Variation in polyphenolic profile and in vitro antioxidant activity of eastern teaberry (Gaultheria procumbens L.) leaves following foliar development

Seasonal dynamics in the polyphenolic composition, antioxidant activity, and their relationships during plant development were evaluated for eastern teaberry (Gaultheria procumbens L.) leaves, a traditional herbal medicine of North American natives. With the complementary UHPLC-PDA-ESI-MS³, HPLC-PDA-fingerprint, Folin-Ciocalteau, and n-butanol/HCl assays of methanol-water (75:25, v/v) extracts, the dried leaf samples harvested monthly across the growing season under Polish climate conditions were found rich in structurally diverse polyphenols (149.2–210.7 mg/g DW) including the dominating salicylates (64.6–107.5 mg/g DW), proanthocyanidins (53.0–66.8 mg/g DW), and flavonoids (17.3–25.3 mg/g DW), and the accompanying chlorogenic acid isomers (2.4–4.4 mg/g DW) and simple phenolic acids (0.9–1.1 mg/g DW). Among 28 detected analytes, gaultherin (64.6–107.5 mg/g DW), miquelianin (14.6–21.1 mg/g DW), procyanidin A-type trimer (5.5–9.5 mg/g DW), and (–)-epicatechin (5.8–7.8 mg/g DW) were the most abundant. The phenolic levels and antioxidant activity parameters in the DPPH (EC₅₀, 15.0–18.2 μg DW/mL; 0.95–1.16 mmol Trolox equivalents/g DW) and FRAP (2.3–3.4 mmol Fe ²⁺/g DW; 0.86–1.26 mmol Trolox equivalents/g DW) assays showed parallel seasonal trends with maxima in September and October. As the subsequent correlation studies confirmed the determinative impact of polyphenols on the leaf antioxidant activity and its seasonal fluctuations, the Fall season could be recommended as optimal for harvesting the plant material for medicinal purposes and cost-effective production of natural health products.     

Phytochemical and antioxidant properties of unconventional leafy vegetables consumed in southern Africa

Indigenous leafy vegetables possess high horticultural potential based on their long utilisation history by local communities across Africa. Phytochemical and antioxidant properties of 50% aqueous methanol and water extracts of three indigenous as well as two commercial leafy vegetables commonly consumed in southern Africa were evaluated. The total extractable phenolic content was highest for Amarathus dubius (5.16 ± 0.12 mg GAE/g DW) followed by Cleome gynandra (3.94 ± 0.09 mg GAE/g DW). Total flavonoid concentration was highest for A. dubius (3.89 ± 0.28 mg CE/g DW) followed by C. gynandra (2.19 ± 0.11 mg CE/g DW) and Cucurbita maxima (1.55 ± 0.04 mg CE/g DW). No proanthocyanidins were detected in C. maxima and Brassica napus cv Covo whereas low concentrations were recorded in other vegetables. Total saponins were variable across the evaluated extracts, with the highest concentrations recorded for B. napus cv Covo (83.2 ± 16.58 mg DE/g DW). Total iridoid content was highest for C. gynandra (9.14 ± 0.20 mg HE/g DW). More potent DPPH radical scavenging activities were exhibited by 50% aqueous methanol extracts compared to water extracts. A similar trend was observed in the ferric-reducing antioxidant power assay. The antioxidant activity based on the rate of β-carotene bleaching was higher for water extracts compared to 50% aqueous methanol extracts. The indigenous vegetables evaluated in this study had higher levels of phytochemicals and also exhibited more potent antioxidant activity compared to the commercial varieties. These findings not only suggest the importance of the indigenous vegetables in a healthy diet, but also provide a motivation for exploring their horticultural potential.     

Optimization of extraction method for LC–MS based determination of phenolic acid profiles in different Impatiens species

