Ageratina adenophora causes intestinal integrity damage in goats via the activation of the MLCK/ROCK signaling pathway.

As a global toxin invasive species, the whole herb of Ageratina adenophora (A. adenophora) contains various sesquiterpenes, which can cause various degrees of toxic reactions characterized by inflammatory damage when ingested by animals. Current studies on the toxicity of A. adenophora have focused on parenchymatous organs such as the liver and spleen, but few studies have been conducted on the intestine as the organ that is first exposed to A. adenophora and digests and absorbs its toxic components. In this study, after feeding goats with 40 % A. adenophora herb powder for 90 d, we found that the intestinal structure of goats showed pathological changes characterized, and the damage to the small intestinal segments was more severe than that of the large intestine. The MLCK/ROCK signaling pathway was activated, the cytoskeleton underwent centripetal contraction, the composition of tight junctions between intestinal epithelial cells was altered table, Occludin, Claudin-1 and Zonula occluden (ZO-1) amount was decreased, and the intestinal mechanical barrier was disrupted. The intestinal damage markers diamine oxidase (DAO) and D-lactate (D-LA) levels were elevated. In addition, we also found that intestinal bacteria translocate and enter the portal vein to colonize the liver and mesenteric lymph nodes. The expression of intestinal pro-inflammatory factors and anti-inflammatory factors was changed, the intestinal immune function was disrupted. The present study is the first to analyze the mechanism of poisoning of A. adenophora from the intestinal tract in compound-gastric animals.

Phytochemically analysed extract of Ageratina adenophora (Sprengel) R.M.King & H. Rob. initiates caspase 3-dependant apoptosis in colorectal cancer cell: A synergistic approach with chemotherapeutic drugs.

ETHNOPHARMACOLOGICAL RELEVANCE: Ageratina adenophora (Sprengel) R.M.King & H.Rob. has been used as traditional indigenous medicine all across the globe for its diverse therapeutic applications such as anticancer, analgesic, antipyretic, thermogenic, antiseptic, antimicrobial as well as astringent. The various ethnic groups of India use plant parts to treat cuts and wounds, venomous insect bites, skin lesions, blisters, scabies and other skin irritations, gastritis and indigestion problems, cough, stomach ache and dysentery. The Portuguese traditionally extract the juice from the plant and use it for cancer, diabetes, liver disorder, gallbladder and stomach ailments. Nigerian healers use different parts of the plant to treat diabetes, fever and inflammation. AIM OF THE STUDY: The aim of this study is to investigate the cytotoxic potential of A. adenophora hydroalcoholic leaves extract (AHL) on Colorectal cancer (CRC) cell lines (HCT-116, HCT-15 and HT-29), synergistic potential with chemotherapeutic drugs 5FU and Cisplatin as well as reactive oxygen species (ROS) generation, based on the sample collected from Mao district of Manipur, India. Identification of bioactive phytocompounds in AHL was also performed by HRLCMS. METHODS: The AHL was evaluated for its cytotoxic as well as antiproliferative activities by 3-(4, 5-dimethylthiazol-2-yl)-2, 5 diphenyltetrazolium bromide (MTT) assay, clonogenic and cell migration assays. The total phenolic content (TPC) and total flavonoid content (TFC) were quantified by Folin-ciocalteu and Aluminium chloride assays respectively. Caspase 3 activation was evaluated using Caspase-3 Assay Kit. Apoptosis detection by flow cytometry was carried out using annexin V-FITC/PI apoptosis detection kit. The apoptotic cells were also visualized by Giemsa and 4',6-Diamidino-2-phenylindole (DAPI) staining. The intracellular Reactive oxygen species (ROS) generation was also evaluated using fluorescent probe 2',7'-dichlorodihydrofluorescein di-acetate (H2DCFDA) in flow cytometry. The combination effects of AHL with chemotherapeutic drugs 5FU and Cisplatin were also evaluated. The identification of phytochemical constituents of AHL were analysed by HR-LCMS. RESULTS: The AHL induced cytotoxic activity significantly in HCT-116 with IC50 of 65.65 ± 2.10 µg/mL, but non-cancerous cell HeK-293 was least cytotoxic. Colony formation and cell migration were inhibited in a dose and time dependent manner. The cell morphology upon AHL treatment was significantly altered with apoptotic features. The extract was rich in total phenolic (82.09 ± 0.35mgGAE/g) and total flavonoid (58.31 ± 0.55 mgQAE/g) contents. AHL induced apoptosis as detected by AnnexinV/PI, via activation of caspase 3 and elevated production of Reactive oxygen species (ROS). AHL in combination with 5FU and Cisplatin acts synergistically and potentiates the therapeutic properties of the extract. Sesquiterpenes, phenolic as well as flavonoid derivatives with anticancer properties were detected in AHL by HRLCMS, and these phytoconstituents may be attributed for anticancer property of AHL. CONCLUSION: The present study evaluates the effectiveness of AHL against Colorectal cancer cell lines. AHL is cytotoxic and induces apoptosis in HCT-116 cells by caspase 3 activation and increased ROS production that can be attributed to sesquiterpenoids. Thus, the plant A. adenophora has therapeutic potential for Colorectal cancer and can be further exploited for developing anticancer drug.

