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Ethnopharmacological relevance Flowers of Inula montana L. (Asteraceae), commonly known as “Arnica de Provence”, are used in the traditional medicine of Provence in France with the same indication as Arnica montana, for the relief of bruises, as an anti-inflammatory agent. Aims of the study The aim of our study is to evaluate its anti-inflammatory properties and to justify its traditional uses. Its potential valorization is evaluated in order to propose Inula montana as an alternative to Arnica montana. Materials and methods Bio-guided fractionation of ethanolic extract allowed the isolation of compounds responsible of the inhibition of NO production. The fractionation was realized using chromatographic techniques and structure elucidation was conducted by ESI-MS and NMR spectral data. Anti-inflammatory effect of ethanolic extract, different fractions and isolated pure compounds was studied in vitro on immortalized mouse macrophages RAW 264.7. An analytical UHPLC-DAD-ESI-MS/MS method was developed for the identification of these compounds in the herbal drug. This UHPLC-DAD method was validated and was used to compare the phenolic profile and content in plant material from the two collection sites: Bonnieux and Merindol. Results Eleven compounds were identified by UHPLC-MS. Chlorogenic acid (1), Luteolin (2), Nepetin (3), 3,5-O-Dicaffeoylquinic acid (4), 1,5-O-Dicaffeoylquinic acid (5), Nepitrin (6), Hispiduloside (7) and Jaceosid (8) were isolated and identified by NMR. Compounds 9, 10 and 11 were confirmed to be 6-Hydroxykaempferol 3,7-dimethyl ether, Hispidulin and Chrysosplenol C, respectively by comparing retention times and MS/MS data with those of the authentic substances. Six compounds: 1 and 4–8 are reported for the first time in Inula montana L. Compounds 2–8 showed promising anti-inflammatory activity with the release of NO with IC50 value Conclusion Flowers extracts and isolated compounds present promising anti-inflammatory activity which provides a scientific basis for the traditional use of Inula montana and may be proposed in the same indications as Arnica montana. The developed and validated simple, accurate and rapid UHPLC method can be used for the quality control of the herbal drug.

An undescribed unusual sesquiterpene trimer and three sesquiterpene dimers were isolated from the flowers of Inula japonica. Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data as well as HRESIMS data. Inulajaponicolide A has an undescribed carbon skeleton comprising of one xanthanolide and two guaianolide units with the linkage mode of C-11/C-3ʹ and C-11ʹ/C-1ʹʹ via a Diels-Alder cycloaddition reaction. Inulajaponicolides C and D exhibited moderate cytotoxic activity against A 549 and NCI-H460 human cancer cell lines with IC50 values ranging from 8.5 to 17.8 μM. Inulajaponicolides A-D and lineariifolianoid A possessed significant inhibitory potency against nitric oxide production in LPS-induced RAW264.7 cells with IC50 values ranging from 1.0 to 4.1 μM.

Inula helenium L., commonly known as Elecampane, has been extensively used for many countries in the folk medicine. Its root is a rich source of sesquiterpene lactones, which possess various pharmacological activities. To develop the phytomedicine including sesquiterpene lactones, we prepared hexane fraction from I. helenium (HFIH) and examined the inhibitory effect of HFIH on signal transducers and activators of transcription 3 (STAT3) activation in human breast cancer MDA-MB-231 cells. Additionally, detailed chemical investigation was done to pinpoint the most active sesquiterpene lactones responsible for its anticancer activity. HFIH selectively suppressed STAT3 phosphorylation at tyrosine 705, not affecting its upstream kinases. HFIH downregulated the expression of STAT3 target genes including cyclin D1 , c-myc, and bcl-2 and induced caspase-mediated apoptosis. Moreover, sesquiterpene lactones of HFIH clearly suppressed STAT3 activation. The in vivo results further supported that HFIH inhibits the growth of human breast xenograft tumors. Our results suggest that HFIH possesses potential anticancer activity, which is mainly mediated through STAT3 signaling pathway. These findings provide the potential of HFIH as a promising phytomedicine for the treatment and prevention of triple-negative breast cancer.

Isoalantolactone and alantolactone are the main sesquiterpene lactones in Radix Inulae (dried root of Inula helenium L. or I. racemosa Hook. F.), which is a frequently utilized herbal medicine. They also occur in several plants and have various pharmacologic effects. However, they have been found to have poor oral bioavailability in rats. To understand the intestinal absorptive characteristics of isoalantolactone and alantolactone as well specific influx and efflux transporters in their absorption. Bidirectional permeabilities of isoalantolactone and alantolactone were investigated across Caco-2 cell monolayers. Transport assays were performed using different concentrations of two lactones and specific inhibitors of ATP-binding cassette transporters and influx transporters. The absorption permeability of isoalantolactone and alantolactone was high at the tested concentrations (5, 20 and 80 μmol/l), and the major permeation mechanism of both lactones was found to be passive diffusion with active efflux mediated by multidrug resistance-associated proteins (MRPs) and breast cancer resistance protein (BCRP). Our results demonstrated that the absorption permeability of isoalantolactone and alantolactone was good in the Caco-2 cell model. The isoalantolactone and alantolactone absorption elucidated in this study provides useful information for further pharmacokinetics studies. Since low intestinal absorption can now be ruled out as a cause, further studies are needed to explain the low oral bioavailability of the two sesquiterpene lactones.

To explore the reason causing low yield, poor quality of Inula helenium I., this paper investigated the influence of different remained stem heights on the yield, quality and water consumption of Inula helenium I. in Gannan plateau area using field single factor randomized block method. Research results showed that Inula helenium I. which was cut before blooming period in the last ten-day of July with remained stem height of 25 cm had the lowest water consumption, the best underground root traits (including main root length, root diameter, and root dry weight per plant), and the highest yield which was higher that control group by 18.73% (P

The daunorubicin and doxorubicin anthracycline antibiotics were modified with the Inula helenium L. sesquiterpene lactones (alantolactone, isoalantolactone, and alloalantolactone) and with their epoxy derivatives. Antiproliferative properties of these conjugates were studied on tumor and normal cell lines. The daunorubicin conjugates with the sesquiterpene lactones (isoalantolactone, allantolactone, and alloalantolactone) and with their epoxy derivatives were found to exhibit the higher activity against human tumor cell lines than the corresponding doxorubicin conjugates. The daunorubicin conjugate with epoxyisoalantolactone proved to be the most effective compound, because it was more cytotoxic than daunorubicin towards a number of cell lines, including those daunorubicin-resistant, and did not affect normal human cells.