The main aim of presented study was the comparison of various extraction methods for the quantitative and qualitative analysis (LC-ESI–MS/MS) of phenolic acids present in extracts obtained from leaves, flowers, and roots of Impatiens glandulifera. The accelerated solvent extraction (ASE) at three temperature ranges (80° C, 100° C, and 120° C), ultrasound assisted extraction (USAE) at 60° C, and traditional extraction in Soxhlet apparatus were used. Taking into account the extraction yield, and the diversity of the individual compounds, ultrasound assisted extraction proved to be the most efficient method, and it was used to determine the content of phenolic acids in leaves of four other Impatiens species, including I. balsamina, I. noli-tangere, I. parviflora, and I. walleriana. Eleven phenolic acids were identified in all examined species. These were protocatechuic, gentisic, 4- hydroxybenzoic, vanillic, trans-caffeic, syringic, trans-p-coumaric, trans- and cis-ferulic, salicylic, and 3-hydroxycinnamic acids. In the extract from the leaves of I. balsamina and I. walleriana, gallic and cis-p-coumaric acids were found additionally. The most abundant compounds in all examined extracts were protocatechuic and 3-hydroxycinnamic acids. The latest acid was found in the highest yield in I. noli-tangere (266.12 μg/g DW). In the leaves of I. glandulifera a great amount of 4-hydroxybenzoic (41.44 μg/g DW), vanillic (61.50 μg/g DW), and trans-p-coumaric (58.42 μg/g DW) acids was also observed. Our results indicate that protocatechuic, 4-hydroxybenzoic, vanillic, trans-p-coumaric, trans-ferulic, and 3-hydroxycinnamic acids were most characteristic of Impatiens species.Additionally, various phenolic-rich extracts from leaves, flowers, and roots of Impatiens glandulifera were tested for antioxidant activity. The highest antiradical activity was detected for roots using Soxhlet extraction (EC50 = 0.055 mg [DE/ml]).The study demonstrated that members of the genus Impatiens, and in particular Impatiens glandulifera, and Impatiens noli-tangere, contain significant amounts of phenolic acids. In addition, extracts from various parts of I. glandulifera could be interesting as novel sources of natural antioxidants.     

Variation in phenolic compounds and antioxidant activity in apple seeds of seven cultivars

Polyphenols are the predominant ingredients in apple seeds. However, few data are available on the phenolic profile or antioxidant activity in apple seeds in previous researches. In this study, low-molecular-weight phenolic compounds and antioxidant activity in seeds, peels, and flesh of seven apple cultivars grown in northwest China were measured and analyzed using HPLC and FRAP, DPPH, ABTS assays, respectively. HPLC analysis revealed phloridzin as the dominant phenolic compound in the seeds with its contents being 240.45–864.42 mg/100 gDW. Total phenolic content (TPC) measured by the Folin–Ciocalteu assay in apple seed extracts of seven cultivars ranged from 5.74 (Golden Delicious) to 17.44 (Honeycrisp) mgGAE/gDW. Apple seeds showed higher antioxidant activity than peels or flesh; antioxidant activity in seeds varied from 57.59 to 397.70 μM Trolox equivalents (TE)/g FW for FRAP, from 37.56 to 64.31 μM TE/g FW for DPPH, and from 220.52 to 708.02 μM TE/g FW for ABTS. TPC in apple seeds was significantly correlated with all three assays. Principal component analysis (PCA) indicated that Honeycrisp was characterized with high contents of total polyphenols and phloridzin. Our findings suggest that phenolic extracts from apple seeds have good commercial potential as a promising antioxidant for use in food or cosmetics.     

Biological activities of two macroalgae from Adriatic coast of Montenegro

In the present investigation the acetone extracts of macroalgae Ulva lactuca and Enteromorpha intestinalis were tested for antioxidant, antimicrobial and cytotoxic potential. Antioxidant activity was evaluated by measuring the scavenging capacity of tested samples on DPPH and superoxide anion radicals, reducing the power of samples and determination of total phenolic and flavonoid compounds in extracts. As a result of the study, U. lactuca extract was found to have a better free radical scavenging activity (IC₅₀ = 623.58 μg/ml) than E. intestinalis extract (IC₅₀ = 732.12 μg/ml). Moreover, the tested extracts had effective ferric reducing power and superoxide anion radical scavenging. The total content of phenol in extracts of U. lactuca and E. intestinalis was 58.15 and 40.68 μg PE/mg, while concentrations of flavonoids were 39.58 and 21.74 μg RE/mg, respectively. Furthermore, among the tested species, extracts of U. lactuca showed a better antimicrobial activity with minimum inhibitory concentration values ranging from 0.156 to 5 mg/ml, but it was relatively weak in comparison with standard antibiotics. Bacillus mycoides and Bacillus subtilis were the most susceptible to the tested extracts. Contrary to this Aspergillus flavus, Aspergillus fumigatus and Penicillium purpurescens were the most resistant. Finally, cytotoxic activity of tested extracts was evaluated on four human cancer cell lines. Extract of E. intestinalis expressed the stronger cytotoxic activity towards all tested cell lines with IC₅₀ values ranging from 74.73 to 155.39 μg/ml.     

