First report on phytochemical investigation, antioxidant and antidiabetic activities of Helianthemum getulum.

A new flavonoid, 5,7,2',4',5'-pentahydroxyflavone 3-O-ß-D-galactopyranoside (12) and twelve known derivatives: an aryltetralin-lignan (3), seven flavonoids (4-5, 7-10, 13) and four phenolic acids (1-2, 6, 11) have been isolated from the aerial parts of Helianthemum getulum Pomel. (Cistaceae family) an endemic species to the septentrional Sahara that is being studied for the first time. Structure elucidation of the isolated compounds was established by means of spectroscopic methods especially NMR and Mass Spectrometry. In vitro antioxidant (DPPH, ABTS, GOR and CUPRAC assays) and antidiabetic (micro-dilution method) activities of the crude extract, fractions and isolated compounds were performed. The new flavonol (12) and Compounds (2, 3, 7, 9) were found to be the most active, some of them exhibiting better activity than the antioxidant standards. Compounds 7, 9 and 3 showed higher α-glucosidase inhibitory activity compared to standard acarbose (IC50= 2.70 ± 0.03 µM, 3.09 ± 0.03 µM, 37.28 ± 1.20 µM and 275.43 ± 1.59 µM, respectively).

Screening of Plant Pollen Sources, Polyphenolic Compounds, Fatty Acids and Antioxidant/Antimicrobial Activity from Bee Pollen.

In this study, the botanical origin, total flavonoid and phenolic content, antioxidant activity, phenolic profile and fatty acid composition of mixed bee pollen loads collected in Bayburt, Turkey, were determined. In addition to these assays, antibacterial activity of bee-collected pollen extract (BCPE) against a variety of food-borne pathogenic bacteria was determined in vitro. Pollen loads were classified into five botanical families based on their color: Asteraceae, Fabaceae, Campanulaceae, Cistaceae and Rosaceae. Total flavonoid, total phenolic, CUPRAC and CERAC concentrations were 173.52 mg GAE/g, 79.21 mg QE/g, 85.59 mg Trolox/g and 118.13 mg Trolox/g, respectively. Twenty-three phenolic compounds were scanned in bee pollen extract by LC-MS/MS, with rutin being the most abundant. Cis-4,7,10,13,16,19 docosahexaenoic acid was the predominant fatty acid, followed by cis-11-eicosenoic acid, palmitic acid, and alfa linolenic acid. In addition, the agar well diffusion (AWD) and micro-broth dilution methods were used to determine of the antibacterial activity of the BCPE sample. MIC values were observed to vary between 2.5-5 mg/mL for Gram-positive bacteria and 5-10 mg/mL for Gram-negative bacteria. These findings indicate that bee pollen could be a potential source of antioxidants and antimicrobials.

Results of a randomized controlled phase III trial: efficacy of polyphenol-containing cystus® tea mouthwash solution for the reduction of mucositis in head and neck cancer patients undergoing external beam radiotherapy.

PURPOSE: To determine the effect of Cystus® tea (Naturprodukte Dr. Pandalis GmbH & Co. KG) as mouthwash compared to sage tea on oral mucositis in patients undergoing radio(chemo)therapy for head and neck cancer. METHODS: In this randomized, prospective phase III study, 60 head and neck cancer patients with primary or postoperative radio(chemo)therapy were included between 04/2012 and 06/2014. They received either sage or Cystus® tea for daily mouthwash under therapy. Mucositis was scored twice a week following the Radiation Therapy Oncology Group and the European Organization for Research and Treatment Cancer (RTOG/EORTC) scoring system. Dental parameters were also recorded. Statistical evaluation of the primary endpoint was performed using t­test and log rank test. RESULTS: Data from 57 patients could be evaluated. Patient characteristics showed no significant difference between the two groups (n = 27 sage; n = 30 Cystus®). A total of 55 patients received the prescribed dose (60-66 Gy postoperative; 70-76.8 Gy primary). Mucositis grade 3 was observed in 23 patients (n = 11 sage; n = 12 Cystus®) and occurred between day 16 and 50 after start of therapy. There was no significant difference between the two groups in latency (p = 0.75) and frequency (p = 0.85) of the occurrence of mucositis grade 3. The self-assessment of the oral mucosa and the tolerability of the tea also showed no significant differences. Occurrence of dental pathologies appeared to increase over time after radiotherapy. CONCLUSION: Cystus® and sage tea have a similar effect on the occurrence of radiation-induced mucositis regarding latency and incidence. Cystus® tea mouthwash solution is tolerated well and can be applied in addition to intensive oral care and hygiene along with the application of fluorides.