Background Inula helenium L. is an herb whose anti-inflammatory properties are attributed to its active components, the sesquiterpene lactones (SLs). Our previous study demonstrated that the total sesquiterpene lactones isolated from Inula helenium L. (TSL-IHL), consisting mainly of alantolactone (AL) and isoalantolactone (IAL), may have potential in the prevention and treatment of rheumatoid arthritis (RA). However, the effect of TSL-IHL on atopic dermatitis (AD) has not been studied yet. Aim of the study The present study evaluates the potential of TSL-IHL as a treatment for AD. Methods/Study designs The effects of TSL-IHL on the expression of inflammatory genes and the activation of NF-κB signaling pathway in HaCat cells were examined by quantitative real-time PCR and western blotting, respectively, and compared with those of AL and IAL. The protective effect of TSL-IHL against AD was tested in a mouse model induced by 2,4-dinitrochlorobenzene (DNCB), in which AD-like skin lesions were induced in ICR mice by sensitizing once with 100 µl of 7% DNCB painted on their shaved back skin and then challenging with 20 µl of 0.2% DNCB five times on their right ears at 3 day intervals starting on day 5 post-sensitization. Results TSL-IHL, as well as AL and IAL, could all inhibit TNF-α-induced activation of NF-κB and the expression of TNF-α, IL-1, and IL-4 in HaCat cells in a dose-dependent manner in the range of 0.6–2.4 µg/ml. The topical application of TSL-IHL (1% W/W in emollient cream) attenuated DNCB-induced dermatitis severity and right ear swelling. The serum levels of IgE, TNF-α and IFN-γ in TSL-IHL-treated mice were reduced by 81.39%, 89.69%, and 87.85%, respectively, while the mRNA levels of IL-4, IL-5 and IL-13, in the back-skin lesions of TSL-IHL-treated mice were reduced by 39.21%, 40.62% and 48.12%, respectively, compared with the untreated controls. Histopathological examination showed that TSL-IHL treatment reduced epidermis/dermis thickening and dermal inflammatory infiltration in both ear and back skins. Conclusions We suggest that TSL-IHL inhibited the development of AD-like skin symptoms by regulating cytokine expression and may be an effective alternative therapy for AD.

The anthracycline antibiotic daunorubicin was modified by alantolactone, isoalantolactone, and alloahntolactone, i.e., sesquitapene lactones from Inula helenium L. The antiproliferative properties of the synthesized conjugates were studied using tumor and normal cell lines. It was found that the conjugate of daunorubicin with alantolactone possessed more pronounced antiproliferative properties than daunorubicin for several tumor cell cultures without increasing the toxicity for normal cells.

The effect of preventive administration of the complex phytopreparation "tetraphyton" has been studied in experiments on rats. Tetraphyton developed on the base of Tibetan formula is the dry extract derived from species of plant material: roots of Inula helenium L., roots of Zingiber officinale Roscoe, fruits of Elletaria cardamomum (L.) Maton, offshoots of Caragana spinosa (L.) Wall. ex Hornem., characterized for the content of phenolic compounds, flavonoids, terpenoids. The preventive course introduction of tetraphyton in a dose 100 mg/kg for 7 days before experiment increases the potential for physical performance, increasing overall physical endurance against a background of intense physical exertion by 73 % (p < 0.05), prevents fatigue. It is shown that increase in the physical performance of rats in the experimental group is due to increase in the efficiency of the energy supply system in skeletal muscles and myocardium: a significant increase in the content of ATP (by 13 % and 2.5 times); decrease in lactate and pyruvate content (by 33 and 30 %; p < 0.05); increased glycogen content in the liver by 23 %, blood glucose by 19 %. The increase in catalase, SOD, and gSh levels (5 %, 19 %, 79 %; p < 0.05) indicates an increase in the power of the antioxidant defense of the organism and causes the cytoprotective effect of tetraphyton, confirmed by a decrease in fermentemia (lactate dehydrogenase, kreatinphosphokinase - 49 % and 43 %; p < 0.05). The main molecular-cellular mechanisms of the actoprotective action of the phytoadaptogen is the inhibition of the lipid peroxidation of cell membranes and a significant increase in the capacity of the endogenous antioxidant enzyme system that promotes the preservation of the integrity of skeletal muscles and the myocardium. This is due to the presence of phenolic compounds in the chemical composition that have direct antiradical action and increase the power endogenous antioxidant system.

Britannin (Bri), isolated from Inula aucheriana, is a sesquiterpene lactone (SL), a class of secondary metabolites. Previous studies have suggested the anti-cancer potential of Bri; however, the molecular mechanism remains elusive. The present study investigated the effects of Bri on liver cancer progression. Our findings indicated that Bri significantly suppressed the growth of liver cancer cell lines. Mechanistic researches revealed that Bri induced apoptosis through the extrinsic and intrinsic apoptotic pathways, as evidenced by the increase of Caspase-8, -9 and -3 cleavages. In addition, Bri-triggered autophagy in liver cancer cells, supported by the up-regulation of light chain 3 (LC3) II, p62, autophagy-related 5 (ATG5) and Beclin 1, as well as the occurrence of autophagic vacuoles. Importantly, Bri increased AMPK activation, while decreased the activity of its down-streaming signal, mTOR. Of note, suppression of AMP-activated protein kinase (AMPK) activation using its inhibitor, Compound C, could inhibit both apoptosis and autophagy induced by Bri. Furthermore, Bri was found to induce reactive oxygen species (ROS) generation in hepatic cancer cells. Notably, reducing ROS production by its scavenger, N-acetyl cysteine (NAC), could down-regulate p-AMPK levels, while up-regulate the phosphorylated mechanistic target of rapamycin (p-mTOR) expressions, accompanied with the restored cell viability, as well as the reduced apoptosis and autophagy in Bri-treated liver cancer cells. Finally, Bri inhibited the tumor growth in vivo without side effects. In conclusion, our study illustrated that Bri could induce apoptosis and autophagy by activating AMPK regulated by ROS in liver cancer cells, supplying molecular bases for developing Bri into an effective candidate against liver cancer.

