A new single-reference quantitative method using liquid chromatography with relative molar sensitivity based on 1H-qNMR for khellactone esters from Peucedanum japonicum root extract.

Khellactone ester (KLE) quantification using the absolute calibration method is difficult owing to the unavailability of standard reagents that can guarantee purity. Herein, a new method was developed to quantify KLEs from Peucedanum japonicum root extracts using liquid chromatography (LC) without utilizing standards. This method used relative molar sensitivity (RMS) and 7-ethoxy-4-methylcoumarin as a single-reference (SR) compound instead of KLE standards. RMS is the sensitivity ratio of SR to analytes, determined using an offline combination of quantitative NMR and LC. LC was performed using a triacontylsilyl silica gel column of superficially porous particles with a ternary mobile phase. The range of the method was 2.60-509 µmol/L. The accuracy and precision were reasonable. This is the first study to apply the RMS method to both conventional LC and ultra-high-performance liquid chromatography using the same mobile phase and column. This method may aid the quality assurance of foods containing KLEs.

Pteryxin suppresses osteoclastogenesis and prevents bone loss via inhibiting the MAPK/Ca2+ signaling pathways mediated by ROS.

Osteoporosis, as a severe public health problem worldwide, causes systemic damage to bone mass, strength, and microstructure with an increased propensity for fragility fractures. Given the inherent adverse effects associated with long-term use of current prescription medications for osteoporosis treatment, identifying natural alternatives to existing treatment methods is imperative. Pteryxin (PTX), a natural coumarin, is isolated from the Peucedanum species belonging to the family Apiaceae. PTX has been reported to have antioxidant, anti-inflammatory and anti-obesity properties. However, its effect on osteoporosis has not been clarified. Our study confirmed that PTX could attenuate the formation of osteoclasts and bone resorption on a dose-dependent basis in vitro. Consistently, in vivo ovariectomy (OVX)-induced osteoporosis models simulating the physiological characteristics of postmenopausal women showed that PTX could partially reverse the bone loss caused by OVX. Further study of its mechanism revealed that PTX might block the MAPK and Ca2+-calcineurin-NFATc1 signaling pathways by decreasing the reactive oxygen species (ROS) level in osteoclasts to dampen the expression of critical transcriptional NFATc1 and downstream osteoclast-specific genes. Overall, PTX may present a new or alternative treatment option for osteoporosis.

Cytoprotective Effect of Pteryxin on Insulinoma MIN6 Cells Due to Antioxidant Enzymes Expression via Nrf2/ARE Activation.

The low-level antioxidant activity of pancreatic islets causes type 1 diabetes due to oxidative stress, which is also the cause of failure in the pancreatic islets' isolation and cell transplantation. In our previous study, pteryxin was found to be a natural product as a nuclear factor-erythroid-2-related factor (Nrf2) activator. This study focused on elucidation that the potentiality of pteryxin can activate the antioxidant enzymes, even under oxidative stress, by hydrogen peroxide (H2O2). Pteryxin treated with mouse insulinoma MIN6 cells was enhanced the antioxidant gene expressions in the ARE (antioxidant response element) region for HO-1 (Heme Oxygenase-1), GCLC (Glutamate-cysteine ligase catalytic subunit), SOD1 (Super Oxide dismutase1), and Trxr1 (Thioredoxin reductase1), and those enzymes were also expressed during the nuclei transference of cytoplasmic Nrf2. In fact, the cells exposed to H2O2 concentrations of a half-cell lethal in the presence of pteryxin were then induced main antioxidant enzymes, HO-1, GCLC, and Trxr1 in the ARE region. The increased glutathione (GSH) levels associated with the GCLC expression also suggested to be cytoprotective against oxidative stress by activating the redox-metabolizing enzymes involving their increased antioxidant activity in the cells. In addition, Akt is a modulator for Nrf2, which may be responsible for the Nrf2 activation. These results allowed us to consider whether pteryxin or its synthesized congeners, an Nrf2 activator, is a potential preservative agent against islet isolation during cell transplantation.

