Quorum sensing inhibitors from Leucetta chagosensis Dendy, 1863

Sponges are a rich source for investigation of bioactive small molecules. They have been mostly investigated for the search of new pharmacological models or therapeutic agents for the treatment of human diseases. Micro-organisms can also represent a virulent pathogen for marine invertebrates such as sponges, which need to protect themselves against these microbes. Sponges' self defence mechanisms involving dialogue molecules thus represent a pertinent research track for potent anti-infective and anti-biofilm activities such as quorum sensing inhibitors (QSIs). The investigation of the QSI crude extract of Leucetta chagosensis Dendy, 1863 led to the isolation of three new alkaloids, isonaamine D, di-isonaamidine A and leucettamine D, along with the known isonaamine A and isonaamidine A. Isonaamidine A and isonaamine D were identified as inhibitors of the three quorum sensing pathways of Vibrio harveyi (CAI-1, AI-2 and harveyi auto inducer), but isonaamidine A displayed the strongest activity on AI-2 biosensor. Both compounds are new examples of natural QSIs of V. harveyi. These results outline the importance of these secondary metabolites for their producing organisms themselves in their natural environment, as well as the potential of the marine resource for aquaculture needs.

Extraction and purification of theobromine using ionic liquids

Theobromine is an alkaloid present in cocoa and it is used in the treatment of atherosclerosis, hypertension, angina, among others. Due to its importance, the aim of this work consists on the development of an efficient and sustainable technology for the extraction of theobromine from cocoa beans. For the development of a purification technique for theobromine extracted from cocoa, aqueous biphasic systems (ABS) composed of ionic liquids (ILs) were initially studied to infer on the most promising systems. Cholinium-based ILs, based on a non-toxic and biocompatible cation, were used combined with two polymers (PPG 400 and PEG 400) and an inorganic salt (K3PO4). The respective phase diagrams at 298 K and atmospheric pressure were determined, as well as their extraction efficiencies for theobromine. The results obtained indicate that K3PO4 has a greater ability to induce the formation of ABS compared to PEG 400 and PPG 400. ABS consisting of K3PO4 also have a high potential for the extraction of theobromine, with extraction efficiencies ranging between 96.4 and 99.9 %. Based on the most promising ILs for the purification step, they were further used in aqueous solution to extract theobromine from cocoa beans, with extraction yields ranging between 4.5% and 6.5 wt%. Finally, ABS were applied to the aqueous solutions containing theobromine from the cocoa extract, with extraction efficiencies ranging between 96.7 and 99.0%.

The tolerance of steers (Bos taurus) to sorghum ergot (Claviceps africana) in a feedlot during the cooler months in sub-tropical Queensland

Two experiments tested the tolerance of steers (Bos taurus) to sorghum ergot (Claviceps africana) during cooler months in south-east Queensland. Sorghum grain containing 2.8% ergot and 28 mg/kg ergot alkaloids (84% dihydroergosine, 10% dihydroelymoclavine, 6% festuclavine) was incorporated into feedlot rations. In a previous study in summer–autumn, ergot (1.1–4.4 mg alkaloids/kg ration) severely reduced performance in steers when the temperature–humidity index (THI; dry bulb temperature °C + 0.36 dew-point temperature °C + 41.2) was ~70, whereas a THI of ~79 was tolerated by steers fed ergot-free rations. Experiment 1 was conducted in winter–spring, with rations containing 0, 2.8, 5.6, 8.2 or 11.2 mg ergot alkaloids/kg ration. All ergot inclusions depressed feed intake (14% average reduction) and growth rate (34% average reduction), even when the weekly average daily THI was less than 65. Rectal temperatures were occasionally elevated in ergot-fed steers (P < 0.05), primarily when the THI exceeded ~65. All ergot inclusions depressed plasma prolactin concentrations in steers. Experiment 2 was predominantly carried out in winter, with weekly average daily THI <65 throughout the experiment. Rations containing 0, 0.28, 0.55 or 1.1 mg ergot alkaloids/kg were fed for 4 weeks but produced no significant effect on feed intakes and growth rates of steers. Alkaloid concentrations were then changed to 0, 2.1, 4.3 and 1.1 mg/kg, respectively. Subsequently, feed intakes declined by 17.5% (P < 0.05), and growth rates by 28% (P > 0.05) in the group receiving 4.3 mg/kg alkaloid, compared with Controls. Plasma prolactin concentrations were depressed, relative to the Controls, by dietary alkaloid inclusion greater than 1.1 mg/kg, with alkaloid intake of 4.3 mg/kg causing the greatest reduction (P < 0.05). Cattle performance in these studies shows steers can tolerate up to ~2 mg ergot alkaloid/kg (0.2% ergot) in feedlot rations under low THI conditions (< ~60–65), but previous findings indicate a much lower threshold will apply at higher THI (>65).

