Evaluation of Opuntia spp. bioactive products as promising natural chemotherapeutical agents- an in vitro approach

Dissertation to obtain a Master Degree in Biotechnology

Revalorization of cactus pear (Opuntia spp.) wastes as a source of antioxidants

Recently, an increased interest in antioxidant activity and health-improving capacity of cactus pear has been registered. The antioxidant capacity of the pulp of cactus-pear fruits has been previously assessed. In this work, total phenolics, flavonoids and tannins of peel and seeds of four cactus pear cultivars were examined as well as their antioxidant capacity. Tannins were the major phenolics in cactus pear seeds accounting for almost fifty percent for all cultivars. Analysis of variance revealed that ripeness, cultivar, and its interaction had highly significant effect on the total phenolics, tannin, and flavonoid contents of cactus pear peel. With regard to the seeds, only the stage of ripeness and interaction (ripeness stage x cultivar) were significant on total phenolics and tannins contents. The flavonoid content in seeds was not affected by any of the factors or their interactions. The antioxidant capacity was higher in the peel than in the seeds. Generally, fruits with light-green or yellow-brown peel have higher antiradical activity and Trolox equivalent antioxidant capacity (TEAC) values compared with those with red-purple peel. Cactus pear by-products can indeed be exploited as a good and cheap source of natural antioxidants.

Caracterización, ordenación y clasificación númerica en nopal (Opuntia spp.) mediante atributos morfológicos y físico-químicos por Mariano Molina Velázquez

Tesis (Maestría en Ciencias Agrícolas) U.A.N.L.

Estimación de las características biológicas y químicas en el volumen radical de nopal (opuntia spp.) y maguey (agave spp.) y evaluaciones forrajeras.

Tesis (Doctor en Ciencias Agrícolas) UANL, 2011.

Nova ocorrência da cochonilha-do-carmim em Opuntia spp. (Cactaceae) em Santa Catarina, Brasil.

2009

Ácido ascórbico, betalaínas, e fenóis totais em ecótipos de Opuntia spp.

O género Opuntia spp. pertence à família Cactaceae, sendo a espécie Opuntia ficus-indica (OFI) a que tem maior importância económica. Em frutos de vinte populações provenientes de quatro espécies do género Opuntia spp. (OFI, O. robusta, O. dillenii and O. elata), com origem em Portugal, foram estudadas as características cromáticas, a acidez, o pH, o teor em sólidos solúveis totais (SST) e ainda os teores em ácido ascórbico (AA), betalaínas e fenóis totais (FT). As cultivares Italianas de OFI ‘Bianca’, ‘Gialla’ e ‘Rossa’ foram incluídas como termo de comparação. Os valores mais elevados de acidez foram registados nos frutos de O. dillenii e O. elata e os menores em OFI. Os frutos de O. dillenii apresentaram os teores mais elevados de betalaínas e FT, ao passo que as concentrações mais elevadas de AA foram registadas nos frutos de O. elata. Os ecótipos de OFI mostraram variação na concentração de compostos bioativos. Em OFI, a cv. de polpa vermelha ‘Rossa’ é a que apresenta maior concentração de betalaínas, seguida pelos ecótipos de polpa laranja e, finalmente, os ecótipos de polpa branca. Os valores mais elevados de FT foram encontrados nos ecótipos de polpa branca. O género Opuntia spp. é uma fonte de compostos bioativos, pelo que o consumo dos seus frutos representa uma boa forma de ingestão de compostos de elevado valor nutricional.

Bioactivity, proximate, mineral and volatile profiles along the flowering stages of Opuntia microdasys (Lehm.): defining potential applications

Opuntia spp. flowers have been traditionally used for medical purposes, mostly because of their diversity in bioactive molecules with health promoting properties. The proximate, mineral and volatile compound profiles, together with the cytotoxic and antimicrobial properties were characterized in O. microdasys flowers at different maturity stages, revealing several statistically significant differences. O. microdasys stood out mainly for its high contents of dietary fiber, potassium and camphor, and its high activities against HCT15 cells, Staphylococcus aureus, Aspergillus versicolor and Penicillium funiculosum. The vegetative stage showed the highest cytotoxic and antifungal activities, whilst the full flowering stage was particularly active against bacterial species. The complete dataset has been classified by principal component analysis, achieving clearly identifiable groups for each flowering stage, elucidating also the most distinctive features, and comprehensively profiling each of the assayed stages. The results might be useful to define the best flowering stage considering practical application purposes.

