Machine Learning-Based Analysis Reveals Triterpene Saponins and Their Aglycones in Cimicifuga racemosa as Critical Mediators of AMPK Activation.

Cimicifuga racemosa (CR) extracts contain diverse constituents such as saponins. These saponins, which act as a defense against herbivores and pathogens also show promise in treating human conditions such as heart failure, pain, hypercholesterolemia, cancer, and inflammation. Some of these effects are mediated by activating AMP-dependent protein kinase (AMPK). Therefore, comprehensive screening for activating constituents in a CR extract is highly desirable. Employing machine learning (ML) techniques such as Deep Neural Networks (DNN), Logistic Regression Classification (LRC), and Random Forest Classification (RFC) with molecular fingerprint MACCS descriptors, 95 CR constituents were classified. Calibration involved 50 randomly chosen positive and negative controls. LRC achieved the highest overall test accuracy (90.2%), but DNN and RFC surpassed it in precision, sensitivity, specificity, and ROC AUC. All CR constituents were predicted as activators, except for three non-triterpene compounds. The validity of these classifications was supported by good calibration, with misclassifications ranging from 3% to 17% across the various models. High sensitivity (84.5-87.2%) and specificity (84.1-91.4%) suggest suitability for screening. The results demonstrate the potential of triterpene saponins and aglycones in activating AMP-dependent protein kinase (AMPK), providing the rationale for further clinical exploration of CR extracts in metabolic pathway-related conditions.

AMPK Activation by Cimicifuga racemosa Extract Ze 450 Is Associated with Metabolic Effects and Cellular Resilience against Age-Related Pathologies in Different Tissue Cell Types.

Cimicifuga racemosa extracts (CREs) have gained well-established use for the treatment of menopausal symptoms such as hot flushes and excessive sweating, and weight gain. While the clinical effects of CREs have been well documented, the mechanisms underlying these effects are largely unknown. More recently, the metabolic effects of the CRE Ze 450 were demonstrated in cultured cells in vitro and in mouse models of obesity in vivo. At the molecular level, metabolic regulation, enhanced insulin sensitivity, and increased glucose uptake were linked to the activation of AMP-activated protein kinase (AMPK). Therefore, we tested the effects of Ze 450 on AMPK phosphorylation and thus activation in cells from different tissues, i.e., murine C2C12 myoblast cells, human HEPG2 liver cells, mouse HT22 neuronal cells, and in murine 3T3L1 adipocytes. Using a FRET-based HTRF-assay, we found that Ze 450 induced AMPK phosphorylation and the activation of this key enzyme of metabolic regulation in cells from various different tissues including C2C12 (muscle), HEPG2 (liver), HT22 (hippocampal), and 3T3-L1 (adipocyte) cells. In C2C12 muscle cells, enhanced AMPK activation was accompanied by reduced mitochondrial respiration and enhanced glucose uptake. Further, Ze 450 enhanced the resilience of the cells against oxidative death induced by ferroptosis inducers erastin or RSL3. Our findings suggest a general effect of Cimicifuga racemosa on AMPK activation in different tissues and across species. This may have a significant impact on expanded therapeutic applications of Ze 450, since AMPK activation and the related metabolic effects have been previously associated with anti-aging effects and the prevention of the metabolic syndrome.

Inhibition of SARS-CoV-2 infection and replication by Petasites hybridus CO2-extract (Ze 339).

BACKGROUND: The intensified search for low-threshold herbal anti-viral drugs would be of great advantage in prevention of early stages of infection. Since the SARS-CoV-2 Omicron variant has prevailed in western countries, the course has only been mild, but there are still no widely available drugs that can alleviate or shorten disease progression and counteract the development of Long-COVID. This study aimed to investigate the antiviral effects of a CO2-extract from Petasites hybridus (Ze 339). METHODS: We analyzed the infection and replication rate of SARS-CoV-2 in primary normal human bronchial epithelial cells (NHBEs) using a GFP-expressing version of the wild-type SARS-CoV-2 virus and live cell imaging. Upon infection with a clinical isolate of the Omicron variant, viral RNA content was quantified, and plaque assays were performed. In addition, the human transcriptome was analyzed after 4- and 24-hours post infection using whole genome microarrays. RESULTS: Ze 339 had a protective effect on primary airway epithelial cells during SARS-CoV-2 infection and impeded SARS-CoV-2 infection and replication in NHBE. Notably, Ze 339 inhibited the expression of infection-induced IFNA10 by 8.6-fold (p < 0.05) and additionally reduced a wide range of other interferons (IFNA6, IFNA7, IFNA8, IFNA21, IFNE, IFNW1). CONCLUSION: Thereby, Ze 339 attenuated epithelial infection by SARS-CoV-2 and modeled the IFN response. In conclusion, this study highlights Ze 339 as a potential treatment option for COVID-19 that limits infection-associated cell intrinsic immune responses.

Impact of St. John's wort extract Ze 117 on stress induced changes in the lipidome of PBMC.

