A yeast-based screening system identified bakkenolide B contained in Petasites japonicus as an inhibitor of interleukin-2 production in a human T cell line.

Ca2+ signaling is related to various diseases such as allergies, diabetes, and cancer. We explored Ca2+ signaling inhibitors in natural resources using a yeast-based screening method and found bakkenolide B from the flower buds of edible wild plant, Petasites japonicus, using the YNS17 strain (zds1Δ erg3Δ pdr1/3Δ). Bakkenolide B exhibited growth-restoring activity against the YNS17 strain and induced Li+ sensitivity of wild-type yeast cells, suggesting that it inhibits the calcineurin pathway. Additionally, bakkenolide B inhibited interleukin-2 production at gene and protein levels in Jurkat cells, a human T cell line, but not the in vitro phosphatase activity of human recombinant calcineurin, an upstream regulator of interleukin-2 production. Furthermore, bakkenolide A showed weak activity in YNS17 and Jurkat cells compared with bakkenolide B. These findings revealed new biological effects and the structure-activity relationships of bakkenolides contained in P. japonicus as inhibitors of interleukin-2 production in human T cells.

Immunostimulatory Potential of Extracellular Vesicles Isolated from an Edible Plant, Petasites japonicus, via the Induction of Murine Dendritic Cell Maturation.

Extracellular vesicles (EVs) have recently been isolated from different plants. Plant-derived EVs have been proposed as potent therapeutics and drug-delivery nanoplatforms for delivering biomolecules, including proteins, RNAs, DNAs, and lipids. Herein, Petasites japonicus-derived EVs (PJ-EVs) were isolated through a series of centrifugation steps and characterized using dynamic light scattering and transmission electron microscopy. Immunomodulatory effects of PJ-EVs were assessed using dendritic cells (DCs). PJ-EVs exhibited a spherical morphology with an average size of 122.6 nm. They induced the maturation of DCs via an increase in the expression of surface molecules (CD80, CD86, MHC-I, and MHC-II), production of Th1-polarizing cytokines (TNF-α and IL-12p70), and antigen-presenting ability; however, they reduced the antigen-uptake ability. Furthermore, maturation of DCs induced by PJ-EVs was dependent on the activation and phosphorylation of MAPK and NF-κB signal pathways. Notably, PJ-EV-treated DCs strongly induced the proliferation and differentiation of naïve T cells toward Th1-type T cells and cytotoxic CD8+ T cells along with robust secretion of IFN-γ and IL-2. In conclusion, our study indicates that PJ-EVs can be potent immunostimulatory candidates with an ability of strongly inducing the maturation of DCs.

In vitro and in vivo evaluation of antioxidant activity of Petasites japonicus Maxim. flower buds extracts.

The antioxidant activity of Petasites japonicus flower buds cultivated in Tokushima, Japan, was examined in vitro and in vivo. The flower bud extracts were assayed using either oxygen radical absorbance capacity or 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity. Antioxidants in the 80% ethanol extract were investigated using online high-performance liquid chromatography-DPPH and were identified as caffeic acid, 3-O-caffeoylquinic acid, fukinolic acid, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid using liquid chromatography-mass spectrometry. Fukinolic acid was the most active compound based on its activity and abundance. Administering the extracts orally to ICR mice prior to iron injection significantly suppressed plasma thiobarbituric acid reactive substance (TBARS) production. Moreover, TBARS and triglyceride concentrations in the plasma of C57BL/6 mice fed with a high fat diet were also significantly decreased by the extract. The results suggest that antioxidative compounds in P. japonicus can be used in the management of oxidative stress.

Antioxidant compounds of Petasites japonicus and their preventive effects in chronic diseases: a review.

