Anti-ice nucleation activity in xylem extracts from trees that contain deep supercooling xylem parenchyma cells

Boreal hardwood species, including Japanese white birch (Betula platyphylla Sukat. var. japonica Hara), Japanese chestnut (Castanea crenata Sieb. et Zucc.), katsura tree (Cercidiphyllum japonicum Sieb. et Zucc.), Siebold’s beech (Fagus crenata Blume), mulberry (Morus bombycis Koidz.), and Japanese rowan (Sorbus commixta Hedl.), had xylem parenchyma cells (XPCs) that adapt to subfreezing temperatures by deep supercooling. Crude extracts from xylem in all these trees were found to have anti-ice nucleation activity that promoted supercooling capability of water as measured by a droplet freezing assay. The magnitude of increase in supercooling capability of water droplets in the presence of ice-nucleation bacteria, Erwinia ananas, was higher in the ranges from 0.1 to 1.7 °C on addition of crude xylem extracts than freezing temperature of water droplets on addition of glucose in the same concentration (100 mosmol/kg). Crude xylem extracts from C. japonicum provided the highest supercooling capability of water droplets. Our additional examination showed that crude xylem extracts from C. japonicum exhibited anti-ice nucleation activity toward water droplets containing a variety of heterogeneous ice nucleators, including ice-nucleation bacteria, not only E. ananas but also Pseudomonas syringae (NBRC3310) or Xanthomonas campestris, silver iodide or airborne impurities. However, crude xylem extracts from C. japonicum did not affect homogeneous ice nucleation temperature as analyzed by emulsified micro-water droplets. The possible role of such anti-ice nucleation activity in crude xylem extracts in deep supercooling of XPCs is discussed.     

Deep supercooling xylem parenchyma cells of katsura tree (Cercidiphyllum japonicum) contain flavonol glycosides exhibiting high anti-ice nucleation activity

Xylem parenchyma cells (XPCs) of boreal hardwood species adapt to sub-freezing temperatures by deep supercooling to maintain a liquid state of intracellular water near −40 °C. Our previous study found that crude xylem extracts from such tree species exhibited anti-ice nucleation activity to promote supercooling of water. In the present study, thus, we attempted to identify the causative substances of supercooling. Crude xylem extracts from katsura tree ( Cercidiphyllum japonicum ), of which XPCs exhibited deep supercooling to −40 °C, were prepared by methanol extraction. The crude extracts were purified by liquid–liquid extraction and then by silica gel column chromatography. Although all the fractions obtained after each purification step exhibited some levels of anti-ice nucleation activity, only the most active fraction was retained to proceed to the subsequent level of purification. High-performance liquid chromatography (HPLC) analysis of a fraction with the highest level of activity revealed four peaks with high levels of anti-ice nucleation activity in the range of 2.8–9.0 °C. Ultraviolet (UV), mass and nuclear magnetic resonance (NMR) spectra revealed that these four peaks corresponded to quercetin-3- O - β -glucoside (Q3G), kaempferol-7- O - β -glucoside (K7G), 8-methoxykaempferol-3- O - β -glucoside (8MK3G) and kaempferol-3- O - β -glucoside (K3G). Microscopic observations confirmed the presence of flavonoids in cytoplasms of XPCs. These results suggest that diverse kinds of anti-ice nucleation substances, including flavonol glycosides, may have important roles in deep supercooling of XPCs.     

Change of supercooling capability in solutions containing different kinds of ice nucleators by flavonol glycosides from deep supercooling xylem parenchyma cells in trees

