Triterpenoids from Protorhus longifolia Exhibit Hypocholesterolemic Potential via Regulation of Cholesterol Biosynthesis and Stimulation of Low-Density Lipoprotein Uptake in HepG2 Cells.

The increasing incidence of hypercholesterolemia-related diseases even in the presence of the currently available cholesterol-lowering drugs indicates a need to discover new therapeutic drugs. This study aimed to investigate the hypocholesterolemic potential of two triterpenoids isolated from Protorhus longifolia stem bark. In silico techniques and in vitro enzyme assays were used to evaluate the potential inhibition of cholesterol esterase and HMG-CoA reductase by the triterpenoids (ARM-2 and RA-5). The toxicity, modulation of low-density lipoprotein (LDL) uptake, and associated gene expression were determined in HepG2 hepatocytes. In silico molecular docking revealed that ARM-2 compared with RA-5 has a relatively stronger binding affinity for both enzymes. Both triterpenoids further demonstrated promising in silico drug-likeness properties and favorable ADMET profiles characterized by high intestinal absorption and lack of CYP450 enzyme inhibition. The compounds further showed, to varying degrees of efficacy, inhibition of cholesterol micellization as well as both cholesterol esterase and HMG-CoA reductase activities with IC50 values ranging from 16.4 to 41.1 µM. Moreover, enhanced hepatic cellular LDL uptake and the associated upregulation of the LDL-R and SREBP-2 gene expression were observed in the triterpenoid-treated HepG2 cells. It is evident that the triterpenoids, especially ARM-2, possess hypocholesterolemic properties, and these molecules can serve as leads or structural templates for the development of new hypocholesterolemic drugs.

The Antimethanogenic Potentials of Plant Extracts: Their Yields and Phytochemical Compositions as Affected by Extractive Solvents.

Plant phytochemicals are an important area of study in ruminant nutrition, primarily due to their antimethanogenic potentials. Plant extract yields, their bioactive compounds and antimethanogenic properties are largely dependent on the nature of the extractive solvents. This study evaluated the yields and phytochemical constituents of four plant extracts, as affected by the aqueous-methanolic (H2O-CH3OH) extraction and their antimethanogenic properties on the in vitro methane production. The plant extracts included Aloe vera, Jatropha curcas, Moringa oleifera, and Piper betle leaves with three levels of extractions (70, 85, and 100% CH3OH). The crude plant extract yields increased with the increasing amount of water. M. oleifera crude extracts yields (g/10 g) increased from 3.24 to 3.92, A. vera, (2.35 to 3.11) J. curcas (1.77 to 2.26), and P. betle (2.42 to 3.53). However, the identified and quantified metabolites showed differing degrees of solubility unique to their plant leaves in which they exist, while some of the metabolites were unaffected by the extraction solvents. The methane mitigating potentials of these extracts were evaluated as additives on Eragrostis curvula hay at a recommended rate of 50 mg kg−1 DM. The plant extracts exhibited antimethanogenic properties to various degrees, reducing (p < 0.05) in vitro methane production in the tested hay, A. vera, J. curcas, M. oleifera and P. betle reduced methane emission by 6.37−7.55%, 8.02−11.56%, 12.26−12.97, and 5.66−7.78 respectively compared to the control treatment. However, the antimethanogenic efficacy, gas production and organic matter digestibility of the plant extracts were unaffected by the extraction solvents. Metabolites, such as aloin A, aloin B and kaempferol (in A. vera), apigenin, catechin, epicatechin, kaempferol, tryptophan, procyanidins, vitexin-7-olate and isovitexin-7-olate (in J. curcas), alkaloid, kaempferol, quercetin, rutin and neochlorogenic acid (in M. oleifera) and apigenin-7,4'-diglucoside, 3-p-coumaroylquinic acid, rutin, 2-methoxy-4-vinylphenol, dihydrocaffeic acid, and dihydrocoumaric acid (in P. betle) exhibited a methane reducing potential and hence, additional studies may be conducted to test the methane reducing properties of the individual metabolites as well as their combined forms. Plant extracts could be more promising, and hence, further study is necessary to explore other extraction methods, as well as the encapsulation of extracts for the improved delivery of core materials to the target sites and to enhance methane reducing properties. Furthermore, the use of 70% aqueous extraction on M. oleifera leaf is recommended for practical use due to the reduced cost of extractive solvents, the lower cost and availability of Moringa plants in South Africa, especially in Gauteng Province. Furthermore, 70% aqueous-methanolic extractions of A. vera, J. curcas, and P. betle are recommended for practical use in regions where they exist in abundance and are cost effective.

