Mass spectrometry-based metabolomics for the elucidation of alkaloid biosynthesis and function in invasive Vincetoxicum rossicum populations.

The genus Vincetoxicum includes a couple of highly invasive vines in North America that threaten biodiversity and challenge land management strategies. Vincetoxicum species are known to produce bioactive phenanthroindolizidine alkaloids that might play a role in the invasiveness of these plants via chemical interactions with other organisms. Untargeted, high-resolution mass spectrometry-based metabolomics approaches were used to explore specialized metabolism in Vincetoxicum plants collected from invaded sites in Ontario, Canada. All metabolites corresponding to alkaloids in lab and field samples of V. rossicum and V. nigrum were identified, which collectively contained 25 different alkaloidal features. The biosynthesis of these alkaloids was investigated by the incorporation of the stable isotope-labelled phenylalanine precursor providing a basis for an updated biosynthetic pathway accounting for the rapid generation of chemical diversity in invasive Vincetoxicum. Aqueous extracts of aerial Vincetoxicum rossicum foliage had phytotoxic activity against seedlings of several species, resulting in identification of tylophorine as a phytotoxin; tylophorine and 14 other alkaloids from Vincetoxicum accumulated in soils associated with full-sun and a high-density of V. rossicum. Using desorption-electrospray ionization mass spectrometry, 15 alkaloids were found to accumulate at wounded sites of V. rossicum leaves, a chemical cocktail that would be encountered by feeding herbivores. Understanding the specialized metabolism of V. rossicum provides insight into the roles and influences of phenanthroindolizidine alkaloids in ecological systems and enables potential, natural product-based approaches for the control of invasive Vincetoxicum and other weedy species.

Specialized pollination by cecidomyiid flies and associated floral traits in Vincetoxicum sangyojarniae (Apocynaceae, Asclepiadoideae).

Specialized pollination systems frequently match a particular set of floral characteristics. Vincetoxicum spp. (Apocynaceae, Asclepiadoideae) have disk-shaped flowers with open access to rewards and reproductive organs. Flowers with these traits are usually associated with generalized pollination. However, the highly modified androecium and gynoecium that characterize asclepiads are thought to be associated with specialized pollinators. In V. sangyojarniae, we investigated floral biology, pollination, and the degree of pollinator specialization in two localities in Thailand. We examined floral traits that target legitimate pollinators. Flowers of V. sangyojarniae opened only at night, emitted floral scents containing mainly (E)-ß-ocimene, 1-octen-3-ol, (E)-4,8-dimethyl-1,3,7-nonatriene (E-DMNT) and N-(3-methylbutyl)acetamide, and provided sucrose-dominated nectar openly to insect visitors. Assessment of pollinator effectiveness indicated that V. sangyojarniae is functionally specialized for pollination by cecidomyiid flies. Although various insects, particularly cockroaches, frequently visited flowers, they did not carry pollinaria. Our results suggest that V. sangyojarniae attracts its fly pollinators by emitting floral volatiles bearing olfactory notes associated with the presence of fungi or, less likely, of prey captured by predatory arthropods (food sources of its pollinators) but offers a nectar reward upon insect arrival. Hence, there is a mismatch between the advertisement and the actual reward. Our results also suggest that the size of floral parts constitutes a mechanical filter where reciprocal fit between flower and insect structures ensures that only suitable pollinators can extract the pollinaria, a prerequisite for successful pollination.

Molecular phylogeny of Vincetoxicum (Apocynaceae, Asclepiadoideae) from Thailand and integrative taxonomy corroborating a new cryptic species within Vincetoxicum kerrii.

An updated phylogeny of the genus Vincetoxicum s.l. based on DNA sequences of the nuclear internal transcribed spacer (ITS) region and three plastid markers is presented. In total, 21 accessions newly sequenced from Thailand were added to the dataset of the homologous sequences of 75 other Vincetoxicum taxa downloaded from GenBank. In our analysis, the relationships between the well-supported clades largely correspond to those revealed in previous studies. With some exceptions, the phylogenetic positions of the Thai taxa in relation to other conspecifics and congeners generally reflect the geographic distributions of taxa. Moreover, recent extensive sampling throughout Thailand and in-depth investigation have revealed V. kerrii, a slender twiner widespread from South China to Indo-China, to be a species complex. A combination of molecular, morphological, anatomical, ultrastructural and ecological evidence allowed us to reveal a new cryptic species hidden within V. kerrii, described here under the name V. simplex. A comprehensive description, illustrations, photographs, and comparison with the morphologically similar species are provided. Although V. simplex and V. kerrii s.s. resemble one another in various aspects of vegetative and reproductive structures, the latter is phylogenetically closely related to V. irrawadense, which is much less similar morphologically to both V. simplex and V. kerrii s.s. than the latter two are to each other. In addition to the new cryptic species recognized in the present study, a new record for Thailand, V. microstachys, is also reported.

