Chemical characterization, multivariate analysis and comparison of biological activities of different parts of Fraxinus mandshurica.

Fraxinus mandshurica (Oleaceae) is used as a traditional medicinal plant for the treatment of red eyes, menstrual disorders, excessive leucorrhea, chronic bronchitis and psoriasis. To perform chemical characterization of the secondary metabolites of F. mandshurica roots, bark, stems and leaves, 32 samples were collected from eight provinces in this study. A total of 64 chemical components were detected from four different parts of F. mandshurica by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Meanwhile, a total of nine secoiridoids were obtained by natural product chemical extraction, isolation and identification methods. Quantitative analysis by high-performance liquid chromatography-diode array detection-mass spectrometry showed the highest total content of secoiridoids in the bark, which is also consistent with the traditional medicinal parts. The results of methodological validation showed that the correlation coefficient (R2) values were all >0.9993, indicating a good linear range of the standard curve, while the relative standard deviations of precision, reproducibility and stability were <3%, and the spiked recoveries ranged from 98.22 to 102.27%, indicating that the experimental method was reliable and stable. In addition, fingerprinting and a heatmap were established to demonstrate the content trends of F. mandshurica more visually from different origins. Multivariate analysis, including principal component analysis and partial least squares discriminant analysis, was performed to determine the chemical characteristics of different parts of F. mandshurica, and six characteristic secoiridoids that could be used to distinguish different origins were screened. Finally, the inhibition of tyrosinase, α-glucosidase, acetylcholinesterase and pancreatic lipase activities by the nine characteristic compounds and extracts from different parts were investigated, and the results showed that they all exhibited different degrees of enzyme activity inhibition and thus have potential applications in whitening and blemish removal, hypoglycemia, anti-Alzheimer's disease and anti-obesity as a new source of natural enzyme activity inhibitors. This study establishes an identification and evaluation method applicable to phytochemistry of different origins, which is a guideline for quality control, origin evaluation and clinical application of traditional medicinal plants. This is also an unprecedented study on the identification of the chemical composition of different parts of F. mandshurica, characteristic compounds and the inhibition of enzyme activity of extracts from different parts.

Fraxini cortex: Progresses in phytochemistry, pharmacology and ethnomedicinal uses.

ETHNOPHARMACOLOGICAL RELEVANCE: Fraxini cortex, which has been widely used as a traditional Chinese medicine for 2000 years, is made from the dried bark of four plant species: Fraxinus chinensis subsp. rhynchophylla (Hance) A.E.Murray, Fraxinus chinensis Roxb., Fraxinus chinensis subsp. chinensis and Fraxinus stylosa Lingelsh.. In Chinese traditional medicine, it possesses the properties of heat-clearing and dampness-drying, asthma relief and cough suppression, as well as vision improvement. It is utilized for treating bacterial disorders, enteritis, leukorrhea, chronic bronitis, painful red eyes with swelling, lacrimation due to windward exposure, psoriasis, and other diseases or related symptoms. AIM OF THE STUDY: Fraxini cortex is abundant in chemical constituents and has garnered significant attention from plant chemists, particularly regarding coumarins, as evidenced by the recently identified three coumarin compounds. Considering the current dearth of systematic reporting on studies pertaining to Fraxini cortex, herein we provide a comprehensive summary of the advancements in phytochemistry, pharmacology, detection methods, and ethnomedicinal applications of Fraxini cortex. MATERIALS AND METHODS: We conducted a comprehensive search across online data sources (Web of Science, Public Medicine (PubMed), China National Knowledge Infrastructure (CNKI), as well as Chinese dissertations) and traditional Chinese medicine classics to gather the necessary literature resources for this review. RESULTS: Briefly, The Fraxini cortex yielded a total of 132 phytochemicals, including coumarins, lignans, secoiridoids, phenylethanol glycosides, flavonoids, triterpenoids, and other compounds. Among them, the main active ingredients are coumarins which possess a diverse range of pharmacological activities such as anti-inflammatory effects, anti-tumor properties, prevention of tissue fibrosis and oxidation damage as well as cardioprotective effects. CONCLUSIONS: All types of research conducted on Fraxini cortex, particularly in the field of ethnopharmacology, phytochemistry, and pharmacology, have been thoroughly reviewed. However, certain traditional applications and pharmacological activities of Fraxini cortex lack scientific evaluation or convincing evidence due to incomplete methodologies and ambiguous results, as well as a lack of clinical data. To validate its pharmacological activity, clinical efficacy, and safety profile, a systematic and comprehensive research evaluation is imperative. As an important traditional Chinese medicine, Fraxini cortex should be further explored to facilitate the development of novel drugs and therapeutics for various diseases. Greater attention should be given to how it can be better utilized.

