Dynamics of Polyphenol Biosynthesis by Calli Cultures, Suspension Cultures and Wild Specimens of the Medicinal Plant Ligaria cuneifolia (Ruiz & Pav.) Tiegh. (Loranthaceae). Analysis of Their Biological Activity.

Ligaria cuneifolia (R. et P.) Tiegh. (Loranthaceae) is a South American hemiparasitic species with antioxidant, antitumoral, antimicrobial, and antilipidemic activities attributed to its polyphenolic content. We studied the polyphenolic pattern of L. cuneifolia during different phenological stages: flowering, fruiting, and post-fruiting. The highest total phenolic content was found in stems at post-fruiting (214 ± 12.1 mg gallic acid eq·g-1 DW) and fruiting (209 ± 13.7 mg gallic acid eq·g-1 DW), followed by post-fruiting leaves (207 ± 17.5 mg gallic acid eq·g-1 DW). Flavonoids accumulated at higher levels in leaves and hydroxycinnamic acids in leaves at flowering and post-fruiting. The polyphenolic pattern was similar between organs from wild plants and in vitro cultures, although at a significantly lower level in the latter ones. The performance of calli growing under a 16 h photoperiod in a modified White medium with 1-naphthalene acetic acid (2.50 µM) and Kinetin (9.20 µM) was better than in the dark. When calli grew in media only with auxins (IAA, NAA, and 2,4-D, all at 2.50 µM concentration), its growth and polyphenolic content improved. Cell suspensions with 2.50 µM NAA and 9.20 µM KIN grew slowly and produced very small amounts of polyphenols. As for the antioxidant activity, it was detected in all samples (approximately 1000 µmol trolox eq·g-1 DW) except fruits, where a lower value was found (328 µmol trolox eq·g-1 DW). In vitro cultures have the lowest antioxidant activity when compared to methanolic extracts from organs of wild specimens. Finally, antimutagenic or mutagenic activity in wild plants and in vitro culture extracts was not detected by the Ames test.

In vitro inhibitory effect of Hydrocotyle bonariensis Lam. extracts over Chlamydia trachomatis and Chlamydia pneumoniae on different stages of the chlamydial life cycle.

Chlamydial infections in humans are widely distributed and are responsible for a variety of acute and chronic diseases. Both Chlamydia trachomatis and Chlamydia pneumoniae can lead to chronic conditions that have been linked to complications and sequelae. This study aimed to develop a culture method in order to detect in vitro antichlamydial activity of different extracts obtained from native Argentinian plants used as antimicrobials in local ethnomedicine and to evaluate their inhibitory activity over Chlamydia trachomatis and Chlamydia pneumoniae growth. The inhibitory activity over different stages of the chlamydial life cycle on cell culture was assessed: the entry, the inclusion developing after entry, and the exponential growth stage. Also, the capability of rendering the cell refractory to chlamydial infection by pre-incubation with the extracts was assayed. Inhibitory activity of water-based and organic-based extracts obtained from Hydrocotyle bonariensis Lam. (Araliaceae), Lithraea molleoides (Vell.) Engl. (Anacardiaceae) and Hybanthus parviflorus (Mutis ex L.f.) Baill. (Violaceae) were tested against five strains of Chlamydia trachomatis (L2/434/BU and four clinical isolates form both neonatal conjunctivitis and adult genital infections, genotypes D, E, and K) and against Chlamydia pneumoniae AR39. The Hydrocotyle bonariensis dichloromethane extract showed a broad inhibitory activity over the exponential growth stage of Chlamydia trachomatis and Chlamydia pneumoniae independently from the chlamydial strain and the cell line. These results suggest a high inhibitory potential on both Chlamydiae species. In order to characterize the Hydrocotyle bonariensis dichloromethane active extract, an 1H-NMR was performed. The 1H-NMR characterization showed a spectrum with characteristic signals of the fatty acid moiety of lipids or cerebrosides, volatile phenolics, phytosterols, methyl triterpenes signals, and glucose moiety of the cerebrosides.

Use of the Distress Thermometer in Cancer Survivors: Convergent Validity and Diagnostic Accuracy in a Spanish Sample.

OBJECTIVES: To explore the performance of the National Comprehensive Cancer Network Distress Thermometer (DT) as a distress screening tool in cancer survivors. SAMPLE & SETTING: 236 Spanish adult-onset cancer survivors who visited the Fundación Instituto Valenciano de Oncología in Valencia, Spain, for follow-up appointments. METHODS & VARIABLES: Survivors completed the DT and the Brief Symptom Inventory 18 (BSI-18), which has established a cutoff score for identifying clinically significant distress. RESULTS: Receiver operating characteristic curve analysis of the DT scores relative to the BSI-18 cutoff score showed good overall accuracy. For a score of 5 or greater, sensitivity, specificity, positive predictive value, negative predictive value, and clinical utility indexes indicated that the DT appeared to be satisfactory for screening but had restricted use for case finding. IMPLICATIONS FOR NURSING: Screening for and responding to distress is considered an important part of nursing practice. The DT is suitable for use as a first-stage, quick-detection instrument in a two-step screening process to rule out noncases among Spanish post-treatment cancer survivors.