Aims: The aim of this study is to explore the antioxidant and antiproliferative activities of aqueous extract from aerial parts of Inula helenium (L.) against human U-87 MG glioma cell line. Materials and Methods: The 3'-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase assays were used to study antiproliferative and cytotoxic activities against U-87 MG cell after 48 h exposure. In addition, to assess the oxidative effects, total antioxidant capacity and total oxidant status levels were also measured. Results: Finally, the aqueous extracts displayed antiproliferative and cytotoxic activities at high concentrations tested, particularly at 200 μg/ml, without causing to oxidative stress. Conclusion: The results strengthen the evidence that I. helenium could be considered a natural resource of potential antitumor agents for brain cancer. In addition, this study is expected to expand the existing information on the anticancer activity of I. helenium and to assist in a more focused design of further research as chemotherapeutic agents.

Roots of Inula racemosa are used as a cardio protective in Ayurveda in India, being prescribed as a medicine for precordial chest pain, cough and dyspnoea, both singly and as a poly herbal.Evaluation of Phytoestrogenic activity of the root extracts of Inula racemosa and compounds isolated therefrom in vivo, in silico and in vitro.Alcohol (IrA) and hexane (IrH) extracts characterized by HPTLC/GC-MS analysis respectively and processed for compound isolation were evaluated for estrogenic activity (100250mg/kg bw) by the Immature rat uterotrophic assay using ethinylestradiol (EE -30µg/kg bw) as standard drug. Alantolactone (ALT), Isoalantolactone (IALT) and Stigmasterolglucoside (SG) isolated from the extracts were characterized and screened in silico for ERα, ERβ binding affinity, assessed in vitro for growth modulatory effects on MCF-7 cells by MTT assay and cell cycle distribution analysis using Flow cytometry. RT-PCR analysis evaluated the mRNA expression of pS2 in these cells post exposure to ALT, IALT and SG.In the IrA treated groups there has been a statistically significant increase (P0.05) in absolute and normalised uterine weight, uterine diameter, endometrial thickness, luminal epithelial cell height,diameter of ovary and in the number of primary and secondary ovarian follicles relative to untreated controls. Presence of ciliated epithelial cells in the oviduct, elevated number of early growing follicles characterized by an increased oocyte diameter, and signs of vascularization in the cortex of ovarian sections in this group relative to EE treated group are indicative of pervasive activation of follicular growth and initiation. Virtual docking demonstrated ERα affinity for IALT, ERβ affinity for ALT, while SG showed a high binding affinity to both with a relatively greater ERβ binding affinity. Dose dependent decrease in cell viability mediated by IALT and SG in the MTT assay is corroborated by a statistically significant increase (p0.05) in sub G0-G1 cells by SG at 200 and 400µM in cell cycle analysis and there has been an induction of pS2 by IALT and SG in the ER regulated MCF-7 cells.Demonstration of classical morphological changes induced by estrogen stimulation mediated by IrA in vivo at both the tested doses, isolation of the antioxidant SG from IrA and its dose dependent growth inhibitory effect on estrogen sensitive MCF-7 cells through apoptotic induction and an up regulation of pS2 are suggestive of an anti-estrogenic effect through estrogen receptor binding affinity, typical of phytoestrogens that bind to ER but do not elicit a full estrogenic response. The observed estrogenic effect of IrA suggests a multi mechanistic molecular action involving antioxidant as well as redox signalling pathways acting in consonance with their anti-estrogenic effects owing to the weak estrogen like competitive receptor binding of SG.

Inulae Flos was widely distributed throughout Europe, Africa, and Asia, and was commonly used as a folk medicine in clinic for treating various respiratory diseases, including cough, asthma, bronchitis, pulmonary fibrosis, and pneumonia. However, the ingredients responsible for the pharmacology effects of I. Flos and the underlying mechanisms remain unclear. In this study, the effects of 16 known sesquiterpene lactones and flavonoids from I. Flos on TGF-β1-induced fibroblast activation were assessed by phenotypic high-content screening. Among those sixteen compounds, 1β-hydroxy alantolactone (HAL), the main characteristic sesquiterpene lactone from I. Flos, exhibited remarkable inhibitory activity. The further studies showed that HAL significantly inhibited the proliferation and induced the apoptosis of human fibroblast cell lines HELF and MRC-5 in a concentration-dependent manner. It also reduced intracellular ROS production, suppressed the mRNA expressions of E-cad, TGF-β1, Smad3, Col I, α-SMA and TNF-α, and downregulated protein expressions of α-SMA and F-actin. Furthermore, HAL significantly reduced the levels of HA, LN, PC-III and IV-C in serum, TNF-α and IL-6 in BALF, and TGF-β1, HYP and Col I in lung tissues of bleomycin (BLM)-treated rats. HAL significantly downregulated the expressions of p-JNK, FOXO1, p-p65, α-SMA, p-smad3 and Col I but upregulated p-FOXO1, which could be reversed by JNK agonist anisomycin. These results demonstrated that HAL induced the apoptosis of lung fibroblast cells activated by TGF-β1 and improved BLM-induced lung fibrosis in rats via inhibiting JNK/FOXO1/NF-κB pathway.

Ethnopharmacological relevance The genus Inula has been traditionally used as folk medicine in treating different illnesses such as kidney stones, urethra infection, jaundice, bronchitis, respiratory diseases and cancer. Aim of the study Gaillardin, a sesquiterpene lactone isolated from Inula oculus-christi, seems to have great potential as an anti-cancer agent. This study was carried out to evaluate the anti-cancer properties of Gaillardin in gastric cancer cells and also its possible underlying mechanism. Methods The colorimetric MTT assay was used to assess metabolic activity of cells as an indicator of viability and cytotoxicity. Flow cytometry using Annexin V-FITC/PI was applied to detect and quantify the level of apoptosis. Detection of activated caspase-3, as a biochemical marker of apoptosis, was done using caspase-3 assay kit. Activation of NF-κB pathway was determined by western blotting. The mRNA expression levels of NF-κB target genes were measured using quantitative RT-PCR. Moreover, intracellular reactive oxygen species (ROS) production was evaluated. Results Gaillardin significantly reduced cell viability in a time and dose-dependent manner. The inhibitory effect of Gaillardin was attributed to induction of apoptosis. Investigation about the underlying mechanism revealed that Gaillardin exerts its action through inhibition of NF-κB activation and subsequently down-regulation of genes (COX-2, MMP-9, TWIST-1, and BCl-2) regulated by NF-κB. Moreover, Gaillardin caused remarkable elevation in ROS production in AGS and MKN45 cells. Conclusion We provided evidences about the role of NF-κB pathway in the induction of apoptosis by the sesquiterpene lactone Gaillardin in AGS and MKN45 cells for the first time, which suggest Gaillardin could be considered as a promising natural agent for further investigations to identify new potent anticancer drugs in the future.

