Cell death induction and nitric oxide biosynthesis in white poplar (Populus alba) suspension cultures exposed to alfalfa saponins

The present work reports on the biological activity of alfalfa (Medicago sativa) saponins on white poplar (Populus alba, cultivar ‘Villafranca’) cell suspension cultures. The extracts from alfalfa roots, aerial parts and seeds were characterized for their saponin content by means of thin layer chromatography (TLC) and electrospray ionisation coupled to mass spectrometry. The quantitative saponin composition from the different plant extracts was determined considering the aglycone moieties and determined by gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) analyses. Only soyasapogenin I was detected in the seed extract while several other saponins were found in the root and leaf extracts. Actively proliferating white poplar cell cultures were challenged with the different saponin extracts. Only alfalfa root saponins, at 50 µg ml^?1, induced significant cell death rates (75.00 ± 4.90%). Different cell subpopulations with peculiar cell death morphologies were observed and the programmed cell death (PCD)/necrosis ratio was reduced at increasing saponin concentrations. Enhancement of nitric oxide (NO) production was observed in white poplar cells treated with root saponins (RSs) at 50 µg ml^?1 and release of reactive oxygen species (ROS) in the culture medium was also demonstrated. Saponin‐induced NO production was sensitive to sodium azide and N^G‐monomethyl‐l ‐arginine, two specific inhibitors of distinct pathways for NO biosynthesis in plant cells.     

Aframomum melegueta: a potential botanical pesticide against Sitotroga cerealella infestation on two paddy varieties

This study was conducted to investigate the potential of Aframomum melegueta leaf and seed as biopesticide against Sitotroga cerealella infestation on two paddy varieties at ambient temperature of 28 °C ± 2 °C and relative humidity of 75?±?5%. The two paddy varieties used were FARO 44 and FARO 52. Leaf and seed powders of A. melegueta were made at 0.1, 0.2, 0.4, 0.6 and 0.8 g while the extracts were made at 1, 2, 3, 4 and 5%. Mortality of the insect on the two paddy varieties was record after 24, 48 and 72?h of application of the powders and extracts of this plant. The highest number of the insect was observed on FARO 52 treated with seed powders and extracts of A. melegueta. Only the seed extracts were able to achieve 100% moth mortality within 72 h of application at 4% concentration and were significantly (p?<?0.05) different from others. The powders and extracts of the plant significantly reduced or prevented the adult emergence of the insect and increased their developmental period as well reduced or prevented seed weight loss with seed powders and extracts having the greatest effect on the paddy variety FARO 44. The antinutritional components present in the two paddy varieties include phytate, oxalate, total phenol and tannin cyanide, and the amylase content was also determined, with FARO 44 having the highest value of 17.31, 0.18, 0.43, 10.46 and 5.49 for phytate, oxalate, tannin, cyanide and amylase content, respectively. Powders and extracts of A. melegueta could be introduced into pest management techniques since they are effective against S. cerealella.     

Saponins from Swartzia langsdorffii: biological activities

The presence of saponins and the molluscicidal activity of the roots, leaves, seeds and fruits of Swartzia langsdorffii Raddi (Leguminosae) against Biomphalaria glabrata adults and eggs were investigated. The roots, seeds and fruits were macerated in 95% ethanol. These extracts exerted a significant molluscicidal activity against B. glabrata, up to a dilution of 100 mg/l. Four mixtures (A2, B2, C and D) of triterpenoid oleanane type saponins were chromatographically isolated from the seed and fruit extracts. Two known saponins (1 and 2) were identified as beta-D-glucopyranosyl-[alpha-L-rhamnopyranosyl-(1->3)- beta-D-glucuronopyranosyl-(1->3)]-3beta-hydroxyolean-12-ene-28 -oate, and beta-D-glucopyranosyl-(1->3)-beta-D-glucuronopyranosyl-(1 ->3)]-3beta-hydroxyolean-12-ene-28-oate, respectively. These two saponins were present in all the mixtures, together with other triterpenoid oleane type saponins, which were shown to be less polar, by reversed-phase HPLC. The saponin identifications were based on spectral evidence, including H- H two-dimensional correlation spectroscopy, nuclear Overhauser and exchange spectroscopy, heteronuclear multiple quantum coherence, and heteronuclear multiple-bond connectivity experiments. The toxicity of S. langsdorffii saponins to non-target organisms was prescreened by the brine shrimp lethality test.     