Relationships between Agronomic Traits of Moringa Accessions and In Vitro Gas Production Characteristics of a Test Feed Incubated with or without Moringa Plant Leaf Extracts.

The use of medicinal plants and their extracts has recently attracted the attention of many researchers as a methane (CH4) mitigation strategy. This study evaluated the relationship of agronomic traits of Moringa accessions with in vitro gas production measurements and feed digestibility from ruminants. Twelve Moringa accessions were grown at the Roodeplaat experimental site of the Agricultural Research Council in Pretoria, South Africa. Agronomic traits, such as seedling survival rate, leaf yield, canopy and stem diameter, plant height, number of primary branches, plant vigor, greenness, chlorosis, disease and pest incidences were recorded. The leaves were harvested in the fifth month after transplanting to the field. Freeze-dried leaves were extracted with methanol, and their total phenolic and total flavonoid contents were determined. The extract was applied at a dose of 50 mg/kg of dry matter (DM) feed for in vitro gas production studies. Most of the growth and agronomic traits, i.e., seedling survival rate, leaf yield, canopy diameter, plant height, number of primary branches, the score of plant vigor, and greenness, total phenolics and flavonoids were significantly different among the accessions except for stem diameter and chlorosis score. All accession leaf extracts significantly reduced the total gas and CH4 production compared with the control with equal or higher in vitro organic matter digestibility. Higher CH4 inhibition was obtained in Moringa oleifera (M. oleifera) A3 (28.4%) and A11 (29.1%), whereas a lower inhibition was recorded in A1 (17.9%) and A2 (18.2%). The total phenolic (0.62) and total flavonoid (0.71) contents as well as most agronomic traits of the accessions were positively correlated with the CH4 inhibition potential of the accessions. Moringa oleifera accessions A3, A8 and A11 resulted in higher in vitro CH4 inhibition potential and improved organic matter digestibility of the feed with equal or higher adaptability performances in the field. Thus, there is a possibility of selecting Moringa accessions for higher antimethanogenic activity without compromising the feed digestibility by selecting for higher total phenolics, total flavonoids and agronomic performances traits. There is a need for further study to determine the long-term adaptability of promising accessions in the study area with concurrent antimethanogenesis efficacy when used in the diet of ruminant animals.

Screening of Candidate Bioactive Secondary Plant Metabolite Ion-Features from Moringa oleifera Accessions Associated with High and Low Enteric Methane Inhibition from Ruminants.

This study evaluated the relationship of secondary bioactive plant metabolite ion-features (MIFs) of Moringa oleifera accessions with antimethanogenesis to identify potential MIFs that were responsible for high and low methane inhibition from ruminants. Plant extracts from 12 Moringa accessions were evaluated at a 50 mg/kg DM feed for gas production and methane inhibition. Subsequently, the accessions were classified into low and high enteric methane inhibition groups. Four of twelve accessions (two the lowest and two the highest methane inhibitors), were used to characterize them in terms of MIFs. A total of 24 samples (12 from lower and 12 from higher methane inhibitors) were selected according to their methane inhibition potential, which ranged from 18% to 29%. Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) and untargeted metabolomics with univariate and multivariate statistical analysis with MetaboAnalyst were used in the study. Although 86 MIFs showed (p < 0.05) variation between higher and lower methane inhibition groups and lay within the detection ranges of the UPLC-MS column, only 14 were significant with the volcano plot. However, Bonferroni correction reduced the candidate MIFs to 10, and their R2-value with methane production ranged from 0.39 to 0.64. Eventually, MIFs 4.44_609.1462 and MIF 4.53_433.1112 were identified as bioactive MIFs associated with higher methane inhibition, whereas MIF 9.06_443.2317 and 15.00_487.2319 were associated with lower methane inhibition with no significant effect on in vitro organic matter digestibility of the feed. These MIFs could be used by plant breeders as potential markers to develop new M. oleifera varieties with high methane inhibition characteristics. However, further investigation on identifying the name, structure, and detailed biological activities of these bioactive metabolites needs to be carried out for future standardization, commercialization, and application as dietary methane mitigation additives.