Effects of cytokinins were studied on multiple shoot regeneration in Inula royleana DC. Callus from leaf of I. royleana was treated with different concentrations of BAP and in order to find out the limiting concentrations of both BAP and Kn at which maximum shoot regeneration took place. Among different concentrations 1 mg/l BAP and 0.8 mg/l Kn proved to be optimum. Rooting was obtained in White’s medium. Regenerated plantlets were successfully acclimatized in pots containing vermicompost.Plant Tissue Cult. & Biotech. 27(2): 189-194, 2017 (December)

Dried flowers of Inula britannica commercially serve as pharmaceutical/nutraceutical herbs in the manufacture of medicinal products and functional tea that has been reported to possess extensive biological property. However, the neuroprotective constituents in I. britannica flowers are not known. In the current study, phytochemicals of sesquiterpenoid-enriched I. britannica flowers extract and their potential multifunctional neuroprotective effects were investigated. Nineteen structurally diverse sesquiterpenoids, including two new sesquiterpenoid dimers, namely, inubritanolides A and B (1, 2), and four new sesquiterpenoid monomers (3–6), namely, 1-O-acetyl-6-O-chloracetylbritannilactone (3), 6-methoxybritannilactone (4), 1-hydroxy-10β-methoxy-4αH-1,10-secoeudesma-5(6),11(13)-dien-12,8β-olide (5) and 1-hydroxy-4αH-1,10-secoeudesma-5(6),10(14),11(13)-trien-12,8β-olide (6), as well as 13 known congeners (7–19) were isolated from this source. The structures of compounds 1–6 were elucidated by 1D- and 2D- NMR and HR-ESI-MS data, and their absolute configurations were discerned by electronic circular dichroism (ECD) data analysis and single crystal X-ray diffraction. Interestingly, inubritannolide A (1) is a new type [4 + 2] Diels-Alder dimer featuring a hepta-membered cycloether skeleton. Most of the compounds showed potential multifunctional neuroprotective effects, including antioxidative, anti-neuroinflammatory, and microglial polarization properties. Specifically, 1 and 6 displayed slight strong neuroprotective potency against different types of neuronal cells mediated by various inducers including H2O2, 6-hydroxydopamine (6-OHDA), and lipopolysaccharide (LPS). Overall, this is the first report on multifunctional neuroprotective effects of sesquiterpenoid-enriched I. britannica flowers extract, which supports its potential pharmaceutical/nutraceutical application in neurodegenerative diseases.

Twenty-one eudesmane-type sesquiterpenes, including five new compounds, were isolated from the roots of Inula helenium. The structures of the new compounds (1–5) were determined by extensive spectroscopic data interpretation, single-crystal X-ray diffraction analysis and ECD calculations. Six compounds can synergistically enhance cisplatin effect against ovarian cancer cells, the structure − activity relationship for the synergistic effect of these compounds with cisplatin was revealed for the first time, which provides useful clues to develop novel sensitizers to overcome drug resistance in cancer. In addition, fifteen sesquiterpenes exhibited significant anti-inflammatory activity, which provided promising candidates for development of anti-inflammatory agent.

Soluble epoxide hydrolase (sEH) is a potential drug target to treat inflammation and neurodegenerative diseases. In this study, we found that the extract of Inula britanica exhibited significantly inhibitory effects against sEH, therefore, we investigated its phytochemical constituents to obtain seven new compounds together with sixteen known ones (1-20), including two pairs of novel enantiomers, (2S,3S)-britanicafanin A (1a), (2R,3R)-britanicafanin A (1b), (2R,3S)-britanicafanin B (2a), and (2S,3R)-britanicafanin B (2b), and three new lignans britanicafanins C-E (3–5). Their structures were determined by HRESIMS, 1D and 2D NMR, and electronic circular dichroism (ECD) spectra as well as quantum chemical computations. All the isolates were evaluated for their inhibitory effects against sEH, compounds 1–3, 5–7, 9, 10, 13, 14, and 17–20 showed significant inhibitory effects against sEH with IC50 values from 3.56 μM to 26.93 μM. The inhibition kinetics results indicated that compounds 9, 10, 13, and 19 were all uncompetitive inhibitors, and their inhibition constants (Ki) values were 7.11, 1.99, 4.06, and 8.78 μM, respectively. Their potential interactions were analyzed by molecular docking and molecular dynamics (MD), which suggested that amino acid residues Asp335 and Asn359, especially Gln384, played an important role in the inhibition of compounds 10 and 13 on sEH, and compounds 10 and 13 could be considered as the potential candidates for the development of sEH inhibitors.

A phytochemical study performed on Inula japonica led to isolation of a new 1,10-seco-sesquiterpene dimer Neolinulicin A (1) and 1,10-seco-sesquiterpene Neolinulicin B (2), together with nine known sesquiterpenes (3−11). Among them, Neolinulicin A (1), which has a new carbon skeleton, was a Diels-Alder [4 + 2] adduct of two sesquiterpene moieties. Their structures were established by extensive spectroscopic analysis. All of the isolated compounds showed inhibition of NO production in RAW 264.7 macrophages. The findings might supply information for the future design of anti-inflammatory agents from I. japonica.