The tolerance of steers (Bos taurus) to sorghum ergot (Claviceps africana) in a feedlot during the cooler months in sub-tropical Queensland

Two experiments tested the tolerance of steers (Bos taurus) to sorghum ergot (Claviceps africana) during cooler months in south-east Queensland. Sorghum grain containing 2.8% ergot and 28 mg/kg ergot alkaloids (84% dihydroergosine, 10% dihydroelymoclavine, 6% festuclavine) was incorporated into feedlot rations. In a previous study in summer–autumn, ergot (1.1–4.4 mg alkaloids/kg ration) severely reduced performance in steers when the temperature–humidity index (THI; dry bulb temperature °C + 0.36 dew-point temperature °C + 41.2) was ~70, whereas a THI of ~79 was tolerated by steers fed ergot-free rations. Experiment 1 was conducted in winter–spring, with rations containing 0, 2.8, 5.6, 8.2 or 11.2 mg ergot alkaloids/kg ration. All ergot inclusions depressed feed intake (14% average reduction) and growth rate (34% average reduction), even when the weekly average daily THI was less than 65. Rectal temperatures were occasionally elevated in ergot-fed steers (P < 0.05), primarily when the THI exceeded ~65. All ergot inclusions depressed plasma prolactin concentrations in steers. Experiment 2 was predominantly carried out in winter, with weekly average daily THI <65 throughout the experiment. Rations containing 0, 0.28, 0.55 or 1.1 mg ergot alkaloids/kg were fed for 4 weeks but produced no significant effect on feed intakes and growth rates of steers. Alkaloid concentrations were then changed to 0, 2.1, 4.3 and 1.1 mg/kg, respectively. Subsequently, feed intakes declined by 17.5% (P < 0.05), and growth rates by 28% (P > 0.05) in the group receiving 4.3 mg/kg alkaloid, compared with Controls. Plasma prolactin concentrations were depressed, relative to the Controls, by dietary alkaloid inclusion greater than 1.1 mg/kg, with alkaloid intake of 4.3 mg/kg causing the greatest reduction (P < 0.05). Cattle performance in these studies shows steers can tolerate up to ~2 mg ergot alkaloid/kg (0.2% ergot) in feedlot rations under low THI conditions (< ~60–65), but previous findings indicate a much lower threshold will apply at higher THI (>65).

Chemical characterization and in vitro cyto- and genotoxicity of ‘legal high’ products containing Kratom (Mitragyna speciosa)

Kratom is a popular ‘legal high’ mainly constituted by alkaloids extracted from the Mitragyna speciosa plant with mitragynine (MG) as the dominant active substance. The increasing use of Kratom for recreational purposes has alerted risk assessment bodies of the lack of information on the real composition and its potential health risks. The present study aimed to determine and compare the MG composition of 13 commercial products of Kratom sold online and in “smartshops”, by gas chromatography–mass spectrometry. For the first time, the cytotoxicity induced by pure MG and Kratom, extracts was evaluated in in vitro models of human intestinal (Caco-2) and neuronal (SH-SY5Y) cells after 6 and 24 h. Genotoxicity was also evaluated in intestinal Caco-2 cells following 24 h of exposure to subtoxic concentrations using the comet assay. The obtained results revealed an inconsistency between the information (‘power’) provided in labels and the MG content. Cytotoxicity tests revealed a concentration-dependent decrease in cell viability in both cellular models, with the SH-SY5Y cells being more sensitive to the Kratom extracts. The resin and the ‘powered extracts’ were the most cytotoxic samples, with IC50 values significantly lower than the leaf extracts and pure MG (P < 0.0001 vs. leaf extracts and MG). In addition, significant DNA damage was observed in Caco-2 cells exposed to these extracts but not to pure MG, which suggests that other substances present in the extracts or interactions involving Kratom components might be responsible for the observed effects.

