<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">In vivo</Emphasis> Antioxidant Properties of Extracts from <Emphasis Type="Italic">Coptis chinensis</Emphasis> Inflorescence

The inflorescence of cultivated Coptis chinensis has been valued for tea production for many years in China. The antioxidant activities of C. chinensis inflorescence extracts prepared by various solvents were investigated by using several established in vitro systems: 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS), α,α-diphenyl-β-picrylhydrazyl (DPPH) and superoxide radical scavenging assays, reducing power assay, and ferrothiocyanate (FTC) and thiobarbituric acid (TBA) assays. The results showed that the 70% ethanol extract (EE) had the strongest antioxidant activity in vitro among the various extracts. Based on the in vitro results, EE was used to evaluate the antioxidant activity of C. chinensis inflorescence in vivo. The liver and kidney of intoxicated animals showed a significant decrease in superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) levels, while the malondialdehyde (MDA) level showed a significant increase. These changes were significantly reversed after treatment with EE and the standard vitamin E. Thus, the C. chinensis inflorescence may be a valuable natural source that can be applicable to food industries.     

Comparative Study of Lipophilic and Hydrophilic Antioxidants from <Emphasis Type="Italic">In vivo</Emphasis> and <Emphasis Type="Italic">In vitro</Emphasis> Grown <Emphasis Type="Italic">Coriandrum sativum</Emphasis>

Coriander is commonly used for medicinal purposes, food applications, cosmetics and perfumes. Herein, the production of antioxidants in vegetative parts (leaves and stems) of in vivo and in vitro grown samples was compared. In vitro samples were clone A- with notorious purple pigmentation in stems and leaves and clone B- green. Seeds were also studied as they are used to obtain in vivo and in vitro vegetative parts. Lipophilic (tocopherols, carotenoids and chlorophylls) and hydrophilic (sugars, ascorbic acid, phenolics, flavonols and anthocyanins) compounds were quantified. The antioxidant activity was evaluated by radical scavenging activity, reducing power and lipid peroxidation inhibition. The in vivo sample showed the highest antioxidant activity mainly due to its highest levels of hydrophilic compounds. Otherwise, in vitro samples, mainly clone A, gave the highest concentration in lipophilic compounds but a different profile when compared to the in vivo sample. Clones A and B revealed a lack of β-carotene, β- and δ-tocopherols, a decrease in α-tocopherol, and an increase in γ-tocopherol and clorophylls in comparison to the in vivo sample. In vitro culture might be useful to explore the plants potentialities for industrial applications, controlling environmental conditions to produce higher amounts of some bioactive products.     

Rooting Characteristics of <Emphasis Type="Italic">Solanum chacoense</Emphasis> and <Emphasis Type="Italic">Solanum tuberosum in Vitro</Emphasis>

The precise level of environmental control in vitro may aid in identifying genetically superior plant germplasm for rooting characteristics (RC) linked to increased foraging for plant nitrogen (N). The objectives of this research were to determine the phenotypic variation in root morphological responses of 49 Solanum chacoense (chc), 30 Solanum tuberosum Group Phureja – Solanum tuberosum Group Stenotomum (phu-stn), and three Solanum tuberosum (tbr) genotypes to 1.0 and 0.5 N rate in vitro for 28 d, and identify genotypes with superior RC. The 0.5 N significantly increased density of root length, surface area, and tips. All RC were significantly greater in chc than in either phu-stn or tbr. Based upon clustering on root length, surface area, and volume, the cluster with the greatest rooting values consisted of eight chc genotypes that may be utilized to initiate a breeding program to improve RC in potato.     