ABOVE-GROUND BIOMASS ESTIMATION OF OPUNTIA FICUS-INDICA (L.) MILL. FOR FORAGE CROP IN A MEDITERRANEAN ENVIRONMENT BY USING NON-DESTRUCTIVE METHODS

In marginal lands Opuntia ficus-indica (OFI) could be used as an alternative fruit and forage crop. The plant vigour and the biomass production were evaluated in Portuguese germplasm (15 individuals from 16 ecotypes) by non-destructive methods, 2 years following planting in a marginal soil and dryland conditions. Two Italian cultivars (Gialla and Bianca) were included in the study for comparison purposes. The biomass production and the plant vigour were estimated by measuring the cladodes number and area, and the fresh (FW) and dry weight (DW) per plant. We selected linear models by using the biometric data from 60 cladodes to predict the cladode area, the FW and the DW per plant. Among ecotypes, significant differences were found in the studied biomass-related parameters and several homogeneous groups were established. Four Portuguese ecotypes had higher biomass production than the others, 3.20 Mg ha−1 on average, a value not significantly different to the improved ‘Gialla’ cultivar, which averaged 3.87 Mg ha−1. Those ecotypes could be used to start a breeding program and to deploy material for animal feeding and fruit production.

Isolation and encapsulation of a natural colourant using green technologies

The use of natural pigments instead of synthetic colourants is receiving growing interest in the food industry. In this field, cactus pears (Opuntia spp.) have been identified to be a promising betalainic crops covering a wide coloured spectrum. The aim of this work was to develop adequate clean and mild methodologies for the isolation and encapsulation of betacyanins, from cactus pear fruits (Opuntia spp.). Firstly, two different emerging technologies, namely PLE (Pressurized Liquid Extraction) and HPCDAE (High Pressure Carbon Dioxide-Assisted Extraction), were exploited to isolation of betacyanins form cactus pear fruits. Different process conditions were tested for the maximum recovery of betacyanins. Results showed that highest extraction yields were achieved for HPCDAE and mass ratio of pressurized carbon dioxide vs. acidified water was the parameter that most affected the betacyanins extraction. At optimum conditions of HPCDAE, Opuntia spp. extract presented a total betacyanin content of 211 ± 10 mg/100 g whereas extracts obtained using conventional extraction, PLE in static and in dynamic mode presented a total betacyanin content of 85 ± 3, 191 ± 2 and 153 ± 5 mg/100 g, respectively. HPCDAE has proven to be a successful technology to extract betacyanins from Opuntia spp. fruits. Afterward, Supercritical Fluid Technology was exploited to develop lipidic particles of betalain-rich extract. A betacyanin-rich conventional extract was encapsulated by PGSS® (Particles from Gas Saturated Solutions) technique. Different process conditions were tested in order to model the encapsulation of betacyanins. The pressure had a negative effect on betacyanin encapsulation. Lower pressures leads to an increase in the betacyanin encapsulation. This effect was more pronounced at higher temperatures and lower equilibrium time. At these conditions, Opuntia spp. particles presented 64.4 ± 4.5 mg/100 g and high antioxidant capacity. When compared with the Opuntia spp. dried extract, lipidic particles contributed to a better homogenization of the pink colour after incorporation in ice cream.