BACKGROUND: Membrane lipids have an important function in the brain as they not only provide a physical barrier segregating the inner and outer cellular environments, but are also involved in cell signaling. It has been shown that the lipid composition effects membrane fluidity which affects lateral mobility and activity of membrane-bound receptors. METHODS: Since changes in cellular membrane properties are considered to play an important role in the development of depression, the effect of St. John's wort extract Ze 117 on plasma membrane fluidity in peripheral blood mononuclear cells (PBMC) was investigated using fluorescence anisotropy measurements. Changes in fatty acid residues in phospholipids after treatment of cortisol-stressed [1 µM] PBMCs with Ze 117 [10-50 µg/ml] were analyzed by mass spectrometry. RESULTS: Cortisol increased membrane fluidity significantly by 3%, co-treatment with Ze 117 [50 µg/ml] counteracted this by 4.6%. The increased membrane rigidity by Ze 117 in cortisol-stressed [1 µM] PBMC can be explained by a reduced average number of double bonds and shortened chain length of fatty acid residues in phospholipids, as shown by lipidomics experiments. CONCLUSION: The increase in membrane rigidity after Ze 117 treatment and therefore the ability to normalize membrane structure points to a new mechanism of antidepressant action of the extract.

Effect of St. John's wort extract Ze 117 on the lateral mobility of ß1-adrenergic receptors in C6 cells.

Depression has been associated with altered signal transduction of serotonergic, dopaminergic and adrenergic neurotransmitter systems in the brain. Signaling relies on receptor-ligand interactions and subsequent regulatory processes, but also on lateral receptor mobility. The aim of this study was to investigate the effect of the St. John's wort extract Ze 117 on the lateral mobility of SNAP-tagged ß1-adrenergic receptors (ß1AR) in the plasma membrane of C6 cells under both, non-stimulating and isoprenaline-stimulating conditions. Single particle tracking (SPT) was used, whereby the registered trajectories were evaluated by variational Bayesian treatment of a hidden Markov model (vbSPT) and packing coefficient (Pc) analysis with respect to diffusion coefficients, receptor state occupancies and confinement. Three different diffusion states were identified, differing in their diffusion coefficients. Treatment with Ze 117 [25 µg/ml] decreased the mobility of the ß1AR, which was manifested by a relative increase in the slow-diffusing state S1 (0.21-0.30) compared to control and by an increase in receptor confinement (79.4-68.1 nm). After isoprenaline stimulation of control cells, the slow-diffusing state was more pronounced, whereas confinement was not affected. In summary, SPT has been shown to be a powerful method to analyze lateral receptor mobility. Furthermore, the present study identified a correlation between Ze 117 treatment and ß1AR mobility.

Treat more than heat-New therapeutic implications of Cimicifuga racemosa through AMPK-dependent metabolic effects.

BACKGROUND: Cimicifuga racemosa extracts (CRE) have obtained a "well-established use status" in the treatment of postmenopausal (i.e., climacteric) complaints, which predominantly include vasomotor symptoms such as hot flushes and sweating, as well as nervousness, irritability, and metabolic changes. Although characteristic postmenopausal complaints are known for a very long time and the beneficial effects of CRE on climacteric symptoms are well accepted, both the pathophysiology of postmenopausal symptoms and the mechanism of action of CREs are not yet fully understood. In particular, current hypotheses suggest that changes in the α-adrenergic and serotonergic signaling pathways secondary to estrogen depletion are responsible for the development of hot flushes. PURPOSE: Some of the symptoms associated with menopause cannot be explained by these hypotheses. Therefore, we attempted to extend our classic understanding of menopause by integrating of partly age-related metabolic impairments. METHODS: A comprehensive literature survey was performed using the PubMed database for articles published through September 2021. The following search terms were used: (cimicifuga OR AMPK) AND (hot flush* OR hot flash* OR menopaus* OR osteoporos* OR cancer OR antioxida* OR cardiovasc*). No limits were set with respect to language, and the references cited in the articles retrieved were used to identify additional publications. RESULTS: We found that menopause is a manifestation of the general aging process, with specific metabolic changes that aggravate menopausal symptoms, which are accelerated by estrogen depletion and associated neurotransmitter dysregulation. Cimicifuga extracts with their metabolic effects mitigate climacteric symptoms but may also modulate the aging process itself. Central to these effects are effects of CRE on the metabolic key regulator, the AMP-activated protein kinase (AMPK). CONCLUSIONS: As an extension of this effect dimension, other off-label indications may appear attractive in the sense of repurposing of this herbal treatment.

The Petasites hybridus CO2 Extract (Ze 339) Blocks SARS-CoV-2 Replication In Vitro.