Antioxidant and Pulmonary Protective Potential of Fraxinus xanthoxyloides Bark Extract against CCl4 -Induced Toxicity in Rats.

Fraxinus xanthoxyloides is a perennial shrub belonging to family Oleaceae, traditionally used for malaria, jaundice, pneumonia, inflammation, and rheumatism. Our study is aimed to assess the total phenolics (TPC), flavonoids (TFC), terpenoids contents (TTC) and antioxidant profiling of F. xanthoxyloides methanol bark extract (FXBM) and its fractions, hexane, chloroform, ethyl acetate and aqueous, along with high-performance liquid chromatography with diode-array detection (HPLC-DAD). Further, the antioxidant and pulmonary protective potential was explored against carbon tetrachloride (CCl4 )-induced CCl4-induced pulmonary tissue damage in rats. The highest TPC, TFC and TTC were found in FXBM (133.29±4.19 mg/g), ethyl acetate fraction (279.55±10.35 mg/g), and chloroform fraction (0.79±0.06 mg/g), respectively. The most potent antioxidant capacity was depicted by FXBM (29.21±2.40 µg/mg) and ethyl acetate fraction (91.16±5.51 µg/mg). The HPLC-DAD analysis revealed the predominance of gallic, chlorogenic, vanillic and ferulic acid in FXBM. The administration of CCl4 induced oxidative stress, suppressed antioxidant enzymes' activities including catalase, peroxidase, superoxide dismutase, glutathione peroxidase, glutathione-S-transferase, and glutathione reductase. Further, it increased thiobarbituric acid reactive substances (TBARS) and H2 O2 levels, induced DNA injuries and reduced the total protein and glutathione content in lung tissues. The treatment of rats with FXBM restored these biochemical parameters to the normal level. Moreover, the histopathological studies of lung tissues demonstrated that FXBM protected rats' lung tissues from oxidative damage restoring normal lung functions. Thus, F. xanthoxyloides bark extract is recommended as adjuvant therapy as protective agent for patients with lung disorders.

Bio-Guided Isolation of Compounds from Fraxinus excelsior Leaves with Anti-Inflammatory Activity.

Inflammation is the first physiological defence mechanism against external and internal stimuli. The prolonged or inappropriate response of the immune system may lead to the persistent inflammatory response that can potentially become a basis for chronic diseases e.g., asthma, type II diabetes or cancer. An important role in the alleviation of inflammatory processes, as an adjunct to traditional pharmacological therapy, is attributed to phytotherapy, especially to raw materials with a long tradition of use, e.g., ash leaves. Despite their long-term use in phytotherapy, the specific mechanisms of action have not been confirmed in a sufficient number of biological or clinical studies. The aim of the study is a detailed phytochemical analysis of infusion and its fractions, isolation of pure compounds from the leaves of Fraxinus excelsior and evaluation of their effect on the secretion of anti-inflammatory cytokines (TNF-α, IL-6) and IL-10 receptor expression in an in vitro model of monocyte/macrophage cells isolated from peripheral blood. Methods: Phytochemical analysis was carried out by the UHPLC-DAD-ESI-MS/MS method. Monocytes/macrophages were isolated from human peripheral blood using density gradient centrifugation on Pancoll. After 24 h incubation with tested fractions/subfractions and pure compounds, cells or their supernatants were studied, respectively, on IL-10 receptor expression by flow cytometry and IL-6, TNF-α, IL-1ß secretion by the ELISA test. Results were presented with respect to Lipopolysaccharide (LPS) control and positive control with dexamethasone. Results: The infusion, 20% and 50% methanolic fractions and their subfractions, as well as their dominating compounds, e.g., ligstroside, formoside and oleoacteoside isolated from the leaves, show the ability to increase the IL-10 receptor expression on the surface of monocyte/macrophage cells, stimulated by LPS, and to decrease the secretion of pro-inflammatory cytokines, e.g., TNF-α, IL-6.

Phytochemical study and immunomodulatory activity of Fraxinus excelsior L.

OBJECTIVES: Fraxinus excelsior L. (FE) is traditionally used to treat inflammatory and pain disorders. This study aimed to identify the constituents of FE leaves and evaluate the effects of its n-hexane (FEH), ethyl acetate (FEE), methanol (FEM) extracts and constituents on the viability of THP-1 cells and their ability to release pro-inflammatory cytokines. METHODS: THP-1 cell viability was assessed using an MTT assay. The immunomodulatory activity was evaluated by measuring tumour necrosis factor-alpha (TNF-α) and interleukin 12 (IL-12) released by lipopolysaccharide-stimulated THP-1 cells using enzyme-linked immunosorbent assays. KEY FINDINGS: Triterpenes, tyrosol esters, alkanes, phytyl and steryl esters, pinocembrin and bis(2-ethylhexyl)phthalate were isolated from FE. The tyrosol esters showed no significant effect on THP-1 cell viability. FEH, FEE, FEM, and pinocembrin, ursolic acid, oleanolic acid had IC50 values of 56.9, 39.9, 124.7 µg/ml and 178.6, 61.5 and 199.8 µM, respectively. FE extracts, ursolic acid, oleanolic acid and pinocembrin significantly reduced TNF-α/IL-12 levels. The tyrosol esters did not significantly affect TNF-α/IL-12 production. CONCLUSIONS: FE was able to reduce pro-inflammatory cytokine production indicating a mechanistic focus in its use for inflammation and pain. Further investigations are warranted to unravel the mode of action of the tested constituents and discover other potentially active compounds in FE extracts.