New 9-Terpenyl(7-Terpenyl)Purines: synthesis and cytotoxicity

The purine ring system is one of the most widely distributed N-heterocycles in Nature [1] and many structurally modified purine nucleosides and nucleotides have activities ranging from antineoplastic and antiviral to antihypertensive, antiasthmatic, antituberculosis, etc [2]. Among the purine derivatives, we have put our attention on natural N-alkylpurines such as the asmarines or agelasimines, a group of secondary metabolites isolated from marine sponges with very interesting biological properties [3]. They have a diterpenoid moiety attached to the N-7 nitrogen atom of an adenine and are usually isolated in very small quantities, which limited their structure-activity relationship studies. Our research group has been involved for years in the design, synthesis and biological evaluation of cytotoxic compounds related to natural products, including the chemoinduction of bioactivity on inactive terpenoids [4]. These diterpenoid include compounds such as communic or cupressic acids that bear decaline moieties very close to those present in the above-mentioned marine natural products. These facts prompted us to design and prepare new terpenylpurine derivatives starting from natural monoterpenoids and diterpenoids, commercially available or isolated from their natural sources and transformed into appropriate alkylated agents. Thus, we have prepared purines alkylated at N-7 and N-9 positions with isoprenoids, monoterpenoids and diterpenoids, using two different synthetic approaches: from 6-chloropurine or from 4,5-diamine-6-chloropyrimidine. The structure of the synthesized purines are shown in the following figure. The purine analogues synthesized have been evaluated for their cytotoxicity against four tumour human cell lines (breast, non-small lung, cervical and hepatocellular carcinoma) and non-tumour cells (porcine liver primary cells). The most cytotoxic derivatives were those with a diterpenoid rest on the purine. The results obtained allowed to draw conclusions on the structure-activity relationship of the compounds in order to evaluate the influence of the terpenyl size on their cytotoxic properties.

Chemotaxonomic studies on Schwenckia americana LINN.

This study investigated the chemotaxonomic studies on Schwenckia americana Linn., a member of the family Solanaceae predominantly found mostly in low grass fields, Nigeria. The habit is annual herbaceous weed with slender stem characterized with free branching and growing up to 45cm in height.They are used mainly as medicine. The leaves are simple, entire, elliptic to ovate in shape, smooth, variable, petiolate and larger at the lower region of stem and narrowing to smaller almost sessile and oblanceolate towards the apical regions which are alternately arranged and acrescently structured from the top to the base upto 3.7 ± 1.5cm long and 2.4±0.6cm wide. The inflorescence is a panicle of 15 or more flowers occurring at stem terminal.The flowers are whitish tubular structures measuring up to 1.0±0.2cm in length and 0.1cm in diameter. The petals are whitish up to 0.9 ±0.2cm in length and sepals are greenish up to 0.1cm in length. The berry fruit is greenish when unripe and pale yellowish when ripe up to 0.3±0.1cm in diameter. The seeds are blackish and spherical or triangular shaped with rough edges measuring up to 0.1cm in diameter. The epidermal studies reveal anomocytic stomata whereas the trichomes are simple uniseriate forms wth bulge heads. The anatomy of mid-ribs and petioles showed bicollateral vascular systems. There are three vascular traces and the node is unilacunar. The petioles have 2 rib traces at primary growth phase. At secondary growth phase, the mid-rib and petiole revealed vascular arcs and the stem has a ring of open vascular system. The cytological studies showed a diploid chromosome number of 2n = 24 and n = 12 for the haploids. Alkaloids, saponins, tannins, phlobatannins, flavonoids, combined anthraquinones, free anthraquinones and cardiac glycosides are present.

Anti-nociceptive and anti-inflammatory potentials of fractions from the extract of Tetracarpidium conophorum leaf in rats and mice

Tetracarpidium conophorum (TC) (Euphorbiaceae) is a perennial woody climbing shrub in low bush forest of some parts of West Africa and used among the natives for relief of ailments accompanying pain and inflammation. In this study, the analgesic and anti-inflammatory effects of the methanolic extract (METC) and fractions (ethyl acetate, F1 and n-hexane, F2) of Tetracarpidium conophorum leaf were evaluated in rat and mice. The analgesic activity was evaluated using acetic acid-induced writhing, formalin-induced paw licking and hot plate test models. Carrageenan-induced paw oedema was used to assess anti-inflammatory activity in rats. The mechanism of action of (TC) was explored by the use of naloxone, a non-selective opioid receptor blocker. The highest analgesic effect was observed in F2 extract at 57.21% inhibition and was further studied on various analgesic and anti-inflammatory models in graded doses. F2 significantly inhibited the late phase of formalin-induced paw licking and prolong hot plate latency at 55±1°C. The n-hexane fraction also significantly inhibited carrageenan-induced paw oedema in rats at 100 and 200mg/kg doses significantly (p< 0.001) and reduced paw licking response by 85.08% compared with control. Naloxone, an opioid receptor antagonist, did not significantly affect the changes observed with n-hexane fraction, thus ruling out the possibility of the involvement of opioid receptors in the analgesic actions of Tetracarpidium conophorum. Phytochemical screening showed that the leaf extracts contain alkaloids, tannins, saponins and cardenolides. The investigations showed that Tetracarpidium conophorum possesses significant anti-nociceptive and anti-inflammatory activities that should be explored.