Bioactivity Improvement of <Emphasis Type="Italic">Olea europaea</Emphasis> Leaf Extract Biotransformed by <Emphasis Type="Italic">Wickerhamomyces anomalus</Emphasis> Enzymes

Olive leaves represent a quantitatively significant by-product of agroindustry. They are rich in phenols, mainly oleuropein, which can be hydrolyzed into several bioactive compounds, including hydroxytyrosol. In this study, water extract from olive leaves ‘Biancolilla’ was analyzed for polyphenol profile, DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity and protective effect on differentiated Caco-2 cells. The efficacy of two enzymatic treatments in promoting the release of bioactive phenols was investigated: a) enzymatic extract from Wickerhamomyces anomalus, characterized by β-glucosidase and esterase activities; b) commercial β-glucosidase. Composition and bioactivity of the resulting extracts were compared. The results showed that the yeast-treated extract presented hydroxytyrosol content and DPPH radical scavenging activity comparable to those obtained using commercial β-glucosidase; however, it was showed the additional presence of hydroxycinnamic acids. In experiments on Caco-2 cells, the leaf extracts promoted the recovery of cell membrane barrier at different minimum effective concentrations. The high specificity of W. anomalus enzymatic extract may represent an effective tool for the release of bioactive phenols from olive by-products.     

<Emphasis Type="Italic">In vitro</Emphasis> Antioxidant Activity of Aged Extracts of some Italian <Emphasis Type="Italic">Allium</Emphasis> Species

Antioxidant activity of fresh Allium sativum L. (garlic) is well known and is mainly due to unstable and irritating organosulphur compounds. Fresh garlic extracted over a prolonged period (up to 20 months) produces odourless aged garlic extract (AGE) containing stable and water soluble organosulphur compounds that prevent oxidative damage by scavenging free radicals. The aim of this study was to investigate the in vitro antioxidant activity of aged (up to 20 months) 15% hydroethanolic extracts of different parts (bulbs, bulblets, flower bulblets, flowers, and leaves) of three Allium spontaneous species which are endemic for Italian flora: Allium neapolitanum Cyr., Allium subhirsutum L., Allium roseum L. and to compare it with the in vitro antioxidant activity of aged 15% hydroethanolic extracts of bulbs and leaves of garlic. The antioxidant potential of aged extracts of all species has been evaluated using two different spectrophotometric assays: 2,2-diphenylpicrylhydrazyl (DPPH) test and the ferric reducing/antioxidant power (FRAP) assay. Furthermore the polyphenol content was determined. The aged extracts obtained from the leaves showed the best antioxidant activity, followed by flowers and then by bulbs in both used tests, while flower bulblets and bulblets exhibited lower results or no activity. The polyphenol content was generally directly correlated with antioxidant/antiradical activity. This study confirms the data obtained in previous researches, the wild-type species of Allium and in particular organs other than bulbs are more active and efficacious than garlic bulb. Surely leaves of these Allium spp. deserve special attention.     

Hypolipidemic Effect of Avocado (<Emphasis Type="Italic">Persea americana</Emphasis> Mill<Emphasis Type="Italic">)</Emphasis> Seed in a Hypercholesterolemic Mouse Model

Avocado seed contains elevated levels of phenolic compounds and exhibits antioxidant properties. We investigated the effect of Avocado Seed Flour (ASF) on the lipid levels in mice on a hyperlipidemic diet. The concentration of phenols was determined by high-performance liquid chromatography, antioxidant activity was evaluated using the Trolox equivalent antioxidant capacity method, and dietary fiber was measured using the Association of Official Analytical Chemists (AOAC) method. The LD₅₀ of ASF was determined using Lorke’s method and hypolipidemic activity was evaluated in a hypercholesterolemic model in mice. Protocatechuic acid was the main phenolic compound found in ASF, followed by kaempferide and vanillic acid. The total phenolic content in the methanolic extract of ASF was 292.00 ± 9.81 mg gallic acid equivalents/g seed dry weight and the antioxidant activity resulted in 173.3 μmol Trolox equivalents/g DW. In addition, a high content of dietary fiber was found (34.8%). The oral LD₅₀ for ASF was 1767 mg/kg body weight, and treatment with ASF significantly reduced the levels of total cholesterol, LDL-C, and prediction of the atherogenic index. Therefore, the antioxidant activity of phenolic compounds and dietary fiber in ASF may be responsible for the hypocholesterolemic activity of ASF in a hyperlipidemic model of mice.     