Halimium halimifolium: From the Chemical and Functional Characterization to a Nutraceutical Ingredient Design.

Halimium halimifolium (Hh) is a shrub used in Algerian folk medicine to treat gastrointestinal pain. An UHPLC-PDA-ESI/MSn method was developed to identify the metabolic profile of the traditionally used infusion (Hh-A) from the aerial parts. The structures of flavanols were confirmed by NMR analysis after the isolation procedure from a hydrohalcolic extract (Hh-B) that also allowed for the identification of phenolic acids, an aryl butanol glucoside, and different derivatives of quercetin, myricetin, and kaempferol. Tiliroside isomers were the chemical markers of Hh-A and Hh-B (54.33 and 36.00 mg/g, respectively). Hh-A showed a significant scavenging activity both against the radicals 1,1-diphenyl-2-picrylhydrazyl and 2,2'-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (EC50 = 10.49 µg/mL and TEAC value = 1.98 mM Trolox/mg infusion) and the lipopolysaccharide-induced reactive oxygen species release in A375 and HeLa cells. Moreover, the antihyperglycemic properties, by inhibiting the α-amylase and α-glucosidase enzymes (IC50 = 0.82 mg/mL and 25.01 µg/mL, respectively), were demonstrated. To upgrade the therapeutic effect, a microencapsulation process is proposed as a strategy to optimize stability, handling, and delivery of bioactive components, avoiding the degradation and loss of the biological efficacy after oral intake. Hh-loaded microparticles were designed using cellulose acetate phthalate as the enteric coating material and spray drying as a production process. The results showed a satisfactory process yield (67.9%), encapsulation efficiency (96.7%), and micrometric characteristics of microparticles (laser-scattering, fluorescent, and scanning electron microscopy). In vitro dissolution studies (USPII-pH change method) showed that Hh-loaded microparticles are able to prevent the release and degradation of the bioactive components in the gastric tract, releasing them into the intestinal environment.

Real-time carbon allocation into biogenic volatile organic compounds (BVOCs) and respiratory carbon dioxide (CO2) traced by PTR-TOF-MS, 13CO2 laser spectroscopy and 13C-pyruvate labelling.

Our understanding of biogenic volatile organic compound (BVOC) emissions improved substantially during the last years. Nevertheless, there are still large uncertainties of processes controlling plant carbon investment into BVOCs, of some biosynthetic pathways and their linkage to CO2 decarboxylation at central metabolic branching points. To shed more light on carbon partitioning during BVOC biosynthesis, we used an innovative approach combining δ13CO2 laser spectroscopy, high-sensitivity proton-transfer-reaction time-of-flight mass spectrometry and a multiple branch enclosure system in combination with position-specific 13C-metabolite labelling. Feeding experiments with position-specific 13C-labelled pyruvate, a central metabolite of BVOC synthesis, enabled online detection of carbon partitioning into 13C-BVOCs and respiratory 13CO2. Measurements of trace gas emissions of the Mediterranean shrub Halimium halimifolium revealed a broad range of emitted BVOCs. In general, [2-13C]-PYR was rapidly incorporated into emitted acetic acid, methyl acetate, toluene, cresol, trimethylbenzene, ethylphenol, monoterpenes and sesquiterpenes, indicating de novo BVOC biosynthesis of these compounds. In contrast, [1-13C]-pyruvate labelling substantially increased 13CO2 emissions in the light indicating C1-decarboxylation. Similar labelling patterns of methyl acetate and acetic acid suggested tightly connected biosynthetic pathways and, furthermore, there were hints of possible biosynthesis of benzenoids via the MEP-pathway. Overall, substantial CO2 emission from metabolic branching points during de novo BVOC biosynthesis indicated that decarboxylation of [1-13C]-pyruvate, as a non-mitochondrial source of CO2, seems to contribute considerably to daytime CO2 release from leaves. Our approach, combining synchronised BVOC and CO2 measurements in combination with position-specific labelling opens the door for real-time analysis tracing metabolic pathways and carbon turnover under different environmental conditions, which may enhance our understanding of regulatory mechanisms in plant carbon metabolism and BVOC biosynthesis.