Anti-Melanogenic Effects of Fractioned Cynanchum atratum by Regulation of cAMP/MITF Pathway in a UVB-Stimulated Mice Model.

Based on traditional pharmacological applications and partial in vitro data, Cynanchum atratum (CA) is proposed to act on skin whitening. However, its functional evaluation and underlying mechanisms have yet to be identified. This study aimed to examine the anti-melanogenesis activity of CA fraction B (CAFB) on UVB-induced skin hyperpigmentation. Forty C57BL/6j mice were exposed to UVB (100 mJ/cm2, five times/week) for eight weeks. After irradiation, CAFB was applied to the left ear once a day for 8 weeks (the right ear served as an internal control). The results showed that CAFB significantly reduced melanin production in the ear skin, as indicated by the gray value and Mexameter melanin index. In addition, CAFB treatment notably decreased melanin production in α-MSH-stimulated B16F10 melanocytes, along with a significant reduction in tyrosinase activity. Cellular cAMP (cyclic adenosine monophosphate), MITF (microphthalmia-associated transcription factor), and tyrosinase-related protein 1 (TRP1) were also noticeably downregulated by CAFB. In conclusion, CAFB is a promising ingredient for treating skin disorders caused by the overproduction of melanin and its underlying mechanisms involving the modulation of tyrosinase, mainly mediated by the regulation of the cAMP cascade and MITF pathway.

Natural glycosidic antioxidants from Cynanchum atratum roots.

Two new rhamnosides, 18-O-α-l-rhamnopyranosylabietic acid (1) and (E)-3,5-dimethoxystilben-4'-O-α-l-rhamnopyranoside (2), five known glucosides (3-7) along with three others were isolated from Cynanchum atratum roots. The structures of new compounds were elucidated by physical data analyses such as NMR, UV, IR, HR-ESI-MS, as well as acid hydrolysis. All of them were assessed for their antioxidant activities through 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) radical ion (ABTS•+), 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH•) and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO•) assay, with l-ascorbic acid used as the positive control. As a result, compounds 3-5 exhibited obvious antioxidant activities. These bioactive components could be promising antioxidants.

Isolation and characterization of antioxidative monoterpenes from Cynanchum atratum roots.

One novel monoterpene rhamnoside (1) and 7 known monoterpenes (2-8) were isolated from the ethanol extract of Cynanchum atratum for the first time. Their structures were identified by comprehensive spectroscopic data analysis such as nuclear magnetic resonance, high-resolution electrospray ionization mass spectra, optical rotatory dispersion, and acid hydrolysis. In the subsequent antioxidant assay, compound 8 exhibited obvious 2,2-diphenyl-2-picrylhydrazyl hydrate radical scavenging activity.

Does the Presence of the Biological Control Agent, Hypena opulenta (Lepidoptera: Erebidae) on Swallow-Worts Deter Monarch Oviposition?

Invasive black and pale swallow-worts (Vincetoxicum nigrum (L.) Moench, and Vincetoxicum rossicum Kelopow), which are related to milkweeds, can act as ecological traps for monarch butterflies (Danaus plexippus L. (Lepidoptera: Nymphalidae)) as they lay eggs on them that fail to develop. A recently approved biological control agent against swallow-worts, Hypena opulenta Christoph, occupies the same feeding guild on swallow-worts as monarch larvae and could be perceived as a competitor to monarchs. We tested how the presence of this defoliating moth on swallow-worts may influence monarch host selection. In a two-year field experiment, we placed pale swallow-wort plants that were either infested with H. opulenta or noninfested as well as common milkweed (Asclepias syriaca L.), into monarch habitats to assess oviposition rates. In the laboratory, monarchs were either given a choice or not between milkweeds and black swallow-worts with or without H. opulenta. While monarchs strongly preferred common milkweed in the field, up to 25% of the eggs we observed were laid on pale swallow-wort, without preference for swallow-wort with (10.7%) or without (14.3%) H. opulenta. In laboratory choice and no-choice tests, monarchs did not lay any eggs on black swallow-wort, likely because of the long-term laboratory rearing on common milkweeds. Our results confirm that pale swallow-wort may act as an oviposition sink to monarchs in Michigan as well. Since the biological control program is still in its infancy, the nature of interactions between monarchs and H. opulenta may change as the biocontrol agent becomes more widespread.

