Two New Eremophilane-Type Sesquiterpenoids from Ligularia sagitta.

Two new eremophilane-type sesquiterpenoids, sagittacins F and G (1 and 2), together with one known isomer of sagittacin F (3) were isolated from the leaves and stems of Ligularia sagitta. Their structures were elucidated by interpretation of spectroscopic data and the absolute configurations of 1 and 3 were determined by X-ray spectroscopy. Compound 1 belongs to a rare class of eremophilane-type sesquiterpenoid featuring an α-oriented hydroxy group at C-1. A nitric oxide (NO) production inhibitory assay was applied to evaluate their anti-inflammatory activities by using LPS-induced RAW 264.7 cells. Compounds 2 and 3 exhibited modest NO production inhibitions with IC50 values of 45.15±2.72 and 49.83±2.34 µM, respectively.

Four new fatty acid derivatives from Diaporthe sp. T24, an endophytic fungus isolated from Ligularia fischer.

The endophytic fungus Diaporthe sp. is known to contain many secondary metabolites, but fatty acid derivatives have rarely been found. In this study, four new fatty acid derivatives (1-4), together with four known compounds (5-8), were isolated from Diaporthe sp., which was obtained from the stem of Ligularia fischeri. The absolute configurations of the new compounds 1-4 were deduced based on spectroscopic technique and J-based coupling constant analysis. Moreover, compound 1 exhibited cytotoxic activities against HCT-8 and MCF-7 cancer cells, and compounds 3 and 4 showed modest selectivity for HCT-8 cells by MTT assay.

Chemical and Genetic Diversity of Ligularia kanaitzensis in the Hengduan Mountains Area. Chemical Relationship with L. subspicata.

Root chemicals and the sequences of the internal transcribed spacers (ITSs) were analyzed for 9 Ligularia kanaitzensis and 3 L. subspicata samples collected in northwestern Yunnan and southwestern Sichuan, China. Subspicatins A and C were isolated from two L. kanaitzensis samples. Introgression of genes responsible for these compounds from L. subspicata was suggested by their strong connection with L. subspicata/L. lamarum and the geographical proximity of the samples to L. subspicata. DNA analysis of a set of 27 L. kanaitzensis samples including those analyzed previously showed that they belong to two clades, designated A and B. Together with the presence/absence of furanoeremophilane, the 27 samples were sorted into three groups: clade A/furan, clade B/furan, and clade B/non-furan. The ancestral plant presumably belonged to clade B/non-furan, because furanoeremophilanes are biosynthesized from eremophilan-8-ones. 1ß-Angeloyloxyfukinone, a likely intermediate between fukinone and subspicatin C, was isolated for the first time. This finding allowed us to propose plausible biosynthetic pathways of subspicatins A and C.

Soil and vegetation characteristics during the formation of typical Ligularia virgaurea degraded grassland.

To understand the formation process of typical poisonous plant degraded grassland, we studied the cha-racteristics of vegetation and soil during the gradual expansion of Ligularia virgaurea into the native grassland of Qinghai-Tibet Plateau. The results showed that population density, plant height, coverage, and biomass of L. virgaurea increased during the formation of L. virgaurea degraded grassland. In comparison with native grassland, the degraded grassland had higher total aboveground biomass (113.9%), soil total nitrogen concentration (61.0%), NH4+-N (77.9%), organic carbon concentration (45.3%), available phosphorus concentration (78.8%) as well as soil microbial biomass carbon (42.1%) and nitrogen (47.4%), but lower NO3--N (40.1%) and species richness (28.5%) and aboveground biomass (45.7%) of other species beyond L. virgaurea. The extremely strong abilities of interspecific inhibition and morphological plasticity of L. virgaurea, as well as efficient nutrient accumulation and utilization were the keys to its successful expansion, which facilitated the formation of typical L. virgaurea degraded grassland.

Modulation of feed digestibility, nitrogen metabolism, energy utilisation and serum biochemical indices by dietary Ligularia virgaurea supplementation in Tibetan sheep.

