A novel in vitro whole plant system for analysis of polyphenolics and their antioxidant potential in cultivars of Ocimum basilicum

Plants are an important source for medicinal compounds. Chemical screening and selection is critical for identification of compounds of interest. Ocimum basilicum (Basil) is a rich source of polyphenolics and exhibits high diversity, therefore bioprospecting of a suitable cultivar is a necessity. This study reports on the development of a true to type novel "in vitro system" and its comparison with a conventional system for screening and selection of cultivars for high total phenolics, individual polyphenolics, and antioxidant content. We have shown for the first time using online acidic potassium permanganate chemiluminescence that extracts from Ocimum basilicum showed antioxidant potential. The current study identified the cultivar specific composition of polyphenolics and their antioxidant properties. Further, a distinct relationship between plant morphotype and polyphenolic content was also found. Of the 15 cultivars examined, "Holy Green", "Red Rubin", and "Basil Genovese" were identified as high polyphenolic producing cultivars while "Subja" was determined to be a low producer. The "in vitro system" enabled differentiation of the cultivars in their morphology, polyphenolic content, and antioxidant activity and is a cheap and efficient method for bioprospecting studies.

Synthesis and biological activities of polyphenolic hybrids - evaluation of anti-cancer, anti-inflammatory and antioxidant activities

Os compostos polifenólicos constituem uma classe de metabolitos secundários de plantas, mas existe também uma enorme quantidade de derivados sintéticos ou semi-sintéticos contendo múltiplas unidades fenólicas. Estes compostos apresentam importantes características biológicas, que dependem das suas estruturas básicas. Certos derivados desta família de compostos, tais como flavonoides, cromonas e cumarinas contribuem para os benefícios da dieta humana, e partilham o núcleo de benzopiran-(2 e 4)-ona ou benzofuran-3-ona. A presente dissertação inclui uma introdução geral e três capítulos que descrevem as novas rotas sintéticas estabelecidas para a preparação de novos híbridos de diversos compostos polifenólicos, assim como a sua elucidação estrutural e termina com a presentação dos resultados da avaliação biológica desses mesmos compostos. No segundo capítulo discute-se a preparação de híbridos de pirimidina- e imidazolidina-polifenóis, especialmente a síntese diastereoseletiva de novos híbridos benzofuran-3-ona-hidantoína e derivados de uracilo. A rota sintética envolve a ação de carbodiimidas sobre os ácidos cromona-(2- e 3)-carboxílicos num só passo ou em dois passos sequenciais, catalisada por uma base orgânica ou inorgânica. O terceiro capítulo descreve reações do tipo adições conjugadas 1,4 - hetero-ciclisações em cascata de compostos 1,3-dicarbonílicos em ácido cromona-3-carboxílico catalisadas por uma base orgânica, que originaram novas cromonas, cromanonas e flavonas polissubstituídas. As bispiranonas [bispiran-2 e 4)-onas] foram elaboradas numa reacção de acoplamento da 4-hidroxicumarina ou da lactona do ácido triacético com o ácido cromona-3-carboxílico ou precursores formil-funcionalizados (ω-formil-2’-hydroxy acetofenonas e cromona-3-carbaldeídos) utilizando organocatálise básica. Finalmente, alargou-se o estudo das adições conjugadas 1,4 para uma variedade de 4-hidroxipiran-2-onas e cetonas α,β-insaturadas para originar novos análogos de warfarina. Obteve-se uma variedade de estruturas complexas por hibridação das unidades de 4-hidroxicumarina ou da lactona do ácido triacético com os novos derivados de cromonas polissubstituídas. Todos as reações foram executadas em condições suaves e ambientalmente favoráveis, utilizando a 4-pirrolidinopiridina como organocatalisador básico. As estruturas dos novos híbridos polifenólicos foram caracterizados por técnicas espectroscópicas de alta resolução, incluindo espectroscopia de ressonância magnética nuclear (1D e 2D) e por difractometria de raios-X, que nos permitiram resolver o complexidade estrutural dos compostos sintetizados. O quarto capítulo apresenta os resultados da avaliação biológica obtidos com os híbridos polifenólicos sintetizados neste trabalho, mostrando a possibilidade de seu envolvimento na terapia do cancro. A maioria dos compostos foram avaliados quanto ao seu efeito sobre a citotoxicidade e proliferação de células leucémicas e ao seu envolvimento na regulação de via pró-inflamatória NF-kB, na qual, os híbridos de biscumarinas exibiram actividades elevadas (IC50 = 6-19 μM para inibição de NF-kB depois de 8 horas de incubação e IC50 = 15-39 μM para efeitos citotóxicos em células cancerosas, após 24 horas de incubação). Uma inibição moderada das enzimas HDAC e Cdc25 foi induzida pelos derivados de benzofuran-3-ona-hidantoína. Catorze dos novos derivados polifenólicos polissubstituídos, tendo como estrutura básica a benzopiran-4-ona, foram avaliados pela sua actividade quimiopreventiva do cancro mediada pela indução de sinalização citoprotectora Nrf2 (fator 2 relacionado com o fator nuclear da proteína E2) e capacidade para inibir a proliferação das células de cancro da mama. Os derivados da classe das cromanonas foram identificados como os indutores mais potentes da actividade Nrf2. As concentrações necessárias para aumentar a actividade de luciferase em 10 vezes (C10) foram de 2,8-21,3 μM. Todos os novos híbridos polifenólicos que apresentam atividade citotóxica e anti-proliferativa não afectam o crescimento de células saudáveis periféricas do sangue (PBMC) (IC50 > 50 μM), indicando a sua seletividade para as células cancerosas e sugerindo que alguns deles são estruturalmente interessantes para posteriores análises. A avaliação da atividade antioxidante utilizando os testes do radical livre DPPH e o poder redutor do ião férrico FRAP foram realizados em algumas estruturas híbridas polifenólicas.