A review of the ethnopharmacology, phytochemistry and pharmacology of Cynanchumatratum.

ETHNOPHARMACOLOGICAL RELEVANCE: The dried roots and rhizomes of Cynanchum atratum Bunge is named 'Baiwei' according to traditional Chinese medicine theory. It is also named Cynanchi atrati Radix in Chinese Pharmacopoeia. Cynanchi atrati Radix is famous for its medicinal value of clearing away heat, relieving drenching, detoxifying and treating abscesses. It was commonly used in some Asian countries for the treatment of fever, vasoconstrictive syncope, lymphangitis and other diseases, obviously due to the effect of C21 steroidal glycosides. THE AIM OF THE REVIEW: The review concentrates on the botany, ethnopharmacology, phytochemistry, pharmacology and toxicology of Cynanchum atratum. We also discuss expectations for prospective research and implementation of this herb. MATERIALS AND METHODS: Relevant information about C. atratum was gained from ancient books and records, Doctoral and master's Theses, Science Direct, Pubmed, Wiley, CNKI, WanFang DATA, Google Scholar and other domestic and foreign literature. Some electronic databases have been included. RESULTS: As a member of the Apocynaceae family, C. atratum possesses its up-and-coming biological characteristics. It is widely reported for treating of postpartum fatigue, vomiting, urine drops, nephritis, urinary tract infection, edema, bronchitis and rheumatic low back pain. By now, over 100 compounds have been identified from C. atratum, including C21 steroidal glycosides, acetophenones, alkaloids, volatile oil and other ingredients. Activities such as anti-inflammatory, anti-tumor, anti-virus, antibacterial, anti-forgetful and others have been corroborated in vivo and in vitro. In addition, many of the active ingredients, such as Cynatratoside A, Cynanversicoside A, B, D, G, p-hydroxyacetophenone, 2,4-dihydroxyacetophenone and some volatile oils have been used as quality markers. CONCLUSION: All kinds of research conducted on C.atratum, especially in field of ethnopharmacological use, phytochemicals and pharmacology have been reviewed. The herb has been used over the years in treating nephritis, urinary tract infection, bronchitis and rheumatic lumbocrural pain. Many studies have been carried out to identify compounds that play a leading role in drug activity. However, the mechanism of drug therapy remains unclear. The evidence used to prove the quality standard of medicinal materials is obviously inadequate. Besides, safety evaluation is necessary for clinical medication. Similarly, the separation of steroidal saponins and the development of new drugs will also need further discussion.

Structure characterization of an arabinogalactan from Cynanchum atratum and its immune stimulatory activity on RAW264.7 cells.

In the present study, a water-soluble neutral polysaccharide (CAPW-1) with an average molecular weight of 64 kDa was purified from the root of Cynanchum atratum Bunge (Apocynaceae). The monosaccharide residue analysis revealed that CAPW-1 was composed of arabinose and galactose with a relative molar ratio of 7: 3. The backbone of CAPW-1 was consisted of 1,3-Galp and 1,3,6-Galp, the branches were attached to the O-6 of 1,3-Galp, and the side chains contained 1,6-Galp, 1,3,6-Galp, 1,5-linked, 1,3-linked, 1,3,5-linked, and terminal-Araf, which was attached to the O-3 of side 1,6-Galp. The bioactivity study indicated CAPW-1 could stimulate the proliferation of RAW264.7 cells and promote the secretion of nitric oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) with no cytotoxicity. The results suggested a potential application of CAPW-1 as an immunostimulant for the treatment of diseases such as infection and tumor.

Molecular networking based dereplication of AChE inhibitory compounds from the medicinal plant Vincetoxicum funebre (Boiss. & Kotschy).