Phytochemical Profiling,in Vitro Biological Activities and in Silico Molecular Docking Studies of the Crude Extract of Crambe orientalis,an Endemic Plant in Turkey

In this study, total phenolic and flavonoid analyses of flower, leaf, and stem aqueous extracts of C. orientalis were performed. Total phenolic contents of C. orientalis extracts ranged from 12.2±0.06 to 19.0±0.07 mg GAE/g extract. Total flavonoid values range between 2.0±0.11 and 6.6±0.19 mg CE/g extract. Urease, collagenase, tyrosinase, and α-glucosidase inhibition activities were determined in vitro and the relationship between them was examined. IC₅₀ results for all enzymes were obtained between 0.18 and 3.53 μg/mL. The aqueous extract of the plant C. orientalis showed potent cytotoxic effects against the human colon cancer cell lines DLD-1. As the extract concentration increases, cell death increases. The main fatty acid composition by GC/MS analysis is erucic acid (36.5 %). The potential binding modes of the fatty acids in the plant extract to the enzymes and possible inhibition mechanisms were determined by molecular docking calculation.     

Analysis of volatile aroma constituents of wine produced from Indian mango (<Emphasis Type="Italic">Mangifera indica</Emphasis> L.) by GC-MS

Volatile aroma compounds are synthesized by wine yeast during wine fermentation. In this study the volatile aroma composition of two varieties of mango wine were determined to differentiate and characterize the wines. The wine was produced from the fruits of two varieties of mango cultivars namely Banginapalli and Alphonso. The volatile compounds formed in mango wine were analyzed by gas chromatography coupled with mass spectrometry (GC-MS). Thirty-two volatile compounds in wines were determined of which four were new and unidentified present in lower concentration. Apart from the ethanol (8.5 ± 0.28 and 7.2 ± 0.28% v/v), 1-propanol (54.11 ± 0.33 and 42.32 ± 0.57 mg/l), isobutyl alcohol (102 ± 1.57 and 115.14 ± 2.88 mg/l) and isoamyl alcohol (123 ± 2.88 and 108.40 ± 0.23 mg/1) were found to be the major flavouring higher alcohols in the mango wines produced from the fruits of Banginapalli and Alphonso respectively. Ethyl acetate (35 ± 0.57 and 30.42 ±1.15 mg/l) was the major ester component in both wines produced. Besides, other esters like ethyl octonoate, ethyl hexanoate and ethyl decanoate were also present in the wines. Cyclohexane methanol (1.45 ± 0.11 mg/l) was present only in wine made from Banginapalli and β-phenylethyl butanoate (0.62 ± 0.01 mg/1) was found only in Alphonso wine. The results demonstrate that the wine prepared from Banginapalli variety had better aroma composition and good taste than that from the Alphonso variety.     

O‐glycoside sequence of pentacyclic triterpene saponins from Phytolacca bogotensis using HPLC‐ESI/multi‐stage tandem mass spectrometry