Ageratina adenophora damages the rumen epithelium via inducing the expression of inflammatory factors in goats.

The aim of this experiment was to investigate the effects of Ageratina adenophora on the expression of epithelium tight junction proteins and inflammatory factors in the rumen of goats. Twelve goats were randomly divided into three groups. The first group was the blank control group (n = 3, C) which was fed normal diet. The second group was fistulas control group (n = 3, RFC), which was fitted with rumen fistulas, and fed normal diet. The third group was the A. adenophora test group (n = 6, AA), which was fitted with rumen fistulas and fed a mixture of 60% of normal diet and 40% of A. adenophora grass powder. The feeding experiment lasted for 90 d, after which all goats were sacrificed and samples were collected from the rumen dorsal sac and ventral sac. The relative expression of mRNA of inflammatory factors in the rumen epithelium (tumor necrosis factor alpha [TNF-α], interferon gamma [IFN-γ], interleukin 1 beta [IL-1ß], IL-2, IL-4, IL-6, and IL-10) and tight junction protein genes (occludin, claudin-1, and ZO-1) was measured by quantitative real-time fluorescence PCR. Expression of tight junction proteins in the rumen epithelium was measured by Western blot. A correlation was established between the expression of inflammatory factors and tight junction protein genes using Graph Pad Prism. The results showed that A. adenophora caused a significant increase in the mRNA expression levels of TNF-α, IFN-γ, IL-1ß, IL-2, IL-6, and IL-10 in the rumen epithelial (P < 0.05 or P < 0.01). The expression of tight junction proteins at both gene and protein levels was significantly decreased (P < 0.05 or P < 0.01). Furthermore, the correlation analysis revealed that the changes in tight junction protein expression in the test group were closely related to the upregulation of the expression of inflammatory factors TNF-α and IFN-γ in rumen epithelial cells. In conclusion, the expression of inflammatory factors was increased and the expression of tight junction proteins was decreased in goats after feeding on A. adenophora, which caused some damage to the rumen epithelium.

The article aims to investigate the toxic effects of Ageratina adenophora, an invasive plant on the integrity of the rumen epithelium by measuring the changes in the expression of inflammatory factors and tight junction proteins after the consumption of A. adenophora in goats. The results showed that A. adenophora causes damage to the rumen epithelium by increasing the expression of pro-inflammatory markers like TNF-α and IFN-γ and reducing the expression of tight junction proteins such as occludin and claudin-1 in goats.

Anti-Inflammatory Activity and Mechanism of Cryptochlorogenic Acid from Ageratina adenophora.