Sesquiterpene lactones (STLs) are C15 terpenoid natural products with α-methylene γ-lactone moiety. A large proportion of STLs in Asteraceae species is derived from the central precursor germacrene A acid (GAA). Formation of the lactone rings depends on the regio-(C6 or C8) and stereoselective (α- or β-)hydroxylations of GAA, producing STLs with four distinct stereo-configurations (12,6α-, 12,6β-, 12,8α-, and 12,8β-olide derivatives of GAA) in nature. Curiously, two configurations of STLs (C12,8α and C12,8β) are simultaneously present in the Chinese medicinal plant, Inula hupehensis. However, how these related yet distinct STL stereo-isomers are co-synthesized in I. hupehensis remains unknown. Here, we describe the functional identification of the I. hupehensis cytochrome P450 (CYP71BL6) that can catalyze the hydroxylation of GAA in either 8α- or 8β-configuration, resulting in the synthesis of both 8α- and 8β-hydroxyl GAAs. Of these two products, only 8α-hydroxyl GAA spontaneously lactonizes to the C12,8α-STL while the 8β-hydroxyl GAA remains stable without lactonization. Chemical structures of the C12,8α-STL, named inunolide, and 8β-hydroxyl GAA were fully elucidated by nuclear magnetic resonance analysis and mass spectrometry. The CYP71BL6 displays 63-66% amino acid identity to the previously reported CYP71BL1/2 catalyzing GAA 6α- or 8β-hydroxylation, indicating CYP71BL6 shares the same evolutionary lineage with other stereoselective cytochrome P450s, but catalyzes hydroxylation in a non-stereoselective manner. We observed that the CYP71BL6 transcript abundance correlates closely to the accumulation of C12,8-STLs in I. hupehensis. The identification of CYP71BL6 provides an insight into the biosynthesis of STLs in Asteraceae.

The present study was carried out to evaluate analgesic and anti-inflammatory activities in Inula cuspidata stem and root extracts along with heavy metals estimation in stem and root powder. Stem and roots were extracted with chloroform (ICSCE, ICRCE) and methanol (ICSME, ICRME). Acute oral toxicity of all extracts was determined by OECD guidelines 425. Analgesic activity was investigated by using hot plate and acetic acid induced writhing models. Anti-inflammatory activity (acute) of all extracts was evaluated by carrageenan induced paw edema model. In addition, root and stem powder was screened for heavy metals (As, Pb, Cd, Hg) estimation using atomic absorption spectroscopy. In acute toxicity study no mortality was observed when each extract was orally administered with 2.0 g/kg. At the doses (100 and 200 mg/kg) ICRME followed by ICSME showed significant and dose dependent analgesic and anti-inflammatory effects compared with chloroform extracts. The heavy metals concentration in stem and root powders was found to be within the permissible limits as recommended by WHO for herbal raw materials. The findings of the present study validated the folkloric use of Inula cuspidata as analgesic and anti-inflammatory. In addition, the results intimate that heavy metals present in raw material were found to be within the defined limits, and it exhibits that the therapeutic efficacy of plant may not be effected, which can be otherwise possibly effected if the plant sequester high concentration of heavy metals from the polluted environment as well as from the soil rich in pesticides and sewage sludge etc.

The aim of the present work was to analyze the impact of salinity on the plant response to Cd toxicity in the Mediterranean halophyte species Inula crithmoides. For this purpose, cuttings were cultivated hydroponically during 21d in the presence of 0, 25 or 50 μM CdCl2 combined or not with 0, 100, 200 and 400 mM NaCl. The obtained data demonstrated that, in the absence of Cd, NaCl strongly increased plant growth (the maximal dry weight being observed at 100 mM) and enhanced the Na+/K+ ratio in the shoot. Cd alone strongly affected plant growth in this halophyte. However, in Cd-treated plants, NaCl protected Inula crithmoides from Cd toxicity and contributed to reduce Cd absorption and translocation. Small aliphatic polyamine (putrescine, spermidine, spermine) increased in response to both NaCl and CdCl2, the highest concentration in plants being observed when both agents are present in the medium. The recorded increase preferentially concerned the polyamine bound fraction, which might be related to their involvement in the protection of endogenous cellular structures. The aromatic monoamine tyramine also strongly increased in response to Cd toxicity and its putative role is discussed in relation to conjugation processes. Salinity and Cd increased ammonium/nitrate ratio in leaves and roots and the involvement of stress-induced modification of N nutrition on polyamine oversynthesis is also discussed.

In this study we investigated the size-dependent reproductive pattern of Inula racemosa and I. royleana (Asteraceae) growing at different reaches in the environs of the Kashmir Himalaya. Size effects on reproductive pattern were evaluated by determining the size-dependency of flowering probability and reproductive effort. The results showed that the probability of flowering increased significantly with the size of the plant in all populations, indicating that individuals do not flower until they reach a threshold size and considerable between-site differences were found in the slope and the intercept of the regression between plant size and flower production. In I. racemosa, populations at high altitudes had significantly lower threshold sizes for reproduction and showed sharp increase in flowering probability with plant size, compared to other populations at lower altitudes. However, no pattern in size-dependent flower production was found relative to the altitude in I. royleana, as flower production at some sites, at high altitudes, increased more steeply with plant size than at other sites. For both taxa, reproductive effort decreased allometrically as adults grew, as can be interpreted from the allometric relationship between reproductive and vegetative biomass. Further studies are required to determine whether population differentiation in size-dependent reproductive pattern is maintained by selection.

Background: Inula viscosa L. (Asteraceae) is a medicinal plant widely used as a folk medicine in oriental Morocco, to treat hypertension. The antihypertensive effect of the methanolic extract obtained from I. viscosa leaves was evaluated in hypertensive L-NAME rats. Systolic blood pressure (SBP) was measured using a non-invasive indirect tail-cuff plethysmographic method. Four groups of rats were used: a control group; a hypertensive group treated with L-NAME (32 mg/kg/day); a positive control group treated with L-NAME plus enalapril (15 mg/kg/day) as a reference antihypertensive agent; and a group treated with L-NAME plus MeOH-extract (40 mg/kg/day). Methods: Treatment with L-NAME alone caused a progressive increase in SBP. After 4 weeks, the value of SBP reached 160 ± 2 mmHg which shows the installation of hypertension. Enalapril prevented the increase in SBP, which remained normal at 123 ± 1 mmHg after 4 weeks of treatment. The administration of MeOH-extract along with L-NAME prevented the increase in SBP as well, which remained constant at 115 ± 1 mmHg after 4 weeks of treatment. In ex-vivo models, MeOH-extract produced a relaxation of pre-contracted ring aorta (54 ± 2% of relaxation at 3 g/L). But, when the rings were denuded, MeOH-extract failed to relax pre-contracted rings of aorta. Phytochemical study of I. viscosa MeOH-extract revealed the presence of phenolic compounds, such as cynarin and chlorogenic acid. Results: The present results suggest that I. viscosa MeOH-extract has an antihypertensive, predominantly mediated by an endothelium-dependent vasodilatory effect. Cynarin and chlorogenic acid, which have a strong vasorelaxant effect may be involved in the antihypertensive effect of the plant extract. The bioinformatic POM analysis confirms the therapeutic potential of cynarin and chlorogenic acids as inhibitors of various biotargets. Based on the results, the coordination of these phytochemicals with calcium and transition metals should be studied, for better scope at antihypertensive drug development.