Introduction – The lack of pharmacopoeial methodologies for the quality control of plants used for therapeutic purposes is a huge problem that impacts directly upon public health. In the case of saponins, their great structural complexity, weak glycoside bonds and high polarity hinder their identification by conventional techniques. Objective – To apply high‐performance liquid chromatography–electrospray tandem mass spectrometry (HPLC‐ESI/MSⁿ) to identify the O‐glycoside sequence of saponins from the roots of Phytolacca bogotensis. Methodology – Saponins were isolated by preparative HPLC and characterised by NMR spectroscopic experiments. Collision‐induced dissociation (CID) of isolated saponins was performed producing typical degradation reactions that can be associated with several glycosidic bonds as empirical criteria. A method using solid‐phase extraction (SPE) and HPLC/ESI‐MSⁿ for the characterisation of saponins and identification of novel molecules is described. Results – Three saponins reported for the first time in P. bogotensis were isolated and characterised by NMR spectroscopy. Characteristic cross ring cleavage reactions have been used as empirical criteria for the characterisation of the glycosidic bonds most frequently reported for Phytolacca saponins. One new saponin was proposed on the basis of empirical criteria, and other five saponins were identified for the first time for P. bogotensis using HPLC‐ESI/MSⁿ. Conclusion – Electrospray ionisation in combination with tandem mass spectrometry has been established as a powerful tool for the profiling of saponins from roots of P. bogotensis. CID proved to be a useful tool for the characterisation and identification of known and novel saponins from the plant family Phytolaccaceae and can be used for quality control purposes of crude plant extracts. Copyright © 2009 John Wiley & Sons, Ltd.     

Effect on lycopene, β-carotene,ascorbic acid and phenolic content in tomato fruits infected by Alternaria alternata and its toxins (TeA,AOH and AME)

Tomato is considered as one of the most important sources of nutrients such as lycopene, β-carotene, flavonoids, ascorbic acid (vitamin C) and hydroxyl-cinnamic acid derivatives. The quality and quantity of nutrients in tomato fruits were decreased during the severe infection of Alternaria alternata. The present study deals with the estimation of lycopene, β-carotene, phenolic and ascorbic acid content in tomato fruits which were infected with A. alternata and its toxins such as tenuazonic acid (TeA), alternariol (AOH) and alternariol monomethyl ether (AME). The lycopene, β-carotene, ascorbic acid and phenolic content were found lowest in pathogen-infected fruits i.e. (0.66 ± 0.03 mg/g), (0.14 ± 0.01 mg/g), (1.89 ± 0.2 mg/g) and (0.58 ± 0.05 mg/g), respectively, followed by toxins-treated samples as compared to the control. The results concluded that A. alternata mostly affects the nutritional values of tomato fruits due to the combined effect of the toxins.     

Identification of quantitative trait loci for specific mechanisms of resistance to <Emphasis Type="Italic">Orobanche crenata</Emphasis> Forsk. in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

Crenate broomrape (Orobanche crenata) is the major constraint for pea cultivation in the Mediterranean Basin and Middle East. Cultivation of resistant varieties would be the most efficient, economical and environmentally friendly way to control this parasite. However, little resistance is available within cultivated pea. Promising sources of resistance have been identified in wild peas but their use in breeding programs is hampered by the polygenic nature of the resistance. The identification of molecular markers linked to the resistance would allow tracking of the underlying genes, facilitating their introgression into pea cultivars. The main objective of this study was the identification of genomic regions associated with resistance to O. crenata. A RIL (Recombinant Inbred Lines) population derived from a cross between a resistant accession of the wild pea Pisum sativum ssp. syriacum, and a susceptible pea variety was screened for resistance to O. crenata under field conditions during two seasons. In addition, resistance reactions at different stages of the O. crenata infection cycle were assessed using a Petri dish method. The approach allowed the identification of four Quantitative Trait Loci (QTL) associated with field resistance, assessed as the number of emerged broomrape shoots per pea plant under field conditions. These identified QTLs explained individually from 10 to 17% of the phenotypic variation. In addition QTLs governing specific mechanisms of resistance, such as low induction of O. crenata seed germination, lower number of established tubercles per host root length unit, and slower development of tubercles were also identified. Identified QTLs explained individually from 8 to 37% of the variation observed depending on the trait. Host plant aerial biomass and root length were also assessed and mapped. Both traits were correlated with the level of O. crenata infection and three out of the four QTLs controlling resistance under field conditions co-localized with QTLs controlling plant aerial biomass or root length. The relationship observed among these traits and resistance is discussed.     