Development of a novel dual CD-MEKC system for the systematic flavonoid fingerprinting of Ligaria cuneifolia (R. et P.) Tiegh.-Loranthaceae-extracts.

The present work deals with the development and validation of a novel dual CD-MEKC system for the systematic flavonoid fingerprinting of Ligaria cuneifolia (R. et P.) Tiegh.-Loranthaceae-extracts. The BGE consisted of 20 mM pH 8.3 borate buffer, 50 mM SDS, a dual CD system based on the combination of 5 mM ß-CD and 2% w/v S-ß-CD, and 10% v/v methanol. The proposed method has been successfully applied to the comparative analysis of extracts from aerial parts and different hosts, geographical areas, and extraction procedures in order to establish the flavonoid fingerprint of L. cuneifolia. The method was validated according to international guidelines. LOD and LOQ, intra and interday precision, and linearity were determined for catechin, epicatechin, procyanidin B2, rutin, quercetin-3-O-glucoside, quercetin-3-O-xyloside, quercetin-3-O-rhamnoside, quercetin-3-O-arabinofuranoside, quercetin-3-O-arabinopyranoside, and quercetin. The CD-MEKC methodology emerges as a suitable alternative to the traditional HPLC for quality control, fingerprinting, and standardization of L. cuneifolia extracts from different sources.

Impaired GABAB receptor signaling dramatically up-regulates Kiss1 expression selectively in nonhypothalamic brain regions of adult but not prepubertal mice.

Kisspeptin, encoded by Kiss1, stimulates reproduction and is synthesized in the hypothalamic anteroventral periventricular and arcuate nuclei. Kiss1 is also expressed at lower levels in the medial amygdala (MeA) and bed nucleus of the stria terminalis (BNST), but the regulation and function of Kiss1 there is poorly understood. γ-Aminobutyric acid (GABA) also regulates reproduction, and female GABAB1 receptor knockout (KO) mice have compromised fertility. However, the interaction between GABAB receptors and Kiss1 neurons is unknown. Here, using double-label in situ hybridization, we first demonstrated that a majority of hypothalamic Kiss1 neurons coexpress GABAB1 subunit, a finding also confirmed for most MeA Kiss1 neurons. Yet, despite known reproductive impairments in GABAB1KO mice, Kiss1 expression in the anteroventral periventricular and arcuate nuclei, assessed by both in situ hybridization and real-time PCR, was identical between adult wild-type and GABAB1KO mice. Surprisingly, however, Kiss1 levels in the BNST and MeA, as well as the lateral septum (a region normally lacking Kiss1 expression), were dramatically increased in both GABAB1KO males and females. The increased Kiss1 levels in extrahypothalamic regions were not caused by elevated sex steroids (which can increase Kiss1 expression), because circulating estradiol and testosterone were equivalent between genotypes. Interestingly, increased Kiss1 expression was not detected in the MeA or BNST in prepubertal KO mice of either sex, indicating that the enhancements in extrahypothalamic Kiss1 levels initiate during/after puberty. These findings suggest that GABAB signaling may normally directly or indirectly inhibit Kiss1 expression, particularly in the BNST and MeA, and highlight the importance of studying kisspeptin populations outside the hypothalamus.

Antinociceptive activity of aqueous extract and isolated compounds of Lithrea molleoides.

ETHNOPHARMACOLOGICAL RELEVANCE: Lithrea molleoides (Vell.) Engl. (Anacardiaceae) is a medicinal plant commonly used in traditional medicine in South America. AIM OF THE STUDY: In the present study, the in vivo antinociceptive effect of L. molleoides' aqueous extract and its isolated compounds has been investigated. MATERIALS AND METHODS: Antinociceptive activity was evaluated through writhing, formalin and hot plate tests in mice. The phytochemical analysis was performed. RESULTS: The extract produced significant inhibition on nociception induced by acetic acid (ED50: 8.7 mg/kg, i.p.) and formalin (ED50: 7.7 mg/kg, i.p.) administered intraperitoneally and also orally. Yohimbine diminished the activity of the extract in the acetic acid test meanwhile haloperidol enhanced its effect. Two majority compounds, shikimic and vanillic acid were active in chemical nociceptive models used in this work, producing the highest inhibition of the writhing response at a dose of 30 mg/kg i.p. (55.4% and 57.1%, respectively) meanwhile at 100 mg/kg p.o. produced a slight response (23.3% and 23.9%, respectively). CONCLUSIONS: These results suggest that L. molleoides' aqueous extract produced antinociception possibly related to the presence of shikimic and vanillic acid. The adrenergic and dopaminergic systems seem to be involved in the mechanism of antinociception of the extract.

Potato CONSTANS is involved in photoperiodic tuberization in a graft-transmissible manner.