Inhibition of α-glucosidase and α-amylase by herbal compounds for the treatment of type 2 diabetes: A validation of in silico reverse docking with in vitro enzyme assays.

BACKGROUND: α-Amylase and α-glucosidase are important therapeutic targets for the management of type 2 diabetes mellitus. The inhibition of these enzymes decreases postprandial hyperglycemia. In the present study, compounds found in commercially available herbs and spices were tested for their ability to inhibit α-amylase and α-glucosidase. These compounds were acetyleugenol, apigenin, cinnamic acid, eriodictyol, myrcene, piperine, and rosmarinic acid. METHODS: The enzyme inhibitory nature of the compounds was evaluated using in silico docking analysis with Maestro software and was further confirmed by in vitro α-amylase and α-glucosidase biochemical assays. RESULTS: The relationships between the in silico and in vitro results were well correlated; a more negative docking score was associated with a higher in vitro inhibitory activity. There was no significant (P > .05) difference between the inhibition constant (Ki ) value of acarbose, a widely prescribed α-glucosidase and α-amylase inhibitor, and those of apigenin, eriodictyol, and piperine. For α-amylase, there was no significant (P > .05) difference between the Ki value of acarbose and those of apigenin, cinnamic acid, and rosmarinic acid. The effect of the herbal compounds on cell viability was assessed with the sulforhodamine B (SRB) assay in C2C12 and HepG2 cells. Acetyleugenol, cinnamic acid, myrcene, piperine, and rosmarinic acid had similar (P > .05) IC50 values to acarbose. CONCLUSIONS: Several of the herbal compounds studied could regulate postprandial hyperglycemia. Using herbal plants has several advantages including low cost, natural origin, and easy cultivation. These compounds can easily be consumed as teas or as herbs and spices to flavor food.

Metabolomic Fingerprinting of Potato Cultivars Differing in Susceptibility to Spongospora subterranea f. sp. subterranea Root Infection.

Plants defend themselves from pathogens by producing bioactive defense chemicals. The biochemical mechanisms relating to quantitative resistance of potato to root infection by Spongospora subterranea f. sp. subterranea (Sss) are, however, not understood, and are not efficiently utilized in potato breeding programs. Untargeted metabolomics using ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was used to elucidate the biochemical mechanisms of susceptibility to Sss root infection. Potato roots and root exudate metabolic profiles of five tolerant cultivars were compared with those of five susceptible cultivars, following Sss inoculation, to identify tolerance-related metabolites. Comparison of the relative metabolite abundance of tolerant versus susceptible cultivars revealed contrasting responses to Sss infection. Metabolites belonging to amino acids, organic acids, fatty acids, phenolics, and sugars, as well as well-known cell wall thickening compounds were putatively identified and were especially abundant in the tolerant cultivars relative to the susceptible cultivars. Metabolites known to activate plant secondary defense metabolism were significantly increased in the tolerant cultivars compared to susceptible cultivars following Sss inoculation. Root-exuded compounds belonging to the chemical class of phenolics were also found in abundance in the tolerant cultivars compared to susceptible cultivars. This study illustrated that Sss infection of potato roots leads to differential expression of metabolites in tolerant and susceptible potato cultivars.

Evaluation of the Anti-Diabetic Activity of Some Common Herbs and Spices: Providing New Insights with Inverse Virtual Screening.

