Hippophae rhamnoides L. leaf and twig extracts as rich sources of nutrients and bioactive compounds with antioxidant activity.

Plants have served for centuries as sources of compounds useful for human health such as antioxidant, anti-diabetic and antitumor agents. They are also rich in nutrients that improve the human diet. Growing demands for these compounds make it important to seek new sources for them. Hippophae rhamnoides L. is known as a plant with health-promoting properties. In this study we investigated the chemical composition and biological properties of bioactive components of ethanol extracts from leaves and twigs of H. rhamnoides L. Chemical components such as the total content of phenolic compounds, vitamins and amino acids and the antioxidant activities of these compounds in cellular and cell-free systems were assessed. The results suggest that the studied extracts are rich in bioactive compounds with potent antioxidant properties. Cytotoxicity and hemotoxicity assays showed that the extracts had low toxicity on human cells over the range of concentrations tested. Interaction with human serum albumin was investigated and conformational changes were observed. Our results indicate that leaf and twig extracts of H. rhamnoides L. should be considered as a non-toxic source of bioactive compounds which may be of interest to the food, pharmaceutical and cosmetic industries.

Preliminary Phytochemical Analysis and Evaluation of the Biological Activity of Leonotis nepetifolia (L.) R. Br Transformed Roots Extracts Obtained through Rhizobium rhizogenes-Mediated Transformation.

According to the present knowledge, this is the first report on establishing transformed root cultures of Leonotis nepetifolia after Rhizobium rhizogenes-mediated transformation. The preliminary phytochemical analysis showed differences in the content of phenols and flavonoids in transformed and nontransformed roots. The dominant compounds in the analyzed extracts were (+)-catechin (5464 and 6808 µg/g DW), p-coumaric acid (2549 and 4907 µg/g DW), m-coumaric acid (1508 and 2048 µg/g DW) and rosmarinic acid (1844 and 2643 µg/g DW) for nontransformed (LNNR) and transformed (LNTR4) roots, respectively. Initial biological studies carried out on LNNR, and LNTR4 extracts showed a cytotoxic effect on the A549 lung, HCC1937 breast and leukemia NALM-6 cell lines, antioxidants, as well as repair and protection against DNA damage induced by H2O2 in HUVEC cells. Due to the stronger effect of the LNTR4 root extract, which can be a relatively efficient and cheap source of bioactive secondary metabolites, further biological analyses are needed to discover in detail their potentially valuable biological properties.

An Extract of Transgenic Senna obtusifolia L. Hairy Roots with Overexpression of PgSS1 Gene in Combination with Chemotherapeutic Agent Induces Apoptosis in the Leukemia Cell Line.

Many biologically-active plant-derived compounds have therapeutic or chemopreventive effects. The use of plant in vitro cultures in conjunction with modern genetic engineering techniques allows greater amounts of valuable secondary metabolites to be obtained without interfering with the natural environment. This work presents the first findings concerning the acquisition of transgenic hairy roots of Senna obtusifolia overexpressing the gene encoding squalene synthase 1 from Panax ginseng (PgSS1) (SOPSS hairy loot lines) involved in terpenoid biosynthesis. Our results confirm that one of PgSS1-overexpressing hairy root line extracts (SOPSS2) possess a high cytotoxic effect against a human acute lymphoblastic leukemia (NALM6) cell line. Further analysis of the cell cycle, the expression of apoptosis-related genes (TP53, PUMA, NOXA, BAX) and the observed decrease in mitochondrial membrane potential also confirmed that the SOPSS2 hairy root extract displays the highest effects; similar results were also obtained for this extract combined with doxorubicin. The high cytotoxic activity, observed both alone or in combination with doxorubicin, may be due to the higher content of betulinic acid as determined by HPLC analysis. Our results suggest synergistic effects of tested extract (betulinic acid in greater amount) with doxorubicin which may be used in the future to develop new effective strategies of cancer chemosensitization.

Leaf position-dependent effect of Alternaria brassicicola development on host cell death, photosynthesis and secondary metabolites in Brassica juncea.