Divergent evolution of rice blast resistance <Emphasis Type="Italic">Pi54</Emphasis> locus in the genus <Emphasis Type="Italic">Oryza</Emphasis>

Background

The rice blast resistance gene Pi54 was cloned from Oryza sativa ssp. indica cv. Tetep, which conferred broad-spectrum resistance against Magnaporthe oryzae. Pi54 allelic variants have been identified in not only domesticates but also wild rice species, but the majority of japonica and some indica cultivars lost the function.

Results

We here found that Pi54 (Os11g0639100) and its homolog Os11g0640600 (named as #11) were closely located on a 25 kbp region in japonica cv. Sasanishiki compared to a 99 kbp region in japonica cv. Nipponbare. Sasanishiki lost at least six genes containing one other R-gene cluster (Os11g0639600, Os11g0640000, and Os11g0640300). Eight AA-genome species including five wild rice species were classified into either Nipponbare or Sasanishiki type. The BB-genome wild rice species O. punctata was Sasanishiki type. The FF-genome wild rice species O. brachyantha (the basal lineage of Oryza) was neither, because Pi54 was absent and the orientation of the R-gene cluster was reversed in comparison with Nipponbare-type species. The phylogenetic analysis showed that #11gene of O. brachyantha was on the root of both Pi54 and #11 alleles. All Nipponbare-type Pi54 alleles were specifically disrupted by 143 and 37/44?bp insertions compared to Tetep and Sasanishiki type. In addition, Pi54 of japonica cv. Sasanishiki lost nucleotide-binding site and leucine-rich repeat (NBS–LRR) domains owing to additional mutations.

Conclusions

These results suggest that Pi54 might be derived from a tandem duplication of the ancestor #11 gene in progenitor FF-genome species. Two divergent structures of Pi54 locus caused by a mobile unit containing the nearby R-gene cluster could be developed before domestication. This study provides a potential genetic resource of rice breeding for blast resistance in modern cultivars sustainability.

     

Proteomic analysis of the defense response to <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> in rice harboring the blast resistance gene <Emphasis Type="Italic">Piz-t</Emphasis>

Background

Rice blast (caused by Magnaporthe oryzae) is one of the most destructive diseases of rice. While many blast resistance (R) genes have been identified and deployed in rice cultivars, little is known about the R gene-mediated defense mechanism. We used a rice transgenic line harboring the resistance gene Piz-t to investigate the R gene-mediated resistance response to infection.

Results

We conducted comparative proteome profiling of the Piz-t transgenic Nipponbare line (NPB-Piz-t) and wild-type Nipponbare (NPB) inoculated with M. oryzae at 24, 48, 72 h post-inoculation (hpi) using isobaric tags for relative and absolute quantification (iTRAQ) analysis. Comparative analysis of the response of NPB-Piz-t to the avirulent isolate KJ201 and the virulent isolate RB22 identified 114 differentially expressed proteins (DEPs) between KJ201-inoculated NPB-Piz-t (KJ201-Piz-t) and mock-treated NPB-Piz-t (Mock-Piz-t), and 118 DEPs between RB22-inoculated NPB-Piz-t (RB22-Piz-t) and Mock-Piz-t. Among the DEPs, 56 occurred commonly in comparisons KJ201-Piz-t/Mock-Piz-t and RB22-Piz-t/Mock-Piz-t. In a comparison of the responses of NPB and NPB-Piz-t to isolate KJ201, 93 DEPs between KJ201-Piz-t and KJ201-NPB were identified. DEPs in comparisons KJ201-Piz-t/Mock-Piz-t, RB22-Piz-t/Mock-Piz-t and KJ201-Piz-t/KJ201-NPB contained a number of proteins that may be involved in rice response to pathogens, including pathogenesis-related (PR) proteins, hormonal regulation-related proteins, defense and stress response-related proteins, receptor-like kinase, and cytochrome P450. Comparative analysis further identified 7 common DEPs between the comparisons KJ201-Piz-t/KJ201-NPB and KJ201-Piz-t/RB22-Piz-t, including alcohol dehydrogenase I, receptor-like protein kinase, endochitinase, similar to rubisco large subunit, NADP-dependent malic enzyme, and two hypothetical proteins.