Deep supercooling xylem parenchyma cells (XPCs) in Katsura tree contain flavonol glycosides with high supercooling-facilitating capability in solutions containing the ice nucleation bacterium (INB) Erwinia ananas, which is thought to have an important role in deep supercooling of XPCs. The present study, in order to further clarify the roles of these flavonol glycosides in deep supercooling of XPCs, the effects of these supercooling-facilitating (anti-ice nucleating) flavonol glycosides, kaempferol 3-O-β-d-glucopyranoside (K3Glc), kaempferol 7-O-β-d-glucopyranoside (K7Glc) and quercetin 3-O-β-d-glucopyranoside (Q3Glc), in buffered Milli-Q water (BMQW) containing different kinds of ice nucleators, including INB Xanthomonas campestris, silver iodide and phloroglucinol, were examined by a droplet freezing assay. The results showed that all of the flavonol glycosides promoted supercooling in all solutions containing different kinds of ice nucleators, although the magnitudes of supercooling capability of each flavonol glycoside changed in solutions containing different kinds of ice nucleators. On the other hand, these flavonol glycosides exhibited complicated nucleating reactions in BMQW, which did not contain identified ice nucleators but contained only unidentified airborne impurities. Q3Glc exhibited both supercooling-facilitating and ice nucleating capabilities depending on the concentrations in such water. Both K3Glc and K7Glc exhibited only ice nucleation capability in such water. It was also shown by an emulsion freezing assay in BMQW that K3Glc and Q3Glc had no effect on homogeneous ice nucleation temperature, whereas K7Glc increased ice nucleation temperature. The results indicated that each flavonol glycoside affected ice nucleation by very complicated and varied reactions. More studies are necessary to determine the exact roles of these flavonol glycosides in deep supercooling of XPCs in which unidentified heterogeneous ice nucleators may exist.     

Presence of supercooling-facilitating (anti-ice nucleation) hydrolyzable tannins in deep supercooling xylem parenchyma cells in <Emphasis Type="Italic">Cercidiphyllum japonicum</Emphasis>

Xylem parenchyma cells (XPCs) in trees adapt to subzero temperatures by deep supercooling. Our previous study indicated the possibility of the presence of diverse kinds of supercooling-facilitating (SCF; anti-ice nucleation) substances in XPCs of katsura tree (Cercidiphyllum japonicum), all of which might have an important role in deep supercooling of XPCs. In the previous study, a few kinds of SCF flavonol glycosides were identified. Thus, in the present study, we tried to identify other kinds of SCF substances in XPCs of katsura tree. SCF substances were purified from xylem extracts by silica gel column chromatography and Sephadex LH-20 column chromatography. Then, four SCF substances isolated were identified by UV, mass and nuclear magnetic resonance analyses. The results showed that the four kinds of hydrolyzable gallotannins, 2,2′,5-tri-O-galloyl-α,β-d-hamamelose (trigalloyl Ham or kurigalin), 1,2,6-tri-O-galloyl-β-d-glucopyranoside (trigalloyl Glc), 1,2,3,6-tetra-O-galloyl-β-d-glucopyranoside (tetragalloyl Glc) and 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranoside (pentagalloyl Glc), in XPCs exhibited supercooling capabilities in the range of 1.5–4.5°C, at a concentration of 1 mg mL⁻¹. These SCF substances, including flavonol glycosides and hydrolyzable gallotannins, may contribute to the supercooling in XPCs of katsura tree.     

Analysis of supercooling-facilitating (anti-ice nucleation) activity of flavonol glycosides

Deep supercooling xylem parenchyma cells (XPCs) of katsura tree (Cercidiphyllum japonicum) contain four kinds of flavonol glycosides with high supercooling-facilitating (anti-ice nucleation) activities. These flavonol glycosides have very similar structures, but their supercooling-facilitating activities are very different. In this study, we analyzed the supercooling-facilitating activities of 12 kinds of flavonol glycosides in order to determine the chemical structures that might affect supercooling-facilitating activity. All of the flavonol glycosides tested showed supercooling-facilitating activity, although the magnitudes of activity differed among the compounds. It was clear that the combination of the position of attachment of the glycosyl moiety, the kind of attached glycosyl moiety and the structure of aglycone determined the magnitude of anti-ice nucleation activity. However, there is still some ambiguity preventing the exact identification of features that affect the magnitude of supercooling-facilitating activity.     