Petasites japonicus (P. japonicus) is a plant of the Asteraceae family. Its roots and stems have been used for the treatment or the prophylaxis of migraine and tension headache as a traditional Chinese medicine in Japan and Korea. Sesquiterpenoids, lignans, and flavonoids are components of P. japonicus. Regarding the biological activity of P. japonicus, its anti-allergic effect has been researched extensively using IgE antigen-stimulated degranulation of RBL-2H3 cells or passive cutaneous anaphylaxis reaction in experimental animal models. The study of the antioxidant activity of P. japonicus was initiated approximately 15 years ago using in vitro assays. In addition, its in vivo effect has also been examined in animal models with induced oxidative injury. Moreover, recently, many types of antioxidant compounds have been rapidly and simultaneously identified using the liquid chromatography-mass spectrometry technique. The number of reports on the other functions of this plant, such as its neuroprotective and anti-inflammatory effects, has been increasing. In this review, I summarized the studies of functional foods derived from P. japonicus, which may provide a basis for the development of potential functional foods. Finally, I discuss the future research avenues in this field.

Butterbur Leaves Attenuate Memory Impairment and Neuronal Cell Damage in Amyloid Beta-Induced Alzheimer's Disease Models.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease, and is characterized by the accumulation of amyloid beta (Aß) as a pathological hallmark. Aß plays a central role in neuronal degeneration and synaptic dysfunction through the generation of excessive oxidative stress. In the present study, we explored whether leaves of Petasites japonicus (Siebold & Zucc.) Maxim. (PL), called butterbur and traditionally used in folk medicine, show neuroprotective action against Aß25⁻35 plaque neurotoxicity in vitro and in vivo. We found that PL protected Aß25⁻35 plaque-induced neuronal cell death and intracellular reactive oxygen species generation in HT22 cells by elevating expression levels of phosphorylated cyclic AMP response element-binding protein, heme oxygenase-1, and NAD(P)H quinine dehydrogenase 1. These neuroprotective effects of PL were also observed in Aß25⁻35 plaque-injected AD mouse models. Moreover, administration of PL diminished Aß25⁻35 plaque-induced synaptic dysfunction and memory impairment in mice. These findings lead us to suggest that PL can protect neurons against Aß25⁻35 plaque-induced neurotoxicity and thus may be a potential candidate to regulate the progression of AD.

Dual Protective Effects of Flavonoids from Petasites japonicus against UVB-Induced Apoptosis Mediated via HSF-1 Activated Heat Shock Proteins and Nrf2-Activated Heme Oxygenase-1 Pathways.

The leaves of Petasites japonicus are used for their anti-allergic properties in traditional Korean, Japanese, and Chinese medicine. This study aimed to identify bioactive compounds isolated from P. japonicus leaves. All compounds were assessed for their ability of transcriptional activation, induction of phase 2 enzymes and heat shock proteins (HSPs), as well as protection against the UVB-induced apoptotic cell death. Bioactive compounds were isolated from P. japonicus leaves. All compounds were evaluated for their protective effect using human dermal fibroblasts (HDF) and human epidermal keratinocyte cells (HEKC) treated with UVB radiation. Four flavonoids were isolated from the leaves of P. japonicus and identified as kaempferol-3-O-(6″-acetyl)-ß-D-glucoside (1), quercetin-3-O-(6″-acetyl)-ß-D-glucoside (2), kaempferol-3-O-ß-D-glucoside (3), and quercetin-3-O-ß-D-glucoside (4). These compounds activated nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heat-shock response transcription elements (HSE) that resulted in the induction of heme oxygenase-1 (HO-1) and HSP70, respectively. Activation of these pathways provided protection to the skin cells against UVB radiation. The isolated compounds activated the Nrf2 and HSE pathways and could protect against UVB-induced apoptosis.

LC-MS/MS characterization, anti-inflammatory effects and antioxidant activities of polyphenols from different tissues of Korean Petasites japonicus (Meowi).