Analysis of anti-bacterial and anti oxidative activity of Azadirachta indica bark using various solvents extracts

Herbal medications have been used for relief of symptoms of disease. Regardless of the great advances observed in current medicine in recent decades, plants still make a significant contribution to health care. An alarming increase in bacterial strains resistant to a number of antimicrobial agents demands that a renewed effort be made to seek antibacterial agents effective against pathogenic bacteria resistant to or less sensitive to current antibiotics. Anti-bacterial activity of Azadirachta indica stem bark was tested against pathogenic Salmonella paratyphi and Salmonella typhi using various solvent extracts. The in vitro anti-bacterial activity was performed by agar well diffusion method and the results were expressed as the average diameter of zone of inhibition of bacterial growth around the well. The ethanol and methanol extracts showed better anti-bacterial activity with zone of inhibition (20–25 mm) when compared with other tested extracts and standard antibiotic Erythromycin (15 mcg) with zone of inhibition (13–14 mm). Using Fisher’s exact test of significance difference was found between two Salmonella strains sensitivity patterns against tested extracts (P ⩽ 0.035). Extracts of A. indica stem bark also exhibited significant antioxidant activity, thus establishing the extracts as an antioxidant. The results obtained in this study give some scientific support to the A. indica stem bark for further investigation of compounds and in future could be used as drug.     

Enhanced antioxidant and antitumor activities of Antrodia cinnamomea cultured with cereal substrates in solid state fermentation

Growing exclusively on stout camphor trees in Taiwan, Antrodia cinnamomea is known for its extraordinary antioxidant and antitumor activities. As an alternative to the limited supply of natural source, cultured A. cinnamomea from solid state or submerged liquid fermentation still offers many of its medicinal effects. To further enhance the production of functional compounds and corresponding activities, oat, wheat, buckwheat and pearl barley were used as substrates for solid state fermentation of A. cinnamomea in this study. Among these cereal-based culturing, the methanol extract of A. cinnamomea mycelia grown on oats showed stronger overall antioxidant properties. EC₅₀ for the antioxidant activity (conjugated diene method), the DPPH radicals scavenging ability and reducing power were estimated to be around 0.57 mg/mL, 1.07 mg/mL and 0.31 mg/mL, respectively. Incubating cultured cells with 150 ppm of the oat-cultured mycelial extract for 24 h greatly reduced the viability of MCF-7 breast cancer cells and HepG2 hepatocellular carcinoma cells to 29% and 76%, while 3T3 normal fibroblasts were virtually unaffected. In general, cereal-based solid state fermentation of A. cinnamomea produced more of the secondary metabolites and their methanolic extracts showed stronger antioxidant and anti-tumor activities than extracts obtained from liquid fermentation at the same concentration.     

Reactions and resistance ofChenopodium species to tobacco mosaic virus

Chenopodium species react on infection with tobacco mosaic virus by the formation of chlorotic or necrotic lesions and later by the abscission of infected leaves. A transition of local infection into the stem has been observed exceptionally inChenopodium quinoa, C. hybridum, andC. rubrum, but no systemic infection of the leaves followed. Systemic infection was demonstrated only inC. polyspermum andC. murale. The recovery of new sprouts was demonstrated in C.murale in the late chronic phase of infection.     

Studies on the accumulation of phenolic acids and flavonoids in different in vitro culture systems of Schisandra chinensis (Turcz.) Baill. using a DAD-HPLC method

Different in vitro culture systems of the East-Asian origin medicinal plant species − Schisandra chinensis, were tested in order to investigate their potential for the accumulation of two groups of phenolic compounds. In vitro cultures were maintained on the Murashige and Skoog (MS) medium supplemented with 3 mg/l BA and 1 mg/l NAA in an agar system (30- and 60-day growth cycles), and also in two different liquid systems: stationary and agitated. Stationary liquid cultures were grown in batch (30- and 60-day growth cycles) and fed-batch modes. Of the twenty compounds, seven free phenolic acids and of the eleven compounds, five flavonoids were quantified in methanolic extracts from lyophilized biomass and in the growth media using the RP-HPLC-DAD method. For comparison purposes, phytochemical analyses of leaf and fruit extracts from the parent plant were also conducted. The estimated compounds were not detected in the growth media. The highest total amounts of phenolic acids (71.48 mg/100 g DW) and flavonoids (29.36 mg/100 g DW) were found in extracts from the biomass of agar cultures harvested after 30 days of cultivation. The main metabolites in all the tested systems were: protocatechuic acid (max. 35.69 mg/100 g DW), chlorogenic acid (max. 13.05 mg/100 g DW), and quercitrin (max. 27.43 mg/100 g DW).     