Inhibition of microbial growth by silver nanoparticles synthesized from Fraxinus xanthoxyloides leaf extract.

AIMS: Conventional antibiotics have been failed to treat infectious diseases due to emergence of multidrug resistance (MDR) in some common pathogens. The current study aimed to formulate new antimicrobials from greener sources. In the midst of these efforts, nanotechnology is a newly emerged field, in which the synthesis of new nanoparticles through novel and efficient means is on the rise. METHODS AND RESULTS: The current work has been carried out to assess the potential of Fraxinus xanthoxyloides (FX) leaf extract in biosynthesis of silver nanoparticles (FX-AgNPs). This method is economical and simple one-step approach to synthesize AgNPs. Characterization of FX-AgNPs has been done by UV-Visible spectroscopy, scanning electron microscope (SEM), X-ray diffraction (XRD), transmission electronic microscope (TEM) and Fourier transforms infrared spectroscopy (FT-IR). The formation of FX-AgNPs has confirmed through UV-Visible spectroscopy (at 430 nm) by change of colour owing to surface Plasmon resonance. Based on the XRD pattern, the crystalline property of FX-AgNPs has established. Functional groups existing in F. xanthoxyloides leaf extract are confirmed by FT-IR spectrum. SEM and TEM authenticated morphology of the AgNPs. The newly synthesized nanoparticles were evaluated for their antimicrobial potential. Minimum inhibitory concentration was determined against Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA) strains, Pseudomonas aeruginosa and Candida albicans by microtiter plate assay. The lowest inhibition (69%) observed against MRSA was at a concentration of 50 ppm FX-AgNPs and maximum inhibition (81%) observed was against P. aeruginosa. The biosynthesized AgNPs triggered up to 68·6% reduction of the P. aeruginosa biofilm when compared to the control. CONCLUSION: It can be concluded that nanoparticles could be a better alternative of antibiotics with greater efficacies and represent a valuable milestone to fight against infections caused by MDR pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: This study imparts a useful insight into the development of a new antimicrobial agent from a novel source.

Secoiridoid Glucosides and Anti-Inflammatory Constituents from the Stem Bark of Fraxinus chinensis.

Qin Pi (Fraxinus chinensis Roxb.) is commercially used in healthcare products for the improvement of intestinal function and gouty arthritis in many countries. Three new secoiridoid glucosides, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), and 3'',4''-di-O-methyl-demethyloleuropein (3), have been isolated from the stem bark of Fraxinus chinensis, together with 23 known compounds (4-26). The structures of the new compounds were established by spectroscopic analyses (1D, 2D NMR, IR, UV, and HRESIMS). Among the isolated compounds, (8E)-4''-O-methylligstroside (1), (8E)-4''-O-methyldemethylligstroside (2), 3'',4''-di-O-methyldemethyloleuropein (3), oleuropein (6), aesculetin (9), isoscopoletin (11), aesculetin dimethyl ester (12), fraxetin (14), tyrosol (21), 4-hydroxyphenethyl acetate (22), and (+)-pinoresinol (24) exhibited inhibition (IC50 ≤ 7.65 µg/mL) of superoxide anion generation by human neutrophils in response to formyl-L-methionyl-L-leuckyl-L-phenylalanine/cytochalasin B (fMLP/CB). Compounds 1, 9, 11, 14, 21, and 22 inhibited fMLP/CB-induced elastase release with IC50 ≤ 3.23 µg/mL. In addition, compounds 2, 9, 11, 14, and 21 showed potent inhibition with IC50 values ≤ 27.11 µM, against lipopolysaccharide (LPS)-induced nitric oxide (NO) generation. The well-known proinflammatory cytokines, tumor necrosis factor-alpha (TNF-α) and interleukin 6 (IL-6), were also inhibited by compounds 1, 9, and 14. Compounds 1, 9, and 14 displayed an anti-inflammatory effect against NO, TNF-α, and IL-6 through the inhibition of activation of MAPKs and IκBα in LPS-activated macrophages. In addition, compounds 1, 9, and 14 stimulated anti-inflammatory M2 phenotype by elevating the expression of arginase 1 and Krüppel-like factor 4 (KLF4). The above results suggested that compounds 1, 9, and 14 could be considered as potential compounds for further development of NO production-targeted anti-inflammatory agents.