Ageratina adenophora is an invasive plant known for its toxicity to livestock. Current research on this plant has shifted from toxicity prevention to the beneficial utilization of plant resources. This study was performed to investigate the effects and mechanisms of cryptochlorogenic acid (CCGA) isolated from Ageratina adenophora on the inflammatory responses induced by lipopolysaccharide (LPS) in RAW264.7 cells. RAW264.7 cells were pretreated with CCGA (200, 100, and 50 µg/mL) and subsequently stimulated with LPS (1 µg/mL) for 16 h. The cytotoxicity of CCGA was tested using the Cell Counting Kit (CCK8). The mechanism of action of CCGA in attenuating inflammation was also identified using enzyme-linked immunosorbent assay (ELISA), quantitative reverse transcription-polymerase chain reaction, and Western blot. The results showed that CCGA had a maximal safe concentration of 200 mg/mL. Moreover, CCGA reduced the level of nitric oxide (NO) and iNOS in LPS-induced RAW264.7 cells (p < 0.01). In addition, CCGA reduced the levels of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6, and IL-8) and cyclooxygenase-2 (COX-2) in LPS-induced RAW264.7 cells at both the mRNA and protein levels (p < 0.01). CCGA prevented the activation of nuclear factor-kappa B (NF-kB) in LPS-induced RAW264.7 cells via the inhibition of IKK and IκB phosphorylation and the degradation of IκB proteins (p < 0.01). This finding indicated that CCGA isolated from A. adenophora may be a potential candidate for the treatment of inflammation-related diseases.

An invasive plant experiences greater benefits of root morphology from enhancing nutrient competition associated with arbuscular mycorrhizae in karst soil than a native plant.

The Eupatorium adenophorum have widespread invaded the karst ecosystem of southwest China and threatened the regional native community stability. Arbuscular mycorrhizae (AM) plays an important role in promoting growth for host plants via root external mycelia. However, whether AM regulates plant root traits underlying competition between invasive and native species via mycorrhizal networks in karst habitats, remains unclear. An experiment was conducted in a microcosm composed of two planting compartments flanking a competition compartment. The invasive E. adenophorum and native Artemisia annua were each placed in one of the two planting compartments with or without Glomus etunicatum fungus. The nutrient access treatments included the competitive utilization (Cu), single utilization (Su) and non-utilization (Nu) by using different nylon meshes allowed or prevented mycelium passing to acquire nutrients from the competition compartment. Root traits and nutrients of the two species were analyzed. The results showed that AM fungi had differential effects on root traits and nutrients of E. adenophorum and A. annua seedlings, which increased dry weight, length, surface area, volume, tips and branching points in roots, specific root length and volume, root nitrogen (N) and phosphorus (P) contents under Cu, Su and Nu treatments. AM fungus was also associated with decreases in the average diameter for both species. Under the Cu treatment, E. adenophorum had significantly greater length, surface area, volume, tips and branching points of roots, specific root traits, and root N and P than A. annua. AM fungi changed root phenotypes and nutrient uptake for both invasive and native plant species via interconnected mycorrhizal networks. Overall, our results suggest that through mycorrhizal networks, the invasive plant experiences greater benefits than the native plant in the nutrient competition, which fosters root morphological developments in karst soil.

Degradation of euptox A by tannase-producing rumen bacteria from migratory goats.