In vivo screening of mangrove plants for anti WSSV activity in Penaeus monodon, and evaluation of Ceriops tagal as a potential source of antiviral molecules

The objective of the study was to find out a natural way to fight white spot syndrome virus (WSSV) in cultured shrimps, as the present scenario necessitated an organic remedy for the devastating pathogen in crustaceans. Under this research programme seven mangrove plants were collected, identified and aqueous extracts screened for their protective effect on the giant tiger shrimp Penaeus monodon against WSSV. The experimental design consisted two modes of application, such as exposure of the virus to the extract and injection challenge, and oral administration of the extract coated feed followed by oral challenge. All experimental animals were monitored through a nested diagnostic PCR analysis. Of the seven mangrove extracts screened aqueous extract from Ceriops tagal imparted total protection to shrimp from WSSV when challenged by both methods. Shrimps administered with the aqueous extract from C. tagal were devoid of virions. The HPLC fingerprint of the aqueous extracts from C. tagal showed more than 25 peaks and 7 of them were larger and well separated. Preliminary phytochemical analysis revealed the presence of alkaloids, flavonoids, polyphenolics, cardiac glycosides, saponins and sterols. The study indicated suitability of the aqueous extract of C. tagal as a possible prophylaxis for WSSV infection in shrimp. This is the first report on the anti WSSV property of the mangrove plant C. tagal

In vivo screening of mangrove plants for anti WSSV activity in Penaeus monodon, and evaluation of Ceriops tagal as a potential source of antiviral molecules

The objective of the study was to find out a natural way to fight white spot syndrome virus (WSSV) in cultured shrimps, as the present scenario necessitated an organic remedy for the devastating pathogen in crustaceans. Under this research programme seven mangrove plants were collected, identified and aqueous extracts screened for their protective effect on the giant tiger shrimp Penaeus monodon against WSSV. The experimental design consisted two modes of application, such as exposure of the virus to the extract and injection challenge, and oral administration of the extract coated feed followed by oral challenge. All experimental animals were monitored through a nested diagnostic PCR analysis. Of the seven mangrove extracts screened aqueous extract from Ceriops tagal imparted total protection to shrimp from WSSV when challenged by both methods. Shrimps administered with the aqueous extract from C. tagal were devoid of virions. The HPLC fingerprint of the aqueous extracts from C. tagal showed more than 25 peaks and 7 of them were larger and well separated. Preliminary phytochemical analysis revealed the presence of alkaloids, flavonoids, polyphenolics, cardiac glycosides, saponins and sterols. The study indicated suitability of the aqueous extract of C. tagal as a possible prophylaxis for WSSV infection in shrimp. This is the first report on the anti WSSV property of the mangrove plant C. tagal