Alzheimer's disease (AD) is a devastating neurodegenerative disease affecting 47 million people worldwide. While acetylcholinesterase (AChE) inhibitors such as donepezil and galantamine are leading drugs in the symptomatic treatment of AD, new AChE inhibitors continue to be explored for improved potency and selectivity. Herein, a molecular networking approach using high resolution (HR-MS) and tandem mass spectrometry (MS2) has been used for rapid chemical profiling of an extract of the medicinal plant Vincetoxicum funebre Boiss. & Kotschy (Apocynaceae family) that was active against AChE. A total of 44 compounds were identified by combining the MN with traditional natural product methods, including the isolation and identification of five known compounds (13, 41-44) and a novel C13-norisoprenoid (40). In addition, the potential inhibitory activity of all 44 compounds was evaluated against the AChE enzyme via molecular docking to provide further support to the proposed structures. The glycosylated flavonoid querciturone (31) exhibited the highest affinity with a docking score value of -13.43 kJ/mol. Another five compounds showed stronger docking scores against AChE than the clinically used donepezil including the most active isolated compound daucosterol (44), with a binding affinity of -10.11 kJ/mol towards AChE. These findings broaden our understanding of Vincetoxicum metabolites and highlight the potential of glycosylated flavonoids as AChE inhibitors.Communicated by Ramaswamy H. Sarma.

Biomass allocation of Vincetoxicum rossicum and V. nigrum in contrasting competitive environments.

PREMISE: Understanding how drought and biomass allocation patterns influence competitive ability can help identify traits related to invasiveness and guide management. Vincetoxicum nigrum and V. rossicum are increasingly problematic herbaceous perennial vines in the northeastern United States and southeastern Canada. METHODS: Using a greenhouse experiment, we investigated how biomass allocation and competition intensity of Vincetoxicum spp. responded to four competitive regimes at two levels of soil water availability in the presence of conspecific or congeneric neighbors. RESULTS: Soil moisture was the most important influence on growth and biomass allocation. Vincetoxicum nigrum had a greater capacity for growth and reproduction than V. rossicum, especially under drought. Drought reduced the probability of reproduction for V. rossicum. Vincetoxicum rossicum had a higher root-to-shoot ratio than V. nigrum under adequate soil moisture. This difference more than doubled under drought. Under interspecific competition, V. nigrum maximized its biomass, while V. rossicum limited aboveground growth and reproduction. Root-only competition increased shoot and root biomass relative to shoot-only competition. The effects of root and shoot competition were additive under interspecific competition, but interacted under intraspecific competition (negative interaction under drought and positive interaction under sufficient soil moisture). CONCLUSIONS: Management strategies targeting mixed populations of V. rossicum and V. nigrum are most important under ample water availability. Under drought conditions, strategies focused on V. nigrum should effectively limit Vincetoxicum growth and seed reproduction. Phenotypic plasticity and the positive competition intensity associated with drought in monocultures may contribute to drought resistance in these invasive species.

Vincetoxicum arnottianum modulates motility features and metastatic marker expression in pediatric rhabdomyosarcoma by stabilizing the actin cytoskeleton.

BACKGROUND: Prevention of metastatic invasion is one of the main challenges in the treatment of alveolar rhabdomyosarcoma. Still the therapeutic options are limited. Therefore, an anti-tumor screening was initiated focusing on the anti-metastatic and anti-invasion properties of selected medicinal plant extracts and phytoestrogens, already known to be effective in the prevention and treatment of different cancer entities. METHODS: Treatment effects were first evaluated by cell viability, migration, invasion, and colony forming assays on the alveolar rhabdomyosarcoma cell line RH-30 in comparison with healthy primary cells. RESULTS: Initial anti-tumor screenings of all substances analyzed in this study, identified the plant extract of Vincetoxicum arnottianum (VSM) as the most promising candidate, harboring the highest anti-metastatic potential. Those significant anti-motility properties were proven by a reduced ability for migration (60%), invasion (99%) and colony formation (61%) under 48 h exposure to 25 µg/ml VSM. The restricted motility features were due to an induction of the stabilization of the cytoskeleton - actin fibers were 2.5-fold longer and were spanning the entire cell. Decreased proliferation (PCNA, AMT, GCSH) and altered metastasis (e. g. SGPL1, CXCR4, stathmin) marker expression on transcript and protein level confirmed the significant lowered tumorigenicity under VSM treatment. Finally, significant alterations in the cell metabolism were detected for 25 metabolites, with levels of uracil, N-acetyl serine and propanoyl phosphate harboring the greatest alterations. Compared to the conventional therapy with cisplatin, VSM treated cells demonstrated a similar metabolic shutdown of the primary cell metabolism. Primary control cells were not affected by the VSM treatment. CONCLUSIONS: This study revealed the VSM root extract as a potential, new migrastatic drug candidate for the putative treatment of pediatric alveolar rhabdomyosarcoma with actin filament stabilizing properties and accompanied by a marginal effect on the vitality of primary cells.