Our previous studies found that the crude polysaccharides (IJP) from flowers of Inula japonica exhibited significantly anti-diabetic activity in alloxan or MLD-STZ induced diabetic mice. In this study, we will trace an active polysaccharide from IJP and investigate its physico-chemical property and its protective mechanism on islet cell damage. The result showed that an active polysaccharide (IJP-B-1) was isolated from IJP, its molecular mass was 3.7×104Da. IJP-B-1 was composed of rhamnose, arabinose, xylose, mannose, glucose, galactose and galactocuronic acid. Its major backbone structure was (1→3, 6)-linked-galactose and other branched residues. IJP-B-1 could protect pancreatic cells against STZ impairment at 50μg/mL and scavenge OH and O2 radicals to decrease reactive oxygen generation in islet-cells in vitro. These results suggested that IJP-B-1 might be useful for protecting β-cells and against oxidative stress as an anti-diabetic candidate drug in future.

This work reports for the first time the in vitro anti Leishmania infantum activity of acetone and dichloromethane (DCM) extracts from 25 extremophile plants from Southern Portugal. DCM extracts from Inula chritmoides and Spergularia rubra were active against axenic promastigotes and intracellular amastigotes, had anti-inflammatory properties on lipopolysaccharide (LPS)-stimulated macrophages, inhibited acetylcholinesterase and had no haemolytic activity on human erythrocytes. Eleven phenolics were identified by high-performance liquid chromatography with diode-array detection (HPLC-DAD) in I. crithmoides and one phenolic in S. rubra. Isolation and identification of the active molecules is in progress.

Heterorhabditis bacteriophora can represent a model system for herbal medication against gastro-intestinal strongylid parasites in determining the recovery and development due to their unique parasitic infectious cycle. The fact that plant extracts impair nematode development is known but their differential impact on stages of the life cycle of H. bacteriophora has never been investigated. We examined the developmental stages resumed from eggs, young juveniles (J1-3), infective juveniles (IJs), young and adult hermaphrodites of H. bacteriophora upon exposure to crude ethanolic extracts of Inula viscosa, Salix alba, and Quercus calliprinos at concentrations of 600, 1200, and 2400ppm. Our results showed that plant extracts were highly toxic to the survival of the eggs and young juveniles J1 to J3 at all concentrations. The plant extracts inhibited their development and were associated with low reproduction parameters (i.e. fecundity and viability of eggs). The IJs, J4, young and developed hermaphrodites displayed concentration-dependent negative effect on development with less egg count, poor vulval muscle development, loss of egg laying capacity and progeny development by matricidal hatching. Plant extract of I. viscosa at low (600ppm) concentration did not impair vulval development. These results suggest that these plant extracts show potential for the control of parasitic rhabditids.

Inula helenium (IH) is known to possess antifungal, anti-bacterial, anti-helminthic, and anti-proliferation activities. Caesalpinia Sappan (CS) is known to reduce inflammation and improve blood circulation. Based on their folkloric use, these plants are expected to be promising candidates for promoting hair growth and preventing hair loss. Moreover, these plants are rich sources of certain phytochemicals, which have been reported to promote hair growth. In this clinical trial, we investigate the efficacy of a scalp shampoo formulated by mixing extracts of IH and CS in preventing hair loss and promoting hair growth in patients with androgenetic alopecia. Using a phototrichogram (Folliscope 2.8, LeadM, Korea), we compared the hair density and total hair counts in patients receiving the scalp shampoo at baseline, and at 8, 16, and 24 weeks after use of the shampoo. We found a statistically significant increase in the total hair count in the test group (n = 23) after 16 and 24 weeks of using the scalp shampoo (2.17 n/cm2 &plusmn, 5.72, p &lt, 0.05, and 4.30 n/cm2 &plusmn, 6.37, p &lt, 0.01, respectively) as compared to the control subjects. Based on the results of this clinical study, we conclude that the IH and CS extract complex is a promising remedy for preventing hair loss and promoting hair growth.

Three rare squiterpene lactone dimers lineariifolianoids M-O (1–3) were isolated from Inula lineariifolia for the first time. Their structures and absolute configuration were established on the basis of by NMR and MS spectroscopic data and X-ray crystallography. Furthermore, those three compounds exhibited significant inhibitory activity against LPS-induced NO production in RAW 264.7 macrophages with IC50 values of 1.421, 1.087 and 1.243 μM, respectively.

Inula japonica was used as the research object, "3414" fertilization experiment were conducted to study the effects of nitrogen,phosphorus and potassium formula fertilizer on the growth and chemical composition content of I. japonica. The characteristics of fertilizer requirement were preliminarily revealed and the study provided fertilization guidance for artificial cultivation of I. japonica. The results showed that different nitrogen,phosphorus and potassium formula fertilizers had significant effects on plant morphology,physiological and biochemical indexes,dry matter accumulation and chemical composition content. The growth indexes and chemical components of I. japonica showed an upward trend with the increase of fertilization amount,especially the nitrogen fertilizer was the most significant. The indicators were analyzed by membership function. After comprehensive evaluation,the optimal nitrogen,phosphorus and potassium formula fertilization level was N3 P2 K2,namely high level nitrogen fertilizer,medium level phosphorus fertilizer and potassium fertilizer. I. japonica is a high fertilizer demand plant,and the rational fertilization scheme is " applying nitrogen fertilizer again and applying phosphorus and potassium fertilizer properly".

