Pharmacological, Cytotoxic, Immunostimulant, and Auto-Immune Activity of Thalloid Liverworts: An Overview.

Bryophytes have historically been employed as verdant medicine in China, Native America and India. Phenolics, glycosides, fatty acids, other rare aromatic compounds and Terpenoids found in bryophytes may help prevent cancer and other chronic disorders. Liverworts have historically been utilized in traditional medicine and also as immu-nomodulators or immunostimulants. Diterpenoids, Lipophilic mono-, sesqui- and aromatic compounds assisting to the biological activities of liverworts. For their biological functions more than 220 aromatic compounds and 700 terpenoids and other chemicals discovered in liverworts scrutinized for their pharmacological, cytotoxic, immunostimulant and auto-immune efficacies.

Cytotoxicity, antimicrobial and antioxidant activities of mosses obtained from open habitats.

Mosses are mainly the object of ecological and taxonomic research. This group of plants are still underestimated by scientists in other aspects of research. Recent research has shown that these plants contain remarkable and unique substances with high biological activity. Five species of mosses from a large urban ecosystem were identified for present study. In order to determine their biological potential, multifaceted studies were carried out, including: total phenolics content, antioxidant activity, antimicrobial and antifungal study, cytotoxicity evaluation, and scratch assay to assess pro-regenerative effect in the context of their possible use as the ingredients of biologically active cosmetics. Additionally, determination of individual phenolic compounds in selected extracts of the tested mosses was made. Research showed that Ceratodon purpureus and Dryptodon pulvinatus extracts had the greatest potential as antioxidants and antimicrobial activity. The cytotoxicity assessment indicated that the extracts from Dryptodon pulvinatus and Rhytidiadelphus squarossus exerted the strongest negative effect on mouse fibroblast line L929 viability at higher concentrations. While, the extract from Tortulla muralis best stimulated human foreskin fibroblast line HFF-1 proliferation and wound healing. The research on individual phenolic compounds content in the extracts tested indicated over 20 peaks on UPLC chromatograms. The conducted study has shown that mosses, especially so far unexplored species of open ecosystems, and e.g. epilytic habitats, may be a valuable source of biologically active substances and thus may constitute important medical and cosmetic possibilities.

Ecological stoichiometric characteristics of soil-moss C, N, and P in restoration stages of karst rocky desertification.

Rocky desertification is the most serious ecological disaster in karst areas. Comprehensive control of rocky desertification plays an important role in promoting the economic development of karst areas. Studying the stoichiometric characteristics of mosses and soil can provide a powerful reference for the ecological restoration and evaluation of ecosystems experiencing rocky desertification. Soil and mosses were collected from sites representing different stages of ecological restoration (bare rock, grassland, shrubland, and secondary forest), and the contents of carbon (C), nitrogen (N), and phosphorus (P) were detected for ecological stoichiometric analysis. The results indicate that in different restoration stages following karst rocky desertification, the contents of soil organic carbon (SOC), total nitrogen (TN), and total phosphorus (TP) and the stoichiometric ratios in the shrub habitat are higher than those in the bare rock, grassland, and secondary forest habitats. However, the TP and available P contents were low at all stages (0.06 g/kg and 0.62 mg/kg, respectively). The N and P contents and stoichiometric ratios in the mosses showed no significant differences among the succession stages. The C contents in the mosses had a significant positive correlation with SOC and TN and TP content, and the P content had a significant positive correlation with the soil available P. However, there was a significant negative correlation between the C: N and C:P ratios of the bryophytes and soil C: N. In summary, during the process of natural restoration of karst rocky desertification areas, SOC and soil TN contents accumulate with each succession stage. Soil nutrients are higher in shrub habitats than in other succession stages. Mosses have a strong effect on improving soil nutrients in rocky desertification areas.

Characterization of Trace Elements in Atmospheric Deposition Studied by Moss Biomonitoring in Georgia.