Cynanchum atratum Alleviates Non-Alcoholic Fatty Liver by Balancing Lipogenesis and Fatty Acid Oxidation in a High-Fat, High-Fructose Diet Mice Model.

Cynanchum atratum, a medicinal herb, is traditionally used as an antidote, diuretic, and antipyretic in eastern Asia. The current study aimed to investigate the anti-fatty liver capacity of the ethanol extract of Cynanchum atratum (CAE) using a 10-week high-fat, high-fructose diet mouse model. A six-week treatment of CAE (from the fifth week) significantly attenuated the weights of the body, liver, and mesenteric fat without a change in diet intake. CAE also considerably restored the alterations of serum aminotransferases and free fatty acid, fasting blood glucose, serum and hepatic triglyceride, and total cholesterol, as well as platelet and leukocyte counts. Meanwhile, CAE ameliorated hepatic injury and lipid accumulation, as evidenced by histopathological and immunofluorescence observations. Additionally, CAE significantly lowered the elevation of hepatic TNF-α, the TNF-α/IL-10 ratio, fecal endotoxins, and the abundance of Gram-negative bacteria. Hepatic lipogenesis and ß-oxidation-related proteins and gene expression, including PPAR-α, SREBP-1, SIRT1, FAS, CTP1, etc., were normalized markedly by CAE. In particular, the AMPK, a central regulator of energy metabolism, was phosphorylated by CAE at an even higher rate than metformin. Overall, CAE exerts anti-hepatic steatosis effects by reducing lipogenesis and enhancing fatty acid oxidation. Consequently, Cynanchum atratum is expected to be a promising candidate for treating chronic metabolic diseases.

Specialized cockroach pollination in the rare and endangered plant Vincetoxicum hainanense in China.

PREMISE: Species of Apocynaceae are pollinated by a diverse assemblage of animals. Here we report the first record of specialized cockroach pollination in the family, involving an endangered climbing vine species, Vincetoxicum hainanense in China. Experiments were designed to provide direct proof of cockroach pollination and compare the effectiveness of other flower visitors. METHODS: We investigated the reproductive biology, pollination ecology, pollinaria removal, pollinia insertion, and fruit set following single visits by the most common insects. In addition, we reviewed reports of cockroaches as pollinators of other plants and analyzed the known pollination systems in Vincetoxicum in a phylogenetic context. RESULTS: The small, pale green flowers of V. hainanense opened during the night. The flowers were not autogamous, but were self-compatible. Flower visitors included beetles, flies, ants and bush crickets, but the most effective pollinator was the cockroach Blattella bisignata, the only visitor that carried pollen between plants. Less frequent and effective pollinators are ants and Carabidae. Plants in this genus are predominantly pollinated by flies, moths and wasps. CONCLUSIONS: Globally, only 11 plant species are known to be cockroach-pollinated. Because their range of floral features encompass similarities and differences, defining a "cockroach pollination syndrome" is difficult. One commonality is that flowers are often visited by insects other than cockroaches, such as beetles, that vary in their significance as pollinators. Cockroach pollination is undoubtedly more widespread than previously thought and requires further attention.

Gonadoprotective ability of Vincetoxicum arnottianum extract against bisphenol A-induced testicular toxicity and hormonal imbalance in male Sprague Dawley rats.