Ligularia virgaurea is the most widely functional native herbage in the alpine meadow pastures of the Qinghai-Tibet Plateau (QTP) and has multiple pharmacological and biological activities. The effect of L. virgaurea as a dietary component on the digestion and metabolism of sheep was evaluated by conducting feeding trials in metabolic cages. Thirty-two Tibetan yearling rams (29 ± 1.56 kg BW) were randomly allotted to four groups included in a completely randomised design with eight animals per treatment. Sheep were fed a basal diet (freshly native pasture) without the addition of L. virgaurea (control) or with the addition of L. virgaurea (100, 200, or 300 mg/kg BW per day) for 45 days. Addition of L. virgaurea to the diet of Tibetan sheep was found to influence the average daily gain (quadratic [Q], P < 0.001), feed conversion ratio (Q, P = 0.002), CH4 emissions (linear [L], P = 0.029), DM (Q, P = 0.012), neutral detergent fibre (Q, P = 0.017), acid detergent fibre (ADF) (Q, P = 0.027), and ether extract (EE) intake (Q, P = 0.026). Apparently, different levels of L. virgaurea affected the digestibility coefficients of DM, ADF, and EE (L, P > 0.05; Q, P < 0.05). The nitrogen (N) intake (Q, P = 0.001), retained nitrogen (Q, P < 0.001), and N utilisation efficiency (L, P > 0.05; Q, P ≤ 0.001) were also affected by the dietary inclusion of L. virgaurea. Effects of L. virgaurea feeding were also witnessed on methane energy (CH4-E) (L, P = 0.029), gross energy (GE) (Q, P = 0.013), digestible energy (DE) (Q, P = 0.015), and metabolisable energy (ME) intake (Q, P = 0.015). Energy utilisation efficiency expressed as a proportion of GE intake (DE/GE intake, ME/GE intake, ME/DE intake, FE/GE intake, and CH4-E/GE intake) manifested quadratic changes (P < 0.05) with the increase in the L. virgaurea supplementation level. The addition of L. virgaurea increased the activity of superoxide dismutase (Q, P = 0.026) and glutathione peroxidase activity (Q, P = 0.039) in the serum. Overall, the greatest improvement of feed digestibility, N retention, energy utilisation, and antioxidant capacity of Tibetan sheep was yielded by the inclusion of 200 mg/kg BW per day of L. virgaurea. Therefore, the addition of an appropriate amount of L. virgaurea to the diet of Tibetan sheep is safe and natural, and may enhance the sustainability of small ruminant production systems in QTP areas.

Ligularia fischeri ethanol extract: An inhibitor of alpha-melanocyte-stimulating hormone-stimulated melanogenesis in B16F10 melanoma cells.

BACKGROUND: Ligularia fischeri is a perennial herb isolated from plants of the Asteraceae family. Ligularia fischeri is distributed throughout Korea, Japan, eastern Siberia, and China. AIMS: The aim of this study is to examine the intracellular inhibitory effect of Ligularia fischeri ethanol extract on melanin synthesis and expression of tyrosinase and tyrosinase-related protein 1 and 2. In addition, we analyzed the mitogen-activated protein kinase signaling pathway and microphthalmia-associated transcription factor in alpha-melanocyte-stimulating hormone-stimulated B16F10 melanoma cells. METHODS: To assess the inhibition of melanogenesis in alpha-melanocyte-stimulating hormone-stimulated B16F10 melanoma cells, the expression of melanogenesis-related genes was investigated by quantitative real-time polymerase chain reaction, while western blotting was performed to determine protein expression levels. RESULTS: We confirmed that the ethanol extract of Ligularia fischeri inhibited melanin synthesis in vitro by decreasing tyrosinase and tyrosinase-related protein 1 and 2 expression. Furthermore, we revealed that tyrosinase expression was regulated by the suppression of microphthalmia-associated transcription factor expression and activation of extracellular signal-regulated kinase phosphorylation. The ethanol extract of Ligularia fischeri inhibited melanogenesis by activating extracellular signal-regulated kinase phosphorylation and suppressing microphthalmia-associated transcription factor and tyrosinase expression. CONCLUSIONS: Ligularia fischeri ethanol extract may be used as an effective skin whitening agent in functional cosmetics.

Ligulaveitnoid A, a new phenylpropanoid from rhizomes and roots of Ligularia veitchiana.