Pteryxin attenuates LPS-induced inflammatory responses and inhibits NLRP3 inflammasome activation in RAW264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Pteryxin is a natural coumarin compound that is found in "Qianhu", a traditional Chinese medicine, which possesses heat-clearing and detoxifying functions according to the theory of Traditional Chinese Medicine. Despite its medicinal effects, its anti-inflammatory and mechanisms of actions have not been established. AIM OF THIS STUDY: This study aims to evaluate the anti-inflammatory property and reveal the possible anti-inflammatory mechanisms of pteryxin. MATERIAL AND METHODS: LPS-induced RAW 264.7 macrophages and LPS-induced zebrafish model were used for the anti-inflammatory activity determination of pteryxin. The level of NO, PEG2, TNF-α and IL-6 were measured by ELISA. The accumulation of NO and ROS was stained and observed by a fluorescence microscopy. The nuclear translocation of NF-κB p65 and formation of NLRP3 inflammasome complex in LPS-induced RAW 264.7 macrophage cells were analyzed by immunofluorescence assay. The expression level of iNOS, IL-6, COX-2, TNF-α, p-p38, p38, ERK, JNK, p-ERK, p-JNK, IKK, IκB-α, p-IKK, p-IκB-α, p65, NLRP3, p-p65, Caspase 1 (p 20), ASC, and GAPDH were determined by Western blotting. RESULTS: Lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) and nitric oxide (NO) secretions were found to be downregulated by pteryxin. Moreover, pteryxin significantly suppressed inflammatory factor secretion in LPS-treated RAW 264.7 cells. Mechanistically, pteryxin significantly downregulated NF-κB/MAPK activation. Moreover, pteryxin inhibited caspase-1 and NLRP3 activation and formation of ASC specks in RAW 264.7 cells, implying that pteryxin inhibits inflammasome assembly, which is a signal for NLRP3 inflammasome activation. In conclusion, pteryxin blocks NF-κB/MAPK signaling, and suppresses the initiation and activation of NLRP3 thereby preventing inflammation. CONCLUSION: Pteryxin is a potential treatment option for inflammatory-related diseases.

Molecular Effects of Pteryxin and Scopoletin in the 5xFAD Alzheimer's Disease Mouse Model.

BACKGROUND: Alzheimer's disease (AD) is one of the most prevalent diseases with rapidly increasing numbers, but there is still no medication to treat or stop the disease. Previous data on coumarins suggests that scopoletin may have potential benefits in AD. OBJECTIVE: Evaluate the therapeutic potential of the coumarins with natural origin - scopoletin and pteryxin- in a 5xFAD mouse model of AD. METHODS: Both compounds were administered at two doses to 12-month-old mice, which represent severe AD pathology. The effects of coumarins were assessed on cognition in mouse experiments. Changes in the overall brain proteome were evaluated using LCMS/ MS analyses. RESULTS: The Morris water maze test implicated that a higher dose of pteryxin (16 mg/kg) significantly improved learning, and the proteome analysis showed pronounced changes of specific proteins upon pteryxin administration. The amyloid-ß precursor protein, glial fibrillary acid protein, and apolipoprotein E protein which are highly associated with AD, were among the differentially expressed proteins at the higher dose of the pteryxin. CONCLUSION: Overall, pteryxin may be evaluated further as a disease-modifying agent in AD pathology in the late stages of AD.

Protective effect of pteryxin on LPS-induced acute lung injury via modulating MAPK/NF-κB pathway and NLRP3 inflammasome activation.