The present work was conducted to obtain and highlight the first comprehensive baseline data on atmospheric deposition of trace elements and to evaluate the air quality in Georgia. A total of 120 moss samples were collected over accessible territories in Georgia in the period from 2014 to 2017. Hylocomium splendens (Hedw.) Schimp., Hypnum cupressiforme (Hedw.), and Pleurozium schreberi (Brid.) Mitt. moss species were analyzed by two complementary analytical techniques: instrumental neutron activation analysis and atomic absorption spectrometry. Concentrations of 41 elements in mg/kg were determined. The concentrations were compared with the corresponding values in the literature and are in a good agreement, except for the concentration of Mg, Al, K, Ca, Ti, and Fe, which were higher than those reported for other countries. The principal component and discriminant analyses were implemented to extract information about the similar geochemical features and to decipher the provenance of the studied elements. The analysis showed that a considerable association of crustal elements and the provenance of elements can be considered as a mixture of geogenic and anthropogenic sources. In addition, the influence of different latitudinal climate zones on the distribution of elements in the atmospheric deposition was observed. The enrichment factor shows considerable values for Th and Zr. The spatial distribution of the pollution load index identifies four zones (#12, 38, 53, and 64). The potential ecological risk index and the risk index were calculated and it does not pose significant risk except As and Cd. The data obtained can be used as the first dataset of metal characterization of air pollution in Georgia.

Extracts Characterization and In Vitro Evaluation of Potential Immunomodulatory Activities of the Moss Hypnum cupressiforme Hedw.

Recently, there has been an increasing interest in the chemistry and biological potential of mosses, since a large number of biologically active compounds have been found within these species. This study aimed at examining the chemical composition and immunomodulatory potential (antioxidant, antidiabetic, anti-neuroinflammatory/antineurodegenerative, and antitumor activities) of moss Hypnum cupressiforme Hedw. extracts. Corresponding extracts have been obtained applying Soxhlet extractor. The chemical characterization was performed using spectrophotometric assays and liquid chromatography-mass spectrometry (LC-MS). The extracts were analyzed for antioxidant activity and for inhibitory activities on α-glucosidase, α-amylase, acetylcholinesterase, and tyrosinase. Additionally, extracts were tested against four cell lines-MRC-5, BV2, HCT-116, and MDA-MB-231-for antitumor and anti-inflammatory activities. Chemical analysis of extracts revealed the presence of flavonoids, phenolic acids, and triterpenoids. Major compounds identified by LC-MS in H. cupressiforme were kaempferol and five phenolic acids: p-hydroxybenzoic, protocatechuic, p-coumaric, gallic, and caffeic acid. According to biochemical assays the investigated extracts exhibited significant immunomodulatory potential. Significant antiproliferative potential against MDA-MB-231 cells has been observed together with the promising anti-neuroinflammatory application. The obtained data suggest that moss H. cupressiforme is a valuable natural source of biologically active compounds with potential application in the pharmaceutical industry.

Sources and variation of isotopic ratio of airborne radionuclides in Western Arctic lichens and mosses.

This research concerned radioactivity of lichens and mosses from coastal zones of the Canadian Arctic and Alaska. Over 50 samples were collected from 7 positions during two scientific expeditions in 2012 and 2013. The tundra contamination caused by anthropogenic radionuclides was relatively low, reaching mean values with SD's of: 17.4 ±â€¯3.5 Bq/kg for 90Sr, 14.0 ±â€¯2.9 Bq/kg for 134Cs, 38.4 ±â€¯7.5 Bq/kg for 137Cs, 0.86 ±â€¯0.24 Bq/kg for 239+240Pu, 0.065 ±â€¯0.017 Bq/kg for 238Pu and 0.50 ±â€¯0.13 Bq/kg for 241Am. The increase of activity concentration with increasing latitudes was noticed mostly in regard to 90Sr, Pu isotopes and 241Am. The analysis of isotopic ratios exhibited dominant contribution of the global fallout (+SNAP 9A satellite re-entry fallout) for the presence of plutonium isotopes and 241Am. The Fukushima fallout signature was identified in a few lichens from Alaska. However, the influence of additional unknown factor on the occurrence of 90Sr and 137Cs has been detected in western part of Canadian Arctic. Natural radioisotopes of thorium and uranium were found throughout the entire investigated region and the average values of activity concentration with SD's were as follows: 2.92 ±â€¯0.47 Bq/kg for 230Th, 2.61 ±â€¯0.48 Bq/kg for 232Th, 4.32 ±â€¯0.80 Bq/kg for 234U and 3.97 ±â€¯0.71 Bq/kg for 238U. Examined Western Arctic tundra was not affected with any technically enhanced natural radioactivity.