Composition, antimicrobial and antioxidant activity of the extracts of Eryngium palmatum Pančić and Vis. (Apiaceae)

The chemical composition, antimicrobial and antioxidant activity of Eryngium palmatum, an endemic plant species from the Balkan Peninsula, were investigated. The flavonoids apigenin (9.5±0.3 mg g^?1) and apigenin 7-O-glucoside (2.4±0.1 mg g^?1) were determined in a methanol extract of aerial parts using HPLC analysis. The methanol extract of roots contained catechin (5.0±0.1 mg g^?1), epicatechin (2.9±0.1 mg g^?1), chlorogenic acid (1.6±0.0 mg g^?1), gallic acid (0.9±0.0 mg g^?1) and rosmarinic acid (0.9±0.2 mg g^?1). GC-FID and GCMS analysis of a chloroform extract of aerial parts showed that the main volatile constituents were falcarinol, linoleic acid, hexadecanoic acid and methyl linoleate (comprising 32.6%; 24.4%; 19.9; 13.2% of the volatile fraction, respectively), while octanoic acid, tetradecanol and dodecanol dominated in the chloroform extract of the roots (34.9%; 25.8%; 22.2% of the volatile fraction, respectively). Investigation of antimicrobial activity by broth microdilution showed that the methanol and chloroform extracts of aerial parts and roots exerted a significant effect (MIC 3.5–15.6 μg mL^?1) against tested Gram-positive and Gram-negative bacteria. The methanol extracts of aerial parts or roots exerted moderate ferric reducing antioxidant power, DPPH radical scavenging activity and hydroxyl radical scavenging activity.     

Relationship between Maturity Stage,Triterpenoid Content and Cytotoxicity of Sorbus intermedia (EHRH.) PERS. Fruits – A Chemometric Approach

In this study, two ursane-type triterpenes, i. e., ursolic acid (UA) and 3-O-β-acetoxyursolic acid (AUA), were isolated and quantified in Sorbus intermedia (EHRH.) PERS. fruits. UA and AUA levels differed slightly throughout fruit maturation, and both triterpenes showed similar dynamics of accumulation with the highest levels found in ripe fruits (up to 6.33±0.56 and 1.17±0.18 mg/g dw. of UA and AUA, respectively). The extracts derived from unripe fruits displayed significant cytotoxic effects against WM793, DU-145, PC3, 8505C, Caco2 and A549 cells, but no correlation between UA and AUA levels and cytotoxicity was found. On the other hand, extracts from mature fruits were not active towards almost all of the tested cell lines. The chemometric approach showed that the extracts derived from fruits harvested earlier clustered to form a clearly distanced group from extracts prepared from more-mature fruits. The extracts at higher concentrations formed separate groups, which indicated the concentration-dependent effect of these extracts on the cells.     

Flavonoid content and antioxidant activity of Artemisia vulgaris L. “hairy” roots

We investigated the effect of Agrobacterium rhizogenes-mediated transformation on antioxidant activity of Artemisia vulgaris “hairy” roots. It appeared that transformation may increase flavonoid content as well as DPPH-scavenging activity and ability to reduce Fe³⁺ as compared to the non-transformed plants. Some “hairy” roots accumulated flavonoids up to 73.1?±?10.6?mg RE/g DW (while the amount of flavonoids in the leaves of non-transformed plants was up to 49.4?±?5.0?mg RE/g DW). DPPH-scavenging activity of some “hairy” root lines was 3–3.8 times higher than such one of the roots of the control plants. The Fe³⁺-reducing power of most transgenic root extracts exceeded such power of the extracts of the roots of the control plants. The decrease in SOD activity was found in the most “hairy” root lines compared to the control roots. The increase of flavonoid content correlated with the increase of ability of extracts to scavenge DPPH*- radical and Fe³⁺ - reducing power. No correlation between SOD activity of extracts and concentration of flavonoids was found (p?≥?0.2).Thus, transformation has led to the alteration in flavonoid accumulation and antioxidant activity in A. vulgaris “hairy” roots. Transgenic roots with high-antioxidant properties can be selected after A. rhizogenes-mediated transformation.     