Anti-Proliferative Activity of A Hydrophilic Extract of Manna from Fraxinus angustifolia Vahl through Mitochondrial Pathway-Mediated Apoptosis and Cell Cycle Arrest in Human Colon Cancer Cells.

Manna is produced from the spontaneous solidification of the sap of some Fraxinus species, and, owing its content in mannitol, is used in medicine as a mild laxative. Manna is also a rich source of characteristic bio-phenols with reducing, antioxidant and anti-inflammatory properties. This study assesses the activity of a hydrophilic extract of manna (HME) on cellular and molecular events in human colon-rectal cancer cells. HME showed a time- and concentration-dependent anti-proliferative activity, measured by MTT assay, in all the cell lines examined, namely Caco-2, HCT-116 and HT-29. The amounts of HME that caused 50% of cell death after a 24 h treatment were 8.51 ± 0.77, 10.73 ± 1.22 and 28.92 ± 1.99 mg manna equivalents/mL, respectively; no toxicity was observed in normally differentiated Caco-2 intestinal cells. Hydroxytyrosol, a component of HME known for its cytotoxic effects on colon cancer cells, was ineffective, at least at the concentration occurring in the extract. Through flow-cytometric techniques and Western blot analysis, we show that HME treatment causes apoptosis, assessed by phosphatidylserine exposure, as well as a loss of mitochondrial membrane potential, an intracellular formation of reactive oxygen species (ROS), increases in the levels of cleaved PARP-1, caspase 3 and Bax, and a decrease in Bcl-2 expression. Moreover, HME interferes with cell cycle progression, with a block at the G1/S transition. In conclusion, the phytocomplex extracted from manna exerts an anti-proliferative activity on human colon cancer cells through the activation of mitochondrial pathway-mediated apoptosis and cell cycle arrest. Our data may suggest that manna could have the potential to exert chemo-preventive effects for the intestine.

Light Limitation Impacts Growth but Not Constitutive or Jasmonate Induced Defenses Relevant to Emerald Ash Borer (Agrilus planipennis) in White Fringetree (Chionanthus virginicus) or Black Ash (Fraxinus nigra).

White fringetree is a host for the invasive emerald ash borer (EAB) but is of lower quality than the related and highly susceptible black ash. Field observations suggest that host trees grown in full sun are more resistant to EAB than those in shade, however the impact of light limitation on chemical defenses has not been assessed. We quantified constitutive and jasmonate-induced phloem defenses and growth patterns of white fringetree and black ash under differential light conditions and related them to EAB larval performance. White fringetree had significantly lower constitutive and induced activities of peroxidase, polyphenol oxidase, ß-glucosidase, chitinase and lignin content, but significantly higher gallic acid equivalent soluble phenolic, soluble sugar, and oleuropein concentrations compared to black ash. Multivariate analyses based on tissue chemical attributes displayed clear separation of species and induced defense responses. Further, EAB performed significantly worse on white fringetree than black ash, consistent with previous studies. Light limitation did not impact measured defenses or EAB larval performance, but it did decrease current year growth and increase photosynthetic efficiency. Overall our results suggest that phenolic profiles, metabolite abundance, and growth traits are important in mediating white fringetree resistance to EAB, and that short-term light limitation does not influence phloem chemistry or larval success.

Anti-Obesity and Gut Microbiota Modulation Effect of Secoiridoid-Enriched Extract from Fraxinus mandshurica Seeds on High-Fat Diet-Fed Mice.

Previously we conducted a phytochemical study on the seeds of Fraxinus excelsior and isolated nine secoiridoid compounds with adipocyte differentiation inhibitory activity and peroxisome proliferator activated receptor alpha (PPARα) activation effects. However, the bioactive constituents and functions of Fraxinus mandshurica seeds have not been studied. In the present study, we investigated the secoiridoid compounds in F. mandshurica seed extract (FM) using column chromatography, 1H-NMR, 13C-NMR and HPLC-DAD methods. The pancreatic lipase inhibitory activities of isolated compounds were evaluated in vitro. Additionally, the anti-obesity and gut microbiota modulation effect of FM on high-fat diet-induced obesity in C57BL/6 mice were also studied in vivo. The results showed that 19 secoiridoids were isolated from FM and identified. The total content of secoiridoids in FM reached 181.35 mg/g and the highest content was nuzhenide (88.21 mg/g). All these secoiridoid compounds exhibited good pancreatic lipase inhibitory activity with inhibition rate ranged from 33.77% to 70.25% at the concentration of 100 µM. After obese mice were administrated with FM at 400 mg/kg.bw for 8 weeks, body weight was decreased by 15.81%. Moreover, FM could attenuate the lipid accumulation in serum and liver, relieve the damage in liver and kidney, and extenuate oxidative stress injury and inflammation caused by obesity in mice. FM could also modulate the structural alteration of gut microbiota in obese mice, increasing the proportion of anti-obesity gut microbiota (Bacteroidetes, Bacteroidia, S24-7 and Allobaculum), and reducing the proportion of obesogenic gut microbiota (Firmicutes and Dorea). This study suggests that F. mandshurica seeds or their secoiridoids may have potential for use as a dietary supplement for obesity management.