Ellagitannin-rich bioactive extracts of Tuberaria lignosa: insights into the radiation-induced effects in the recovery of high added-value compounds.

Ellagitannins are polyphenols responsible for a number of bioactivities and health-promoting effects. These industrially important molecules can be affected by post-harvest treatments and recovery processes, but little is known about the irradiation-induced effects on their integrity, bioactivity and extractability. Herein, the impact of gamma radiation on the production of ellagitannin-rich extracts was investigated using Tuberaria lignosa as a case study. These effects were compared with those induced in flavonoids and organic acids. The extracts were particularly rich in hydrophilic antioxidants (measured by in vitro assays). The recovery of different phytochemicals was favoured by longer extraction times. Ellagitannins (mainly punicalagin derivatives) were extracted better from samples irradiated at 5 kGy and were not significantly affected by the 10 kGy dose. However, the total contents of flavonoids and organic acids were decreased by the consequent increase in irradiation dose. Therefore, this study supports the use of gamma radiation for processing T. lignosa, aiming to obtain ellagitannin-rich bioactive extracts.

Phytochemical Profile and Biological Activities of Helianthemum canum l. baumg. from Turkey.

In the current study, antioxidant, antibacterial activities, and the phenolic compositions of extracts from Helianthemum canum L. Baumg. (Apiaceae) aerial parts were investigated for the first time. The H. canum was extracted with 70% methanol (HCMeOH) and water (HCW). Both extracts were determined by total phenolic contents (3 mg/ml), flavonoids (1.5 mg/ml), flavonols (1.5 mg/ml), qualitative-quantitative compositions, iron (II) chelation activities (0.1 - 5 mg/ml), free radical scavenging activities (DPPH• : 0.01 - 0.6 mg/ml and ABTS+• : 0.125 - 0.5 mg/ml) and the effect upon inhibition of ß-carotene/linoleic acid co-oxidation (1 mg/ml). The peroxidation level was also determined using the thiobarbituric acid method (0.01 - 1.5 mg/ml). The results of the activity tests given as IC50 values were estimated from non-linear algorithm and compared with standards. Antibacterial activities of extracts and standards were evaluated against Gram-negative and -positive ten standard strains using disc diffusion and broth microdilution methods. The MIC results (312.5 - 2500 µg/ml) against tested microorganisms varied from 625 to 2500 µg/ml. In HPLC analysis, 3,5-dihydroxybenzoic acid was found as the main substance in both extracts. These results showed that HCMeOH was richer in phenolic compounds (284.13 ± 0.30 mg GAE/g extract) from HCW (244.55 ± 0.35 mg GAE/g extract). In conclusion, H. canum extracts showed in vitro antibacterial and antioxidant activities.

On-Line Screening, Isolation and Identification of Antioxidant Compounds of Helianthemum ruficomum.