AIMS: The gut microbiota capable of degrading plant biomass and antinutritional phytometabolites are of immense importance. This study reports isolation and characterization of tannase-producing rumen bacteria that could also degrade euptox A (9-oxo-10,11-dehydroageraphorone) present in Eupatorium adenophorum (Spreng). METHODS AND RESULTS: Migratory Gaddi goats were selected as source of inoculums for isolating rumen bacteria with ability to produce tannase which catalyses degradation of hydrolysable tannins (HTs). Three rumen bacterial isolates producing tannase were studied, and identified as Klebsiella variicola strain PLP G-17 LC, K. variicola strain PLP S-18 and Klebsiella pneumoniae strain PLP G-17 SC. The isolates exhibited optimal tannase activity at 40°C, and pH 6·0. The bacteria could also degrade euptox A, a potent hepatotoxin in E. adenophorum Spreng, a widely distributed noxious weed. CONCLUSIONS: The rumen bacteria could degrade antinutritional HTs and euptox A. Culture-independent metagenomic interventions are envisioned to completely decipher the rumen microbial ecology and exploit its genetic and metabolic potential. SIGNIFICANCE AND IMPACT OF THE STUDY: The bacteria producing tannase which catalyses degradation of HTs, and concurrently degrading euptox A, may have potential as microbial feed additives to increase utilization of plant biomass containing antinutritional phytometabolites.

TeA is a key virulence factor for Alternaria alternata (Fr.) Keissler infection of its host.

A toxin-deficient mutant strain, HP001 mutant of Alternaria alternata, whose mycelium is unable to infect its host, produces little tenuazonic acid (TeA) toxin. How TeA plays a role in initiating host infection by A. alternata remains unclear. In this research we use Imaging-PAM based on chlorophyll fluorescence parameters and transmission electron microscopy to explore the role of TeA toxin during the infection process of A. alternata. Photosystem II damage began even before wild type mycelium infected the leaves of its host, croftonweed (Ageratina adenophora). Compared with the wild type, HP001 mutant produces morphologically different colonies, hyphae with thinner cell walls, has higher reactive oxygen species (ROS) content and lower peroxidase activity, and fails to form appressoria on the host surface. Adding TeA toxin allows the mutant to partially recover these characters and more closely resemble the wild type. Additionally, we found that the mutant is able to elicit disease symptoms when its mycelium is placed on leaves whose epidermis has been manually removed, which indicates that TeA may be determinant in the fungus recognition of its plant host. Lack of TeA toxin appears responsible for the loss of pathogenicity of the HP001 mutant. As a key virulence factor, TeA toxin not only damages the host plant but also is involved in maintaining ROS content, host recognition, inducing appressoria to infect the host and for allowing completion of the infection process.

Toxicity of white snakeroot (Ageratina altissima) and chemical extracts of white snakeroot in goats.

White snakeroot (Ageratina altissima) is a sporadically toxic plant that causes trembles in livestock and milk sickness in humans that drink tainted milk. The putative toxin in white snakeroot is tremetone and possibly other benzofuran ketones, even though it has not been demonstrated in vivo. Toxic white snakeroot was dosed to goats, and they developed clinical signs of poisoning, exercise intolerance, significant increases in serum enzyme activities, and histological changes. Tremetone and the other benzofuran ketones were extracted with hexane; the extracts and residues were analyzed for tremetone and dosed to goats at tremetone and benzofuran ketone concentrations similar to the original plant material. However, none of the dosed goats developed the disease. The results demonstrate for the first time that white snakeroot is a potent myotoxin in goats and that other compound(s), which may be lost or modified during the extraction process, could be involved in causing trembles and milk sickness.

[Composition of phenolic allelochemicals in Eupatorium adenophorum root zone soils and its effects on soil-borne pathogens].

The allelochemicals released by Eupatorium adenophorum roots is an important factor inducing the changes of the weed soil environment, which provides favorable conditions for the successful invasion of the weed. By using GC/MS technique, the components and their relative contents of phenolic allelochemicals in aphid-infested and non-infested E. adenophorum root zone soils were analyzed, with unplanted soil as the control. Less difference was observed in the components of phenolic allelochemicals among the test soils, but their relative contents differed significantly. The relative contents of benzoic acid and 4-hydroxybenzoic acid in aphid-infested E. adenophorum root zone soil were significantly higher than the control; whereas that of 4-hydroxylcinnamic acid was in reverse. In the test three soils, the proportions of benzoic acid, 4-hydroxybenzoic acid, and 4-hydroxylcinnamic acid were different. The bacteriostatic test with the three acids and their combinations based on their proportions in test soils showed that at lower concentrations (50-150 mg x L(-1)), the three acids had significant inhibitory effects on five kinds of soil-borne pathogens, but their combinations had different inhibitory effects on the five pathogens, with the effects being significantly higher for the combinations with the proportions in non-infested E. adenophorum root zone soil and the control.