Antioxidative effect of Iranian Pulicaria gnaphalodes L. extracts in soybean oil

This study was aimed to evaluate antioxidative activities of the ethanol, methanol and water extracts of Pulicaria gnaphalodes in vegetable oil during the storage period. Different concentrations (0, 200, 400 and 800 ppm) of ethanol, methanol and water extracts and beta-hydroxy toluene (BHT; 100, 200 ppm) were added to soybean oil and incubated for 35 days at 65 °C. Peroxide values (PVs) and thiobarbituric acid-reactive substance (TBARS) levels were measured every week during the period of the study. Moreover, antioxidant capacities of the extracts were determined using DPPH and β-carotene–linoleic acid methods. Values were compared among groups in each incubation time points using ANOVA. Results showed that DPPH and β-carotene–linoleic acid assay findings on the P. gnaphalodes extracts were comparable to those found on BHT. Moreover, during incubation time, P. gnaphalodes extracts lowered PVs and TBARS levels when compared to the control (p < 0.001). In this respect, water extract was more potent than the ethanol and methanol extracts. It seems that water extract of P. gnaphalodes is a potent antioxidant which makes it as a potential antioxidant for oil and oily products during storage.     

Concurrent purification and antioxidant activity of phycobiliproteins from Lyngbya sp. A09DM: An antioxidant and anti-aging potential of phycoerythrin in Caenorhabditis elegans

The present study probes into the purification of phycobiliproteins, and characterization of their in vitro anti-oxidant activity. Moreover, the study also demonstrates the use of antioxidant virtue of phycoerythrin in moderating the phenomenon of aging in Caenorhabditis elegans. Phycoerythrin, phycocyanin and allophycocyanin were purified successfully from Lyngbya sp. A09DM by ammonium sulfate fractionation appended with Triton X-100 intercession. The success of protocol was examined by a series of biochemical characterization like SDS-PAGE, native-PAGE, UV–visible spectroscopy and fluorescence spectroscopy ensuring purity, integrity and functionality of purified phycoerythrin, phycocyanin and allophycocyanin. Purified phycobiliproteins were evaluated for antioxidant and metal ion chelating activity by various in vitro antioxidant assay systems. Results showed significant and dose-dependent antioxidant as well as metal chelating potential of all phycobiliproteins in decreasing order of phycoerythrin > phycocyanin > allophycocyanin. Expansion in lifespan and improvement in pharyngeal pumping of C. elegans were noticed upon pre-treatment with phycoerythrin (100 μg ml⁻¹). Moreover, phycoerythrin mediated increase in worm survival under oxidative stress revealed that the life expansion effect of phycoerythrin on nematode is in part by an action of its antioxidant virtue. These results collectively added up evidence in favor of the ‘free-radical theory of aging’. The present report, for the first time, describes antioxidant potential of phycoerythrin and its use in extending life-span of C. elegans.     

Comparative analysis of bioactive phenolic compounds composition from 26 medicinal plants

Bioactive phenolic compounds are powerful antioxidants in traditionally used medicinal and industrial crop plants and have attracted increased interest in the last years in their application and role in non-destructive methodology for pre-screening analysis of some stress factors. In this study the qualitative target was linked with future possible applications of received data for improving non-destructive methodology as well as for improving existing knowledge regarding antioxidant content in some plant species. Comparative analysis of total phenolics, flavonoid contents, phenolic acid composition, and antioxidant activity in known east central Europe medicinal and industrial crop plants of 26 species of families Asteraceae, Rosaceae and Lamiaceae was done. Among the investigated leaf extracts the highest total phenolic, total flavonoid contents and antioxidant activity have been seen for Stachys byzantine L. (Lamiaceae), Calendula officinalis L. (Asteraceae) and for Potentilla recta L. (Rosaceae). The highest syringic acid content has been found in the leaf extracts of plant family Asteraceae – in the range from 0.782 to 5.078 mg g^?1 DW. The representative’s family Rosaceae has a higher content of p-anisic acid in the range 0.334–3.442 mg g^?1DW compared to the leaf extracts of families Lamiaceae and Asteraceae. The comparative study showed significant differences of content of phenolic acids in the leaf extracts of different representative’s families Rosaceae, Asteraceae and Lamiaceae. We suggest that the presence of some phenolic acids can be used as a possible marker for family botanical specifications of representative families Asteraceae and Rosaceae. It was supposed that some pharmacological effects can be connected with the analyzed data.     