Conclusions

Our results provide a valuable resource for discovery of complex protein networks involved in the resistance response of rice to blast fungus.

     

An <Emphasis Type="Italic">In Vitro</Emphasis> Assay Method for Resistance to Bacterial Wilt (<Emphasis Type="Italic">Ralstonia solanacearum</Emphasis>) in Potato

To develop an in vitro assay method for bacterial wilt resistance in potato, resistant and susceptible standard genotypes were grown in vitro, and different conditions of inoculation with Ralstonia solanacearum phylotype I/biovar 4 were examined. The optimal condition was the inoculation of 6–8 leaf stage plants with a bacterial concentration of 10² CFU ml^?1 and an incubation temperature of 28 °C. Evaluation of stem wilting was more reliable than that of leaf wilting. Using this method, nine genotypes with different resistance levels in the field were evaluated. Lower disease indices were obtained for genotypes with high resistance levels in the field, suggesting that this assay is useful for evaluating bacterial wilt resistance in a controlled environment.     

Maturity-Adjusted Resistance of Potato (<Emphasis Type="Italic">Solanum tuberosum</Emphasis> L.) Cultivars to Verticillium Wilt Caused by <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

Verticillium wilt is a fungal disease of potato caused by two species of Verticillium, V. dahliae and V. albo atrum. The pathogen infects the vascular tissue of potato plants through roots, interfering with the transport of water and nutrition, and reducing both the yield and quality of tubers. We have evaluated the reaction of 283 potato clones (274 cultivars and nine breeding selections) to inoculation with V. dahliae under greenhouse conditions. A significant linear correlation (r = 0.4, p < 0.0001) was detected between plant maturity and partial resistance to the pathogen, with late maturing clones being generally more resistant. Maturity-adjusted resistance, that takes into consideration both plant maturity and resistance, was calculated from residuals of the linear regression between the two traits. Even after adjusting for maturity, the difference in the resistance of clones was still highly significant, indicating that a substantial part of resistance cannot be explained by the effect of maturity. The highest maturity-adjusted resistance was found in the cv. Navajo, while the most susceptible clone was the cv. Pungo. We hope that the present abundance of data about the resistance and maturity of 283 clones will help potato breeders to develop cultivars with improved resistance to V. dahliae.     

Seasonal deviation effects foliar endophyte assemblage and diversity in <Emphasis Type="Italic">Asparagus racemosus</Emphasis> and <Emphasis Type="Italic">Hemidesmus indicus</Emphasis>

Background

Fungal endophytes are the living symbionts which cause no apparent damage to the host tissue. The distribution pattern of these endophytes within a host plant is mediated by environmental factors. This study was carried out to explore the fungal endophyte community and their distribution pattern in Asparagus racemosus and Hemidesmus indicus growing in the study area.

Results

Foliar endophytes were isolated for 2 years from A. racemosus and H. indicus at four different seasons (June–August, September–November, December–February, March–May). A total of 5400 (675/season/year) leaf segments harbored 38 fungal species belonging to 17 genera, 12 miscellaneous mycelia sterile from 968 isolates and 13 had yeast like growth. In A. racemosus, Acremonium strictum and Phomopsis sp.1, were dominant with overall relative colonization densities (RCD) of 7.11% and 5.44% respectively, followed by Colletotrichum sp.3 and Colletotrichum sp.1 of 4.89% and 4.83% respectively. In H. indicus the dominant species was A. strictum having higher overall RCD of 5.06%, followed by Fusarium moniliforme and Colletotrichum sp.2 with RCD of 3.83% and 3%, respectively. Further the overall colonization and isolation rates were higher during the wet periods (September–November) in both A. racemosus (92.22% and 95.11%) and H. indicus (82% and 77.11%).