Antidiabetic properties of polysaccharide- and polyphenolic-enriched fractions from the brown seaweed Ascophyllum nodosum

We screened seaweed species from Atlantic Canada for antidiabetic activity by testing extracts for α-glucosidase inhibitory effect and glucose uptake stimulatory activity. An aqueous ethanolic extract of Ascophyllum nodosum was found to be active in both assays, inhibiting rat intestinal α-glucosidase (IC50 = 77 μg/mL) and stimulating basal glucose uptake into 3T3-L1 adipocytes during a 20-minute incubation by about 3-fold (at 400 μg/mL extract). Bioassay-guided fractionation of the A. nodosum extract showed that α-glucosidase inhibition was associated with polyphenolic components in the extract. These polyphenolics, along with other constituents appeared to be responsible for the stimulatory activity on glucose uptake. However, attempts to further concentrate this activity through fractionation techniques were unsuccessful. A crude polyphenol extract (PPE), an enriched polyphenolic fraction (PPE-F1) and a polysaccharide extract (PSE) were prepared from commercial A. nodosum powder and administered to streptozotocin-diabetic mice for up to 4-weeks by daily gavage at 200 mg/kg body mass. PPE and PPE-F1 improved fasting serum glucose level in diabetic mice; however, the effect was only statistically significant at day 14. In addition, PPE-F1 was shown to blunt the rise in blood glucose after an oral sucrose tolerance test in diabetic mice. Mice treated with PPE and PPE-F1 had decreased blood total cholesterol and glycated serum protein levels compared with untreated diabetic mice, whereas PPE also normalized the reduction in liver glycogen level that occurred in diabetic animals. All 3 A. nodosum preparations improved blood antioxidant capacity.

On a établit une recherche d’un produit anti-diabétique, parmi les algues locales de la région Atlantique du Canada, en examinant la capacité d’un effet inhibiteur de l’enzyme α-glucosidase et une stimulation de l’incorporation cellulaire du glucose. Un extrait éthanol-aqueux de Ascophyllum nodosum nous a donné une activité positive chez les deux essais, une inhibition de l’α-glucosidase provenant de l’intestin du rat (IC50 = 77 μg/mL) et puis une stimulation triple, à une concentration de 400 μg/mL, de l’incorporation du glucose dans les adipocytes 3T3-L1 durant une période de 20 minutes. L’extrait de A. nodosum a été divisé, guidé par les résultats biologiques, et a ainsi démontré la présence d’éléments polyphénoliques associé à l’inhibition de l’α-glucosidase. Ces éléments polyphénoliques ainsi que d’autres semblent être responsables de l’incorporation stimulée du glucose. Il a été impossible de raffiner cette activité lors d’une division des composants. Un extrait brut polyphénolique (PPE), un extrait enrichi polyphénolique (PPE-F1) et puis un extrait polysaccharide (PSE) furent préparés d’une poudre commerciale de A. nodosum et utilisés dans une étude utilisant des souris, rendues diabétiques par injections de streptozotocin, traitées par un gavage journalier de l’extrait 200 mg/kg du poids corporel durant une période de 4 semaines. Le taux du glucose sanguin à jeun des souris fut moins élevé en présence des extraits qu’en leurs absence. Cependant, l’effet était seulement significatif au jour 14. Les résultats étant toutefois variables. En plus, lors d’un essai oral de la tolérance au sucrose chez la souris diabétique, l’extrait PPE-F1 a empêché l’augmentation du taux du glucose sanguin. Les extraits PPE et PPE-F1 ont réduit le taux de cholestérol sanguin et les niveaux de glycation des protéines en comparaison de ces niveaux en absence des extraits tandis que l’extrait PPE a présenté une réduction du niveau de glycogène du foie chez les souris diabétiques. Les trois extraits de A. nodosum ont tous amélioré la capacité antioxidante du sang.