Many Helianthemum species (Cistaceae) are recognized for their various medicinal virtues. Helianthemum ruficomum is an endemic species to the septentrional Sahara on which no report is available so far. The purpose of this work was to investigate the chemical composition and the radical scavenging capacity of this species and its isolated components. Collected from Mougheul (south-west of Algeria), the aerial parts were macerated with 80% EtOH/H2O, after evaporation, the remaining extract was diluted with H2O and extracted with petroleum ether, chloroform, ethyl acetate and n-butanol. EtOAc and n-BuOH extracts were evaluated for their free radical scavenging capacity by on-line HPLC-ABTS•+ assay. The obtained data which were confirmed by TEAC and ORAC assays, allowed guiding the fractionation of these extracts by CC, TLC and reverse phase HPLC. Among the components, 14 were isolated and identified by spectroscopic analyses: protocatechuic acid (1), trans-tiliroside (2), cis-tiliroside (3), astragalin (4), picein (7), vanillic acid 4-O-ß-d-glucopyranoside (8), lavandoside (9), 4-hydroxybenzoic acid 4-O-ß-d-glucopyranoside (10), nicotiflorin (11), rutin (12), vicenin-2 (13), narcissin (14) and stigmasterol (5) and ß-sitosterol (6) as a mixture (71% and 29%, respectively). Compounds 5, 7, 8, 9, 10 and 14 were new for the genus Helianthemum. The antioxidant power of all the isolated compounds was also evaluated by HPLC-ABTS•+, TEAC and ORAC assays. The results clearly indicated high antioxidant potential of the extracts and tested compounds of this species especially, compounds 1, 4, 8, 9, 10 and 12.

Antioxidant and antibacterial activities and polyphenolic constituents of Helianthemum sessiliflorum Pers.

In this study, the various extracts of aerial parts of Helianthemum sessiliflorum Pers. were examined in vitro for possible source of antioxidants and for antibacterial activity. The antioxidant activity was performed by DPPH radical scavenging method which showed that ethyl acetate extract possessed the best antioxidant potential (IC50 = 32.75 ± 2.07 µg/mL). The significant linear correlation was realised between the values of the total phenolic/flavonoid content and antioxidant activity of plant extracts. The ethyl acetate and n-butanol extracts showed moderate antibacterial activity. In addition, the phytochemical study of n-butanol extract afforded nine known phenolic compounds (1-9). This is the first report of six of them (1, 3, 5-8) in Cistaceae family. The structural identification of the isolated compounds was achieved using several spectroscopic methods.

Kaempferol inhibits Entamoeba histolytica growth by altering cytoskeletal functions.

The flavonoid kaempferol obtained from Helianthemum glomeratum, an endemic Mexican medicinal herb used to treat gastrointestinal disorders, has been shown to inhibit growth of Entamoeba histolytica trophozoites in vitro; however, the mechanisms associated with this activity have not been documented. Several works reported that kaempferol affects cytoskeleton in mammalian cells. In order to gain insights into the action mechanisms involved in the anti-amoebic effect of kaempferol, here we evaluated the effect of this compound on the pathogenic events driven by the cytoskeleton during E. histolytica infection. We also carried out a two dimensional gel-based proteomic analysis to evidence modulated proteins that could explain the phenotypical changes observed in trophozoites. Our results showed that kaempferol produces a dose-dependent effect on trophozoites growth and viability with optimal concentration being 27.7 µM. Kaempferol also decreased adhesion, it increased migration and phagocytic activity, but it did not affect erythrocyte binding nor cytolytic capacity of E. histolytica. Congruently, proteomic analysis revealed that the cytoskeleton proteins actin, myosin II heavy chain and cortexillin II were up-regulated in response to kaempferol treatment. In conclusion, kaempferol anti-amoebic effects were associated with deregulation of proteins related with cytoskeleton, which altered invasion mechanisms.

Lethal effects of Helianthemum lippii (L.) on Acanthamoeba castellanii cysts in vitro.

Acanthamoeba spp. commonly cause Acanthamoeba keratitis which is typically associated with the wear of contact lenses. Therefore, finding an economic, efficient, and safe therapy of natural origin is of outmost importance. This study examined the in vitro lethal potential of ethyl acetate and methanol extracts of Helianthemum lippii (L.) (sun roses) against Acanthamoeba castellanii cysts isolated from patients with amoebic keratitis. Both extracts proved to be potent as regard to their lethal effects on A. castellanii cysts with comparable results to chlorhexidine. The ethyl acetate was more promising with cumulative lethality. It showed a highly significant lethal percentage along the duration of treatment. The analysis of the more potent ethyl acetate extract revealed the presence of 2.96 mg/100 g of total phenolics, 0.289 mg/100 ml of total flavonoids and 37 mg/100 mg of total tannins which highlighted their phytomedicinal role.