Apoptosis in Leukemic Cells Induced by Anti-proliferative Coumarin Isolated from the Stem Bark of Fraxinus rhynchophylla.

Esculetin 6-O-ß-D-arabinofuranosyl-(1→6)-ß-D-glucopyranoside (EAG) is a coumarin glycoside isolated from the stem bark of Fraxinus rhynchophylla. This study scrutinized the anti-proliferative activity of EAG on blood cancer-derived Jurkat leukemic cells. Cell viability assays in leukemic cancer cells determined that EAG possesses potent anti-proliferative effects. Moreover, treatment with EAG increased the proportion of apoptotic cells, resulted in cell cycle arrest being induced at the subG0/ G1 phase, and reduced the proportion of cells present in the S phase. In addition, mitochondrial membrane potential was reduced by EAG in Jurkat cells. Additionally, EAG triggered apoptosis that was mediated by the downregulation of BCL-XL, p-IκBα, and p-p65 expressions in addition to the upregulation of cleaved Caspase 3 and BAX expressions. These findings revealed that the toxic effect of EAG was mediated by intracellular signal transduction pathways that involved a mechanism in which reactive oxygen species (ROS) were upregulated. Thus, this study concludes that EAG could potentially serve as a therapeutic agent for leukemia.

Antimutagenic, antigenotoxic and antiproliferative activities of Fraxinus angustifolia Vahl. leaves and stem bark extracts and their phytochemical composition.

In recent years, chronic degenerative diseases such as certain types of cancers, are becoming an evident issue. DNA damage has been for long recognized as a causal factor for cancer development because mutations or chromosomal aberrations affect oncogenes and tumor suppressor genes leading cells to malignant transformation and to the subsequent cancerous growth. Medicinal plants are often used for the prevention or treatment of various diseases with great scientific interest. Among the medicinal plants distributed in the Mediterranean region, Fraxinus angustifolia Vahl. has been used in traditional medicine for its remarkable curative properties. However, in spite of this popularity, little works have been performed on the activity so that further studies should be performed to investigate in depth the antimutagenic, antigenotoxic and antiproliferative activities of the plant. Thus, the present study was aimed to the evaluation of the potential antimutagenic, antigenotoxic and antiproliferative properties of leaves and stem bark extracts of this well-known tree. Antimutagenic activity was evaluated by Salmonella mutagenicity assay in Salmonella typhimurium TA98 and TA100 strains. The antigenotoxic potential was assessed by umu test in the strain of S. typhimurium TA1535/pSK1002. Antiproliferative activity was studied on human hepatoblastoma (HepG-2) and on breast adenocarcinoma (MCF-7) cell lines by MTT assay. Furthermore, the antiproliferative activity observed on cancer cells was compared with that on the human normal-like fibroblasts (TelCOFS02MA) and the selectivity index was calculated to understand if extracts were able to exert selective toxicity towards cancer cells. Moreover, phenolic compounds are plant substances with a large spectrum of biochemical activities with antioxidant, antimutagenic and anticarcinogenic effects. Based on the strong evidence of biological activities of phenolic compounds, the study was focused on the determination of total phenolics and flavonoids contents, and the phytochemical composition of the extracts assessed by LC/MS. The ethanol extracts of both leaves and stem barks showed significant from moderate to strong antimutagenic and antigenotoxic effects. In addition, selective cytotoxicity towards cancer cells was shown by ethanolic leaves extract and aqueous/chloroform leaves and stem bark extracts. The latter showed high levels of total phenolic contents among all the other extracts. Identified phenylethanoids (calceolariosides, verbascoside) and secoiridoids (oleuropein and ligstroside) could be responsible for the demonstrated broad spectrum of healthy properties.

Chemical Constituents from Fraxinus hupehensis and Their Antifungal and Herbicidal Activities.