A new phenylpropanoid, ligulaveitnoid A (1), along with four known compounds, (E)-2,3-dihydroconiferyl p-coumarate (2), dihydroconiferyl ferulate (3), 4-hydroxy-3-methoxybenzaldehyde (4) and (E)-p-coumaric acid (5) were isolated from rhizomes and roots of L. veitchiana. All the structures of compounds were identified by the interpretation of their spectroscopic data and comparison with those reported in the literature. The anti-inflammatory activity of the isolates was examined for their inhibitory effects on LPS-induced NO production in macrophage RAW264.7 cells. Among them, compound 2 showed strong inhibitory activities towards the LPS-induced NO production in macrophage RAW264.7 cells with IC50 value of 8.0 µM.

A sesquiterpenoid from Ligularia pleurocaulis modulated macrophages polarisation through TLR4 pathway.

An eremophilane-type sesquiterpenoid (EPS), 3-oxo-eremophila-1,7(11)-dien-12,8ß-olide, has been isolated from anti-inflammatory folk herbs, Ligularia pleurocaulis. The aim of present study is to explore protective effects of EPS on lipopolysaccharide (LPS)-induced inflammatory responses in acute lung injury (ALI). EPS treatments (40 and 80 mg/kg) significantly ameliorated LPS-stimulated pathological changes in lungs. Furthermore, in vivo and in vitro mechanism studies suggest that EPS exerts its protective effects on LPS-induced ALI by regulating macrophage polarisation via suppression of TLR4/MyD88-mediated MAPK and NF-κB signaling pathways, and EPS may be useful for the prevention on ALI in the clinical setting.

Phytochemical study of Ligularia subspicata and valuation of its anti-inflammatory activity.

This report illustrated isolation and identification of 42 compounds comprising five (spicatainoids A-E) undescribed eremophilanolide type sesquiterpenoids and one undescribed nor-eremophilane (spicatainoid F) from Ligularia subspicata.. Among all the isolated new compounds, 4 is reported as the first enantiomeric form of novel eremophilanolide type sesquiterpenoid. Comprehensive analysis of HRESIMS, 1D/2D NMR, experimental circular dichroism (CD), calculated ECD analysis, and X-ray crystallographic (XRD) analysis validated the complete configuration and confirmation of these isolated compounds. All the isolated compounds were tested for the anti-inflammatory potential by measuring the amount of nitric oxide production. Among the tested compounds, 4 was the most effective with 90% NO-inhibition activity. Compounds 1, 2, 3, 9, 10 18, 29, 34, 35 exhibited moderate inhibitory effects against the production of NO, while other compounds displayed no activity even at the concentration of 50 µM. Additionally, compounds 1, 3 and 4 presented moderate anti-inflammatory activity by inhibiting the release of pro-inflammatory cytokines (TNF-α, IL-1ß, and IL-6) in LPS-stimulated N9 cells. The IC50 values of compounds 1, 3 and 4 were calculated 39.6 ± 2.7, 42.5 ± 3.8 and 27.60 ± 1.9 µΜ.

Chemical Constituents of Ligularia Species (Asteraceae) and Their Diversity in East Asia.

More than 100 Ligularia species and those of several related genera (Cremanthodium, Cacalia, Senecio, and others) in the plant family Senecioneae grow in East Asia. For many years, researchers have studied the chemical constituents of these plants, and terpenoids, flavonoids, sterols, alkaloids, and aromatic compounds have been isolated. Among these, in particular, numerous sesquiterpenoids were reported. In this contribution, relevant chemical studies are described mainly from literature reports appearing since 2000, inclusive of investigations performed by the present authors, on the diversity in secondary metabolites of Ligularia growing in the Hengduan Mountains area of China, focusing on eremophilane sesquiterpenoids and other metabolites. Terpenoids and aromatic compounds (totaling 1049), both new and known, are listed. Genetic studies and synthesis investigations are also reviewed briefly.

Two new eremophilane Sesquiterpenoides from Ligularia dictyoneura.