Conclusion

Study samples treated with 0.2% HgCl₂ and 75% EtOH for 30 s and 1 min, respectively, confirmed most favorable method of isolation of the endophytes. Owing to high mean isolation and colonization rates, September–November season proved to be the optimal season for endophyte isolation in both the study plants. Assessing the bioactive potential of these endophytes, may lead to the isolation of novel natural products and metabolites.

     

Comparative Study on the Chemical Composition,Antioxidant Properties and Hypoglycaemic Activities of Two <Emphasis Type="Italic">Capsicum annuum</Emphasis> L. Cultivars (<Emphasis Type="Italic">Acuminatum</Emphasis> small and <Emphasis Type="Italic">Cerasiferum</Emphasis>)

The present study aimed to evaluate for the first time the phenols, flavonoids, carotenoids, capsaicin and dihydrocapsaicin content and the antioxidant and hypoglycemic properties of Capsicum annuum var. acuminatum small and C. annuum var. cerasiferum air-dried fruits. The ethanol extract of C. annuum var. acuminatum small, characterized by the major content of total poliphenols, flavonoids, carotenoids and capsaicinoids, showed the highest radical scavenging activity (IC₅₀ of 152.9 μg/ml). On the contrary, C. annuum var. cerasiferum showed a significant antioxidant activity evaluated by the β-carotene bleaching test (IC₅₀ of 3.1 μg/ml). The lipophilic fraction of both C. annuum var. acuminatum and C. annuum var. cerasiferum exhibited an interesting and selective inhibitory activity against α-amylase (IC₅₀ of 6.9 and 20.1 μg/ml, respectively).     

Competition between the invasive macrophyte <Emphasis Type="Italic">Caulerpa taxifolia</Emphasis> and the seagrass <Emphasis Type="Italic">Posidonia oceanica</Emphasis>: contrasting strategies

Background  

Plant defense strategy is usually a result of trade-offs between growth and differentiation (i.e. Optimal Defense Theory – ODT, Growth Differentiation Balance hypothesis – GDB, Plant Apparency Theory – PAT). Interaction between the introduced green alga Caulerpa taxifolia and the endemic seagrass Posidonia oceanica in the Mediterranean Sea offers the opportunity to investigate the plausibility of these theories. We have accordingly investigated defense metabolite content and growth year-round, on the basis of an interaction gradient.     

The Low Potential of Teff (<Emphasis Type="Italic">Eragrostis tef</Emphasis>) as an Inoculum Source for <Emphasis Type="Italic">Verticillium dahliae</Emphasis>

Teff (Eragrostis tef) is a fine stemmed annual grass and gluten free small grain that is of interest as a forage, cover, or a rotation crop. Little is known about the susceptibility of teff to many diseases. Teff could be grown in rotation with potato in the northwestern United States provided teff cultivation is economical and does not increase soil populations for pathogens affecting rotation crops such as Verticillium dahliae. Verticillium dahliae infects a wide range of dicotyledonous plants, making it one of the most important fungal pathogens of crop plants in North America, including potato. The objective of this study was to quantify the susceptibility of teff to eight V. dahliae isolates and compare the susceptibility of teff to eggplant. Teff was confirmed as a host for V. dahliae, as indicated by the presence of microsclerotia in teff stems and roots after artificial inoculation in two years of greenhouse studies. The number of microsclerotia produced in teff did not differ between mint and potato pathotypes of V. dahliae. No V. dahliae isolate produced significantly greater numbers of microsclerotia than any of the seven other isolates tested in a two-year study. Microsclerotia production of V. dahliae in teff was consistently less than in susceptible eggplant cv. Night shadow in both greenhouse experiments (P?<?0.02). It is unlikely that teff infected by V. dahliae will proliferate microsclerotia of mint or potato-aggressive pathotypes, especially when compared to susceptible eggplant cultivars.     