This study aimed to evaluate the protective effect of igalan, a sesquiterpene lactone isolated from Inula helenium (L.), on inhibiting inflammation, regulating the epidermal differentiation gene expression, and reactive oxygen species scavenging in atopic dermatitis (AD)-like inflammatory keratinocytes. HaCaT human keratinocytes were treated with igalan at indicated concentrations before being activated by a combination of TNF-α and IFN-γ or IL-4 representative for T-helper 1 and T-helper 2 cell cytokines, which are associated with AD pathogenesis. By inhibiting the NF-κB pathway as well as the STAT activation, igalan could downregulate several marker inflammatory genes in AD, such as TARC/CCL17, MDC/CCL22, and RANTES/CCL5. In contrast, igalan, acting as JAK inhibitor, could promote the mRNA expression levels of the genes FLG, LOR, KRT10, and DSC1, which encode for essential proteins responsible for keratinocyte differentiation, by inhibiting STAT3 signaling. Furthermore, igalan exerts its antioxidant effect through activating the Nrf2 pathway, triggering the expression of some enzymes that contribute to preventing intracellular ROS generation during inflammation. These findings indicate that igalan, via suppressing JAK/STAT3 signaling, could impair the production of pro-inflammatory chemokines and enhance expression levels of several genes involved in keratinocyte differentiation in AD-like stimulated keratinocytes.

Gaillardin (GLN) is a sesquiterpene lactone isolated from the chloroform extract of

Nepetin derived from the flowers of Inula japonica, Inulae flos, has been reported to exert several biological activities, including anti-inflammatory responses. In this study, we evaluated the anti-allergic property of nepetin with its molecular mechanisms in bone marrow-derived mast cells (BMMC) and mice. In this in vitro study, we investigated the inhibitory effects of nepetin on degranulation and generation of leukotriene C4 (LTC4) and prostaglandin D2 (PGD2) in IgE/antigen (Ag)-stimulated BMMC. The effect of nepetin on passive cutaneous anaphylaxis (PCA) reaction was also studied in mice. Nepetin reduced degranulation and LTC4 generation in BMMC. The IgE/Ag-mediated signaling pathway demonstrated that nepetin suppressed intracellular Ca2+ level and activation of PLCγ1 and cPLA2. However, MAPKs were not affected by nepetin in BMMC. In addition, nepetin treatment reduced PGD2 production and suppressed cyclooxygenase-2 protein expression via the inhibition of the Akt and nuclear factor-κB signaling pathways. With respect to the local allergic response in vivo, oral administration of nepetin suppressed mast cell-dependent PCA reaction in a dose-dependent manner. The results of this study suggest that nepetin might have an anti-allergic potential related to mast cell-mediated inflammatory diseases.

Background: Hepatocellular carcinoma is cancer with many new cases and the highest mortality rate. Chemotherapy is the most commonly used method for the clinical treatment of hepatocellular carcinoma. Natural products have become clinically important chemotherapeutic drugs due to their great potential for pharmacological development. Many sesquiterpene lactone compounds have been proven to have antitumor effects on hepatocellular carcinoma. Objective: Britanin is a sesquiterpene lactone compound that can be considered for the treatment of hepatocellular carcinoma. The present study aimed to investigate the antitumor effect of britanin. Methods: BEL 7402 and HepG2 cells were used to study the cytotoxicity and antitumor effects of britanin. Preliminary studies on the nuclear factor kappa B pathway were conducted by western blot analysis. A BEL 7402-luc subcutaneous tumor model was established for the in vivo antitumor studies of britanin. In vivo bioluminescence imaging was conducted to monitor changes in tumor size. Results: The results of the cytotoxicity analysis showed that the IC50 values for britanin in BEL 7402 and HepG2 cells were 2.702μM and 6.006μM, respectively. The results of the colony formation demonstrated that the number of cells in a colony was reduced significantly after britanin treatment. And the results of transwell migration assays showed that the migration ability of tumor cells was significantly weakened after treatment with britanin. Tumor size measurements and staining results showed that tumor size was inhibited after britanin treatment. The western blot analysis results showed the inhibition of p65 protein expression and reduced the ratio of Bcl-2/Bax after treatment. Conclusion: A series of in vitro and in vivo experiments demonstrated that britanin had good antitumor effects and provided an option for hepatocellular carcinoma treatment.

Toll-like receptors (TLRs) are a group of pattern-recognition receptors (PRRs) that are at the core of innate and adaptive immune responses. TLRs activation triggers the activation of two downstream signaling pathways, the myeloid differential factor 88 (MyD88)- and toll-interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF)-dependent pathways. To evaluate the therapeutic potential of DHL, a natural sesquiterpene lactone derived from Inulahelenium L. and Saussurea lappa, we examined its effect on signal transduction via the TLR signaling pathways. DHL inhibited the activation of nuclear factor-κB (NF-κB) and interferon regulatory factor 3 (IRF3), the representative transcription factors involved in the inflammatory response, induced by TLR agonists, as well as the expression of cyclooxygenase-2 and interferon inducible protein-10. DHL also inhibited the activation of NF-κB and IRF3 induced by the overexpression of downstream signaling components of the TLRs signaling pathways. All results suggest that DHL might become a new therapeutic drug for a variety of inflammatory diseases.

Blossoms of Inula nervosa Wall. (BINW) are traditionally used as an analgesic and antitussive in China. In this study, in vitro anticomplementary activities of crude extract from BINW in 21 batches and of extracts of four monomeric compounds were evaluated by the classical pathway. The effect of the region of origin on the quality of BINW was evaluated by fingerprint analysis for the first time. Furthermore, chemometric methods including similarity analysis and principal component analysis were employed to evaluate the quality of BINW. The nine major monomeric compounds were quantitated by ultra-high-performance liquid chromatography. All nine analytes demonstrated excellent linearity with recoveries ranging from 97.25% to 102.76%. The limits of detection and quantification were 0.07-12.20 μg/mL and 0.22-40.27 μg/mL, respectively. Results indicate that different regions of origin have a significant effect on the quality of BINW. Fingerprint analysis in combination with chemometrics and multi-ingredient determination is an efficient and reliable approach for quality evaluation. The BINW samples from Yunnan had the highest ratio of 1,5-dicaffeoylquinic acid and thymol; they also exhibited significantly higher anticomplementary activity than those from three other areas. This study successfully established a rapid and efficient method to evaluate the quality and biological activity of BINW.