Water Extract of Cryphaea heteromalla (Hedw.) D. Mohr Bryophyte as a Natural Powerful Source of Biologically Active Compounds.

Bryophytes comprise of the mosses, liverworts, and hornworts. Cryphaea heteromalla, (Hedw.) D. Mohr, is a non-vascular lower plant belonging to mosses group. To the date, the most chemically characterized species belong to the liverworts, while only 3.2% and 8.8% of the species belonging to the mosses and hornworts, respectively, have been investigated. In this work, we present Folin-Ciocalteu and oxygen radical absorbance capacity (ORAC) data related to crude extracts of C. heteromalla obtained by three different extraction solvents: pure water (WT), methanol:water (80:20 v/v) (MET), and ethanol:water (80:20 v/v) (ETH). The water extract proved to be the best solvent showing the highest content of biophenols and the highest ORAC value. The C. heteromalla-WT extract was investigated by HPLC-TOF/MS (High Performance Liquid Chromatography-Time of Flight/Mass Spectrometry) allowing for the detection of 14 compounds, five of which were phenolic compounds, derivatives of benzoic, caffeic, and coumaric acids. Moreover, the C. heteromalla WT extract showed a protective effect against reactive oxygen species (ROS) generation induced by tert-butyl hydroperoxide (TBH) on the murine NIH-3T3 fibroblast cell line.

Distribution of radionuclides in moss-lichen cover and needles on the same grounds of landscape-climatic zones of Siberia.

The radiation status of the landscape and climatic zones of Siberia at the turn of the 20th and 21st centuries is characterized using bioindicators/biomonitors: lichens, mosses, and needles, according to the results obtained at the sites of their joint growth. The maximal activity of 137Cs in these components is observed in the forest-tundra landscaped zone, polluted during the period of nuclear tests from the nuclear test site "Novaya Zemlya" and also due to slow migration of these elements to the soil under the arctic conditions. In the southern territories the specific activity of radiocesium in the moss-lichen cover and needles of conifers corresponds to the regional background, in the forest-tundra zone it sometimes exceeds it, but in general does not pose a threat to human health. Determined differences in the contents of radioactive elements in lichens and mosses that grow together on sites in different landscape zones of Siberia statistically not significant within one or two standard deviations, and recorded only in the range of 3Ï­ at the level of significance 0.05. Specificity of radionuclide distribution in lichens, mosses and needles (differences for epigeals and epiphytic lichens; different species selected at one site, annual and perennial needles, etc.) made it possible to identify the causes of variations in their activities in different zones, along with landscape features of these zones. In the needles of conifers potassium concentration exceeds the content in lichens, at lower levels of thorium and 137Cs. The contents of uranium and thorium in the studied components in all landscape-climatic zones correspond to the natural ones, except for the single local territories, because of the possible anthropogenic influence.

Regulation of the NRF2 transcription factor by andrographolide and organic extracts from plant endophytes.