The coronavirus disease 2019 (COVID-19), caused by a novel coronavirus (SARS-CoV-2), has spread worldwide, affecting over 250 million people and resulting in over five million deaths. Antivirals that are effective are still limited. The antiviral activities of the Petasites hybdridus CO2 extract Ze 339 were previously reported. Thus, to assess the anti-SARS-CoV-2 activity of Ze 339 as well as isopetasin and neopetasin as major active compounds, a CPE and plaque reduction assay in Vero E6 cells was used for viral output. Antiviral effects were tested using the original virus (Wuhan) and the Delta variant of SARS-CoV-2. The antiviral drug remdesivir was used as control. Pre-treatment with Ze 339 in SARS-CoV-2-infected Vero E6 cells with either virus variant significantly inhibited virus replication with IC50 values of 0.10 and 0.40 µg/mL, respectively. The IC50 values obtained for isopetasin ranged between 0.37 and 0.88 µM for both virus variants, and that of remdesivir ranged between 1.53 and 2.37 µM. In conclusion, Ze 339 as well as the petasins potently inhibited SARS-CoV-2 replication in vitro of the Wuhan and Delta variants. Since time is of essence in finding effective treatments, clinical studies will have to demonstrate if Ze339 can become a therapeutic option to treat SARS-CoV-2 infections.

Complete hematological and major molecular response through treatment with low-dose Interferon alpha 2a in high-risk polycythemia vera patient: a case report.

Low-dose interferon-α 2a treatment may be considered as an alternative to cytoreductive therapy with hydroxyurea or regularly dosed interferon in high-risk polycythemia vera patients.

Cimicifuga racemosa Extract Ze 450 Re-Balances Energy Metabolism and Promotes Longevity.

Recently, we reported that the Cimicifuga racemosa extract Ze 450 mediated protection from oxidative cell damage through a metabolic shift from oxidative phosphorylation to glycolysis. Here, we investigated the molecular mechanisms underlying the effects of Ze 450 against ferroptosis in neuronal cells, with a particular focus on mitochondria. The effects of Ze 450 on respiratory complex activity and hallmarks of ferroptosis were studied in isolated mitochondria and in cultured neuronal cells, respectively. In addition, Caenorhabditis elegans served as a model organism to study mitochondrial damage and longevity in vivo. We found that Ze 450 directly inhibited complex I activity in mitochondria and enhanced the metabolic shift towards glycolysis via cMyc and HIF1α regulation. The protective effects against ferroptosis were mediated independently of estrogen receptor activation and were distinct from effects exerted by metformin. In vivo, Ze 450 protected C. elegans from the mitochondrial toxin paraquat and promoted longevity in a dose-dependent manner. In conclusion, Ze 450 mediated a metabolic shift to glycolysis via direct effects on mitochondria and altered cell signaling, thereby promoting sustained cellular resilience to oxidative stress in vitro and in vivo.

St John's wort extract influences membrane fluidity and composition of phosphatidylcholine and phosphatidylethanolamine in rat C6 glioblastoma cells.

BACKGROUND: Chronic stress, an important factor in the development of depressive disorders, leads to an increased formation of cortisol, which causes a hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. In addition, cortisol mediates an adaptive effect on plasma membrane fluidity which may affect signal transduction of membrane-bound receptors and contribute to pathophysiological changes. METHODS: Membrane fluidity was measured by fluorescence anisotropy using DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene). Changes in cellular content of phosphatidylcholine species was determined by pulse-chase experiments using deuterated choline and mass spectrometry. Single molecule tracking was used to examine the lateral mobility of ß1-adrenoceptors and changes in cAMP formation were measured by ELISA. RESULTS: Chronic exposure (6 - 8 days) of C6 cells to cortisol dose-dependently decreased DPH and TMA-DPH fluorescence anisotropy, reflecting increased membrane fluidity. In contrast, cells pretreated with St. John's wort extract Ze117 showed increased DPH and TMA-DPH fluorescence anisotropy values, indicating a membrane rigidification effect which was mediated at least by the constituents hypericin, hyperforin, quercetin, amentoflavone and biapigenin. The observed membrane fluidizing effect of cortisol could be reversed by cotreatment with Ze117. The membrane rigidification of Ze117 was in line with the in parallel observed decrease in the phosphatidylcholine/phosphatidylethanolamine ratio determined in whole cell lipid extracts. Interestingly, pulse-chase experiments demonstrated, that Ze117 inhibited the incorporation of choline-D9 in phosphatidylcholine species with saturated or monounsaturated fatty acids compared to control cells, while the synthesis of phosphatidylcholine species with polyunsaturated fatty acids was not affected. C6 cells whose membranes have become more rigid by Ze117 showed altered lateral mobility of ß1-adrenoceptors as well as reduced cAMP formation after stimulation with the ß1-adrenoceptor agonist dobutamine. CONCLUSION: Obviously, the signaling of ß1-adrenoceptors depends on the nature of the membrane environment. It can therefore be assumed that Ze117 has a normalizing effect not only on the membrane fluidity of "stressed" cells, but also on lateral mobility and subsequently on the signal transduction of membrane-associated receptors.