Approaching invasive species in Madagascar

While a number of plants, animals, and insects in Madagascar have been called 'invasive', the topic of invasive species has until recently received less attention here than in other island contexts. Some species, often alien to Madagascar and introduced by humans, have expanded their range rapidly and have had both negative and positive effects on landscapes, on native biodiversity, and on livelihoods. Examples include the prickly pear (raketa), the silver wattle (mimosa), and, recently, the Asian common toad (radaka boka). Building on a conceptual approach to 'invasive species', this paper emphasizes the importance of inclusive and deliberative site- and population - specific management of invasive species. It analyses three separate concepts commonly used in definitions of invasion: the origin, behaviour, and effects of particular species. It places these concepts in their broader social and ecological context, with particular attention to local perspectives on invasive species. We illustrate these concepts with Malagasy examples and data. The examples demonstrate that while invasions can have dramatic consequences, there can be multiple, often competing, interests as well as site - specific biophysical, environmental, and cultural considerations that need to be taken into account when designing policy and management interventions. We conclude with a number of lessons learned. RESUME FRANCAIS Contrairement à la plupart des autres îles, et en dépit du qualificatif 'invasif' rattaché depuis longtemps à certaines espèces qui s'y sont naturalisées, les réflexions autour de l'approche des espèces invasives à Madagascar demeurent récentes. L'opuntia (Opuntia spp.) figure certes parmi les plus anciens exemples d'espèces traités dans la littérature sur les invasions biologiques. Mais ce n'est vraiment qu'avec le retentissement médiatique autour de la détection en 2011 de la présence du crapaud masqué (Duttaphrynus melanostictus) et la recherche d'une parade appropriée que s'est affirmée la nécessité de traiter cette question des espèces invasives en tant que telle. Une posture nativiste et uniforme qui ignorerait la spécificité des contextes biophysiques et socio - économiques locaux, mais aussi la pluralité des formes d'invasion biologique et des défi- nitions qui s'y rattachent, ne saurait être privilégiée. L'article montre qu'il s'agit de situer les réflexions dans un contexte insulaire socio - économique dans lequel les espèces allogènes tiennent depuis longtemps une large place. Il défend en outre la nécessité d'envisager les espèces invasives non pas selon une forme de perception unique et autoritariste, mais selon une diversité de points de vue, conforme aux conflits d'intérêts qui se manifestent parfois, et mettant plutôt en avant le caractère exogène des espèces invasives, leurs effets (négatifs, mais aussi positifs) sur le milieu, ou leur mode de fonctionnement (disper- sion, dominance) dans des contextes spécifiques et locaux. Il convient en particulier d'observer qu'aux coûts générés par les invasions biologiques peuvent s'ajouter des bénéfices économiques, et que les impacts écologiques néfastes peuvent se combiner avec des incidences heureuses, y compris auprès d'espèces indigènes en situation critique. En outre, le point de vue des populations humaines, leur connaissance d'espèces invasives quotidiennement rencontrées, leur réticence à scin- der le vivant en espèces indigènes et allogène, mais aussi leur vision pragmatique, ne sauraient être mésestimés, et moins encore oubliés. Enfin, l'article invite à prendre du recul face aux effets rhétoriques liés aux discours conventionnels sur les inva- sions biologiques, à éviter les amalgames et les généralisations excessives, à tenir compte des contraintes environnementales mais aussi des aspirations socio - économiques des populations locales, et à prendre en compte la diversité des spécificités locales, qu'elles soient biophysiques ou sociales. En conclusion, il est sans doute heureux que Madagascar n'ait rejoint que très récemment la mouvance internationale des réflexions sur les espèces invasives : cela lui permet en effet d'être en mesure de disposer d'une position équilibrée, déjouant certains discours catastrophistes, et préférant une approche résolument contextualisée, à l'échelle nationale comme aux échelles régionales.

Identification Of Corynebacterium Spp. Isolated From Bovine Intramammary Infections By Matrix-assisted Laser Desorption Ionization-time Of Flight Mass Spectrometry.

Corynebacterium species (spp.) are among the most frequently isolated pathogens associated with subclinical mastitis in dairy cows. However, simple, fast, and reliable methods for the identification of species of the genus Corynebacterium are not currently available. This study aimed to evaluate the usefulness of matrix-assisted laser desorption ionization/mass spectrometry (MALDI-TOF MS) for identifying Corynebacterium spp. isolated from the mammary glands of dairy cows. Corynebacterium spp. were isolated from milk samples via microbiological culture (n=180) and were analyzed by MALDI-TOF MS and 16S rRNA gene sequencing. Using MALDI-TOF MS methodology, 161 Corynebacterium spp. isolates (89.4%) were correctly identified at the species level, whereas 12 isolates (6.7%) were identified at the genus level. Most isolates that were identified at the species level with 16 S rRNA gene sequencing were identified as Corynebacterium bovis (n=156; 86.7%) were also identified as C. bovis with MALDI-TOF MS. Five Corynebacterium spp. isolates (2.8%) were not correctly identified at the species level with MALDI-TOF MS and 2 isolates (1.1%) were considered unidentified because despite having MALDI-TOF MS scores >2, only the genus level was correctly identified. Therefore, MALDI-TOF MS could serve as an alternative method for species-level diagnoses of bovine intramammary infections caused by Corynebacterium spp.

Behavioral Changes Caused By Austrodiplostomum Spp. In Hoplias Malabaricus From The São Francisco River, Brazil.