Vincetoxicum arnottianum (Wight) of family Apocynaceae is a rich source of therapeutic alkaloids, phenolics and flavonoids. Study aims to evaluate the protective potential of methanol extract of Vincetoxicum arnottianum (VAM) on bisphenol A (BPA)-induced testicular toxicity in male Sprague Dawley rat. Quantitative analysis of VAM for total phenolic (TPC), total flavonoid (TFC) and total alkaloid content (TAC) along with HPLC analysis for polyphenolics was carried out. BPA-induced testicular toxicity was determined through analysis of antioxidant enzymes, DNA damages and testicular histopathology along with reproductive hormones in serum of rat. VAM was constituted of TFC (382.50 ± 1.67 µg GAE/mg), TPC (291.17 ± 0.82 µg RE/mg), TAC (16.5 ± 0.5%), ferulic acid (2.2433 µg/mg) and vanillic acid (2.1249 µg/mg). VAM co-administration to BPA-treated rats attenuated the toxic effects of BPA and restored the body and testis weights. Altered level of luteinizing hormone (LH), testosterone and follicle-stimulating hormone (FSH) in serum, and level of antioxidants (GSH, POD, CAT and SOD) and nitric oxide in testis tissues of BPA-induced toxicity were significantly restored by VAM. Histological and comet assay studies also sanctioned the protective potential of VAM in BPA-intoxicated rats. The presence of polyphenols and alkaloids might contribute towards the scavenging and ameliorative potential of VAM in testicular toxicity induced by BPA.

Transcriptome analyses revealed molecular responses of Cynanchum auriculatum leaves to saline stress.

Cynanchum auriculatum is a traditional herbal medicine in China and can grow in saline soils. However, little is known in relation to the underlying molecular mechanisms. In the present study, C. auriculatum seedlings were exposed to 3.75‰ and 7.5‰ salinity. Next, transcriptome profiles of leaves were compared. Transcriptome sequencing showed 35,593 and 58,046 differentially expressed genes (DEGs) in treatments with 3.75‰ and 7.5‰, compared with the control, respectively. Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of these DEGs enriched various defense-related biological pathways, including ROS scavenging, ion transportation, lipid metabolism and plant hormone signaling. Further analyses suggested that C. auriculatum up-regulated Na+/H+ exchanger and V-type proton ATPase to avoid accumulation of Na+. The flavonoid and phenylpropanoids biosynthesis pathways were activated, which might increase antioxidant capacity in response to saline stress. The auxin and ethylene signaling pathways were upregulated in response to saline treatments, both of which are important plant hormones. Overall, these results raised new insights to further investigate molecular mechanisms underlying resistance of C. auriculatum to saline stress.

Vincetoxicum arnottianum ameliorate inflammation by suppressing oxidative stress and pro-inflammatory mediators in rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Aerial parts of Vincetoxicum arnottianum (Wight) Wight (Family Apocynaceae) are used by local communities for inflammation, healing of wound and injuries and also for urticaria. AIM OF STUDY: Extract/fractions of V. arnottianum were evaluated for potential anti-inflammatory activity in rat. METHODS: Methanol extract of aerial parts of V. arnottianum (VAM) was partitioned on polarity for n-hexane (VAH), ethyl acetate (VAE), butanol (VAB) and aqueous (VAA) fractions. The extract/fractions were evaluated during in vitro assay for protection against heat induced protein denaturation and Carrageenan induced paw inflammation in rat. VAM and VAE were evaluated for anti-inflammatory potential against formalin and Freund's complete adjuvant (FCA) induced inflammation in paw of rat while croton oil induced inflammation in ear of rat, respectively. The level of inflammatory mediators; IL-17, IL-1ß, IL-6, TNF-α and nitric oxide (NO) was estimated in serum of rat. RESULTS: All the extract/fractions used in this study exhibited anti-inflammatory activity. However, VAE (300 mg/kg) exhibited potential anti-inflammatory activity in carrageenan (78.06 ± 4.6%), formalin (54.71 ± 0.34%) and croton oil (73.12 ± 1.9%) induced edema in rat. In FCA induced inflammation model VAM and VAE showed admiring proficiencies against alteration of body weight and organ weight indices, paw edema and histological studies. In serum increased level of pro-inflammatory cytokines (IL-1ß, TNF-α, IL-6, IL-17) and NO during adjuvant-induced inflammation were more efficiently restored with VAE treatment to rat. Presence of polyphenolics; rutin, gallic acid, caffeic acid, apigenin, myricetin and quercetin was indicated in VAE. CONCLUSION: The results suggest the presence of anti-inflammatory constituents in V. arnottianum.