The transcription factor NF-E2 Related Factor-2 (NRF2) is an important drug target. Activation of NRF2 has chemopreventive effects in cancer and exerts beneficial effects in a number of diseases, including neurodegenerative diseases, inflammatory diseases, hepatosteatosis, obesity and insulin resistance. Hence, there have been great efforts to discover and characterize novel NRF2 activators. One reported NRF2 activator is the labdane diterpenoid andrographolide. In this study, we identified the mechanism through which andrographolide activates NRF2. We showed that andrographolide inhibits the function of KEAP1, a protein that together with CUL3 and RBX1 forms an E3 ubiquitin ligase that polyubiquitinates NRF2. Andrographolide partially inhibits the interaction of KEAP1 with CUL3 in a manner dependent on Cys151 in KEAP1. This suggests that andrographolide forms Michael acceptor dependent adducts with Cys151 in KEAP1 in vivo, leading to inhibition of NRF2 ubiquitination and consequently accumulation of the transcription factor. Interestingly, we also showed that at higher concentrations andrographolide increases NRF2 protein expression in a Cys151 independent, but likely KEAP1 dependent manner, possibly through modification of other Cys residues in KEAP1. In this study we also screened secondary metabolites produced by endophytes isolated from non-flowering plants for NRF2-inducing properties. One of the extracts, ORX 41, increased both NRF2 protein expression and transcriptional activity markedly. These results suggest that endophytes isolated from non-flowering or other plants may be a good source of novel NRF2 inducing compounds.

Momilactone and Related Diterpenoids as Potential Agricultural Chemicals.

Momilactones are allelochemicals in rice and moss defense. Momilactone-like compounds are therefore considered important secondary metabolites for plant defense. They may serve as promising lead compounds for crop-friendly herbicides as well as antifungal and antibacterial agents. Many of these substances possess potent cytotoxicity property against cancer cell lines as well. The present paper is the first review on these versatile molecules, focusing on the structure, biological activity, chemical synthesis, and biosynthesis of the naturally occurring momilactone-like molecules reported from 1973 to 2017.

Antiproliferative and Antimicrobial Activities of Selected Bryophytes.

One-hundred and sixty-eight aqueous and organic extracts of 42 selected bryophyte species were screened in vitro for antiproliferative activity on a panel of human gynecological cancer cell lines containing HeLa (cervix epithelial adenocarcinoma), A2780 (ovarian carcinoma), and T47D (invasive ductal breast carcinoma) cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and for antibacterial activity on 11 strains using the disc-diffusion method. A total of 99 extracts derived from 41 species exerted ≥25% inhibition of proliferation of at least one of the cancer cell lines at 10 µg/mL. In the cases of Brachythecium rutabulum, Encalypta streptocarpa, Climacium dendroides, Neckera besseri, Pleurozium schreberi, and Pseudoleskeella nervosa, more than one extract was active in the antiproliferative assay, whereas the highest activity was observed in the case of Paraleucobryum longifolium. From the tested families, Brachytheciaceae and Amblystegiaceae provided the highest number of antiproliferative extracts. Only 19 samples of 15 taxa showed moderate antibacterial activity, including the most active Plagiomnium cuspidatum, being active on 8 tested strains. Methicillin-resistant Staphylococcus aureus (MRSA) and Staphylococcus aureus were the most susceptible to the assayed species. This is the first report on the bioactivities of these 14 species.

Protection against UV-induced oxidative stress and DNA damage by Amazon moss extracts.

Amazon mosses, such as Holomitriopsis laevifolia and Leucobryum sp. are naturally exposed to high levels of solar ultraviolet (UV) radiation. Theoretically, under environmental stress conditions these mosses have developed protective chemical and metabolic strategies against UV damage, by way of biosynthesis of secondary metabolites, such as flavonoids. The present paper aimed to evaluate the free-radical scavenging activity, and the photoprotective, mutagenic and photomutagenic potencies of the methanolic (ME), aqueous (AE), hydroalcoholic (HE), ethanolic (EE) extracts of H. laevifolia and Leucobryum sp. The phenolic contents were evaluated by spectrophotometry and by High-Performance Liquid Chromatography (HPLC). The present findings showed that the AE and HE of H. laevifolia and the AE of Leucobryum sp. presented the highest phenolic contents. The HPLC analysis indicated the presence mainly of phenolic and cinnamic acids, flavonols, flavones and flavanones. The AE and EE of H. laevifolia and the AE and HE of Leucobryum sp. efficiently scavenged the 2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical. All extracts showed significant values of in vitro Sun Protection Factor alone, and HE of Leucobryum sp. showed a synergistic effect in association with benzophenone-3. None of the extracts induced mutagenicity in the auxotrophic strains for histidine of Salmonella typhimurium, and photomutagenicity of the TA102 and TA104 strains was not detected after exposure to UV-A radiation. Besides, all extracts showed photoprotective activity against UV-A radiation for the TA104 strain, including synergistic protection in association with BP-3. Thus, the constituents in H. Laevifolia and Leucobryum sp. could be good candidates for cosmetic and dermatological applications, particularly in association with synthetic UV filters, since the concentration of the filters in the final product could be reduced.

