids have attracted the attention of researchers in the fight against cancer. Calycopterin and xanthomicrol, are two polymethoxylated flavonoids found in the aerial parts of Dracocephalum kotschyi Bioss.. We have recently shown that these compounds possess antiangiogenic activity and may be of value as potential anticancer agents. In order to demonstrate putative in vivo antitumor effect of these compounds we needed preliminary information on both pharmacokinetics and toxicological properties of these two agents. METHOD: A new online SPE HPLC method for measurement of calycopterin and xanthomicrol in rat plasma was developed. Pharmacokinetic parameters of calycopterin and xanthomicrol, after i.v. administration in rats, were determined. RESULTS: The plasma half-life for both agents was around 4 h, however, the volume of distribution of calycopterin appeared to be about 8 times greater than xanthomicrol. This was probably due the greater hydrophobicity of the former which had other consequences such as much smaller maximum plasma concentration of calycopterin compared to its less methoxylated congener. Preliminary toxicological study of xanthomicrol failed to show any behavioral, histological and biochemical adverse effects after repeated administrations of high doses. Pharmacokinetics of xanthomicrol in rats.

A new bioactive monoterpene-flavonoid from Satureja khuzistanica.

A new monoterpene-flavonoid, saturejin (3'-(2,5-dihydroxy-p-cymene) 5,7,4'-trihydroxy flavone) (4), together with twelve known flavonoids consist of two flavanonols (aromadendrin (8) and taxifolin (12)), two flavanones (naringenin (3) and 5,7,3',5'-tetrahydroxy flavanone (9)) and eight flavones (xanthomicrol (1), acacetin (2), cirsimaritin (5), 7-methoxy luteolin (6), apigenin (7), cirsilineol (10), diosmetin (11) and 6-hydroxyluteolin 7,3'-dimethyl ether (13)), were isolated from an ethyl acetate extract and identified for the first time in the dried aerial parts of Satureja khuzistanica Jamzad, an endemic medicinal plant traditionally used as dental anesthetic, oral antiseptic and anti-inflammatory among the nomadic inhabitants of southwestern Iran. The structures of these compounds were determined using the usual spectroscopic methods including 2D-NMR and MS analyses. Saturejin showed a significant ß-glucosidase inhibitory activity at concentration of 10 µg as well as positive antioxidant activity at the amount of 1 µg. These results could be correlated with the in vitro and in vivo anti-inflammatory, anti-oxidant and anti-diabetic properties reported from this medicinal plant. Similar activities were also described for some of the other isolated compounds.

Antiangiogenic activity of xanthomicrol and calycopterin, two polymethoxylated hydroxyflavones in both in vitro and ex vivo models.

Our previous studies had shown xanthomicrol and calycopterin, two plant-derived flavonoids, to have selective antiproliferative activity against some malignant cell lines. The present study is focused on the investigation of antiangiogenic potential of these two flavonoids, using in vitro and ex vivo models. Xanthomicrol and calycopterin were found to have potent inhibitory effects on microvessel outgrowth in the rat aortic ring assay. Xanthomicrol was able to completely block microvessel sprouting at 10 µg/mL, and calycopterin suppressed microvessel outgrowth by 89% at 5 µg/mL. Suramin and thalidomide, used at 20 µg/mL as positive controls, inhibited microvessel formation by 23% and 64%, respectively. The flavones also inhibited endothelial cell tube formation and human umbilical vein endothelial cell proliferation at 0.5, 5, and 10 µg/mL. In order to delineate the underlying mechanisms of antiangiogenic activity of these flavones, we investigated the influences of xanthomicrol and calycopterin on expression of vascular endothelial growth factor (VEGF) and basic-fibroblast growth factor (b-FGF) in endothelial cells. These flavones were able to inhibit VEGF expression at 0.5, 5, and 10 µg/mL, but they had little or no effect on b-FGF expression. These findings suggest that xanthomicrol and calycopterin possess potent antiangiogenic activities, which may be due to their inhibitory influences on VEGF expression.

Overproduction of valuable methoxylated flavones in induced tetraploid plants of Dracocephalum kotschyi Boiss.

BACKGROUND: Ploidy manipulation is considered an efficient method to increase production potential of medicinally important compounds. Dracocephalum kotschyi Boiss. is an endangered medicinal plant of Iran. Various concentrations of colchicine (0.05, 0.10, 0.20, and 0.50% w/v) were applied to shoot apical meristems of D. kotschyi seedlings in two and four-leaf stages to induce tetraploidy. RESULTS: According to the results, 0.5% (w/v) of colchicine can be effective for polyploidy induction in D. kotschyi. Putative tetraploids were selected by morphological and microscopic characteristics and their ploidy level was confirmed by flow cytometry analysis and chromosome counting. The chromosome number of original diploid plant was confirmed to be 2n = 2× = 20 whereas that of the tetraploid plant was 2n = 4× = 40. Tetraploid and mixoploid plants showed different morphological, physiological and microscopic characteristics from those of diploid counterparts. The total content of flavonoids was increased from 1583.28 in diploids to 1890.07 (µg/g DW) in stable tetraploids. CONCLUSION: High-Performance Liquid Chromatography with Diode-Array Detection (HPLC-DAD) confirmed over accumulation of methoxylated hydroxyflavones in solid tetraploid plants of D. kotschyi.