During the first 24 hours of infection, Alternaria brassicicola developmental parameters such as conidial germination, germ tubes and appressoria formation on each of the five mature Brassica juncea leaves, correlated with a leaf position showing stronger development of the pathogen on older leaves than on young ones. As a consequence of fungal development, the black spot disease was observed during 96 hours of infection on a macroscopic scale, as well as via confocal microscopy. Degradation of the chloroplast thylakoids and plastoglobule appearance during infection, followed by the decrease in chlorophyll a fluorescence parameters i.e. maximum quantum yield of PSII (Fv /Fm ), non-photochemical quenching (NPQ) and chlorophyll a:b ratio, have been observed. Also, after an initial increase of carbohydrates (glucose, fructose and sucrose), content far below the respective control values was found. The content of secondary metabolites such as flavonoids and glucosinolates increased in a leaf position-dependent manner in infected leaves, with a lower level in older leaves than in younger ones. Although, the total phenolic compounds (TPCs) content did not differ significantly in infected leaves compared to control leaves, TPCs level in both control and infected leaves was leaf position-dependent. To the best of our knowledge, this is the first report on leaf position-dependent effect on the B. juncea biochemical response to A. brassicicola infection.

Induction of apoptosis by in vitro and in vivo plant extracts derived from Menyanthes trifoliata L. in human cancer cells.

Menyanthes trifoliata L. has been used in traditional medicine for centuries. It exists in Asia, Europe, North America and in Morocco and is exploited as a remedy for anemia and lack of appetite. This plant shows many pharmacological properties, but its most interesting one is its anti-cancer potential. The present study examines the induction of apoptosis in grade IV glioma cells after treatment with the extracts from aerial part and root of M. trifoliata plants derived from in vitro (MtAPV and MtRV, respectively) and from soil (MtAPS and MtRS, respectively) and presents the first comparison of the biological effects of four different extracts of M. trifoliata against glioblastoma cells. The root extracts of M. trifoliata plants were found to exhibit cytotoxic effects against grade IV glioma cells, but not normal human astrocytes. HPLC analysis demonstrated the presence of various polyphenolic compounds, including sinapinic acid, ferulic acid, syringic acid and vanilic acid. Higher amount of pentacyclic triterpene (betulinic acid) was also found in MtRV extract. The growth inhibition of human grade IV glioma cells mediated by MtRV extract appears to be associated with apoptosis and G2/M phase cell cycle arrest, and altered expression of the pro- and anti-apoptotic genes (Bax, Bcl-2, Cas-3 and TP53) and proteins (Bax, Bcl-2, Cas-3 and p53), as well as decreased mitochondrial membrane potential. Our results indicate that M. trifoliata gives promising results as an anti-cancer agent for human glioblastoma cell lines. However, further research is necessary in view of its therapeutic use.

Over-Expression of AtPAP1 Transcriptional Factor Enhances Phenolic Acid Production in Transgenic Roots of Leonurus sibiricus L. and Their Biological Activities.

This study examines the production of five phenolic acids (chlorogenic acid, neochlorogenic acid, ferulic acid, caffeic acid and p-coumaric acid) following over-expression of AtPAP1 transcription factor by four transgenic root clones of Leonurus sibiricus after Agrobacterium rhizogenes transformation. The AtPAP1 expression level was estimated by quantitative real-time PCR. High levels of phenolic acids were found in the transgenic roots of L. sibiricus and were determined by high-performance liquid chromatography-mass spectrometry analysis. Additionally, transgenic roots showed antimicrobial potential and cytotoxic activity on glioma cells in IV grade. Our results suggest that L. sibiricus transformed roots with AtPAP1 gene over-expression may represent a potential source of phenolic acids.

Transformed Root Extract of Leonurus sibiricus Induces Apoptosis through Intrinsic and Extrinsic Pathways in Various Grades of Human Glioma Cells.

This study determines the influence of transformed root (TR) extract of Leonurus sibiricus L. on various grades (I-III) of human glioma cells derived from patients. This plant occurs in southern Asia and Siberia and is widely used as a medicinal plant with various biological activities. Chromatographic profile of TR extract have revealed the presence of various polyphenolic compounds (4-hydroxybenzoic acid, gentisic acid, vanilic acid, 1,3-dicaffeoylquinic acid, α-resorcylic acid). We found TR root extract to have antiproliferative activity on glioma cells after 24 h of treatment. TR root extract induces apoptosis on various grades (I-III) of human glioma cells by the generation of reactive oxygen species (ROS) along with concurrent loss of mitochondrial membrane potential, enhanced S and G2/M phases of the cell cycle, and altered mRNA levels of Bax, Bcl-2, p53, Cas-3, Cas-8 and Cas-9 factors involved in apoptosis. This work for the first time demonstrate that TR extract from L. sibiricus root has the potential to activate apoptosis in grade I-III human glioma cells through the intrinsic and extrinsic pathways.