Screening of plant resources with anti-ice nucleation activity for frost damage prevention

Previous studies have shown that some polyphenols have anti-ice nucleation activity (anti-INA) against ice-nucleating bacteria that contribute to frost damage. In the present study, leaf disk freezing assay, a test of in vitro application to plant leaves, was performed for the screening of anti-INA, which inhibits the ice nucleation activity of an ice-nucleating bacterium Erwinia ananas in water droplets on the leaf surfaces. The application of polyphenols with anti-INA, kaempferol 7-O-β-glucoside and (–)-epigallocatechin gallate, to the leaf disk freezing assay by cooling at ?4–?6 °C for 3 h, revealed that both the compounds showed anti-INAs against E. ananas in water droplets on the leaf surfaces. Further, this assay also revealed that the extracts of five plant leaves showed high anti-INA against E. ananas in water droplets on leaf surfaces, indicating that they are the candidate resources to protect crops from frost damage.     

Analysis of supercooling activity of tannin-related polyphenols

Based on the discovery of novel supercooling-promoting hydrolyzable gallotannins from deep supercooling xylem parenchyma cells (XPCs) in Katsura tree (see Wang et al. (2012) [38]), supercooling capability of a wide variety of tannin-related polyphenols (TRPs) was examined in order to find more effective supercooling-promoting substances for their applications. The TRPs examined were single compounds including six kinds of hydrolyzable tannins, 11 kinds of catechin derivatives, two kinds of structural analogs of catechin and six kinds of phenolcarboxylic acid derivatives, 11 kinds of polyphenol mixtures and five kinds of crude plant tannin extracts. The effects of these TRPs on freezing were examined by droplet freezing assays using various solutions containing different kinds of identified ice nucleators such as the ice nucleation bacterium (INB) Erwinia ananas, the INB Xanthomonas campestris, silver iodide and phloroglucinol as well as a solution containing only unintentionally included unidentified airborne ice nucleators. Among the 41 kinds of TRPs examined, all of the hydrolyzable tannins, catechin derivatives, polyphenol mixtures and crude plant tannin extracts as well as a few structural analogs of catechin and phenolcarboxylic acid derivatives exhibited supercooling-promoting activity (SCA) with significant differences (p > 0.05) from at least one of the solutions containing different kinds of ice nucleators. It should be noted that there were no TRPs exhibiting ice nucleation-enhancing activity (INA) in all solutions containing identified ice nucleators, whereas there were many TRPs exhibiting INA with significant differences in solutions containing unidentified ice nucleators alone. An emulsion freezing assay confirmed that these TRPs did not essentially affect homogeneous ice nucleation temperatures. It is thought that not only SCA but also INA in the TRPs are produced by interactions with heterogeneous ice nucleators, not by direct interaction with water molecules. In the present study, several TRPs that might be useful for applications due to their high SCA in many solutions were identified.     

Soil salinity and drought alter wood density and vulnerability to xylem cavitation of baldcypress (Taxodium distichum (L.) Rich.) seedlings

We investigated the role of hydraulic conductivity, wood density, and xylem cavitation in the response of baldcypress (Taxodium distichum) seedlings to increased soil salinity and drought. One-year-old, greenhouse-grown seedlings were irrigated daily with a 100 mM (≈6‰) salt solution or once per week with fresh water (drought). Controls were irrigated daily with fresh water. Gas exchange rates of stressed plants were reduced by approximately 50% (salt) and 70% (drought), resulting in a 50–60% reduction in diameter growth for both treatments. Stem-specific hydraulic conductivity (K_{S native}) of stressed plants was 33% (salt) and 66% (drought) lower than controls and we observed a strong positive correlation between K_{S native} and gas exchange. In addition, we found a strong relationship between CO₂ assimilation rate (A) and the soil-to-leaf hydraulic conductance (k_L). The relationship was identical for all treatments, suggesting that our moderate salt stress (as well as drought) did not affect the photosynthetic biochemistry of leaves, but rather reduced A via stomatal closure. Lower K_{S native} of stressed plants was associated with increased wood density and greater resistance to xylem cavitation. Xylem pressures causing 50% loss of hydraulic conductivity (P₅₀) were ?2.88 ± 0.07 MPa (drought), ?2.50 ± 0.08 MPa (salt) and ?2.01 ± 0.04 MPa (controls). P₅₀s were strongly correlated with wood density (r = ?0.71, P < 0.01) and K_{S native} (r = 0.74, P < 0.01). These findings support the hypothesis that there is a significant trade-off between a plant's cavitation resistance and its hydraulic efficiency. The results of the present study indicate that stressed plants partitioned their biomass in a way that strengthened their xylem and reduced vulnerability to xylem cavitation. Hence, these seedlings could be better suited to be planted in environments with elevated soil salinity. For most parameters (especially P₅₀), drought had an even more pronounced effect than salinity. This is important as nurseries could produce “stress-acclimated” seedlings simply by reducing irrigation amounts and would not have to contaminate the soils in their nursery beds with salt applications.     