Two new eremophilane sesquiterpenoides, 6α,9α-dihydroxyeremophilenolide (1), and 1ß,10ß-dihydroxyeremophilenolide (2), along with ten known eremophilane sesquiterpenoides (3-12) were isolated from the aerial parts of Ligularia dictyoneura (Franch.) Hand.-Mazz. Their structures were elucidated by means of extensive spectroscopic analysis. Compounds 3-6 were assessed for their cytotoxicity against five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7 and SW-480), and the result showed that they had no activity.

Protective Effects of Ligularia fischeri and Aronia melanocarpa Extracts on Alcoholic Liver Disease (In Vitro and In Vivo Study).

Hepatic protective effects of Ligularia fischeri (LF) and Aronia melanocarpa (AM) against alcohol were investigated in vitro and in vivo test. LF, AM, and those composed mixing material (LF+AM) were treated in HepG2 cell. Alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities were significantly increased in each singleness extract and mixed composite. The protective effect on alcoholic liver damage was investigated by animal models. Serum alcohol level and acetaldehyde level were significantly decreased by LF+AM treatment in acute experimental model. In the chronic mouse model study, we had found that the increased plasma liver damage index (alkaline phosphatase) by alcohol treatment was declined by oral administration of LF+AM extraction composite. As well as, it was identified that the protection effect was induced by increasing catalase activity and suppressing COX-2, TNF-α, MCP-1, and IL-6 mRNA expressions. CYP2E1 mRNA expression was also increased. These results suggest that oral ingestion of LF and AM mixed composite is able to protect liver against alcohol-induced injury by increasing alcohol metabolism activity and antioxidant system along with decreasing inflammatory responses.

Molecular mechanism underlying the anti-inflammatory effects of volatile components of Ligularia fischeri (Ledeb) Turcz based on network pharmacology.

BACKGROUND: Ligularia fischeri (Ledeb) Turcz (LFT) is a well-known expectorant and active anti-inflammatory agent in Chinese traditional medicine. LFT's expectorant effect is closely related to its anti-inflammatory effects. This study aimed to evaluate the differential composition and anti-inflammatory mechanisms of the volatile components in LFT from different production areas. METHOD: Headspace solid-phase microextraction-gas chromatography-mass spectrometry analysis of volatile components, as well as chemometric methods, including similarity analysis, hierarchical clustering analysis, and principal component analysis, were performed to identify LFT produced in different areas. The molecular mechanism underlying the anti-inflammatory effects of these components was determined by network pharmacology analysis. RESULTS: We observed significant differences in the chemical constituents and percentage contents in samples with different origins. Eighteen volatile components were identified in four different producing areas, among which the highest content of olefinic components was the main component of the aroma of LFT. The mechanisms of these pharmacological effects involved multiple targets and pathways. Twenty-seven potential target proteins and 65 signaling pathways were screened, and a "component-target-disease" interaction network map was constructed. The volatile components of the LFT function mainly by inhibiting the production of inflammatory factors. CONCLUSION: This study provides a theoretical framework for further development and application of LFT used in traditional Chinese medicine.

Ethanol Extract of Ligularia fischeri Inhibits the Lipopolysaccharide-Induced Inflammatory Response by Exerting Anti-Histone Acetyltransferase Activity to Negatively Regulate p65.

Histone acetyltransferase (HAT) activity is well established to regulate inflammatory responses. In contrast, the mechanisms by which natural nutritional extracts influence epigenetic mechanisms to regulate inflammation have not yet been thoroughly investigated. Thus, in the present study, we observed that the anti-HAT activity exerted by an ethanol extract of Ligularia fischeri (ELF) inhibited inflammation. Specifically, we used a cell-free system to show that ELF attenuates HAT activity. We also demonstrated that ELF decreases lipopolysaccharide (LPS)-induced HAT mRNA and protein expression levels in Raw 264.7 cells, and thereby attenuates inflammation-induced patterns of hyperacetylation at nonhistone and histone-H4 proteins. Interestingly, we found that ELF blocked p65 translocation in LPS-stimulated Raw 264.7 cells by attenuating acetylation at lysine residue 310 of p65. Finally, we investigated whether ELF reduces the inflammatory cytokines, IL-6, IL-1ß, and TNFα, using its HAT inhibitor activity. Taken together, these results suggest that ELF negatively regulates inflammatory responses by inhibiting HATs and HAT activity.