The Korean Petasites japonicus is a perennial plant used in folk medicine as a remedy for many diseases and popularly consumed as spring greens. Ten polyphenols were characterized from the leaves, stems and roots of this plant via high-performance liquid chromatography-tandem mass spectrometry. Individual polyphenols were quantified for the first time using calibration curves of six structurally related external standards. Validation data indicated that coefficients of determinations (R2 ) were ≥0.9702 for all standards. Recoveries measured at 50 and 100 mg/L were 80.0-91.9 and 80.3-105.3%, respectively. Precisions at these two concentration levels were 0.7-6.1 and 1.1-5.5%, respectively. The total number of identified components was largest for the leaves and smallest for the stems. The leaf and root polyphenolic extracts showed anti-inflammatory effects by inducing LPS-activated COX-2 and iNOS protein levels in mouse macrophage RAW 264.7 cells. The antioxidant capacity of the polyphenols, when evaluated for DPPH (α,α-diphenyl-ß-picrylhydrazyl)ˑ , ABTS+ [2-2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] and superoxide radical scavenging activities, and in ferric reducing ability of plasma (FRAP) assays, was highest in the leaf and lowest in the stem. This trend suggests that the antioxidant capacities depend primarily on polyphenol concentration in each tissue. The current findings suggest that polyphenols derived from P. japonicas tissues could have potential as functional health foods.

Short-Term Effects of PJE Administration on Metabolic Parameters in Diet-Induced Obesity Mice.

The study investigated the effects of Petasites japonicus (Siebold & Zucc.) Maxim. extract (PJE) and fenofibrate on diet-induced obesity (DIO) in mice. PJE was found to contain various bio-active polyphenolic compounds, including kaempferol, p-hydroxybenzoic acid, ferulic acid, gallic acid, chlorogenic acid, 3,4-dicaffeoylquinic acid, caffeic acid, quercetin, rutin, protocatechuic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, p-coumaric acid, apigenin, and 1,3-dicaffeoylquinic acid. The results showed that PJE treatment up to 1000 µg/mL did not affect the viability of 3T3-L1 cell line, and it reduced the feed efficiency ratio in DIO mice. PJE administration also resulted in a significant reduction in body weight gain and fat accumulation in the liver compared to the DIO control group. Additionally, PJE administration improved the levels of lipid and related parameters, including total cholesterol, triacylglycerol, low-density lipoprotein, very low-density lipoprotein, glucose, insulin, insulin resistance, leptin, and atherogenic or cardiac indexes compared to the DIO control group. The study suggested that PJE may have a beneficial effect on insulin resistance, lipid profiles, atherogenesis, adipokines, and cardiac risk associated with diet-induced obesity.

Petasites japonicus extract exerts anti-malarial effects by inhibiting platelet activation.

BACKGROUND: New antimalarial agents are needed to combat emerging resistance to the currently available drugs. In the pathology of cerebral malaria, platelets play a central role by binding infected and uninfected red cells and the endothelium. Since Petasites japonicus extract was reported as an effective inhibitor of platelet activation, we examined the antimalarial activities of the P. japonicus extract. PURPOSE: This study aimed to evaluate the impact of P. japonicus extract prepared from whole plants on malarial infection. METHODS: The P. japonicus extract were characterized by high-performance liquid chromatography (HPLC) profiling. Antimalarial activity of the P. japonicus ethanolic extract was evaluated in vitro using chloroquine-sensitive (3D7) and chloroquine-resistant (Dd2) P. berghei strains. Also, the in vivo activity of the extract was evaluated in P. berghei-infected mice via oral administration followed by a four-day suppressive test to measure the hematological parameters. In addition, platelet activation signaling induced by the P. japonicus extract in P. berghei infection was evaluated. RESULTS: In HPLC study, catechin, rutin, liquiritin, 3,4-di-O-caffeoylquinic acid, 3,5-di-O-caffeoylquinic acid, and 4,5-di-O-caffeoylquinic acid were identified in P. japonicus extract. Exposure to the P. japonicus extract significantly inhibited both CQ-sensitive (3D7) and resistant (Dd2) strains of P. falciparum with IC50 values of 8.48 ± 1.70 and 7.83 ± 6.44 µg/ml, respectively. Administration of the P. japonicus extract also resulted in potent antimalarial activities in P. berghei-infected mice with no associated toxicity. The treatment also improved the hematologic parameters. In addition, the survived mice from P. berghei infection exhibited the inhibition of collagen-induced platelet aggregation by attenuated glycoprotein VI (GPVI) downstream signaling. CONCLUSION: P. japonicus extracts promote antimalarial effects both in vitro and in vivo. In addition, the effects appear to be induced by the inhibition of collagen-induced platelet activation related to attenuated GPVI downstream signaling. Further studies to identify and characterize the antimalarial compounds in P. japonicus will promote the development of new drugs.