Toxicity of crude extracts from several terrestrial plants to barnacle larvae on mangrove seedlings

This study involved the use of terrestrial plant extracts as substitutes for toxic chemicals to control the major fouling organisms of mangroves, the barnacles. In terms of the solvents used to obtain the plant crude extracts, ethyl acetate was best, ethyl alcohol next and pure water worst, except in the case of Allium cepa (AC), where pure water was better than ethyl alcohol. For different plants, different solvents resulted in different poisonous effects, and plants belonging to different classes showed different levels of activities. In particular, using ethyl acetate crude extracts, Allium sativum (AS), AC and Capsicum annuum (CA) caused massive death of barnacle larvae at a low concentration in the shortest time. The less poisonous organic solvents were able to extract more active materials, and this was advantageous in enhancing the effect. For pure water crude extracts, AC and Zingiber officinale were best, but for 95% ethyl alcohol crude extracts, AS and AC were best. For ethyl acetate crude extracts, the effects of AS, AC and CA were better. Different solvent extracts of AC with 12 h of exposure all had LC₅₀ values <100 mg/L. The concentrations of the effective plant crude extracts used were close to, or even lower than, those used for pesticides referred to in the literature.     

Influence of sampling time and sap extraction methodology on xylem pH values in two grapevine varieties grown under drought conditions

The effect of differences in applied pressure and time of sampling on pH values of xylem sap collected using the leaf pressurization technique was examined in two grapevine varieties originating from contrasting habitats (Vitis vinifera L., cvs. Sabatiano and Mavrodafni) after subjecting them to drought. Three fractions of xylem sap exudates were collected from each leaf according to differences in applied pressure; fractions (I), (II) and (III) corresponding to 1 MPa, 2 MPa and 2.5 MPa pressure, respectively. The pH values in fraction (I) were significantly lower than those in fractions (II) and (III). The sap pH values in fraction (III) seemed to better correspond to changes in leaf apoplastic pH. The time of sampling was found to strongly influence xylem pH values. In particular, a positive relationship between predawn xylem pH values and soil drying was observed. Conversely, xylem pH values measured later during the day (i.e. at 8:00, 9:00 and 10:00 am) were not significantly affected by the reduction in soil water availability in both varieties. It is suggested that the most suitable period for sap sampling in order to better discriminate drought effects on xylem sap pH is at predawn. Furthermore, there were significant differences in pH values as well as in the sensitivity of stomatal conductance to pH between the two varieties. These differences might be related to strategy differences between grapevine varieties for adaptation to drought.     

Immunodetection and immunolocalization of tryptophanins in oat (Avena sativa L.) seeds