[Effects of gap and growth substrate on nitrogen and phosphorus contents of bryophytes in an alpine forest]. / 高山森林林窗和生长基质对苔藓植物氮和磷含量的影响.

Bryophyte plays an important role in nutrient enrichment and cycling in the forest ecosystems. The role of bryophyte in nitrogen (N) and phosphorus (P) cycles might be affected by forest regeneration and growth substrate. To understand the role of bryophyte in N and P cycling in the forest ecosystem, we measured the contents of N and P in the bryophytes that grew on different positions (gap center, gap edge, and closed canopy) and growth substrates (standing tree, fallen log, snag, large dead branch, stump and forest floor) in an alpine forest ecosystem. The results showed that the N content in the bryophyte on the forest floor was 3.12 mg·g-1, which was significantly lower than those on other growth substrates. Although N content in the bryophyte on the snag reached up to 17.41 mg·g-1, no significant differences of N contents in the bryophytes were observed among standing tree, fallen log, large dead branch and snag. The highest and lowest P contents was 1.09 mg·g-1 in the bryophyte on the forest floor and 0.61 mg·g-1 in the bryophytes on the snag, respectively. Furthermore, P content in the bryophyte on the forest floor was significantly higher than that on other growth substrates, but no significant differences of P contents in the bryophytes were detected among standing tree, fallen log, large dead branch and stump. The gap position significantly affected N and P contents in the bryophytes, with the N and P contents in the bryophytes on fallen log and large dead branch at gap center being significantly higher than those at the gap edge. The effects of coarse woody debris (CWD) on the N and P contents in the bryophyte depended on its types and decay classes, with their interaction having much stronger effects on N and P contents in the bryophytes. The N contents in the epiphytic bryophytes on fallen logs with V decay class were significantly higher than those with other decay classes. Similarly, the N contents in the epiphytic bryophytes on large dead branches with III decay class were significantly higher than those with other decay classes. Meanwhile, the P contents in the bryophytes on fallen logs with 2 decay class were significantly higher than those with other decay classes. Moreover, the P contents in the epiphytic bryophytes on the snags with 4 decay class were significantly higher than those with other decay classes. In conclusion, both forest gap regeneration and CWD decay process can affect the N and P contents in the bryophytes, and thereafter manipulate the nutrient cycles in the forest ecosystems.

Trace elements in Polytrichum commune and Polytrichastrum formosum from the Karkonosze Mountains (SW Poland).

The Karkonosze National Park, an unique mountainous biosphere reserve, is influenced by long-distance anthropogenic atmospheric transport of among others trace elements and additionally by local tourist centres, which may be supplementary sources of pollution. Discharged trace elements are non-degradable, and their level must be precisely monitored. Therefore, the concentrations of As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Mo, Ni, Pb, Rb and Zn in Polytrichum commune and Polytrichastrum formosum collected from the Karkonosze sites influenced only by long-range pollution and from sites in the vicinity of local tourist centres were established. P. commune and P. formosum revealed the ability to accumulate higher concentrations of trace elements while growing in locally contaminated sites in comparison with sites free from such pollution. Therefore, both species may be utilised for bioindication in the Karkonosze National Park. Elevated levels of trace elements in both species (except for Hg) in comparison with concentrations typical for mosses from unpolluted sites point to the existence of pollution of this area. P. commune was a better bioindicator of Cd, Co, Cr, Cu, Mo, Ni, Pb and Rb than P. formosum, probably because of the larger gametophytes and its morphology, which appears prone to an increased uptake of trace elements from the atmosphere.

High Content of Dicranin in Anisothecium spirale (Mitt.) Broth., a Moss from Eastern Himalayas and Its Chemotaxonomic Significance.