The Effect of Leonurus sibiricus Plant Extracts on Stimulating Repair and Protective Activity against Oxidative DNA Damage in CHO Cells and Content of Phenolic Compounds.

Leonurus sibiricus L. has been used as a traditional and medicinal herb for many years in Asia and Europe. This species is known to have antibacterial, anti-inflammatory, and antioxidant activity and has demonstrated a reduction of intracellular reactive oxygen species. All tested extracts of L. sibiricus showed protective and DNA repair stimulating effects in Chinese hamster ovary (CHO) cells exposed to H2O2. Preincubation of the CHO cells with 0.5 mg/mL of plant extracts showed increased expression level of antioxidant genes (SOD2, CAT, and GPx). LC-MS/MS and HPLC analyses revealed the presence of nine phenolic compounds in L. sibiricus plant extracts: catechin, verbascoside, two flavonoids (quercetin and rutin), and five phenolic acids (4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, and ferulic acid). The roots and aerial parts of in vitro L. sibiricus plant extracts, which had the strongest antioxidant properties, may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, as well as protecting DNA via enhanced activation of the antioxidant genes (SOD2, CAT, and GPx) regulating intracellular antioxidant capacity. The content of phenolic compounds in in vitro raised plants was greater than the levels found in plants propagated from seeds.

A preliminary study of apoptosis induction in glioma cells via alteration of the Bax/Bcl-2-p53 axis by transformed and non-transformed root extracts of Leonurus sibiricus L.

Leonurus sibiricus L. is a traditional medicinal plant which occurs in southern Siberia, China, Korea, Japan, and Vietnam. The plant shows several pharmacological effects, but the most interesting is its anti-cancer activity. The aim of our study was to examine the induction of apoptosis in malignant glioma cells, the most aggressive primary brain tumors of the central nervous system, following treatment with transformed root (TR) or non-transformed root (NR) L. sibiricus extracts. Both the NR and TR extracts were found to have cytotoxic activity in the glioma primary cells. The human glioblastoma cell lines obtained from patients were confirmed to be tumorogenic by the following three markers: D10S1709, D10S1172, and D22S283. HPLC and MS analysis revealed the presence of polyphenolic compounds (chlorogenic acid, ferulic acid, caffeic acid, p-coumaric acid, ellagic acid, and verbascoside) in both sets of root extracts. In summary, our findings demonstrate that treatment of the glioma cells with NR and TR extracts resulted (a) in significant cell growth inhibition, (b) S- and G2/M-phase cell cycle arrest, and (c) apoptosis in a dose-dependent fashion by changing Bax/Bcl-2 ratio (about 4-fold increase) and p53 (5-fold increase) activation. These findings indicate that NR and TR extracts exhibit anti-cancer activity through the regulation of genes involved in apoptosis. This is the first report to demonstrate the cytotoxic effect of polyphenolic extracts from L. sibiricus roots against glioma cells, but further studies are required to understand the complete mechanism of its apoptosic activity.

Salicylic acid and cysteine contribute to arbutin-induced alleviation of angular leaf spot disease development in cucumber.

Arbutin induced suppression of angular leaf spot disease in cucumber resulting from lower populations of Pseudomonas syringae pv lachrymans in the infected tissues. This study provides insight into mechanisms that may potentially account for this effect. In the absence of the pathogen, exogenous arbutin-induced expression of PR1, the marker of salicylic acid signaling, increased the content of salicylic acid and modulated the cysteine pool. This suggested that arbutin promoted cucumber plants to a "primed" state. When challenged with the pathogen, the arbutin-treated plants showed strongly reduced infection symptoms 7 days after inoculation. At this time point, they were characterized by higher contents of free and protein-bound cysteine due to higher cysteine biosynthetic capacity related to increased activities of serine acetyltransferase and cysteine synthase when compared with plants infected without arbutin treatment. Moreover, in the arbutin-treated and infected plants the contents of free salicylic acid and its conjugates were also increased, partly owing to its biosynthesis via the phenylpropanoid pathway. We suggest that arbutin-induced abrogation of angular leaf spot disease in cucumber could be mediated by salicylic acid and cysteine-based signaling.