Evolutionary tradeoffs for nitrogen allocation to photosynthesis versus cell walls in an invasive plant.

Many studies have shown that individuals from invasive populations of many different plant species grow larger than individuals from native populations and that this difference has a genetic basis. This increased vigor in invasive populations is thought to be due to life history tradeoffs, in which selection favors the loss of costly defense traits, thereby freeing resources that can be devoted to increased growth or fecundity. Despite the theoretical importance of such allocation shifts for invasions, there have been no efforts to understand apparent evolutionary shifts in defense-growth allocation mechanistically. Reallocation of nitrogen (N) to photosynthesis is likely to play a crucial role in any growth increase; however, no study has been conducted to explore potential evolutionary changes in N allocation of introduced plants. Here, we show that introduced Ageratina adenophora, a noxious invasive plant throughout the subtropics, appears to have evolved increased N allocation to photosynthesis (growth) and reduced allocation to cell walls, resulting in poorer structural defenses. Our results provide a potential mechanism behind the commonly observed and genetically based increase in plant growth and vigor when they are introduced to new ranges.

Nitrogen allocation and partitioning in invasive and native Eupatorium species.

There is a trade-off between nitrogen (N) allocation to photosynthesis and to defence. Invasive species may reduce N allocation to defence because of the absence of natural enemies. Thus, I hypothesised that invasive species may allocate a higher fraction of total leaf N to photosynthesis and have higher light-saturated photosynthetic rate (P(max)) and photosynthetic N-use efficiency (PNUE) than closely related native species. To test these hypotheses, invasive Eupatorium adenophorum and native E. chinense and E. heterophyllum were compared in a limestone shrub. Unlike expectation, the invader did not allocate a higher fraction of leaf N to photosynthesis than the natives. However, it was more efficient in photosynthetic N partitioning than the natives. It partitioned a higher fraction of the photosynthetic N to carboxylation and showed higher use efficiency of the photosynthetic N, while the natives partitioned a higher fraction of the photosynthetic N to light-harvesting components. Total leaf N content was not significantly different among the three studied invasive and native species. For the invader, the higher fraction of leaf N allocated to carboxylation resulted in the higher N content in carboxylation and in both carboxylation and bioenergetics, which led to higher P(max), and therefore to higher PNUE, water-use efficiency, respiration efficiency and apparent quantum yield. These physiological advantages of the invader and its higher leaf area ratio may contribute to its invasiveness.

Comparative study on chemical pretreatment methods for improving enzymatic digestibility of crofton weed stem.

In order to utilize and control the invasive weed, crofton weed (Eupatorium adenophorum Spreng), a potential pathway was proposed by using it as a feedstock for production of fermentable sugars. Three chemical pretreatment methods were used for improving enzymatic saccharification of the weed stem. Mild H2SO4 pretreatment could obtain a relatively high yield of sugars in the pretreatment (32.89%, based on initial holocellulose), however, it led to only a slight enhancement of enzymatic digestibility. NaOH pretreatment could obtain a higher enzymatic conversion ratio of cellulose compared with H2SO4 pretreatment. Peracetic acid (PAA) pretreatment seemed to be the most effective for improving enzymatic saccharification of the weed stem in the three chemical pretreatment methods under the same conditions. The conversion ratio of cellulose in the sample pretreated by PAA under the "optimal" condition was increased to 50% by cellulase loading of 80 FPU/g cellulose for 72 h incubation. A number of empirical quadratic models were successfully developed according to the experimental data to predict the yield of sugar and degree of delignification.