The phytochemical investigation of Fraxinus hupehensis led to the isolation and characterization of ten compounds which were identified as fraxin (1), fraxetin (2), esculetin (3), cichoriin (4), euphorbetin (5), kaempferol-3-O-ß-rutinoside (6), oleuropein (7), linoleic acid (8), methyl linoleate (9), and ß-sitosterol (10). Structures of the isolated constituents were characterized by 1H NMR, 13C NMR and HRMS. All the compounds, except compounds 3 and 4, were isolated for the first time from this plant. Further, this was the first report for the occurrence of compound 5 in the Fraxinus species. Antifungal activity evaluation showed that compound 2 exhibited significant inhibitory effects against Bipolaris maydis, Sclerotium rolfsii, and Alternaria solani with EC50 values of 0.31 ± 0.01 mmol/L, 10.50 ± 0.02 mmol/L, and 0.40 ± 0.02 mmol/L respectively, compared to the positive control, Carbendazim, with its EC50 values of 0.74 ± 0.01 mmol/L, 1.78 ± 0.01 mmol/L and 1.41 ± 0.00 mmol/L. Herbicidal activity tests showed that compounds 8-10 had strong inhibitory effects against the roots of Echinochloa crus-galli with EC50 values of 1.16 ± 0.23 mmol/L, 1.28 ± 0.58 mmol/L and 1.33 ± 0.35 mmol/L respectively, more potently active than that of the positive control, Cyanazine, with its EC50 values of 1.56 ± 0.44 mmol/L. However, none of the compounds proved to be active against the tested bacteria (Erwinia carotovora, Pseudomonas syringae, and Ralstonia solanacearum).

Zinc oxide nanoparticles synthesized from Fraxinus rhynchophylla extract by green route method attenuates the chemical and heat induced neurogenic and inflammatory pain models in mice.

Fraxinus rhynchophylla belongs to the family of Oleaceae and also called as Chinese ash wood possesses various pharmacological properties such as neuroprotective, antimicrobial, anti-inflammatory, etc. Therefore we synthesized ZnO nanoparticles using Fraxinus rhynchophylla wood extract as reducing and capping agent. The synthesized nanoparticles were characterized with the aid of UV-Spec, DLS, FT-IR and TEM analysis. Green synthesized ZnO nanoparticles were then assessed for anti-nociceptive property by using various nociception models such as thermal stress-induced, acetic acid, glutamate, capsaicin, and formalin-induced nociception. The sedative effect of synthesized ZnO nanoparticles was evaluated with an open field test. UV-Spectroscopic analysis confirms the formation of ZnO nanoparticles and the characterization studies DLS, FT-IR, and TEM analysis prove it has ideal nanoparticle can be used as a nano-drug. Results of both thermal stress-induced methods hot plate and tail immersion nociception test verified the synthesized ZnO nanoparticles are a potent antinociceptive drug. ZnO nanoparticles effectively reduced the abdominal writhes in acetic acid-induced nociception and it also significantly decreased the nociception activity in another glutamate, capsaicin, and formalin-induced nociception models. Open field experiment proved that synthesized ZnO nanoparticles are less sedative compared to the standard antinociceptive drug morphine. Overall our findings authentically confirm ZnO nanoparticles synthesized from Fraxinus rhynchophylla wood extract is a novel drug that persuasively reduces nociception in different nociceptive induced mice models and can be the best alternative for allopathic drugs which renders severe side effects.

Inhibitory effect of α-amyrin acetate isolated from Fraxinus rhynchophylla on Th17 polarization.

BACKGROUND: T helper 17 (Th17) cells, which are differentiated from CD4+ T cells, drive inflammation, leading to autoimmune diseases such as psoriasis, rheumatoid arthritis, and inflammatory bowel disease. Therefore, inhibiting Th17 polarization could be a therapeutic target for inflammatory diseases. PURPOSE: We investigated the inhibitory effect of Fraxinus rhynchophylla (Oleaceae) on Th17 differentiation and found its active component. STUDY DESIGN: The activity of F. rhynchophylla and its active constituent was verified using CD4+ cells extracted from C57BL/6 mice. METHODS: Micro-environment for Th17 polarization was provided to CD4+ cells and the effect of treatment with samples was measured by enzyme linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), and Western blot. RESULTS: The extract of F. rhynchophylla Hance and its chemical constituent, α-amyrin acetate, which was isolated via bioassay-guided isolation, significantly inhibited Th17 polarization as revealed when interleukin (IL)-17, a characteristic cytokine produced by Th17 cells, was measured. Furthermore, the inhibitory effect of α-amyrin acetate was compared to the amyrin derivatives, α-amyrin and ß-amyrin. All displayed a suppressive effect on Th17 polarization and all reduced the expression of single transducer and activator of transcription 3 (STAT3) and retinoic acid receptor-related orphan receptor γt (RORγt), which are crucial transcription factors regulating Th17 differentiation. α-Amyrin acetate, however, exhibited the most prominent effects, which indicates that the functional group, acetate, might strengthen the inhibitory effect on Th17 differentiation. CONCLUSION: Taken together, these results suggest that the extract of F. rhynchophylla and its active constituent, α-amyrin acetate, could be applied as a potential therapeutic agent for autoimmune disorders such as rheumatoid arthritis.