Tryptophanins (TRPs) are low molecular weight, tryptophan-rich, basic proteins found in oat (Avena sativa L.) seeds. Like their counterpart puroindolines (PINs) from wheat (Triticum aestivum L.), TRPs are thought to be involved in flour softness as well as disease resistance against phytopathogenic fungi. PINs are known to be the major components of ‘friabilin’ associated with the surface of water washed starch grains and possess lipid binding properties. Two polyclonal antisera against puroindoline-a (PIN-a), and puroindoline-b (PIN-b) respectively; and a monoclonal antiserum raised against ‘friabilin’ were used as primary antibodies in immunoblotting experiments. All antisera detected immunoreactive polypeptides, with approximate relative masses of 15–16 kDa, in oat, wheat, and barley (Hordeum vulgare L.) seed extracts but not in rice (Oryza sativa L.), maize (Zea mays L.), bean (Phaseolus vulgaris L.), pea (Pisum sativum L.) and lentil (Lens culinaris Medic.) seed extracts. Immunoreactive polypeptides were detected in aqueous ethanol [52% (v/v) ethanol] seed extracts. Both anti-‘friabilin’ monoclonal and anti-PIN-b polyclonal antisera recognized 15 as well as 16 kDa tryptophanins in oat seeds from different cultivars. On the other hand, anti-PIN-a polyclonal antiserum strongly cross-reacted with 16 kDa TRP and weakly with 15 kDa TRP. Tryptophanins were found to be associated with the surface of starch grains in oat endosperm tissue using both fluorescence and confocal laser scanning microscopies with anti-‘friabilin’ monoclonal antiserum. SDS-PAGE and immunoblotting assays revealed a gradual synthesis of TRPs as early as milk stage in developing oat seeds. On the other hand, TRPs tend to undergo degradation during seed germination.     

Chemical changes in Ulmus minor xylem tissue after salicylic acid or carvacrol treatments are associated with enhanced resistance to Ophiostoma novo-ulmi

Application of endogenous plant hormone salicylic acid (SA) or essential oil component carvacrol (CA) in elms enhances tree resistance to the Dutch elm disease pathogen, although the effect of these compounds on tree metabolism is unknown. The chemical changes induced by SA or CA treatments in Ulmus minor were studied through gas chromatography–mass spectrometry (GC–MS) analysis of xylem tissues. Treatments consisted of fortnightly irrigating seedlings with water, SA or CA at 600 mg L⁻¹. The chemical composition of the xylem tissues sampled from treated trees was significantly altered depending on the treatment type. SA treatment induced an accumulation of the sinapyl alcohol, a precursor of lignin and other phenylpropanoid-derived products. CA treatment induced an accumulation of the methyl esters of palmitic, linoleic and stearic acids. Both treatments resulted in early bud burst and SA significantly reduced sapwood radial growth, possibly as a consequence of a trade-off between tree growth and tree defence. The enhanced resistance provided by these treatments is discussed.     

A new cerebroside from the twigs of Lindera glauca (Sieb. et Zucc.) Blume

Lindera glauca (Sieb. et Zucc.) Blume (Lauraceae) has been used to treat rheumatic arthritis, stroke, and cardiac pain. Phytochemical investigation of twigs of L. glauca (Sieb. et Zucc.) Blume resulted in the isolation and identification of a new cerebroside, glaucerebroside (1). The structure of 1 was elucidated by a combination of extensive spectroscopic analyses, including extensive 2D NMR, HR-MS, chemical reactions, and LC/MS analysis. Compound 1 is a relatively rare cerebroside with l-threo-configuration of the sphingosine part. This is the second example of identification of a cerebroside from the family Lauraceae. Compound 1 significantly inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated BV-2 cells, with an IC₅₀ value of 23.84 μM without inducing cell toxicity. This study suggests that glaucerebroside (1) can be an excellent candidate for development of novel anti-neuroinflammatory agents.     

Evaluating fermentation effects on cell growth and crude extract metabolic activity for improved yeast cell-free protein synthesis

Saccharomyces cerevisiae is a promising source organism for the development of a practical, eukaryotic crude extract based cell-free protein synthesis (CFPS) system. Crude extract CFPS systems represent a snapshot of the active metabolism in vivo, in response to the growth environment at the time of harvest. Therefore, fermentation plays a central role in determining metabolic activity in vitro. Here, we developed a fermentation protocol using chemically defined media to maximize extract performance for S. cerevisiae-based CFPS. Using this new protocol, we obtained a 4-fold increase in protein synthesis yields with extracts derived from wild-type S288c as compared to a previously developed protocol that uses complex growth media. The final luciferase yield in our new method was 8.86 ± 0.28 μg mL⁻¹ in a 4 h batch reaction. For each of the extracts processed under different fermentation conditions, synthesized protein, precursor monomers (amino acids), and energy substrates (nucleotides) were evaluated to analyze the effect of the changes in the growth environment on cell-free metabolism. This study underscores the critical role fermentation plays in preparing crude extract for CFPS reactions and offers a simple strategy to regulate desired metabolic activity for cell-free synthetic biology applications based on crude cell extracts.     