Two New Triterpenoid Saponins Cytotoxic to Human Glioblastoma U251MG Cells from Ardisia pusilla

Two new triterpenoid saponins, ardipusillosides IV and V ( 1 and 2 , resp.), together with one known saponin, ardisiacrispin B ( 3 ), were isolated from the whole plants of Ardisia pusilla A. DC. Their structures were deduced by extensive spectral analysis and chemical evidences. Compound 1 contains a glycosylated glycerol residue which is a very rare structural feature among triterpenoid glycosides and has been so far found only in the genus Ardisia. All the saponins exhibited significant cytotoxicity against human glioblastoma U251MG cells, but did not affect the growth of primary cultured human astrocytes.     

The allelopathic potential of alfalfa root medicagenic acid glycosides and their fate in soil environments

Summary The allelopathic effect of alfalfa (Medicago media Pers.) and red clover (Trifolium pratense L.) root saponins on winter wheat seedling growth and the fate of these chemicals in soil environments were studied. Seed germination, seedling and test fungus growth were suppressed by water and by alcohol extracts of alfalfa roots, and by crude saponins of alfalfa roots, indicating that medicagenic acid glycosides are the inhibitor. Powdered alfalfa roots inhibited wheat seedling growth when added to sand. At concentrations as low as 0.25% (w/w) the root system was completely destroyed whereas seedling shoots suffered little damage. Red clover roots caused some wheat growth inhibition when incorporated to sand, but their effect was much lower than in the alfalfa root treatment. Soil textures had a significant influence on the inhibitory effect of alfalfa roots. The inhibition of seedling growth was more pronounced on light than on heavy soils. This was attribted to the higher sorption of inhibitors by heavy soils. Incubation of alfalfa roots mixed into loose sand, coarse sand, loamy sand and clay loam for a period of 0–8 days resulted in decreased toxicity to bothT. viride and wheat seedlings. This decrease occurred more quickly in heavier soils than in loose sand, due to the hydrolysis of glycosides by soil microorganisms. Soil microbes were capable of detoxifying medicagenic acid glycosides by partial hydrolysis of sugar chain to aglycone. These findings illustrate the importance of medicagenic acid glycosides as an inhibitor of wheat seedling growth, and of their fate in different soil environments.     

A Comprehensive Search of the Primary and Secondary Metabolites and Radical Scavenging Potential of Trillium govanianum Wall. ex D. Don

Trillium govanianum rhizomes are traditionally consumed as a raw powder and decoction for the treatment of health complications. Hence, the present study aimed to investigate whether aqueous and alcoholic extracts of T. govanianum rhizomes under hot and cold extraction conditions have similar or dissimilar chemical, nutrient, and antioxidant profiles. The total phenolics, flavonoids, carbohydrates, proteins, fats, and energy values were estimated in all the conditionally prepared samples. The total phenolics (21.23±1.4 mg GAE/g extract), flavonoids (70.57±3.24 mg RE/g extract) were found higher in hot ethanolic extract (TGHEt), while cold water extract (TGGC) showed higher nutrients including amino acids (10.545±0.219 mg/g) and nucleosides (1.803±0.018 mg/g). The nutrient energy value (2.60 and 2.49 Kcal/g extract) was higher in cold and hot ethanolic extracts. Further, TGHEt scavenged the DPPH^. (IC₅₀; 870±22 μg/mL) and ABTS^.+ (IC₅₀; 80±1.49 μg/mL) effectively and proved its highest antioxidant activity compared to other samples. In LC/MS/MS-based metabolite profiling, twenty-six metabolites (fatty acids, steroidal saponins, triterpene saponins, ecdysteroid hormones) were confirmed with mass fragmentation and literature, while one hundred nine metabolites were identified using the METLIN database. The principal component analysis showed clustering of hot condition extracts while cold extracts were differentially located in quadrants. The heatmaps exhibited the associations and differences between metabolite composition, solvents, and extraction conditions. The identified metabolites speculatively predicted the biosynthesis pathway of T. govanianum. Findings also illustrated that T. govanianum is a source of bioactive nutritional components and saponins. The current metabolite profiling of T. govanianum will help in its agricultural and biotechnological interventions for higher quality produce.     