Chemical constituents from Tribulus terrestris and screening of their antioxidant activity

Two oligosaccharides (1, 2) and a stereoisomer of di-p-coumaroylquinic acid (3) were isolated from the aerial parts of Tribulus terrestris along with five known compounds (4–8). The structures of the compounds were established as O-β-d-fructofuranosyl-(2 → 6)-α-d-glucopyranosyl-(1 → 6)-β-d-fructofuranosyl-(2 → 6)-β-d-fructofuranosyl-(2 → 1)-α-d-glucopyranosyl-(6 → 2)-β-d-fructofuranoside (1), O-α-d-glucopyranosyl-(1 → 4)-α-d-glucopyranosyl-(1 → 4)-α-d-glucopyranosyl-(1 → 2)-β-d-fructofuranoside (2), 4,5-di-p-cis-coumaroylquinic acid (3) by different spectroscopic methods including 1D NMR (¹H, ¹³C and DEPT) and 2D NMR (COSY, TOCSY, HMQC and HMBC) experiments as well as ESI-MS analysis. This is the first report for the complete NMR spectral data of the known 4,5-di-p-trans-coumaroylquinic acid (4).The antioxidant activity represented as DPPH free radical scavenging activity was investigated revealing that the di-p-coumaroylquinic acid derivatives possess potent antioxidant activity so considered the major constituents contributing to the antioxidant effect of the plant.     

Effect of enzymatic and hydrothermal treatments of rapeseeds on quality of the pressed rapeseed oils: Part I: Antioxidant capacity and antioxidant content

Response surface methodology was used to evaluate the quantitative effects of three independent variables: rapeseed moisture content, concentration of the added enzymes and conditioning temperature, on the antioxidant capacity and total phenolic, tocopherol, and phospholipid contents in the enzyme-treated rapeseed oils. The highest antioxidant capacity (1220.0, 964.8 μmol TE/100 g) total phenolic (83.3, 74.0 mg SA/100 g) and phospholipid (12,532, 12,376 mg/kg) contents reveal two rapeseed oils extruded from seeds contained 11% moisture, treated with cellulolytic and pectolytic enzymes (0.05%), respectively, and heated at 120 °C. However, the highest content of total tocopherols was determined in rapeseed oils pressed from seeds with 7% moisture, after addition of cellulolytic (0.05%) and pectolytic (0.1%) enzymes, heated at 90 and 105 °C, respectively. Total phenolic and phospholipid contents in the enzyme-treated rapeseed oils correlated significantly (p < 0.0000001) with antioxidant capacities of oils (R² = 0.8710 and 0.6581, respectively). Experimental results of the antioxidant capacity, total phenolic, tocopherol and phospholipid contents were close to the predicted values calculated from the polynomial response surface models equations (R² = 0.9727, 0.9870, 0.8390 and 0.9706 for the cellulolytic enzyme-assisted rapeseed oils and R² = 0.9148, 0.9489, 0.9426 and 0.9479 for the pectolytic enzyme-assisted rapeseed oils). The optimum rapeseed moisture content, enzyme concentration and conditioning temperature for the cellulolytic and pectolytic enzyme-treated rapeseed oils were 11% and 9.7%, 0.08% and 0.1%, and 120 °C, respectively.     