ETHNOPHARMACOLOGICAL RELEVANCE: Peucedanum praeruptorum seed root is a common medicinal herb with antipyretic, expectorant, antitussive, and therapeutic effects against bronchitis and furuncle. The roots of this herb contain many coumarin compounds, including pteryxin. AIM OF THIS STUDY: To investigate whether pteryxin can alleviate the LPS-induced lung injury and the mechanism involved. MATERIAL AND METHODS: Male BALB/C mice were orally given sodium carboxymethylcellulose (CMC-Na) (0.5%, 1mL/100g) and pteryxin (suspended in CMC-Na; 0.5%) at 5, 10, 25 mg/kg once daily for 7 days. Subsequently, the mice received a single intratracheal instillation of 5 mg/kg LPS or saline as the control. After 8 hours, the mice were sacrificed to collect bronchoalveolar lavage fluid (BALF) and lung tissues. These samples were used to determine the lung W/D (wet/dry) weight ratio, total protein (TP) levels, inflammatory cytokines (IL-6, TNF-α, and IL-1ß) and expression of protein involved in MAPK/NF-κB pathway and NLRP3 inflammasome. H&E staining was carried out on tissue sections to explore the pathological alterations induced by LPS. The protein expression of F4/80 and NLRP3 in lung tissues was analyzed using immunohistochemical staining. The binding of pteryxin to target proteins (MAPK, NF-κB and NLRP3) was determined based on molecular docking tests. RESULTS: Treatment with pteryxin reduced the lung W/D weight ratio, total protein (TP) level and levels of inflammatory cytokines (TNFα, IL-6 and IL-1 ß) significantly. Therefore, it ameliorated LPS-induced inflammatory response in BALB/C mice. Moreover, pteryxin suppressed LPS-induced upregulation of proteins involved in MAPK/NF-κB signaling pathway and NLRP3 inflammasome activation. The expression level of F4/80 and NLRP3 was also downregulated by pteryxin pretreatment in lung tissues. Docking analysis revealed that pteryxin bound to target proteins (MAPK, NF- κB and NLRP3) with a fit-well pattern . CONCLUSION: Pteryxin may attenuate LPS-induced acute lung injury by dampening MAPK/NF-κB signaling and NLRP 3 inflammasome activation.

Induction of Antioxidant Protein HO-1 Through Nrf2-ARE Signaling Due to Pteryxin in Peucedanum Japonicum Thunb in RAW264.7 Macrophage Cells.

This study focused on exploring the nuclear factor-erythroid-2-related factor (Nrf2) active compound to avoid oxidative stress related to various diseases, such as obesity and diabetes mellitus. The activity of the Nrf2-ARE (antioxidant response element) signaling was evaluated by a reporter assay involving over five hundred various edible medicinal herbs, and the highest Nrf2 activity was found in the ethanol extract of Peucedanum japonicum leaves. The active compound in the extract was isolated by high performance liquid chromatography (HPLC), and the chemical structure was identical to pteryxin based on 1H, 13C-NMR spectra and liquid chromatography/time-of-fright mass spectrometer (LC/TOF/MS). From the pteryxin, the transcription factor Nrf2 was accumulated in the nucleus and resulted in the expression of the antioxidant protein, heme oxygenase-1 (HO-1). In addition, the Nrf2 activity involving HO-1 expression due to coumarin derivatives was evaluated together with pteryxin. This suggested that the electrophilicity, due to the α,ß-carbonyl and/or substituted acyl groups in the molecule, modulates the cysteine residue in Keap1 via the Michel reaction, at which point the Nrf2 is dissociated from the Keap1. These results suggest that pteryxin will be a useful agent for developing functional foods.

Pteryxin - A promising butyrylcholinesterase-inhibiting coumarin derivative from Mutellina purpurea.

Pteryxin is a dihydropyranocoumarin derivative found in Apiaceae family. In this study, pteryxin, which was previously isolated from the fruits of Mutellina purpurea, was investigated for its inhibitory potential against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which are the key enzymes in the pathology of Alzheimer's disease (AD). The compound was tested in vitro using ELISA microplate reader at 100 µg/ml and found to cause 9.30 ± 1.86% and 91.62 ± 1.53% inhibition against AChE and BChE, respectively. According to our results, pteryxin (IC50 = 12.96 ± 0.70 µg/ml) was found to be a more active inhibitor of BChE than galanthamine (IC50 = 22.16 ± 0.91 µg/ml; 81.93± 2.52% of inhibition at 100 µg/ml). Further study on pteryxin using molecular docking experiments revealed different possible binding modes with both polar and hydrophobic interactions inside the binding pocket of BChE. Top docking solution points out to the formation of two hydrogen bonds with the catalytic residues S198 and H438 of BChE as well as a strong π - π stacking with W231. Therefore, pteryxin as a natural coumarin seems to be a strong BChE inhibitor, which could be considered as a lead compound to develop novel BChE inhibitors for AD treatment.

Traditional uses, phytochemistry and pharmacological properties of the genus Peucedanum: a review.