Metabolomics Profiling of Tunisian Sonchus oleraceus L. Extracts and Their Antioxidant Activities

Sonchus oleraceus (L.) L. (Asteraceae) is an edible wild plant, known for its uses in traditional medicine. The aim of this study is to explore the phytochemical composition of the aerial parts (AP) and roots (R) of aqueous extracts of Sonchus oleraceus L. growing in Tunisia, using liquid chromatography-tandem mass spectrometry(LC/MS/MS), and determine the content of polyphenols and antioxidant activities. Results showed that aqueous extracts of AP and R contained, respectively, 195.25±33 μg/g and 118.66±14 μg/g gallic acid equivalent (GAE), and 52.58±7 μg/g and 3.2±0.3μg/g quercetin equivalent. AP and R extracts also contained tannins, 581.78±33 μg/g and 948.44±19 μg/g GAE. The AP extract in the 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging activities, hydroxyl radical scavenging (OH−) and in cupric reducing antioxidant activity (CUPRAC) assays were respectively 0.325±0.036 mg/mL, 0.053±0.018 mg/mL, 0.696±0.031 mg/mL and 60.94±0.004 μMTE/g, while the R extract using the same assays showed, 0.209±0.052 mg/mL, 0.034±0.002 mg/mL, 0.444±0.014 mg/mL and 50.63±0.006 μM Trolox equivalent/g, respectively. A total of 68 compounds were tentatively identified by LC/MS/MS in both extracts in which quinic acid, pyrogallol, osthrutin, piperine, gentisic acid, fisetin, luteolin, caffeic acid, gingerol, were the most abundant in the LC/MS/MS spectrum. Many of these metabolites were found for the first time in Tunisian Sonchus oleraceus L. which may take account for the antioxidant activities exhibited by the plant.     

Root cultures of <Emphasis Type="Italic">Hypericum perforatum</Emphasis> subsp. <Emphasis Type="Italic">angustifolium</Emphasis> elicited with chitosan and production of xanthone-rich extracts with antifungal activity

Hypericum perforatum is a well-known medicinal plant which contains a wide variety of metabolites, including xanthones, which have a wide range of biological properties, including antifungal activity. In the present study, we evaluated the capability of roots regenerated from calli of H. perforatum subsp. angustifolium to produce xanthones. Root biomass was positively correlated with the indole-3-butyric acid concentration, whereas a concentration of 1 mg l⁻¹ was the most suitable for the development of roots. High auxin concentrations also inhibited xanthone accumulation. Xanthones were produced in large amounts, with a very stable trend throughout the culture period. When the roots were treated with chitosan, the xanthone content dramatically increased, peaking after 7 days. Chitosan also induced a release of these metabolites into the culture. The maximum accumulation (14.26 ± 0.62 mg g⁻¹ dry weight [DW]) and release (2.64 ± 0.13 mg g⁻¹ DW) of xanthones were recorded 7 days after treatment. The most represented xanthones were isolated, purified, and spectroscopically characterized. Antifungal activity of the total root extracts was tested against a broad panel of human fungal pathogen strains (30 Candida species, 12 Cryptococcus neoformans, and 16 dermatophytes); this activity significantly increased when using chitosan. Extracts obtained after 7 days of chitosan treatment showed high antifungal activity (mean minimum inhibitory concentration of 83.4, 39.1, and 114 μg ml⁻¹ against Candida spp., C. neoformans, and dermatophytes, respectively). Our results suggest that root cultures can be considered as a potential tool for large-scale production of extracts with stable quantities of xanthones.     

The potential of <Emphasis Type="Italic">Rhizobium</Emphasis> strains for biological control of <Emphasis Type="Italic">Orobanche crenata</Emphasis>