Traira (Hoplias malabaricus) is a neotropical fish that is widely distributed in freshwater environments in South America. In the present study, we documented the occurrence of metacercariae of Austrodiplostomum spp. (Diplostomidae) in the eyes and cranial cavity of H. malabaricus and described parasite-induced behavioral changes in the host. The fish were collected from the upper São Francisco River, in the Serra da Canastra mountain range, Minas Gerais, transported alive to the laboratory, observed for 2 weeks, and subsequently examined for parasites. Of the 35 fish examined, 28 (80 %) had free metacercariae in the vitreous humor (mean intensity=95.4; mean abundance=76.3), and 24 (68.57 %) had free metacercariae in the cranial cavity, mainly concentrated below the floor of the brain, at the height of the ophthalmic lobe (mean intensity=12.91; mean abundance=8.85). Specimens of H. malabaricus with a high intensity of infection in the brain displayed changes in swimming behavior.

Molecular Genetic Variability Of Commercial And Wild Accessions Of Passion Fruit (passiflora Spp.) Targeting Ex Situ Conservation And Breeding.

Passiflora species are distributed throughout Latin America, and Brazil and Colombia serve as the centers of diversity for this genus. We performed cross-species amplification to evaluate 109 microsatellite loci in 14 Passiflora species and estimated the diversity and genetic structure of Passiflora cincinnata, Passiflora setaceae and Passiflora edulis. A total of 127 accessions, including 85 accessions of P. edulis, a commercial species, and 42 accessions of 13 wild species, were examined. The cross-species amplification was effective for obtaining microsatellite loci (average cross-amplification of 70%). The average number of alleles per locus (five) was relatively low, and the average diversity ranged from 0.52 in P. cincinnata to 0.32 in P. setacea. The Bayesian analyses indicated that the P. cincinnata and P. setacea accessions were distributed into two groups, and the P. edulis accessions were distributed into five groups. Private alleles were identified, and suggestions for core collections are presented. Further collections are necessary, and the information generated may be useful for breeding and conservation.

Bartonella Spp. Bacteremia In Blood Donors From Campinas, Brazil.

Bartonella species are blood-borne, re-emerging organisms, capable of causing prolonged infection with diverse disease manifestations, from asymptomatic bacteremia to chronic debilitating disease and death. This pathogen can survive for over a month in stored blood. However, its prevalence among blood donors is unknown, and screening of blood supplies for this pathogen is not routinely performed. We investigated Bartonella spp. prevalence in 500 blood donors from Campinas, Brazil, based on a cross-sectional design. Blood samples were inoculated into an enrichment liquid growth medium and sub-inoculated onto blood agar. Liquid culture samples and Gram-negative isolates were tested using a genus specific ITS PCR with amplicons sequenced for species identification. Bartonella henselae and Bartonella quintana antibodies were assayed by indirect immunofluorescence. B. henselae was isolated from six donors (1.2%). Sixteen donors (3.2%) were Bartonella-PCR positive after culture in liquid or on solid media, with 15 donors infected with B. henselae and one donor infected with Bartonella clarridgeiae. Antibodies against B. henselae or B. quintana were found in 16% and 32% of 500 blood donors, respectively. Serology was not associated with infection, with only three of 16 Bartonella-infected subjects seropositive for B. henselae or B. quintana. Bartonella DNA was present in the bloodstream of approximately one out of 30 donors from a major blood bank in South America. Negative serology does not rule out Bartonella spp. infection in healthy subjects. Using a combination of liquid and solid cultures, PCR, and DNA sequencing, this study documents for the first time that Bartonella spp. bacteremia occurs in asymptomatic blood donors. Our findings support further evaluation of Bartonella spp. transmission which can occur through blood transfusions.

Prevalence Of Treponema Spp. In Endodontic Retreatment-resistant Periapical Lesions.

This study investigated the presence of the Treponema species in longstanding endodontic retreatment-resistant lesions of teeth with apical periodontitis, the association of this species with clinical/radiographic features, and the association among the different target species. Microbial samples of apical lesions were collected from twenty-five adult patients referred to endodontic surgery after unsuccessful root canal retreatment. Nested-PCR and conventional PCR were used for Treponema detection. Twenty-three periradicular tissue samples showed detectable levels of bacterial DNA. Treponema species were detected in 28% (7/25) of the cases. The most frequently detected species were T. socranskii (6/25), followed by T. maltophilum (3/25), T. amylovorum (3/25), T. lecithinolyticum (3/25), T. denticola (3/25), T. pectinovorum (2/25) and T. medium (2/25). T. vicentii was not detected in any sample. Positive statistical association was found between T. socranskii and T. denticola, and between T. maltophilum and T. lecithinolyticum . No association was detected between the presence of any target microorganism and the clinical or radiographic features. Treponema spp. are present, in a low percentage, in longstanding apical lesions from teeth with endodontic retreatment failure.