Chemical investigation of Anisothecium spirale (Mitt.) Broth. (Family Dicranaceae), an epiphytic moss from the Darjeeling district hill region of the Eastern Himalayas was performed for the first time. Analysis of neutral lipid and phospholipid classes as well as their respective fatty acids revealed the presence of a significantly high content of octadeca-6-yn-9,12,15-trienoic acid, an acetylenic fatty acid commonly known as dicranin. Dicranin and a less abundant acetylenic fatty acid were detected in neutral lipids. The unique nature of the fatty acid signature supports the view of considering dicranin as chemotaxonomic marker for Dicranaceae members.

Chemical composition, antimicrobial, antioxidant and anthocyanin activities of mosses (Cinclidotus fontinaloides (Hedw.) P.Beauv. and Palustriella commutata (Hedw.) Ochyra) gathered from Turkey.

Cinclidotus fontinaloides (Hedw.) P. Beauv. and Palustriella commutata (Hedw.) Ochyra were used in this study. The chemical composition of mosses in the ethanol and water extract was determined using GC-MS and FT-IR. Antioxidant activities of moss extracts were tested by total phenol and ABTS methods. Trolox equivalent value (TEAC) of mosses was calculated as 26 ± 0.32 and 10 ± 0.22 mg/g. The amounts of the flavonoid compounds were calculated as mg gallic acid. Gallic acid equivalent value of mosses was calculated as 587 ± 0.55 and 496 ± 0.22 mg/g. The extracts antimicrobial activity was tested against Escherichia coli (ATCC 8739), Bacillus subtilis (ATCC 6633), Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 10145) and Candida albicans (ATCC 10231). Significant antibacterial effect was observed for C. fontinaloides and P. commutata in ethanol extract. Total anthocyanin activities of C. fontinaloides and P. commutata were determined as 50 ± 0.17 and 144 ± 0.33 mg/L.

Chemical Composition Analysis, Antimicrobial Activity and Cytotoxicity Screening of Moss Extracts (Moss Phytochemistry).

Mosses have been neglected as a study subject for a long time. Recent research shows that mosses contain remarkable and unique substances with high biological activity. The aim of this study, accordingly, was to analyze the composition of mosses and to screen their antimicrobial and anticancer activity. The total concentration of polyphenols and carbohydrates, the amount of dry residue and the radical scavenging activity were determined for a preliminary evaluation of the chemical composition of moss extracts. In order to analyze and identify the substances present in mosses, two types of extrahents (chloroform, ethanol) and the GC/MS and LC-TOF-MS methods were used. The antimicrobial activity was tested on four bacteria strains, and the anticancer activity on six cancer cell lines. The obtained results show the presence of a high number of primary (fatty acids and amino acids), but mainly secondary metabolites in moss extracts-including, sterols, terpenoids, polyphenols and others-and a high activity with respect to the studied test organisms.

Bryophyte communities as biomonitors of environmental factors in the Goujiang karst bauxite, southwestern China.

Bauxite mining on karst results in several ecological and environmental issues including heavy metal pollution, soil erosion and the destruction of vegetation. In turn, these may affect the distribution of plant communities and endanger human health. In general, bryophytes (mosses, liverworts and hornworts) are pioneer plants, lacking roots, vascular systems and well-developed cuticles. Due to their high sensitivity to the environment, they are often used to monitor air and soil pollution. A total of 25 bryophyte taxa from 19 genera and 9 families were recorded on Goujiang karst bauxite near the city of Zunyi in the Guizhou Province of southwestern China. Eleven principal bryophyte communities were identified, most of which consisted of only one species (monospecific assemblage), although the proportion of these single-species communities differed at the six locations. The levels of heavy metals also differed in soil from the six locations: iron, 8748.9-10,023µg/g; zinc, 146.7-240.9µg/g; copper, 24.6-60.4µg/g; and nickel, 35.6-95.1µg/g. A canonical correspondence analysis (CCA) of the bryophyte communities and environmental variables revealed the effect of gradient (slope), altitude and heavy metals in the soil on the distribution of the principal bryophyte communities. More than 36% of bryophyte taxa identified reproduced asexually by gemmae, as gemmiferous bryophyte communities tolerate substrates with high levels of heavy metals more readily than non-gemmiferous communities do. The distribution of heavy metals in the soil is reflected in the distribution of the bryophyte communities. The distribution characteristics of the principal bryophyte communities and of the gemmiferous bryophyte communities are useful in monitoring heavy metal pollution in karst bauxite.