Orobanche crenata Forsk is a chlorophyll lacking holoparasite that subsists on the roots of plants and causes significant damage to the culture of leguminous plants and, in particular, to peas (Pisum sativum L.). Here, we investigated the potential of Rhizobium strains for biological control of Orobanche crenata using a commercial pea cultivar (Douce de province) and different Rhizobium strains. Firstly, benefit of bacterial inoculation on plant growth and efficiency in N-incorporation were demonstrated with four isolates, P.SOM, P.1001, P.Mat.95 and P.1236. After five Rhizobium strains (three efficient: P.SOM, P.1236, P.Mat.95 and two not efficient: P.OM1.92, P.MleTem.92) were investigated for their ability to control Orobanche crenata using pot and Petri dish experiments. Inoculation of peas with two (P.SOM and P.1236) of the five strains induced a significant decrease in O. crenata seed germination and in the number of tubercles on pea roots. Furthermore, other symptoms, including the non-penetration of the germinated seeds into pea roots followed by radicle browning and death of the parasites, were observed in the presence of these inoculated pea plants. The hypothesis that roots secrete toxic compounds related to Rhizobium inoculation is discussed.     

Peroxidase and chitinase isoenzyme activities during root infection of Chinese cabbage with Plasmodiophora brassicae

Roots of two Chinese cabbage (Brassica campestris L. ssp. pekinensis) varieties, one tolerant and one susceptible, were inoculated with Plasmodiophora brassicae in liquid medium and in soil. Chitinase and peroxidase activities were determined in roots and shoots 1–21 days after inoculation with resting spores of Plasmodiophora and the enzyme activities compared with healthy tissue of the same age. In infected roots of the susceptible variety ‘Granat’ chitinase activity was higher than in the control 10 days after inoculation with spores. In the tolerant variety ‘Parkin’ we detected an increase in chitinase activity at the same time, which was about twice that of ‘Granat’. Chitinase activity in ‘Granat’ was also enhanced on day 13, 14 and 17 after inoculation, whereas chitinase activity in ‘Parkin’ was lower in the infected roots than in the controls during that period. In the shoots no correlation between chitinase activity and infection in the two varieties was observed. Chitinase from Chinese cabbage was further characterized and showed a pH optimum at pH 4.5–5.5 and a temperature optimum at 35–45°C. After isoelectric focusing 7 isoenzymes were discovered, but there were almost no differences between infected and healthy root extracts. Two isoenzymes with pI 8.7 and 8.8 showed cross-reactivity with an antiserum against bean chitinases. The molecular mass of these isoenzymes was determined as 33 kDa. Total peroxidase activity was generally higher in root tissue of both varieties than in the shoots. Peroxidase activity was increased most prominently in infected ‘Granat’ roots on day 13 after inoculation and of both varieties on day 17 compared to the controls. In clubbed tissue of ‘Granat’ a specific peroxidase isoenzyme appeared the first time 21 days after inoculation and was most prominent 28–30 days after inoculation. This isoenzyme had a molecular mass of ca 24 kDa and a pI of ca 8.8. With respect to our results the strategy of the Plasmodiophorales for plant attack is discussed.     

A submerged culture system for rapid micropropagation of the commercially important aquarium plant, ‘Amazon sword’ (Echinodorus ‘Indian Red’)

Echinodorus ‘Indian Red’ is an underwater plant, used worldwide for aquarium ornamentation. An efficient method for in vitro propagation and plantlet acclimatization of this popular aquarium plant was standardized. Surface-disinfected shoot-tips were cultured in submerged conditions in a solid–liquid bilayer medium, consisting of an upper, liquid layer (sterile distilled water) and a lower, solid layer Murashige and Skoog (MS) basal medium supplemented with 3.0% (w/v) sucrose, 0.8% (w/v) agar-agar, and plant growth regulators (PGRs) in different combinations and concentrations. The combination of 2.5 mg L⁻¹ 6-benzylaminopurine and 1.0 mg L⁻¹ α-naphthaleneacetic acid improved the multiplication rate to a maximum of 26.8 ± 0.51 shoots per explant after 60 d of culture. The number of multiplied shoots increased with each regeneration cycle, thus from only 26.8 ± 0.51 shoots per explant (first regeneration cycle), this number increased to 33.5 ± 0.58 (second regeneration cycle), and to 38.3 ± 0.62 for the third regeneration cycle with the same medium composition. The highest number of roots (8.3 ± 0.28) per shoot was induced in the presence of 1.0 mg L⁻¹ indole-3-butyric acid, but further growth of these roots was stunted. The best rooting was achieved on PGR-free ½-strength MS medium, where 6.1 ± 0.21 roots per shoot were induced with 5.8 ± 0.35 cm length after 30 d of culture. The regenerated plantlets were successfully acclimatized to submerged underwater conditions, with 100% survival rate. The present protocol is suitable for the commercial propagation of Echinodorus ‘Indian Red’ for aquarium-industries.