Anti-inflammatory effects of Phytodolor® (STW 1) and components (poplar, ash and goldenrod) on human monocytes/macrophages.

BACKGROUND: Populus tremula L. (Poplar), Fraxinus excelsior L. (ash) and Solidago virgaurea L. (goldenrod) have been used for medicinal purposes through centuries, to treat pain, fever and inflammation, but their mechanisms of action are still not fully understood. The present study was performed to investigate, whether the herbal medicinal product Phytodolor® (STW 1) and its components have anti-inflammatory effects on activated human monocytes and differentiated human macrophages to elucidate their modes of action in comparison with well-known analgesic, non-steroidal anti-inflammatory drug (NSAIDs) as diclofenac. METHODS: Adherent human monocytes obtained from peripheral blood mononuclear cells (PBMCs) were cultured in serum-free medium and pre-treated with 50-100 µg/ml of diclofenac, STW 1, their components, poplar, ash or goldenrod or its combination (0.05% to 2%). Thereafter, monocytes were activated with 0.1 or 1 µg/ml LPS for 24 h. The intracellular expressions of TNF-α or PTGS2 were determined by cell-based ELISA. Apoptotic cells were identified by YO-PRO-1 staining. Protein or total RNA were isolated to perform SDS-PAGE/Western blot and qRT-PCR analyses. PMA-differentiated human THP-1 macrophages were pre-treated with diclofenac (50 µg/ml) or STW1 (0.1%) and afterwards with LPS (1 µg/ml) and the translocation of the intracellular p62 NF-κB subunit was detected by immunofluorescence. RESULTS: STW 1 inhibited the intracellular content of TNF-α and PTGS2 protein, as well as of TNF-α and PTGS2 gene expression and induced apoptosis in LPS-activated human monocytes under serum free conditions. Furthermore, STW 1 inhibited the translocation of the p65 subunit of the redox-regulated NF-κB into the nucleus in LPS-activated human macrophages. CONCLUSION: The present in vitro investigations suggest a significant anti-inflammatory activity of STW 1 and its components by inhibiting pro-inflammatory cytokine as TNF-α and the key enzyme PTGS2 in LPS-activated human monocytes, which is, at least partly mediated through the suppression of NF-κB activation. Our results provide evidence for distinctive anti-inflammatory effects of STW 1 and its components on LPS-activated human monocytes/macrophages and, thus, for the therapeutic use of STW 1 in inflammation and pain related disorders.

Antidepressant Effect of Fraxinus rhynchophylla Hance Extract in a Mouse Model of Chronic Stress-Induced Depression.

Prolonged exposure to stress can affect mood and cognition and lead to mood disorders. Research on stress-associated mood disorders is important in modern society as people are increasingly exposed to unavoidable stressors. We used a mouse model with 2 weeks of exposure to electric foot shock and restraint, to determine the effect of Fraxinus rhynchophylla Hance (FX) extract on chronic stress-induced depression. We measured the effect of FX extract using various physiological, behavioral, and biochemical measures. FX extract ameliorated chronic stress-induced body and relative liver weight loss and improved depressive-like behaviors in the open field and forced swim tests. In addition, plasma cortisol and serotonin levels in stress-induced mice following FX treatment were similar to normal mice, and the elevation of proinflammatory cytokines was prevented. Moreover, FX treatment increased the expression of phosphorylated cyclic adenosine-3',5'-monophosphate response element-binding protein (pCREB)/brain-derived neurotrophic factor (BDNF). Further experiments confirmed the efficacy of FX extract by showing similar results using esculin and esculetin, compounds extracted from FX. Taken together, these results indicate that FX extract has an antidepressant effect on chronic stress-induced depression by associating signaling with neuroinflammation and neurogenesis.

Analysis of Fraxinus pollen seasons and forecast models based on meteorological factors.