Petasin is the main component responsible for the anti-adipogenic effect of Petasites japonicus.

Petasites japonicus is one of the most popular edible wild plants in Japan. Many biological effects of P. japonicus have been reported, including anti-allergy, anti-inflammation, and anticancer effects. Although its anti-obesity effect has been reported in several studies, the most important component responsible for this activity has not been fully elucidated. On screening the components that suppress adipocyte differentiation in 3T3-F442A cells, we found that the extract of the flower buds of P. japonicus has anti-adipogenic effect. Among the known major components of P. japonicus, petasin exhibited a potent anti-adipogenic effect at an IC50 value of 0.95 µM. Quantitative analysis revealed that the active component responsible for most of the anti-adipogenic effects of P. japonicus extract is petasin. Petasin suppressed the expression of markers of mature adipocytes (PPARγ, C/EBPα, and aP2). However, as isopetasin and petasol, analogs of petasin, did not exhibit these effects, it indicates that a double bond at the C11-C12 position and an angeloyl ester moiety were essential for the activity. Petasin affected the late stage of adipocyte differentiation and inhibited the expression of lipid synthesis factors (ACC1, FAS, and SCD1). Additionally, it was revealed that petasin could be efficiently extracted using hexane with minimal amount of pyrrolizidine alkaloids, the toxic components. These findings indicate that P. japonicus extract containing petasin could be a promising food material for the prevention of obesity.

Single and Repeated Oral Dose Toxicity and Genotoxicity of the Leaves of Butterbur.

Butterbur (Petasites japonicus (Siebold & Zucc.) Maxim) leaves are available to consumers in the marketplace, but there is no guarantee that they are safe for human consumption. Previously, we demonstrated that hot water extracts of P. japonicus leaves (KP-1) had anti-inflammatory properties and attenuated memory impairment. However, data regarding KP-1 toxicity are lacking. This study assessed the safety of KP-1 by examining oral and genotoxic effects using in vivo and in vitro tests, respectively. In a single oral dose toxicity and two-week repeated oral dose toxicity study, we observed no toxicologically significant clinical signs or changes in hematology, blood chemistry, and organ weights at any dose during the experiment. Following a thirteen-week repeated oral dose, toxicity, hyperkeratosis, and squamous cell hyperplasia of the limiting ridge in the stomach were observed. The no observable adverse effect level (NOAEL) was found to be 1250 mg/kg/day in male and female rats. However, hyperkeratosis and hyperplasia were not considered to be of toxicological significance when extrapolating the NOAEL to humans because the limiting ridge in the stomach is species-specific to rats. Therefore, in our study, the NOAEL was considered to be 5000 mg/kg/day when the changes in the stomach's limiting ridge were discounted. Moreover, in vitro bacterial reverse mutations and chromosomal aberrations in Chinese hamster lung (CHL) cells and the in vivo micronucleus in Institute of cancer research (ICR) mice assays showed that KP-1 possessed no mutagenicity. Although additional research is required, these toxicological evaluations suggest that KP-1 could be safe for human consumption.

Cytotoxic activities of sesquiterpenoids from the aerial parts of Petasites japonicus against cancer stem cells.