     

Some analytical assays for the determination of bioactivity of exotic fruits

Introduction – The consumption of new exotic fruits, with their high nutritional and sensory value, has significantly increased in the past few years. Among the tropical fruits durian (Durio zibethinus Murr.) is less known than mango (Mangifera indica L.) and avocado (Persea americana). It has been shown that durian, mango and avocado possessed high nutritional and bioactive properties, but these data were determined using different methods. In order to obtain reliable results we investigated samples of durian, mango and avocado of the same stage of ripeness and unified methods were used for determination of the antioxidant potential. As far as we know, no results of such comparative investigation of three tropical fruits (durian, mango and avocado) and the use of such tests for phytochemical control have been published. Objective – Lyophilised durian, mango and avocado samples harvested in 2008 in Thailand and Israel were investigated. Methodology – The contents of crude protein, fat, carbohydrate, dietary fibre, total polyphenols, flavonoids, tannins and flavanols were determined by elemental analysis and UV spectroscopy. The presence of polyphenols (flavonoids and phenolic acids) in the investigated samples was studied by Fourier transform infrared (FT‐IR) spectroscopy and three‐dimensional fluorometry. Four complementary radical scavenging assays were used for antioxidant determination: ferric reducing antioxidant power (FRAP), 2, 2‐azino‐bis (3‐ethyl‐benzothiazoline‐6‐sulfonic acid) diamonium salt (ABTS^?+), 1‐diphenyl‐2‐picrylhydrazyl method (DPPH) and cupric reducing antioxidant capacity (CUPRAC). Chemometrical processing was used for statistical comparison of the fruits. Results – All spectrometric measurements were highly correlated. The contents of total fibre, proteins and fats were significantly higher (p < 0.05) in avocado, and carbohydrates were significantly lower in avocado (p > 0.05) than in the two other fruits. The wavelength numbers of FTIR spectra for three investigated fruits were in the same range (1700–600 cm^?1) as for catechin and gallic acid, used as standards. One main peak could be easily observed at the approximate location of ex/em 275/305 nm and the other one at ex/em 350/430 nm in the methanol polyphenol extracts of investigated fruits in three‐dimensional fluorescence, in contour and cross fluorescence maps. Similarity was found between durian, mango and avocado in polyphenols (9.88 ± 1.0, 12.06 ± 1.3 and 10.69 ± 1.1, mg gallic acid equivalents/g dry weight, d.w.), and in antioxidant assays such as CUPRAC (27.46 ± 2.7, 40.45 ± 4.1 and 36.29 ± 3.7, µM Trolox equivalent (TE)/g d.w.) and FRAP (23.22 ± 2.0, 34.62 ± 3.4 and 18.47 ± 1.9, µM TE/g d.w.), respectively. The multisample median test between all possible pairs of groups is a Tukey–HSD type comparison and denotes the different groups in a case when a pair‐wise test is significant and its q statistical value is greater than the table q parameter. The multisample median test of FRAP values were chosen from the compared fruits triplets as similar or homogenous subsets durian and avocado. Conclusion – Nutritional and bioactive values of durian are comparable with these indices in mango and avocado. These fruits contain high, comparable quantities of basic nutritional and antioxidant compounds, and possess high antioxidant potentials. All fruits show a high level of correlation between the contents of phenolic compounds and the antioxidant potential. The methods used (three‐dimensional fluorescence, FTIR spectroscopy, radical scavenging assays) are suitable for bioactivity determination of these fruits. In order to receive best results, a combination of these fruits has to be included in the diet. The methods used are applicable for bioactivity determination in phytochemical analysis in general. Copyright © 2010 John Wiley & Sons, Ltd.