Polytrichum commune L.ex Hedw ethyl acetate extract-triggered perturbations in intracellular Ca²âº homeostasis regulates mitochondrial-dependent apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Polytrichum commune L.ex Hedw (PCLH), a moss of Bryopsida, has been used as a traditional Chinese medicine and shown to possess anticancer activities. Previous studies have indicated its anti-leukemia effect but the potential mechanisms have not been fully explained. AIM OF THE STUDY: The present study aimed to further investigate the efficacy of PCLH ethyl acetate fraction (PC-EEF) and the associated mechanisms in human leukemia cells. MATERIALS AND METHODS: Phytochemical analysis of PC-EEF was performed by spectrophotometry and HPLC. MTT analysis and trypan blue exclusion assay were adopted to examine its cytotoxicity on a panel of leukemia cells (K562, U937, HL-60 and K562/DOX cells) and non-cancerous cells (human PBMCs). Anti-proliferative effect was monitored by colony formation assay and EdU incorporation assay. Ultrastructural alterations on K562 cell membrane surface were observed by scanning electron microscopy. Changes on plasma membrane integrity, cell membrane potential, mitochondrial membrane potential and apoptosis were analyzed by flow cytometry. Fluorescence microscope was performed to assess [Ca(2+)]i level, mitochondrial injury and cytochrome c release. Apoptosis-associated protein expression was analyzed by western blot. The role of Ca(2+) in PC-EEF-induced cell death was investigated by Ca(2+) chelating reagent BAPTA-AM. RESULTS: PC-EEF possessed relatively high flavonoid content (about 88.84 ± 0.89%) and showed significant cytotoxicity to human leukemia cells. PC-EEF could cause obvious cell morphological deformation, membrane integrity loss and membrane depolarization. Meanwhile, PC-EEF treatment could dramatically potentiate perturbations in cellular Ca(2+) homeostasis. Subsequently, mitochondrial membrane potential (MMP) collapse, cytochrome c release and Bcl-2/Bax down-regulation were all observed. Consistent with these results, PC-EEF treatment resulted in significant activation of caspase 3, poly (ADP-ribose) polymerase (PARP) degradation and apoptosis. Moreover, PC-EEF-caused cytotoxicity, membrane damage, mitochondrial injury and apoptosis were remarkably reversed by BAPTA-AM. CONCLUSIONS: PC-EEF damaged the membrane system and triggered Ca(2+)-dependent mitochondrial apoptosis, which may provide some new insights into its efficacy against human leukemia cells.

Identification of sesquiterpene lactones in the Bryophyta (mosses) Takakia: Takakia species are closely related chemically to the Marchantiophyta (liverworts).

Takakia lepidozioides has been considered to be the most primitive liverwort morphologically and classified initially in the Marchantiophyta (liverworts). However, the Takakia have been reclassified from liverworts to mosses on the basis of the similarity of the male sporophyte of T. ceratophylla to that of some mosses. Reinvestigation of secondary metabolites of fresh T. lepidozioides resulted in identification of eudesmane-type sesquiterpene lactones and hydrocarbon that are significant chemical markers of several liverworts. T. lepidozioides also produces a small amount of hop-22(29)-ene, together with coumarin, which produce the characteristic odor of T. lepidozioides, and 1,4-hydroquinone; these are the predominant volatile components, whereas dihydrocoumarin, 1,4-benzoquinone, dihydrobenzofuran, α-asarone and α-tocopherol are minor components. These chemical results indicated that T. lepidozioides is more closely related to the Marchantiophyta than the Bryophyta. T. lepidozioides is morphologically similar to the liverwort Haplomitrium species. However, both species are totally different chemically.