CONSTANS (CO) is involved in the photoperiodic control of plant developmental processes, including flowering in several species and seasonal growth cessation and bud set in trees. It has been proposed that CO could also affect the day-length regulation of tuber induction in Solanum tuberosum (potato), a plant of great agricultural relevance. To address this question, we examined the role of CO in potato. A potato CO-like gene, StCO, was identified and found to be highly similar to a previously reported potato gene of unknown function. Potato plants overexpressing StCO tuberized later than wild-type plants under a weakly inductive photoperiod. StCO silencing promoted tuberization under both repressive and weakly inductive photoperiods, but did not have any effect under strongly inductive short days, demonstrating that StCO represses tuberization in a photoperiod-dependent manner. The effect of StCO on tuber induction was transmitted through grafts. In addition, StCO affected the mRNA levels of StBEL5 - a tuberization promoter, the mRNA of which moves long distances in potato plants - and StFT/StSP6A, a protein highly similar to FLOWERING LOCUS T (FT), which is a key component of systemic flowering signals in other species. We also found that StFT/StSP6A transcript levels correlate with the induction of tuber formation in wild-type plants. These results show that StCO plays an important role in photoperiodic tuberization and, together with the recent demonstration that StFT/StSP6A promotes tuberization, indicate that the CO/FT module participates in controlling this process. Moreover, they support the notion that StCO is involved in the expression of long-distance regulatory signals in potato, as CO does in other species.

Comparative antioxidant activity of an extract of Lithraea molleoides and an isolated 5-alkyl resorcinol derivative. Effects on the proliferation of normal and tumoral lymphocytes.

Lithraea molleoides (Vell.) Engl. (Anacardiaceae), is known in South America for its medicinal properties: antiarthritic, haemostatic, diuretic, tonic and useful for the treatment of respiratory diseases. Previously the isolation of a new cytotoxic 5-alkyl resorcinol derivative, 1,3-dihydroxy-5-(tridec-4',7'-dienyl) benzene, from a dichloromethane extract (DM) was reported. The aim of this work was to determine and to compare the antioxidant activity of DM and the isolated compound, 1,3-dihydroxy-5-(tridec-4',7'-dienyl) benzene. Moreover, the activity of both on the proliferation of tumoral and normal lymphocytes was determined. The compound was isolated and quantified by HPLC. The compound represented 0.036% of DM. The extract and the compound exerted antioxidant activity by DPPH and FTC methods. On tumoral cells, both exerted antiproliferative action but the compound was more active (EC(50) : 17.4 ± 1 µg/mL). On normal lymphocytes, both exerted a stimulatory effect on cell proliferation inversely related to concentration, the extract was more active than the compound (maximum effect: 300 ± 20% of stimulation). Most of the effects observed with the extract were independent of the isolated compound. DM could be an important source of antioxidant and immunomodulatory compounds to be studied on cancer diseases.

Graft-transmissible induction of potato tuberization by the microRNA miR172.

The photoreceptor phytochrome B (PHYB) and the homeodomain protein BEL5 are involved in the response of potato tuber induction to the photoperiod. However, whether they act in the same tuberization pathway is unknown. Here we show the effect of a microRNA, miR172, on this developmental event. miR172 levels are higher under tuber-inducing short days than under non-inductive long days and are upregulated in stolons at the onset of tuberization. Overexpression of this microRNA in potato promotes flowering, accelerates tuberization under moderately inductive photoperiods and triggers tuber formation under long days. In plants with a reduced abundance of PHYB, which tuberize under long days, both BEL5 mRNA and miR172 levels are reduced in leaves and increased in stolons. This, together with the presence of miR172 in vascular bundles and the graft transmissibility of its effect on tuberization, indicates that either miR172 might be mobile or it regulates long-distance signals to induce tuberization. Consistent with this, plants overexpressing miR172 show increased levels of BEL5 mRNA, which has been reported to be transmissible through grafts. Furthermore, we identify an APETALA2-like mRNA containing a miR172 binding site, which is downregulated in plants overexpressing miR172 and plants in which PHYB is silenced. Altogether, our results suggest that miR172 probably acts downstream of the tuberization repressor PHYB and upstream of the tuberization promoter BEL5 and allow us to propose a model for the control of tuberization by PHYB, miR172 and BEL5.

Induction of apoptosis on human hepatocarcinoma cell lines by an alkyl resorcinol isolated from Lithraea molleoides.

AIM: To study the mechanism of cytotoxicity of a new active 5-alkyl resorcinol [1, 3-dihydroxy-5- (tridec-4', 7'-dienyl) benzene] isolated from Lithraea molleoides leaves on liver tumor cells. METHODS: Human hepatocarcinoma cell lines (HepG2 and Hep3B) in culture were treated with inhibitory concentrations, 50% of the compound, for 24 h. The induction of apoptosis was detected in treated cells by analysis of DNA fragmentation, DNA content, and acridine orange and propidium iodide staining. RESULTS: After 24 h of 5-alkyl resorcinol treatment, both cell lines showed: (1) the typical morphological alterations of apoptosis; (2) DNA fragmentation, detected by laddering and appearance of a subG0 population by flow cytometry; and (3) condensed and fragmented nuclei by acridine orange-propidium iodide staining. CONCLUSION: Based on the results, this compound exerts its cytotoxic effect in both hepatocellular cell lines through apoptotic cell death. For Hep3B, cells with mutated p53 and Fas, apoptosis would proceed by p53- or Fas-independent pathways.