New methylated flavonol glucosides from Fumana montana Pomel.

Three new methylated flavonol glucosides: 3-methoxy-7-O-ß-(6″-galloylgluco-pyranoside) quercetin (1), 3,4'-dimethoxy-7-O-ß-(6″-galloyl-glucopyranoside) quercetin (2) and 3-methoxy-7-O-ß-(6″-galloylgluco-pyranoside) kaempferol (3), in addition to six known flavonols, were isolated from the ethyl acetate extract of Fumana montana Pomel. Their structures were assigned by spectroscopic methods.

Antioxidant activity, ascorbic acid, phenolic compounds and sugars of wild and commercial Tuberaria lignosa samples: effects of drying and oral preparation methods.

The antioxidant activity and phytochemical composition (ascorbic acid, free sugars and phenolic compounds) of decoctions and infusions of wild and commercial samples of Tuberaria lignosa (Sweet) Samp. Aerial parts were evaluated and compared. Among wild samples, the effects of the drying method (freeze or shade-drying) on those parameters were studied. Infusion of the freeze-dried wild sample gave the highest levels of sugars, while infusion of shade-dried wild sample and decoction of the freeze-dried sample presented higher ascorbic acid and phenolic compounds content (including ellagitannins and flavonoids) than the other samples. The last two samples also revealed higher antioxidant activity, in some cases even higher than Trolox. Decoctions gave lower amounts of disaccharides than infusions, which seemed to be hydrolysed, increasing the content of monosaccharides. Commercial samples showed the lowest content in phenolic compounds, mainly in ellagitannins and flavonoids, and also the lowest antioxidant activity. This work gives scientific evidence to the traditional medicinal uses of wild Tuberaria lignosa, highlighting the interest of its decoctions and infusions as a source of bioactive compounds and functional beverages.

Total synthesis of isofregenedadiol.

The first total synthesis of isofregenedadiol, a bicyclic diterpene isolated from H. Viscosum, is reported starting from a D-(-)-pantolactone chiral pool. A one-pot quadruple reaction sequence comprising an enyne ring-closing metathesis/cross-metathesis/Diels-Alder/aromatization for the construction of a target skeleton is the highlight of the present synthesis.

Mycorrhizal association between the desert truffle Terfezia boudieri and Helianthemum sessiliflorum alters plant physiology and fitness to arid conditions.

The host plant Helianthemum sessiliflorum was inoculated with the mycorrhizal desert truffle Terfezia boudieri Chatin, and the subsequent effects of the ectomycorrhizal relationship on host physiology were determined. Diurnal measurements revealed that mycorrhizal (M) plants had higher rates of photosynthesis (35%), transpiration (18%), and night respiration (49%) than non-mycorrhizal (NM) plants. Consequently, M plants exhibited higher biomass accumulation, higher shoot-to-root ratios, and improved water use efficiency compared to NM plants. Total chlorophyll content was higher in M plants, and the ratio between chlorophyll a to chlorophyll b was altered in M plants. The increase in chlorophyll b content was significantly higher than the increase in chlorophyll a content (2.58- and 1.52-fold, respectively) compared to control. Calculation of the photosynthetic activation energy indicated lower energy requirements for CO(2) assimilation in M plants than in NM plants (48.62 and 61.56 kJ mol(-1), respectively). Continuous measurements of CO(2) exchange and transpiration in M plants versus NM plants provided a complete picture of the daily physiological differences brought on by the ectomycorrhizal relationships. The enhanced competence of M plants to withstand the harsh environmental conditions of the desert is discussed in view of the mycorrhizal-derived alterations in host physiology.

Antiestrogenic and antigenotoxic activity of bee pollen from Cystus incanus and Salix alba as evaluated by the yeast estrogen screen and the micronucleus assay in human lymphocytes.