Isolation and characterization of ellagitannins as the major polyphenolic components of Longan (Dimocarpus longan Lour) seeds

Longan (Dimocarpus longan Lour, syn. Euphoria longan Lam.) represents an important fruit in Northern Thailand and has significant economic impact. The fruit is either consumed fresh or as commercially prepared dried and canned products. The canning industry in Thailand produces considerable quantities of waste products, in particular Longan seeds. Because these seeds may be an exploitable source of natural phenolic antioxidants, it was of interest to identify, purify and quantitate the major potential antioxidant phenolics contained therein. The polyphenolic fraction from ground Longan seeds was obtained by extraction with methanol after delipidation with hexane. The hexane extract contained predominantly long-chain fatty acids with major contributions from palmitic (35%) and oleic (28%) acids. The polyphenolic fraction (80.90 g/kg dry weight) was dominated by ellagic acid (25.84 g/kg) and the known ellagitannins corilagin (13.31 g/kg), chebulagic acid (13.06 g/kg), ellagic acid 4-O-α-l-arabinofuranoside (9.93 g/kg), isomallotinic acid (8.56 g/kg) and geraniin (5.79 g/kg). Structure elucidation was performed with mass spectrometry and complete assignment of ¹H and ¹³C NMR signals. The methanol extracts exhibited strong antioxidant capacities with an IC₅₀ of 154 μg/ml for reactive oxygen species attack on salicylic acid and 78 μg/ml for inhibition of xanthine oxidase in the hypoxanthine/xanthine oxidase assay. The extracts were less effective in the 2-deoxyguanosine assay (IC₅₀ = 2.46 mg/ml), indicating that gallates along with ellagic acid and its congeners exert their potential antioxidant effects predominantly by precipitation of proteins such as xanthine oxidase. This was confirmed for the pure compounds gallic acid, methyl gallate, ellagic acid and corilagin.     

Antioxidant activity,acetylcholinesterase and tyrosinase inhibitory potential of Pulmonaria officinalis and Centarium umbellatum extracts

In this study several investigations and tests were performed to determine the antioxidant activity and the acetylcholinesterase and tyrosinase inhibitory potential of Pulmonaria officinalis and Centarium umbellatum aqueous extracts (10% mass) and ethanolic extracts (10% mass and 70% ethanol), respectively. Moreover, for each type of the prepared extracts of P. officinalis and of C. umbellatum the content in the biologically active compounds – polyphenols, flavones and proanthocyanidins was determined. The antioxidant activity was assessed using two methods, namely the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay and reducing power assay. The analyzed plant extracts showed a high acetylcholinesterase and tyrosinase inhibitory activity in the range of 72.24–94.24% (at the highest used dose – 3 mg/mL), 66.96% and 94.03% (at 3 mg/mL), respectively correlated with a high DPPH radical inhibition – 70.29–84.9% (at 3 mg/mL). These medicinal plants could provide a potential natural source of bioactive compounds and could be beneficial to the human health, especially in the neurodegenerative disorders and as sources of natural antioxidants in food industry.     

Production of rhoifolin and tiliroside from callus cultures of Chorisia chodatii and Chorisia speciosa

The current work aims to stimulate the production of rhoifolin and tiliroside as two valuable phytochemicals from Chorisia chodatii Hassl. and Chorisia speciosa A. St.-Hil. callus cultures. A comparison between three explants from the in vitro germinated seedlings of both species for callus induction and accumulation of both flavonoids was carried out. Highly efficient calluses were induced from the leaves, stems and roots of C. chodatii seedlings on Gamborg’s B5 (B5) and Murashige and Skoog (MS) media containing 2.0 mg/l β-naphthalene acetic acid (NAA) and 0.5 mg/l 6-benzyladenin (BA) or kinetin (Kn), while those of C. speciosa seedlings efficiently produced calluses on both media supplemented with 0.5 or 1.0 mg/l NAA and 0.5 mg/l BA. Besides, the highest contents of rhoifolin (1.927 mg/g DW) and tiliroside (1.776 mg/g DW) from C. speciosa cultures were obtained from the calluses of seedlings’ roots and stems maintained on B5 medium containing 1.0 mg/l NAA and 0.5 mg/l BA, respectively. On the other hand, the maximum rhoifolin content (0.555 mg/g DW) from C. chodatii cultures was obtained from the calluses of seedlings’ stems grown on B5 medium supplemented with 2.0 mg/l NAA and 0.5 mg/l BA, whereas the highest tiliroside content (0.547 mg/g DW) was provided by the root explants on B5 medium containing 2.0 mg/l NAA and 0.5 mg/l Kn. Both flavonoids were bioaccumulated in greater amounts than the wild and cultivated intact plants, which provides a promising tool for their future commercial production under a controlled environment, independent of climate and soil conditions.     