Extractos de residuos de la vitivinicultura : concentración de polifenoles

El objetivo del trabajo fue determinar la concentración de polifenoles en extractos de hojas de verano y otoño y de escobajo de variedades tintas de vitis vinifera, obtenidos por distintos métodos.Se trabajó con hojas de verano y de otoño de variedad Syrah y con hojas de otoño variedad Malbec, dividiéndolas por su color en rojas y amarillas, y con escobajo de variedad Malbec. Los extractos se obtuvieron por maceración en agua y baño María hirviente durante tres horas (BM), maceración en agua y agitación, a temperatura ambiente, durante 48 horas (AG). Se utilizó como referencia oleorresina de romero. El orden decreciente respecto de la cantidad de polifenoles expresados en g de quercitina / g de extracto seco fue el siguiente: oleorresina de romero: 56,3 ± 0,3 extracto de: hojas de otoño Syrah por maceración y agitación en agua a temperatura ambiente durante 48 horas: 7,5 ± 0,3; escobajo Malbec por maceración y agitación en agua a temperatura ambiente durante 48 horas: 24 ± 0,3; hojas de otoño rojas Malbec por maceración y agitación en agua a temperatura ambiente durante 48 horas: 22 ± 0,3; escobajo de Malbec: a baño María 21,4 ± 0,3; hojas de otoño Syrah 3 horas a baño María: 21,1 ±0,3; hojas de otoño amarillas Malbec por maceración y agitación en agua a temperatura ambiente durante 48 horas: 17 ± 0,3; hojas de verano Syrah 3 horas a baño María: 16,5 ± 0,3; hojas de otoño rojas Malbec 3 horas a baño María: 13,8 ± 0,3; hojas de otoño amarillas Malbec 3 horas a baño María: 12,4 ± 0,3; hojas de verano Syrah por maceración y agitación en agua a temperatura ambiente durante 48 horas: 12,2 ± 0,3. Se concluyó que las hojas de otoño contienen mayor cantidad de polifenoles que las de verano; la variedad Syrah posee más polifenoles en hojas que la variedad Malbec; el escobajo de Malbec contiene más olifenoles que las hojas de la misma variedad; y el mejor método de extracción para obtener estos compuestos activos fue por maceración en agua y agitación a temperatura ambiente durante 48 horas.

A novel multifunctional O-methyltransferase implicated in a dual methylation pathway associated with lignin biosynthesis in loblolly pine

S-adenosyl-l-methionine (SAM)-dependent O-methyltransferases (OMTs) catalyze the methylation of hydroxycinnamic acid derivatives for the synthesis of methylated plant polyphenolics, including lignin. The distinction in the extent of methylation of lignins in angiosperms and gymnosperms, mediated by substrate-specific OMTs, represents one of the fundamental differences in lignin biosynthesis between these two classes of plants. In angiosperms, two types of structurally and functionally distinct lignin pathway OMTs, caffeic acid 3-O-methyltransferases (CAOMTs) and caffeoyl CoA 3-O-methyltransferases (CCoAOMTs), have been reported and extensively studied. However, little is known about lignin pathway OMTs in gymnosperms. We report here the first cloning of a loblolly pine (Pinus taeda) xylem cDNA encoding a multifunctional enzyme, SAM:hydroxycinnamic Acids/hydroxycinnamoyl CoA Esters OMT (AEOMT). The deduced protein sequence of AEOMT is partially similar to, but clearly distinguishable from, that of CAOMTs and does not exhibit any significant similarity with CCoAOMT protein sequences. However, functionally, yeast-expressed AEOMT enzyme catalyzed the methylation of CAOMT substrates, caffeic and 5-hydroxyferulic acids, as well as CCoAOMT substrates, caffeoyl CoA and 5-hydroxyferuloyl CoA esters, with similar specific activities and was completely inactive with substrates associated with flavonoid synthesis. The lignin-related substrates were also efficiently methylated in crude extracts of loblolly pine secondary xylem. Our results support the notion that, in the context of amino acid sequence and biochemical function, AEOMT represents a novel SAM-dependent OMT, with both CAOMT and CCoAOMT activities and thus the potential to mediate a dual methylation pathway in lignin biosynthesis in loblolly pine xylem.