The estrogenic/antiestrogenic activity and the genotoxicity/antigenotoxicity of bee pollen from Salix alba L. and Cystus incanus L. and its derivative extracts in yeast and human cells was investigated. All samples showed a marked inhibitory effect on the activity of the natural estrogen 17 beta-estradiol (higher than 90% for extracts 2) and failed to cause estrogenic activity and chromosome damage. At least one preparation from each species showed a marked antigenotoxic effect against the action of the anticancer drugs mytomicin C, bleomycin, and vincristine. Bee pollens from C. incanus and S. alba were found to be neither genotoxic nor estrogenic as well as effective estrogen inhibitors, and able to reduce the chromosome damage induced by the three cancer drugs used, thus supporting their use as a safe food supplement and future chemoprotective/chemopreventive agents.

Cistaceae aqueous extracts containing ellagitannins show antioxidant and antimicrobial capacity, and cytotoxic activity against human cancer cells.

Roots and aerial parts of Cistaceae have been used since ancient times in the Mediterranean cultures for its medicinal properties. In this study, phenolic and tannin content of C. ladanifer and C. populifolius leaves aqueous extracts were determined and their antioxidant and antimicrobial activity were fully studied by several in vitro assays. Their major compounds were identified and quantitated by high-performance liquid chromatography with diode array detection coupled to electrospray ion-trap mass spectrometry. Cytotoxicity on a panel of human cancer cells was also determined. C. populifolius extract was stronger antioxidant than C. ladanifer extract in electron transfer reaction based assays but C. ladanifer extract was more effective to inhibit peroxyl radicals. The major compounds in both extracts were ellagitannins, especially punicalagins derivatives, showing C. populifolius a higher content. C. ladanifer showed noteworthy antibacterial activity against Staphylococcus aureus, whereas C. populifolius was effective against Escherichia coli, with MICs values of 154 and 123 microg/mL, respectively. Last, both extracts showed a notorious capacity to inhibit the proliferation of M220 pancreatic cancer cells and MCF7/HER2 and JIMT-1 breast cancer cells. The leaves of these plants suppose a source for water-soluble ellagitannins-enriched polyphenolic extracts with antioxidant and antimicrobial activities. Their cytotoxic activity against several cancer cells may deserve further attention.

Ellagitannins from Tuberaria lignosa as entry inhibitors of HIV.

Screening of plants from the Iberian Peninsula for anti-human immunodeficiency virus (-HIV) activity revealed that aqueous extract of Tuberaria lignosa gave positive results. Following an activity-guided procedure, the crude extract was counterextracted, and the subsequent fractions obtained tested for their anti-HIV activity in vitro. The bioassay-guided fractionation of the extract afforded an ellagitannin enriched fraction (EEF) isolated for the first time from this species. This EEF exhibited antiviral activity against HIV in MT-2 infected cells, with an IC(50) value of 2.33mug/ml (selectivity index greater than 21). Inhibition of HIV infection by EEF appears to be mediated by CD4 down-regulation, the main receptor for HIV entry. CXCR4 and CCR5 receptors were not affected by EEF, explaining why EEF is able to inhibit R5 and X4 infections.

Determination of nickel, calcium and magnesium in xylem sap by flame atomic absorption spectrometry using a microsampling technique.

INTRODUCTION: Knowledge of xylem sap chemical composition is important to the understanding of translocation, detoxification and tolerance mechanisms. However, the small amount of sample available often hampers its characterisation. Hence, low volume consumption techniques are needed for xylem sap analysis. OBJECTIVE: To develop a microsampling technique for the determination of elements in xylem sap from different plants by flame atomic absorption spectrometry (FAAS). METHODOLOGY: The microsampling device was optimised in terms of sample volume and integration time. The analytical characteristics of the microsampling technique (micro-FAAS) were established and compared with those of FAAS with traditional continuous nebulisation. The method was validated by means of an independent technique. RESULTS: Ca, Mg and Ni were determined in a 50 microL aliquot of xylem sap solution/element that was introduced directly into the flame via the microsampling accessory. Good precision was obtained with relative standard deviations of 1.1, 0.6 and 2.3% for Ca, Mg and Ni, respectively. Matrix effects resulting from the physical characteristics of the samples and possible chemical interferences caused by phosphate and/or sulphate were ruled out. CONCLUSION: A simple, rapid and reproducible microsampling technique coupled to FAAS was developed and successfully applied in the determination of Ca, Mg and Ni in xylem sap.