Quantitative analysis of polyphenols and antioxidant activity in four Daphne L. species

The content of biologically active phenolic compounds (total polyphenols, tannins, flavonoids, and phenolic acids) were determined using spectrophotometry in four wild Croatian species of Daphne L. in the family Thymelaeaceae (Daphne alpina, D. cneorum, D. laureola, and D. mezereum). The concentration of total flavonoids (TF) was highest in the leaves of these Daphne species (0.12?C0.51% dry herb weight, DW) whereas the content of other phenolic compounds analyzed were highest in the roots, including total polyphenols (TP; 2.71?C19.03% DW), tannins (T; 1.14?C7.39% DW), and total phenolic acids (TPA; 0.12?C0.87% DW). D. alpina contained the highest amount of polyphenols, with the exception of flavonoids, where maximum concentrations were found in D. laureola. We also examined the antioxidant activity of leaf, stem, and root extracts. All extracts analyzed demonstrated high free radical scavenging activity with the highest concentration in the leaf extracts of D. alpina. Leaf extracts of D. cneorum showed the highest antioxidant activity in a ??-carotene bleaching assay.     

Seed storage and germination in Kumara plicatilis,a tree aloe endemic to mountain fynbos in the Boland,south-western Cape,South Africa

Seed storage under appropriate conditions is a relatively inexpensive means of safeguarding plant genetic material for ex situ conservation. Post-storage germination trials are used to determine the viability of stored seeds, and hence the efficacy of the particular storage treatment. Kumara plicatilis (= Aloe plicatilis) is a tree aloe endemic to mountain fynbos in the Boland, south-western Cape. The viability and germination behaviour of K. plicatilis seeds were assessed for seeds stored for four and nine months at − 80 °C, 4 °C, 25 °C and under ambient conditions in a laboratory. Seeds were germinated under controlled conditions and germination rates and percentages determined. Ungerminated seeds were tested for viability using tetrazolium salt. Seed viability was not significantly reduced during storage. Seeds stored at − 80 °C for four and nine months exhibited the fastest germination rate overall (both 5.9 ± 0.3 weeks, mean ± S.E.), and slowest was for seeds stored under ambient conditions for four and nine months (both 7.8 ± 0.4 weeks). All seed lots showed similar percentage germination after four months of storage (78.0–90.4%). The highest percentage germination overall was for seeds stored at − 80 °C for four months (90.4%) and the lowest was for seeds kept at 4 °C and − 80 °C for nine months (39.2 and 39.6%, respectively). Respective percentage viability for ungerminated seeds in these two treatments was 82% and 87%, respectively, indicating the induction of secondary dormancy. Induced dormancy triggered by protracted cold temperatures may be an adaptation that enables seeds to survive prolonged extreme conditions that are unfavourable for germination. Further research on the long-term storage of aloe seeds would be beneficial for developing long-term seed storage and germination testing protocols for ex situ conservation.     

Freeze tolerance and accumulation of cryoprotectants in the enchytraeid Enchytraeus albidus (Oligochaeta) from Greenland and Europe

The freeze tolerance and accumulation of cryoprotectants was investigated in three geographically different populations of the enchytraeid Enchytraeus albidus (Oligochaeta). E. albidus is widely distributed from the high Arctic to temperate Western Europe. Our results show that E. albidus is freeze tolerant, with freeze tolerance varying extensively between Greenlandic and European populations. Two populations from sub Arctic (Nuuk) and high Arctic Greenland (Zackenberg) survived freezing at −15 °C, whereas only 30% of a German population survived this temperature. When frozen, E. albidus responded by catabolising glycogen to glucose, which likely acted as a cryoprotectant. The average glucose concentrations were similar in the three populations when worms were frozen at −2 °C, approximately 50 μg glucose mg⁻¹ tissue dry weight (DW). At −14 °C the glucose concentrations increased to between 110 and 170 μg mg⁻¹ DW in worms from Greenland. The average glycogen content of worms from Zackenberg and Nuuk were about 300 μg mg⁻¹ DW, but only 230 μg mg⁻¹ DW in worms from Germany showing that not all glycogen was catabolised during the experiment. Nuclear magnetic resonance spectrometry (NMR) was used to screen for other putative cryoprotectants. Proline, glutamine and alanine were up regulated in frozen worms at −2 °C but only in relatively small concentrations suggesting that they were of little significance for freeze survival. The present study confirms earlier reports that freeze tolerant enchytraeids, like other freeze tolerant oligochaete earthworms, accumulate high concentrations of glucose as a primary cryoprotectant.