From the methanolic extract of the aerial parts of Petasites japonicus, six new eremophilane-type sesquiterpenoids, petasitesterpenes I-VI were isolated together with eight known compounds including S-japonin and eremophilenolide. The chemical structures of the isolated new compounds were elucidated based on chemical/physicochemical evidence. For petasitesterpenes I and II, the absolute configurations were established by comparison of experimental and predicted electronic circular dichroism (ECD) data. Among the isolated compounds, petasitesterpenes I, II, VI, and S-japonin showed cytotoxic activity against both human astrocytoma U-251MG cancer cells (non-CSCs) and their cancer stem cells (CSCs) isolated by sphere formation. In addition, cytotoxic activities of these compounds against breast cancer MDA-MB-231 were evaluated, supporting that petasitesterpene II has more effective than other isolated compounds.

Anti-Obesity Effects of Petasites japonicus (Meowi) Ethanol Extract on RAW 264.7 Macrophages and 3T3-L1 Adipocytes and Its Characterization of Polyphenolic Compounds.

Koreans have been consuming Petasites Japonicus (PJ) as food. Although the therapeutic effect of PJ on allergic or inflammatory reactions associated with asthma has been proven, its effect on obesity is unclear. Therefore, the present study was aimed to assess the obesity related anti-inflammatory and anti-adipogenic effects of ethanol extract PJ (EPJ) on the inflammatory response in RAW 264.7 macrophages and on differentiation in 3T3-L1 adipocytes. In addition, the polyphenolic compound was quantitatively characterized from the EPJ using ultra performance liquid chromatography coupled with diode array detector, quadrupole time-of-flight-mass spectrometry (UPLC-DAD-QToF-MS). In RAW 264.7 or 3T3-L1, reduction of nitric oxide (in macrophages) production as well as monocyte chemoattractant protein-1 and tumor necrosis factor-α were observed. Treatment of EPJ in adipocyte differentiation showed an improvement in adiponectin and lipid accumulation and a significant reduction of PPARγ and FABP-4 mRNA expression levels. On the other hand, mRNA expression of UCP-1, PPARα, and ACO increased in the EPJ treated group. In addition, a total of 26 polyphenolic compounds were detected and of which 12 are reported for the first time from PJ. The higher content of diverse polyphenolic compounds presented in EPJ might be responsible for the observed anti-inflammatory and anti-adipogenic effect. These results suggest that PJ is valuable in improving obesity-related inflammatory responses.

Petatewalide B alleviates oxygen­glucose deprivation/reoxygenation­induced neuronal injury via activation of the AMPK/Nrf2 signaling pathway.

Neuronal injury is a common, and critical, occurrence in clinical ischemic strokes, and can cause irreversible brain damage. However, the precise pathological mechanisms underlying this condition and effective treatment remain unclear. Increasing evidence shows that the nuclear factor erythroid 2­related factor 2 (Nrf2)/activated protein kinase (AMPK) signaling pathway serves a significant role in neuronal injury and is involved in neuroprotection. The present study demonstrated that petatewalide B, the active constituent of Petasites japonicus, otherwise known as butterbur, can alleviate oxygen­glucose deprivation/reoxygenation (OGD/R)­induced neuronal death via the adenosine monophosphate­AMPK/glycogen synthase kinase (GSK)­3/ß/Nrf2/antioxidant response element (ARE) signaling pathways in human neuroblastoma SH­SY5Y cells. A neuronal injury model was established by depriving SH­SY5Y cells of oxygen and glucose for 8 h, followed by 24 h of reoxygenation (OGD/R). The results indicated that the OGD/R model exhibited reduced cell viability but increased lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) production and apoptosis. These were accompanied by increased levels of cleaved PARP, cleaved caspase­9, cleaved caspase­3, p53, Bax and p21, as well as decreased Bcl­2 levels. Treatment with petatewalide B was able to strengthen cell viability but reduced LDH release, ROS production and the expression levels of apoptosis­related proteins. Additionally, treatment with petatewalide B activated AMPK in the OGD/R­exposed SH­SY5Y cells and upregulated activation of the downstream transcription factor Nrf2, which accompanied heme oxygenase 1 (HO­1) and NAD(P)H quinone dehydrogenase 1 (NQO1) expression. Furthermore, silencing AMPK, Nrf2, HO­1 and NQO1 expression inhibited petatewalide B's protective effect against apoptosis in the OGD/R­exposed SH­SY5Y cells. Therefore, petatewalide B protected human neuroblastoma cells against OGD/R­induced injury by downregulating apoptosis and oxidative stress via upregulation of the AMPK/Nrf2 signaling pathway, suggesting that petatewalide B may be a prospective protector against neuronal injury, having possible therapeutic and medical implications.