Antimicrobial and cytotoxic activities of the crude extracts of Dietes bicolor leaves,flowers and rhizomes

The crude extracts of Dietes bicolor leaves, flowers and rhizomes were subjected to comparative antimicrobial screening against two Gram-positive, two Gram-negative bacteria and four fungal strains using the agar well diffusion method. The minimum inhibitory concentrations (MIC) of the tested extracts were also determined. Furthermore, the cytotoxic activity was evaluated. D. bicolor extracts generally demonstrated notable broad spectrum antimicrobial activities (MIC values ≤ 500 μg/mL) against all tested pathogens. D. bicolor leaf extract showed potent broad spectrum antimicrobial activity with MIC values ranging between 0.24 and 31.25 μg/mL against all tested pathogens. Moreover, the flowers extract exhibited promising antimicrobial activities, displaying MIC values ranging between 1.95 and 125 μg/mL against the tested bacteria and fungi. However, the rhizomes extract showed moderate antimicrobial activity with MIC values ranging between 31.25 and 500 μg/mL. Despite the potent antimicrobial activity of D. bicolor extracts, they were ineffective as cytotoxic agents against nine tested cancer cell lines, displaying 50% inhibitory concentration (IC₅₀) values above 100 μg/mL. The reported potent antimicrobial activity along with the lack of measurable cytotoxic activity indicated that the antimicrobial activity of D. bicolor crude extracts is mediated through a mechanism other than cytotoxicity. These results suggest that D. bicolor can act as a potential source for natural antibacterial and antifungal agents with a good safety profile at a preliminary level.     

Acridone alkaloids from Vepris verdoorniana (Excell & Mendonça) Mziray (Rutaceae)

Two new acridone alkaloids, verdoocridone A (1) and B (4), together with fifteen known compounds were isolated from methanol extracts of the roots and leaves of Vepris verdoorniana. The structures of all compounds were determined by comprehensive spectroscopic analyses (1D and 2D NMR, EI- and ESI–MS). The ¹³C NMR values of 1,2,3,5-tetramethoxy-N-methylacridone (2) and 5-methoxyaborinine (3) are also reported. The crude extracts and compounds (1-6) were tested for their antimicrobial activity. The test delivered moderate activities for crude extracts and compounds 1, 5 and 6 against the bacterium Staphylococcus aureus and the fungi Mucor meihei and Candida albicans with MIC values between 115 and 180 μg/mL for extracts and between 21.3 and 29.4 μM for compounds, compared to gentamycin with 0.2 μM and nystatin with 5.2 μM against both fungi. The determination of the radical scanvenging activity using 1,1-dephenyl-2-picrylhydrazyl (DPPH) assay gave moderate antioxidant values for all tested compounds, with IC₅₀ between 0.29 and 0.41 μM, compared to the standard 3-t-butyl-4-hydroxyanisole (BHA) displaying 0.03 μM.     

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.     

Antimycobacterial activity of ellagitannin and ellagic acid derivate rich crude extracts and fractions of five selected species of Terminalia used for treatment of infectious diseases in African traditional medicine