EVALUATION OF THE ANTI-FUNGAL PROPERTIES OF EXTRACTS OF Daniella oliveri

The in vitro anti-fungal activity of leaf and stem bark of Daniella oliveri Rolfe was investigated against selected yeasts and moulds including dermatophytes. Water and methanol were used to extract the powdered leaf and stem bark using cold infusion. Antimicrobial activity was assessed by agar-well diffusion. Phytochemical analysis was carried out using standard procedures. The plant extracts were active against the test organisms at concentrations ranging from 3.125-100 mg/mL. The methanol extracts were more active than the aqueous extracts with the highest inhibition against the yeasts, Candida albicans and Candida krusei (MIC values of 3.125 mg/mL and 6.25 mg/mL respectively). Epidermophyton floccosum and Trichophyton interdigitale were the least inhibited of all the fungal strains. Phytochemical screening revealed the presence of tannins, anthraquinones, flavonoids, cardiac glycosides, alkaloids and saponins. The anti-fungal activity of Daniella oliveri as shown in this study indicates that the plant has the potential of utilisation in the development of chemotherapeutic agents for the treatment of relevant fungal infections.

Avaliação de inibidores verdes microemulsionados na inibição à corrosão do aço carbono AISI 1020

In general, among the corrosion inhibitors surfactants are the most commonly used compounds, because they are significantly effective by forming protective films on anodic and cathodic areas. In this study, microemulsions containing he biodegradable saponified coconut oil as surfactant (SME-OCS) was used as green corrosion inhibitors. With this purpose, methanolic extracts of Ixora coccinea Linn (IC) and a polar fraction rich in alkaloids (FA) obtained from Croton cajucara Benth solubilized in the SME-OCS system were examined in the presence of AISI 1020 carbon steel, in saline solution (NaCl 3,5 %). The efficiency of corrosion inhibition of IC and FA were evaluated in the following microemulsions: SME-OCS-IC and SME-OCS-FA. The microemulsion system SME-OCS in the presence and absence of IC and FA was assessed by measurements of weight loss and the electrochemical method of polarization resistance, with variation in the concentration of IC and FA (50 - 400 ppm), showing significant results of corrosion inhibition (83,6 % SME-OCS; 92,2 % SME-OCS-FA; and 95,3 % SME-OCS-IC)

Aplicações biotecnológicas do supressor de silenciamento de cucumber mosaic virus

Tese de Doutoramento, Ciências Agrárias, Faculdade de Ciências e Tecnologia, Universidade do Algarve, 2015

Phytochemical screening and evaluation of cytotoxicity of stem bark extracts of Genus dolichocarpa and Duguetia chrysocarpa (Annonaceae)

Purpose: To investigate the phytochemistry and cytotoxic activity of stem bark extracts from Genus dolichocarpa and Duguetia chrysocarpa - two species of the Annonaceae family. Methods: The crude ethanol bark extracts (EtOH) of the plants were obtained by maceration. The crude extracts were suspended in a mixture of methanol (MeOH) and water (H2O) (proportion 3:7 v/v) and partitioned with hexane, chloroform (CHCl3) and ethyl acetate (AcOEt) in ascending order of polarity to obtain the respective fractions. The extracts were evaluated on thin layer chromatography (TLC) plates of silica gel to highlight the main groups of secondary metabolites. Cytotoxicity was tested against human tumor cell lines - OVCAR-8 (ovarian), SF-295 (brain) and HCT-116 (colon) - using 3- (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Results: The screening results demonstrated that all the extracts were positive for the presence of flavonoids and tannins. The presence of alkaloids also was detected in some extracts. The hexane extract of A. dolichocarpa showed the strongest cytotoxicity against HCT-116 with cell growth inhibition of 89.02 %. Conclusion: The findings demonstrate for the first time the cytotoxic activity of the extracts of A. dolichocarpa and D. chrysocarpa, thus providing some evidence that plants of the Annonaceae family are a source of active secondary metabolites with cytotoxic activity.