INTRODUCTION AND OBJECTIVE: The timings of Fraxinus and Betula flowering and pollen release overlap, which may cause increased allergic reactions in sensitive people. The aim of the present study was to characterize Fraxinus pollen seasons in Lublin (central-eastern Poland) and to identify meteorological factors that most determine the occurrence of airborne pollen of this taxon, as well as obtain forecast models for the basic characteristics of the pollen season. MATERIAL AND METHODS: The study was conducted in Lublin during the period 2001-2016, employing the volumetric method. The seasons were compared by PCA (Principal Component Analysis). To determine relationships between meteorological conditions and the pattern of pollen seasons, regression analysis was used. Data for the period 2001-2015 were used to create forecast models by applying regression analysis, while the 2016 data served to verify these models. RESULTS: Season end date and seasonal peak date were characterized by the lowest variation. The biggest differences were found for peak value and total annual pollen sum. The average dates of occurrence of ash pollen grains in the air of Lublin were between 13 April 13 - 3 May 3, whereas, on average, the pollen peak date occurred on 23 April. The factor loading values for the PC1 variable indicate that it is most strongly correlated with peak value and total pollen sum, while the PC2 variable correlated with the pollen season start date and season duration (a negative correlation). Regression models were developed for the following pollen season characteristics: season start, end and duration, seasonal peak date, and total annual pollen sum. CONCLUSIONS: The fit of the forecast models was at the level of 62-94%. Analysis of the data showed that weather conditions mainly in February were important factors controlling the Fraxinus pollen season.

Nummularic acid, a triterpenoid, from the medicinal plant Fraxinus xanthoxyloides, induces energy crisis to suppress growth of prostate cancer cells.

We recently identified and characterized nummularic acid (NA) as a major chemical constituent of Fraxinus xanthoxyloides, a medicinal plant used for over hundred years in traditional medicine. In this study, we describe its potential anti-cancer activity using prostate cancer (PCa) cells as a model. We found that NA treatment (5-60 µM) significantly reduced the proliferation and colony formation capabilities of PCa DU145 and C4-2 cells in a time and dose dependent manner, reduced the migratory and invasive properties and increased apoptotic cell population. Mechanistically, we found that NA treatment to PCa cells resulted in a sustained activation of adenosine monophosphate-activated protein kinase (AMPK). NA simultaneously increased acetyl CoA carboxylase phosphorylation and decreased pS6 phosphorylation, the two major substrates of AMPK. Further, NA treatment significantly elevated the cellular ADP/ATP ratio and altered glycolytic rate. We further observed a reversible decrease in oxygen consumption rate in NA treated cells when compared to the control. Finally, we performed global untargeted metabolomics which showed that NA treatment alters PCa cell metabolism at multiple sites including glycolysis, tricarboxylic acid, and glutamine metabolism which supported our observation of a possible AMPK activation. In summary, we report NA as a novel small molecule activator of AMPK that alters cellular metabolism to induce energy crisis and ultimately cancer cell death. Because of its unique mechanism NA could be potentially applicable against other cancer types.

Effect of esculetin on bone metabolism in ovariectomized rats.

OBJECTIVE: To determine the effect of an esculetin formulation (at 97.4% purity) on osteoporosis, and to investigate the potential underlying molecular mechanism(s). METHODS: Sixty specific pathogen free-grade female Wistar rats were randomly assigned to three groups: blank control (n = 12), sham (n = 12), and model (n = 36). The model group were bilaterally ovariectomized. The sham group had the tissue surrounding the ovaries removed, while the ovaries were retained. After 3 months, the model group was randomly divided into three subgroups: OVX (n = 12), positive control (n = 12), and esculetin (n = 12). The positive control group and the esculetin group were intragastrically administered diethylstilbestrol (0.046 mg?kg-1?d-1) or esculetin (384 mg?kg-1?d-1), respectively, once per day for 6 consecutive days; medication administration was then stopped for 1 d, before being administered for another 6 consecutive days. All rats were treated for 3 months. Samples were collected at the end of the treatment period. An Osteocore3 Digital 2D bone densitometer was used to test the bone mineral density, and histomorphometric analysis was performed to measure bone mass, bone formation, and bone resorption. Enzyme-linked immunosorbent assay analysis was used to measure the serum concentrations of interleukin-6 (IL-6), osteoprotegerin (OPG), and receptor activator of nuclear factor-kappa B ligand (RANKL). Immunohistochemistry and in situ hybridization were performed to detect the protein and mRNA expressions of OPG and RANKL in osteoblasts and bone marrow stromal cells. RESULTS: Compared with the OVX group, the esculetin group had significantly greater femoral bone mineral density and tibial trabecular bone volume, and significantly smaller trabecular resorption surface. The percentage of trabecular formation surface, average osteoid width, trabecular bone mineralization rate, and cortical bone mineralization rate did not significantly differ between groups. Compared with the sham group, the esculetin group had significantly decreased serum levels of IL-6 and RANKL, and significant downregulation of RANKL protein and mRNA expression levels in osteoblasts and bone marrow stromal cells; however, there was no significant difference between groups in OPG. CONCLUSION: Esculetin can increase bone mass by upregulating RANKL expression in osteoblasts and bone marrow stromal cells, and decreasing serum IL-6 concentration. This indicates that the therapeutic effect of esculetin on osteoporosis occurs via decreased bone resorption.