ETHNOPHARMACOLOGICAL RELEVANCE: The genus Peucedanum (Apiaceae) comprising more than 120 species is widely distributed in Europe, Asia and Africa. The ethnopharmacologial history of this genus indicated that some extracts of aerial and underground parts of several Peucedanum species have been used in folk medicine for treatment of various conditions, such as cough, cramps, pain, rheumatism, asthma and angina. This review focuses on ethnopharmacological uses of Peucedanum species, as well as the phytochemical, pharmacological and toxicological studies on this genus. Through this review, I intend to highlight the known and potential effects of the Peucedanum species or their isolated compounds and show which traditional medicine uses have been supported by pharmacological investigations. METHODS: Information on the Peucedanum species was collected from scientific journals, books, thesis and reports via a library and electronic search (using Google Scholar, Pubmed, Scopus, Web of Science and ScienceDirect). This review covers the available literature from 1970 to the end of September 2013. RESULTS: Although, there are about 120 species in this genus, so far many species have received no or little attention and most of pharmacological studies were performed on just about 20 species. Many phytochemical investigations on this genus confirmed that Peucedanum species are rich in essential oils and coumarins. The present review article shows that Peucedanum species have a wide spectrum of pharmacological activities and the most reported activities of Peucedanum plants come back to the presence of coumarins, flavonoids, phenolics and essential oils. CONCLUSIONS: The present review confirms that some Peucedanum species have emerged as a good source of the traditional medicine for treatment of inflammation, microbial infections, cardiopulmonary diseases and provides new insights for further investigations on isolated compounds, especially on praeruptorins, to find novel therapeutics and aid drug discovery. However, for using Peucedanum species to prevent and treat various diseases, additional pharmacological studies to find the mechanism of action, safety and efficacy of them before starting clinical trials are required.

Pteryxin: a coumarin in Peucedanum japonicum Thunb leaves exerts antiobesity activity through modulation of adipogenic gene network.

OBJECTIVES: Partially purified hexane phase (HP) of Peucedanum japonicum Thunb (PJT) was identified as an antiobesity candidate. However, the compound responsible for the antiobesity activity remained unknown. Thus, in this study we isolated the active compound, to determine the mechanisms related to antiobesity activity in vitro. METHODS: The HP was fractionated, and the effect on the triacylglycerol (TG) content was evaluated in 3T3-L1 preadipocytes and HepG2 hepatocytes. On the basis of comprehensive spectroscopic analyses, the structure of the active compound was identified as pteryxin, a known compound in PJT. However, to our knowledge, its biological activities against obesity have not been reported previously. The dose-dependent effect on the TG content, and the gene expressions related to adipogenesis, fatty acid catabolism, energy expenditure, lipolysis, and lipogenesis due to pteryxin (10, 15, and 20 µg/mL) were examined in vitro. RESULTS: Pteryxin dose dependently suppressed TG content in both 3T3-L1 adipocytes (by 52.7%, 53.8%, and 57.4%, respectively; P < 0.05) and HepG2 hepatocytes (by 25.2%, 34.1%, and 27.4%, respectively; P < 0.05). Sterol regulatory element-binding protein-1 (SREBP-1c), fatty acid synthase (FASN), and acetyl-coenzyme A carboxylase-1 (ACC1) were down-regulated in pteryxin-treated 3T3-L1 adipocytes (by 18%, 36.1%, and 38.2%, P < 0.05) and HepG2 hepatocytes (by 72.3%, 62.9%, and 38.8%, respectively; P < 0.05). The adipocyte size marker gene, paternally expressed gene1/mesoderm specific transcript (MEST) was down-regulated (by 42.8%; P < 0.05), and hormone-sensitive lipase, a lipid catabolizing gene was up-regulated (by 15.1%; P < 0.05) in pteryxin-treated adipocytes. The uncoupling protein 2 (by 77.5%; P < 0.05) and adiponectin (by 76.3%; P > 0.05) were up-regulated due to pteryxin. CONCLUSION: Our study demonstrated that pteryxin in PJT plays the key role in regulating the lipid metabolism-related gene network and improving energy production in vitro. Thus, the results suggest pteryxin as a new natural compound to be used as an antiobesity drug in the pharmaceutical industry.