Ten crude extracts and their solvent partition fractions from five species of Terminalia collected in Tanzania were assessed for antimycobacterial effects using Mycobacterium smegmatis ATCC 14468 as a model organism. We report here, for the first time, on antimycobacterial effects of root and stem bark extracts of Terminalia sambesiaca and Terminalia kaiserana as well as of fruit extracts of Terminalia stenostachya and leaf extracts of Terminalia spinosa. T. sambesiaca gave the best effects of all the investigated species in terms of the sizes of the inhibitory zones of root and stem bark extracts. A crude methanol root extract of T. sambesiaca gave lower MIC values (1250 μg/ml) than its aqueous and butanol soluble fractions (MIC 2500 μg/ml). Our preliminary HPLC–DAD data indicates that methanol and aqueous extracts of T. sambesiaca roots are rich in ellagitannins and ellagic acid glycosides. Particularly, one polar ellagitannin at t_R 10.3–10.9 min dominates the extracts quantitatively and thus may be responsible for their good antimycobacterial effects. In contrast to the more polar fractions, a chloroform soluble fraction of the roots of T. sambesiaca was devoid of antimycobacterial activity. Also crude methanol and aqueous extracts of the stem bark of T. sambesiaca gave promising antimycobacterial effects (MIC 1250 μg/ml). All fractions of T. kaiserana roots, except from the aqueous insoluble gave good antimycobacterial effects (MIC 1250 μg/ml) and the aqueous extract showed the best effects of the fractions in terms of the size of inhibition zones. These results justify the uses of hot water decoctions of the roots of T. kaiserana for treatment of cough, one of the symptoms of TB. According to HPLC–DAD data methanol extracts of T. kaiserana roots and their aqueous fractions are rich in polar ellagitannins and ellagic acid glycosides. Quantitatively, the ellagitannins dominate these extracts and therefore the good antimycobacterial activity of the methanol and aqueous extracts is assumed to be due to these compounds. Sephadex LH-20 CC fractions of a methanol extract of the roots of T. kaiserana inhibited the growth of M. smegmatis, giving MIC values of 1000 μg/ml. Ellagic acid glycosides in these fractions must be responsible for their good antimycobacterial effects since they are present in high concentrations. Good antimycobacterial effects were also obtained with a root extract of Terminalia sericea, and especially the butanol soluble fraction was a good inhibitor of the growth of M. smegmatis (MIC 1562 μg/ml). Our preliminary HPLC–DAD results show that the roots of T. sericea are rich in ellagitannins, ellagic acid glycosides and at least one stilbene compound. Extracts of the fruits of T. stenostachya gave good antimycobacterial effects, butanol extracts being the most active. Also the leaves of T. stenostachya, and especially the butanol soluble extracts, give good antimycobacterial effects. Our HPLC–DAD data indicate that T. stenostachya leaves contain large quantities of gallic acid, ellagitannins and ellagic acid glycosides. Our results indicate that many of the investigated species of Terminalia might contain leads for development of anti-TB drugs. Standardized extracts of T. sambesiaca, T. kaiserana and T. sericea roots could be used as easily available and cheap medicines for treatment of TB in remote regions of East and South Africa.     

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.     

Microbial hydrolysis of 7-xylosyl-10-deacetyltaxol to 10-deacetyltaxol

Enterobacter sp. CGMCC 2487, a bacterial strain isolated from the soil around a Taxus cuspidata Sieb. et Zucc. plant, was able to remove the xylosyl group from 7-xylosyltaxanes. The xylosidase of this strain was an inducible enzyme. In the bioconversion of 7-xylosyl-10-deacetyltaxol (7-XDT) to 10-deacetyltaxol (10-DT), for the purpose of enhancing the conversion efficiency, the effects of NH₄⁺, oat xylan, temperature, pH value, cell density and substrate concentration on the bioconversion have been systematically investigated. 3.0 mM NH₄⁺, 0.6% oat xylan in the media could enhance the yield of 10-DT; the optimum biocatalytic temperature was 26 °C and optimum pH value was 6.0. The highest conversion rate and yield of 10-DT from 7-XDT reached 92% and 764 mg/L, respectively. In addition, the biocatalytic capacity of the cell cultures remained 66.1% after continuous three batches. These results indicate that converting 7-XDT to 10-DT, a useful intermediate for the semisynthesis of paclitaxel or other taxane-based anticancer drugs by a novel bacterial strain, Enterobacter sp. CGMCC 2487, would be an alternative for the practical application in the future.