Berry size and seed number of V. myrtillus and E. hermaphroditum according to treatment, and environmental parameters 1991-2009 at Abisko Research Station

This study investigated the effects of long-term-enhanced UV-B, and combined UV-B with elevated CO2 on dwarf shrub berry characteristics in a sub-arctic heath community. Germination of Vaccinium myrtillus was enhanced in seeds produced at elevated UV-B, but seed numbers and berry size were unaffected. Elevated UV-B and CO2 stimulated the abundance of V. myrtillus berries, whilst UV-B alone stimulated the berry abundance of V. vitis-idaea and Empetrum hermaphroditum. Enhanced UV-B reduced concentrations of several polyphenolics in V. myrtillus berries, whilst elevated CO2 increased quercetin glycosides in V. myrtillus, and syringetin glycosides and anthocyanins in E. hermaphroditum berries. UV-B x CO2 interactions were found for total anthocyanins, delphinidin-3-hexoside and peonidin-3-pentosidein in V. myrtillus berries but not E. hermaphroditum. Results suggest positive impacts of UV-B on the germination of V. myrtillus and species-specific impacts of UV-B x elevated CO2 on berry abundance and quality. The findings have relevance and implications for human and animal consumers plus seed dispersal and seedling establishment.

Acidic potassium permanganate chemiluminescence for the determination of antioxidant potential in three cultivars of Ocimum basilicum

Ocimum basilicum, a member of the family Lamiaceae, is a rich source of polyphenolics that have antioxidant properties. The present study describes the development and application of an online HPLC-coupled acidic potassium permanganate chemiluminescence assay for the qualitative and quantitative assessment of antioxidants in three cultivars of O. basilicum grown under greenhouse conditions. The chemiluminescence based assay was found to be a sensitive and efficient method for assessment of total and individual compound antioxidant potential. Leaves, flowers and roots were found to be rich reserves of the antioxidant compounds which showed intense chemiluminescence signals. The polyphenolics such as rosmarinic, chicoric, caffeic, p-coumaric, m-coumaric and ferulic acids showed antioxidant activity. Further, rosmarinic acid was found to be the major antioxidant component in water-ethanol extracts. The highest levels of rosmarinic acid was found in the leaves and roots of cultivars "holy green" (14.37; 11.52 mM/100 g DW respectively) followed by "red rubin" (10.02; 10.75 mM/100 g DW respectively) and "subja" (6.59; 4.97 mM/100 g DW respectively). The sensitivity, efficiency and ease of use of the chemiluminescence based assay should now be considered for its use as a primary method for the identification and quantification of antioxidants in plant extracts.

Emerging technologies for recovery of value-added components from olive leaves and their applications in food/feed industries

Olive leaves are the most abundant agricultural waste source rich in polyphenolics. Due to the numerous health benefits associated with these compounds, the interest in recovering polyphenols from olive leaves has increased in the scientific community over the last decade. Recent studies have focused on improved extraction techniques and processing methods that are most suited for agro-biological industries involved in the development of nutraceutical and functional products. The major problems in olive leaves processing include bitter taste and the low stability of various phenolic compounds. Oleuropein and hydroxytyrosol are the most important phenolic compounds extracted from olive leaves. The present review highlights the importance of olive leaves, their composition, preparation methods, major phenolic compounds, and commercial applications. This review article focuses on integrating studies on olive leaf extract (OLE) pertinent to nutrition, health, and beauty. The different board categories of delivery systems available for the encapsulation of OLE are given. These novel delivery systems could improve fortification, supplementation, and dietary diversification in food and pharmaceutical products.