A dissection of the effects of ethylene, H2O2 and high irradiance on antioxidants and several genes associated with stress and senescence in tobacco leaves.

Ethylene and hydrogen peroxide are involved in the modulation of stress responses in plants, but their interrelation is not well understood. This work was designed to find differences between the actions of ethylene and H2O2 on antioxidants and senescence markers. Leaves of Nicotiana tabacum were sprayed with H2O2 or with ethephon (precursor of ethylene). To find the possible modulation of responses to acute abiotic stress, ethephon- and H2O2-sprayed leaves were further subjected to high irradiance (HL). The application of H2O2 strongly stimulated ethylene synthesis (ACC). Ethylene and H2O2, as single factors, stimulated the trolox equivalent antioxidant capacity (TEAC) and the activity of catalase (CAT), in contrast to HL alone (stimulation of nonspecific peroxidases and the total glutathione pool). However, after combined treatments (ethylene+HL and H2O2+HL), the stimulatory action of H2O2 was related to TEAC and CAT activity, while the application of ethylene stimulated the total glutathione pool. Hydrogen peroxide enhanced the expression of the three CAT genes (Cat1, Cat2 and Cat3), in contrast to ethylene (Cat2 and Cat3) and HL (Cat1). In regard to the markers of senescence and pathogenesis the most pronounced difference between the actions of ethylene and H2O2, as single factors, was related to NPR1, whereas when leaf spraying was combined with HL, differences were found at WRKY53 and PR1a. HL reversed the stimulatory effects of H2O2/ethylene-driven enhancements of the expression of several genes (Cat1, Cat2, NPR1, WRKY53). These results show that multiple stressors, as usually encountered by plants in nature, may largely change those expression patterns of genes determined in a single factor analysis. Moreover, the actions of HL (often considered the internal H2O2 trigger) and of exogenous H2O2 on gene expression are clearly different.

Involvement of salicylic acid, glutathione and protein S-thiolation in plant cell death-mediated defence response of Mesembryanthemum crystallinum against Botrytis cinerea.

The response of Mesembryanthemum crystallinum plants performing C3 photosynthesis and crassulacean acid metabolism (CAM) to the non-host necrotrophic pathogen Botrytis cinerea was analyzed at the local and systemic levels. The induction of programmed cell death, lignin and callose deposition, changes in salicylic acid, glutathione and cysteinylglycine pools as well as the content of thiolated proteins were studied. The infected C3 and CAM plants exhibited hypersensitive-like defence response, however fluorescence staining with acridine orange and ethidium bromide revealed programmed cell death events in C3 plants only. The local immune response was not related to callose and lignin deposition. In the infected plants, salicylic acid, glutathione and cysteinylglycine, the first product of glutathione catabolism, as well as protein S-thiolation, predominantly S-glutathionylation, contributed to local defence at sites of inoculation. They (except protein thiolation) were also active in the establishment of systemic acclimation response monitored in the non-treated upper leaves. The extent to which they were involved in the local and systemic responses induced by B. cinerea differed in C3 and CAM plants. The accumulation of free salicylic acid, both in treated and upper leaves of the infected plants, was much more pronounced in CAM plants. The results have been discussed with respect to redox regulations in defence against necrotrophic pathogens and to stress acclimation.

Transformation of Nasturtium officinale, Barbarea verna and Arabis caucasica for hairy roots and glucosinolate-myrosinase system production.

Hairy roots of Nasturtium officinale, Barbarea verna and Arabis caucasica with active glucosinolate-myrosinase system were obtained after transformation with Agrobacterium rhizogenes. Hairy roots of N. officinale produced phenylalanine-derived gluconasturtiin and glucotropaeolin (max. 24 and 7 mg g(-1) DW). B. verna and A. caucasica hairy roots produced gluconasturtiin (max. 41 mg g(-1) DW) and methionine-derived glucoiberverin (max. 32 mg g(-1) DW), respectively. Treatment of the roots with amino acid precursors of glucosinolate or/and cysteine biosynthesis increased levels of glucosinolate production, combinations of phenylalanine with cysteine (for gluconasturtiin and glucotropaeolin) and methionine with o-acetylserine (for glucoiberverin) were the most effective.