Quality evaluation of cortex berberidis from different geographical origins by simultaneous high performance liquid chromatography combined with statistical methods

Purpose: To develop an effective method for evaluating the quality of Cortex berberidis from different geographical origins. Methods: A simple, precise and accurate high performance liquid chromatography (HPLC) method was first developed for simultaneous quantification of four active alkaloids (magnoflorine, jatrorrhizine, palmatine, and berberine) in Cortex berberidis obtained from Qinghai, Tibet and Sichuan Provinces of China. Method validation was performed in terms of precision, repeatability, stability, accuracy, and linearity. Besides, partial least squares discriminant analysis (PLS-DA) and one-way analysis of variance (ANOVA) were applied to study the quality variations of Cortex berberidis from various geographical origins. Results: The proposed HPLC method showed good linearity, precision, repeatability, and accuracy. The four alkaloids were detected in all samples of Cortex berberidis. Among them, magnoflorine (36.46 - 87.30 mg/g) consistently showed the highest amounts in all the samples, followed by berberine (16.00 - 37.50 mg/g). The content varied in the range of 0.66 - 4.57 mg/g for palmatine and 1.53 - 16.26 mg/g for jatrorrhizine, respectively. The total content of the four alkaloids ranged from 67.62 to 114.79 mg/g. Moreover, the results obtained by the PLS-DA and ANOVA showed that magnoflorine level and the total content of these four alkaloids in Qinghai and Tibet samples were significantly higher (p < 0.01) than those in Sichuan samples. Conclusion: Quantification of multi-ingredients by HPLC combined with statistical methods provide an effective approach for achieving origin discrimination and quality evaluation of Cortex berberidis. The quality of Cortex berberidis closely correlates to the geographical origin of the samples, with Cortex berberidis samples from Qinghai and Tibet exhibiting superior qualities to those from Sichuan.

Phytopharmacological and ethnomedicinal uses of the Genus Berberis (Berberidaceae): A review

Plants belonging to Berberis are reported in several folklore medicinal pharmacopeias and are used in traditional medicines in Asia and European countries. The plants have been used in the preparation of various traditional and synthetic medicines since pre-historic times for wound healing, fever, eye disease, jaundice, vomiting during pregnancy, rheumatism, kidney and gall balder stones, and several other illnesses. Their healing properties are appear to be due to the presence of secondary metabolites and important alkaloids with different pharmacological activities. Their antibacterial, antifungal, antiviral, anti-diabetic, and anti-tumor activities as well as positive effects on the cardiovascular and body immune systems have been reported. Root extracts of some species of the plant genus contain quinine which acts as a powerful anti-malarial agent. The main chemical constituents of Berberis plants are alkaloids, steroids, glycosides, flavonoids, saponins, terpenoids and reducing sugars. Of these alkaloids, berberine is the most important. The present review focuses on recent advances in phytopharmacological and ethnomedicinal uses of plants belonging to Berberis genus.

I. Synthetic Studies Toward the Total Synthesis of Polycyclic Natural Products – Communesin F, Perophoramidine and Ineleganolide. II. Nickel Catalyzed Intramolecular C–O Bond Formation

Expedient synthetic approaches to the highly functionalized polycyclic alkaloids communesin F and perophoramidine are described using a unified approach featuring a key decarboxylative allylic alkylation to access a crucial and highly congested 3,3-disubstituted oxindole. Described are two distinct, stereoselective alkylations that produce structures in divergent diastereomeric series possessing the critical vicinal all-carbon quaternary centers needed for each synthesis. Synthetic studies toward these challenging core structures have revealed a number of unanticipated modes of reactivity inherent to these complex alkaloid scaffolds. Finally, a previously unknown mild and efficient deprotection protocol for the o-nitrobenzyl group is disclosed – this serendipitous discovery permitted a concise endgame for the formal syntheses of both communesin F and perophoramidine.

In addition, the atroposelective synthesis of PINAP ligands has been accomplished via a palladium-catalyzed C–P coupling process through dynamic kinetic resolution. These catalytic conditions allow access to a wide variety of alkoxy- and benzyloxy-substituted PINAP ligands in high enantiomeric excess.

An efficient and exceptionally mild intramolecular nickel-catalyzed carbon–oxygen bond-forming reaction between vinyl halides and primary, secondary, and tertiary alcohols has been achieved. This operationally simple method allows direct access to cyclic vinyl ethers in high yields in a single step.

Finally, synthetic studies toward polycyclic ineleganolide are described. The entire fragmented carbon framework has been constructed from this work. Highly (Z)-selective olefination was achieved by the method by the Ando group.