Cynanchum atratum Ameliorates Airway Inflammation via Maintaining Alveolar Barrier and Regulating Mast Cell-Mediated Inflammatory Responses.

Asthma is a common allergic airway inflammatory disease, characterized by abnormal breathing due to bronchial inflammation. Asthma aggravates the patient's quality of life and needs continuous pharmacological treatment. Therefore, discovery of drugs for the treatment of asthma is an important area of human health. The aim of the present study was to evaluate whether Cynanchum atratum extract (CAE) modulates the asthma-like allergic airway inflammation and to study its possible mechanism of action using ovalbumin (OVA)-induced airway inflammation and lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice, as well as a mast cell-based in vitro model. The histological analysis showed that CAE reduced the airway constriction and immune cell infiltration. CAE also inhibited release of ß-hexosaminidase and expression of inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-4, and IL-5 in bronchoalveolar lavage fluid and lung tissues. In addition, CAE reduced the OVA-specific immunoglobulin (Ig) E, total IgE, IgG1, and IgG2a levels in the serum. In the LPS-induced ALI model, CAE suppressed the LPS-induced lung barrier dysfunction and the release of proinflammatory cytokines. Because allergic airway inflammatory responses are associated with the activation of mast cells, RBL-2H3 cells were used to evaluate the underlying mechanism of CAE effects. In RBL-2H3 cells, CAE down-regulated release of ß-hexosaminidase and histamine by reducing the intracellular calcium influx. In addition, CAE suppressed the expression of proinflammatory cytokines by inhibiting nuclear translocation of nuclear factor-κB. Taken together, our findings suggest that CAE may help in the prevention or treatment of airway inflammatory diseases.

Genotoxic properties of Betonica officinalis, Gratiola officinalis, Vincetoxicum luteum and Vincetoxicum hirundinaria extracts.

Genotoxicity of B. officinalis, G. officinalis, V. luteum and V. hirundinaria extracts, which demonstrated strong antioxidant capacity, was tested using chromosome aberration, sister chromatid exchange (SCE), cytokinesis-block micronucleus and alkaline single-cell gel electrophoresis (comet) assays in human lymphocytes in vitro and Ames Salmonella/microsome test. All tested extracts were not mutagenic in S. typhimurium strains TA98 and TA100 with and without metabolic activation and did not induce chromosome aberrations in human lymphocytes in vitro. Extract from G. officinalis was the only one, which induced significant increase in micronuclei, indicating possible aneugenic effect. All investigated plant extracts induced DNA damage evaluated by the comet assay, while B. officinalis and V. luteum extracts induced slight increase in SCE values. The determined variation in response might be due to the plant extract tested and donor susceptibility.

A C21-Steroidal Glycoside from Cynanchum atratum Attenuates Concanavalin A-Induced Liver Injury in Mice.

Cynatratoside A (CyA) is a C21 Steroidal glycoside with pregnane skeleton isolated from the root of Cynanchum atratum Bunge (Asclepiadaceae). This study aimed to investigate the effects of CyA on concanavalin A (Con A)-induced autoimmune hepatitis (AIH) and the underlying mechanism. CyA was orally administered to mice at 10 and 40 mg/kg 8 h before and 1 h after Con A treatment. The effects of CyA on Con A-induced spleen and liver in mice were assessed via histopathological changes, T lymphocyte amounts and the expressions of IL-1ß and ICAM-1. Con A-induced L-02 hepatocytes were used to evaluate whether CyA (0.1⁻10 µM) can directly protect hepatocytes from cytotoxicity and the possible mechanism. The results revealed that CyA treatment could significantly improve the histopathological changes of spleen and liver, reduce the proliferation of splenic T lymphocytes, and decrease the expressions of IL-1ß and ICAM-1 in liver. The experiment in vitro showed that CyA inhibited Con A-induced hepatotoxicity in a concentration-dependent manner. CyA (10 µM) significantly increased/decreased the expression of Bcl-2/Bax and reduced the levels of cleaved caspases-9 and -3. Our study demonstrated for the first time that CyA has a significant protective effect on Con A-induced AIH by inhibiting the activation and adhesion of T lymphocytes and blocking hepatocyte apoptosis.