Bio-efficacy of the Essential Oil of Oregano (<Emphasis Type="Italic">Origanum vulgare</Emphasis> Lamiaceae. Ssp. <Emphasis Type="Italic">Hirtum</Emphasis>)

The aim of this study was to investigate the bioactivity of the essential oil isolated from Origanum vulgare L. (EOv). We analyzed the in vivo anti-inflammatory properties in a mouse-airway inflammation model and the in vitro antimicrobial activity, genotoxicity over the anaphase-telophase with the Allium cepa strain and its cytotoxicity/viability in A549 culture cells. In vivo, EOv modified the levels of tumor necrosis factor -α and viable activated macrophages and was capable to mitigate the effects of degradation of conjugated dienes. In vitro, EOv reduced the viability of cultured A549 cells as well as the mitotic index and a number of chromosomal aberrations; however, it did not change the number of phases. We found that EOv presents antimicrobial activity against different Gram (?) and (+) strains, measured by disc-diffusion test and confirmed with a more accurate method, the AutoCad software. We postulate that EOv presents antibacterial, antioxidant and chemopreventive properties and could be play an important role as bioprotector agent.     

Antioxidative and Anticancer Activity of Extracts of Cherry (<Emphasis Type="Italic">Prunus serrulata</Emphasis> var. <Emphasis Type="Italic">spontanea</Emphasis>) Blossoms

Organic solvent (methanol, ethanol, and acetone) extracts and water extracts of cherry (Prunus serrulata var. spontanea) blossoms were prepared, and antioxidant activities of the extracts were evaluated. Methanolic CBE (100 μg/ml) showed the highest total phenol content (104.30 μM), radical scavenging activity (34.2%), and reducing power (0.391). The effect of CBE on DNA damage induced by H₂O₂ in human leukocytes was evaluated by Comet assay. All CBE was a potent dose dependent inhibitor of DNA damage induced by 200 μM of H₂O₂, methanolic CBE showed the most strong inhibition activity. The methanolic CBE of 500 μg/ml showed 38.8% inhibition against growth of human colon cancer cell line HT-29. These results indicated that cherry blossoms could provide valuable bioactive materials.     

Enhanced <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f. sp. <Emphasis Type="Italic">tuberosi</Emphasis> Resistance in Transgenic Potato Expressing a Rice GLP Superoxide Dismutase Gene

Germin like proteins (GLPs) are a large group of related and ubiquitous plant proteins which are considered to be involved in different processes important for plant development and defense. Multiple functional copies of this gene family have been reported in a number of species (wheat, barley, rice, soybean mosses and liverwort), and their role is being evaluated by gene regulation studies and transgenic approaches. To analyze the role of a rice (Oryza sativa) root expressed germin like protein1 OsRGLP1, for its antifungal activity, transgenic potato plants were developed. These transgenic potato plants were molecularly characterized and biologically assessed after inoculation with Fusarium oxysporum f. sp. tuberosi. Functional analysis showed high accumulation of H₂O₂, increased Superoxide Dismutase (SOD) activity and no oxalate oxidase activity (OxO) in transgenics in comparison to nontransformed control. This increased SOD activity, resistance to heat and sensitivity to H₂O₂ suggest it is a Fe-like SOD. OsRGLP1 expression in potato plants exhibited enhanced resistance in comparison to nontransformed wild type plants suggesting its role in providing protection against Fusarium oxysporum f. sp. tuberosi through elevated SOD level. Overall, results suggest that OsRGLP1 is a candidate for the engineering of potato plants with increased fungal tolerance however, the greater height and tuber number was observed. This phenotype associated with the resistance needs to be evaluated to determine if this is a positive or negative feature.