Alantolactone is a sesquiterpene lactone extracted from Inula helenium L. plants possessing many biological activities, including anti-inflammatory, antiproliferation, and antimicrobial. The inhibitory effects and the underlying mechanisms of alantolactone on lung cancer cells NCI-H1299 and Anip973 were investigated in this study. The results showed that alantolactone could decrease cell viability and induce cell apoptosis of NCI-H1299 and Anip973. After the cells were treated with alantolactone, the expression of Bcl-2 decreased, while the expression of Bax increased, the expression of MMP-9, MMP-7, and MMP-2 gradually decreased after alantolactone treatment. Furthermore, results showed that alantolactone could activate p38 MAPK pathway and suppress NF-κB pathway, which are involving in lung cancer development. These results indicated that alantolactone was a potential agent for lung cancer treatment. PRACTICAL APPLICATIONS: Lung cancer is one of the most common contributors of cancer death in the world. Chemoprevention and chemotherapy with natural substances are prospective methods for lung cancer treatment. In recent years, the anti-cancer activity of various sesquiterpene lactones has attracted a great deal of interest. Alantolactone is the major active sesquiterpene lactones isolated from Inula helenium L, which is used as a medicine in ancient Romans due to wide range of pharmacological activities. The results obtained from this study revealed the inhibitory effects of alantolactone on lung cancer cells and might provide some experimental basis for prevention and treatment of lung cancer with alantolactone.

Ethnopharmacological relevance During the epidemic season, over 90% of acute wheezing disease is associated with bronchial inflammation. Both neutrophil- and eosinophil-mediated inflammation have been involved in the pathophysiology of acute bronchitis, but neutrophil cell recruitment has been shown to be dominant. The ongoing inflammation increases the chemotaxis of neutrophils to inflamed site providing to their overaccumulation. The pharmacological reduction of neutrophil migration can be limited by suppression of major chemo-attractants and cytokines (IL-8, IL-1β and TNF-α) release and downregulation of adhesive molecules. Aim of the study During a screening of plants traditionally used in respiratory tracts diseases (e.g. cough, rhinitis, bronchitis, throat infection, fever, influenza) in Europe, we have selected roots of Inula helenium and aerial parts of Grindelia squarrosa as a potential source of compounds limiting neutrophil migration. Materials and methods The effect on IL-8, IL-1β and TNF-α release by neutrophils and respiratory epithelium cell line (A549) was measured by enzyme-linked immunosorbent assay (ELISA). The surface expression of adhesive molecules was analyzed with flow cytometry, and the neutrophil attachment to the epithelial cells was assessed fluorimetrically. Results We confirmed the ability of selected extracts and compounds to suppress neutrophil binding to the epithelium surface via downregulation of β2 integrin. Alantolactone and grindelic acid have shown significant suppression of IL-8, TNF-α and IL-1β release comparable with budesonide, used as a positive control. Conclusions The present study demonstrated that Inula helenium and Grindelia squarrosa, which have been traditionally used in Europe as medicinal plants, are a valuable source of active compounds with anti-inflammatory activity. Our observations justify the traditional use of I. helenium and G. squarrosa for a treatment of inflammation-based diseases in respiratory tract.

Variations in abiotic environmental factors have significant effects on quantity and quality of secondary metabolites, which is particularly important for plant species that possess biologically active compounds. The purpose of this study is determination of the total phenolic content, flavonoid concentration, and antioxidant activity of the different parts of Inula helenium L. (Asteraceae) sampled from different populations and in different time periods. The amounts obtained for the total phenolics varied from 16.73 to 89.85 mg of gallic acid (GA)/g. The concentration of flavonoids ranged from 9.32 to 376.22 mg of rutin (Ru)/g. The IC50 values of antioxidant activity determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical method varied from 161.60 to 1563.02 &mu, g/ml. The inflorescence and roots possessed high concentration of phenolic compounds and significant antioxidant activity, while leaves contained the highest concentration of flavonoids. Additionally, the quantity of the phenolics, as well as antioxidant activity, significantly varied among the different populations due to different impacts of environmental factors. This research showed that I. helenium represents an abundant source of bioactive substances, and that the quantity of these compounds greatly differs among the different populations as well as in the same populations regarding the different time periods as well as plant parts.

The disruption of the MDM2-p53 interaction has been regarded as an attractive strategy for anticancer drug discovery. Here, the natural small-molecule SCY45 was identified as a potent MDM2-p53 interaction inhibitor based on fluorescence polarization and molecular modeling. SCY45 inhibited the MDM2-p53 interaction with an IC50 value of 4.93±0.08 μm. The structural modeling results showed that SCY45 not only had high structural similarity with nutlin-3a, a well-reported MDM2-P53 interaction inhibitor, but also bound to the p53 binding pocket of MDM2 with a binding mode similar to that of nutlin-3a. Moreover, SCY45 reduced the cell viability in cancer cells with MDM2 gene amplification. SCY45 showed the highest inhibition for SJSA-1 cells, which exhibit excessive MDM2 gene amplification, with an IC50 value of 7.54±0.29 μm, whereas SCY45 showed a weaker inhibition for 22Rv1 cells and A549 cells, which have a single copy of the MDM2 gene, with IC50 values of 18.47±0.75 μm and 31.62±1.96 μm, respectively.

To determine the plasma protein binding rate of the nine compounds in Inula cappa extraction by the method of equilibrium dialysis. The proteins in plasma samples were precipitated by methanol, and the ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS) was developed for determination of the concentrations of the nine active compounds, namely chlorogenic acid, scopolin, neochlorogenic acid, cryptochlorogenic acid, 1,3-O-dicaffeoylquinic acid, galuteolin, 3,4-O-dicaffeoylquinic acid, 3,5-O-dicaffeoylquinic acid, 4,5-O-dicaffeoylquinic acid, with the internal standard of puerarin. We found that all components have a good linearity(r≥0.999), and accuracy, precision, extraction recovery and stability conformed to the requirements of determination, without endogenous compounds disturbing within the range of optimum concentration. This suggested that the method was stable and reliable, and could be used for the determination of the plasma protein binding rates of the nine active compounds in rat and human plasma of I. cappa. The plasma protein binding rates of the nine active compounds in rat and human plasma respectively were(41.07±0.046)%-(94.95±0.008)%, and(37.66±0.043)%-(97.46±0.013)%. According to the results, there were differences in the plasma protein binding rates of the nine compounds in I. cappa extraction between rat and human.

This study aimed to improve antioxidant effect and hepatoprotective effect of Inula britannica using fermentation. Epigallocatechin gallate (EGCG) in an I. britannica extract was found to be upregulated from 2.06 to 10.28 μg/mg during fermentation (p