Bakkenolides and Caffeoylquinic Acids from the Aerial Portion of Petasites japonicus and Their Bacterial Neuraminidase Inhibition Ability.

Petasites japonicus have been used since a long time in folk medicine to treat diseases including plague, pestilential fever, allergy, and inflammation in East Asia and European countries. Bioactive compounds that may prevent and treat infectious diseases are identified based on their ability to inhibit bacterial neuraminidase (NA). We aimed to isolate and identify bioactive compounds from leaves and stems of P. japonicas (PJA) and elucidate their mechanisms of NA inhibition. Key bioactive compounds of PJA responsible for NA inhibition were isolated using column chromatography, their chemical structures revealed using 1 H NMR, 13 C NMR, DEPT, and HMBC, and identified to be bakkenolide B (1), bakkenolide D (2), 1,5-di-O-caffeoylquinic acid (3), and 5-O-caffeoylquinic acid (4). Of these, 3 exhibited the most potent NA inhibitory activity (IC50 = 2.3 ± 0.4 µM). Enzyme kinetic studies revealed that 3 and 4 were competitive inhibitors, whereas 2 exhibited non-competitive inhibition. Furthermore, a molecular docking simulation revealed the binding affinity of these compounds to NA and their mechanism of inhibition. Negative-binding energies indicated high proximity of these compounds to the active site and allosteric sites of NA. Therefore, PJA has the potential to be further developed as an antibacterial agent for use against diseases associated with NA.

New otonecine-type pyrrolizidine alkaloid from Petasites japonicus.

One new otonecine-type pyrrolizidine alkaloid secopetasitenine (1), along with petasitenine (fukinotoxine, 2), neopetasitenine (3), and senkirkine (4), was isolated from the whole plant of Petasites japonicus. The structure of 1 was determined by spectroscopic analyses and chemical conversion from the known alkaloid petasitenine (2).

Chemical Constituents of the Leaves of Butterbur (Petasites japonicus) and Their Anti-Inflammatory Effects.

Two new aryltetralin lactone lignans, petasitesins A and B were isolated from the hot water extract of the leaves of butterbur (Petasites japonicus) along with six known compounds. The chemical structures of lignans 1 and 2 were elucidated on the basis of 1D and 2D nuclear magnetic resonance (NMR) spectroscopic data, electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) spectra. Petasitesin A and cimicifugic acid D showed significant inhibitory effects on the production of both prostaglandin E2 (PGE2) and NO in RAW264.7 macrophages. The expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were inhibited by compound 1 in RAW264.7 cells. Furthermore, compounds 1 and 3 exhibited strong affinities with both iNOS and COX-2 enzymes in molecular docking studies.

Isolation, characterization, and neuroprotective activities of sesquiterpenes from Petasites japonicus.

Neuroprotective reagents to protect the nerve cells against oxidative stress and other damages are potentially effective for the medical treatment of Parkinson's disease. Petasites japonicus, a wild vegetable, belongs to the family Compositae and its extract has shown the neuroprotective effects. A further phytochemical investigation of P. japonicus for neuroprotective substances led to the isolation of eight new (1-8) and two known (9 and 10) sesquiterpenes. Their structures were elucidated on the basis of extensive 1D and 2D NMR (HMQC, HMBC, (1)H-(1)H COSY, and NOESY) spectroscopic data analyses, and the structure of 1 was confirmed by X-ray crystallography. The neuroprotective activities of these sesquiterpenes were evaluated against cobalt chloride (CoCl2)-induced neuronal cell death in human dopaminergic SH-SY5Y cells. Five compounds showed a neuroprotective activity.