Culinary herbs and spices are widely used as a traditional medicine in the treatment of diabetes and its complications, and there are several scientific studies in the literature supporting the use of these medicinal plants. However, there is often a lack of knowledge on the bioactive compounds of these herbs and spices and their mechanisms of action. The aim of this study was to use inverse virtual screening to provide insights into the bioactive compounds of common herbs and spices, and their potential molecular mechanisms of action in the treatment of diabetes. In this study, a library of over 2300 compounds derived from 30 common herbs and spices were screened in silico with the DIA-DB web server against 18 known diabetes drug targets. Over 900 compounds from the herbs and spices library were observed to have potential anti-diabetic activity and liquorice, hops, fennel, rosemary, and fenugreek were observed to be particularly enriched with potential anti-diabetic compounds. A large percentage of the compounds were observed to be potential polypharmacological agents regulating three or more anti-diabetic drug targets and included compounds such as achillin B from yarrow, asparasaponin I from fenugreek, bisdemethoxycurcumin from turmeric, carlinoside from lemongrass, cinnamtannin B1 from cinnamon, crocin from saffron and glabridin from liquorice. The major targets identified for the herbs and spices compounds were dipeptidyl peptidase-4 (DPP4), intestinal maltase-glucoamylase (MGAM), liver receptor homolog-1 (NR5A2), pancreatic alpha-amylase (AM2A), peroxisome proliferator-activated receptor alpha (PPARA), protein tyrosine phosphatase non-receptor type 9 (PTPN9), and retinol binding protein-4 (RBP4) with over 250 compounds observed to be potential inhibitors of these particular protein targets. Only bay leaves, liquorice and thyme were found to contain compounds that could potentially regulate all 18 protein targets followed by black pepper, cumin, dill, hops and marjoram with 17 protein targets. In most cases more than one compound within a given plant could potentially regulate a particular protein target. It was observed that through this multi-compound-multi target regulation of these specific protein targets that the major anti-diabetic effects of reduced hyperglycemia and hyperlipidemia of the herbs and spices could be explained. The results of this study, taken together with the known scientific literature, indicated that the anti-diabetic potential of common culinary herbs and spices was the result of the collective action of more than one bioactive compound regulating and restoring several dysregulated and interconnected diabetic biological processes.

Metabolomics for a Millenniums-Old Crop: Tea Plant ( Camellia sinensis).

Tea cultivation and utilization dates back to antiquity. Today it is the most widely consumed beverage on earth due to its pleasant taste and several beneficial health properties attributed to specific metabolites. Metabolomics has a tremendous potential to correlate tea metabolites with taste and health properties in humans. Our review on the current application of metabolomics in the science of tea suggests that metabolomics is a promising frontier in the evaluation of tea quality, identification of functional genes responsible for key metabolites, investigation of their metabolic regulation, and pathway analysis in the tea plant. Furthermore, the challenges, possible solutions, and the prospects of metabolomics in tea science are reviewed.

Exploring African Medicinal Plants for Potential Anti-Diabetic Compounds with the DIA-DB Inverse Virtual Screening Web Server.

Medicinal plants containing complex mixtures of several compounds with various potential beneficial biological effects are attractive treatment interventions for a complex multi-faceted disease like diabetes. In this study, compounds identified from African medicinal plants were evaluated for their potential anti-diabetic activity. A total of 867 compounds identified from over 300 medicinal plants were screened in silico with the DIA-DB web server (http://bio-hpc.eu/software/dia-db/) against 17 known anti-diabetic drug targets. Four hundred and thirty compounds were identified as potential inhibitors, with 184 plants being identified as the sources of these compounds. The plants Argemone ochroleuca, Clivia miniata, Crinum bulbispermum, Danais fragans, Dioscorea dregeana, Dodonaea angustifolia, Eucomis autumnalis, Gnidia kraussiana, Melianthus comosus, Mondia whitei, Pelargonium sidoides, Typha capensis, Vinca minor, Voacanga Africana, and Xysmalobium undulatum were identified as new sources rich in compounds with a potential anti-diabetic activity. The major targets identified for the natural compounds were aldose reductase, hydroxysteroid 11-beta dehydrogenase 1, dipeptidyl peptidase 4, and peroxisome proliferator-activated receptor delta. More than 30% of the compounds had five or more potential targets. A hierarchical clustering analysis coupled with a maximum common substructure analysis revealed the importance of the flavonoid backbone for predicting potential activity against aldose reductase and hydroxysteroid 11-beta dehydrogenase 1. Filtering with physiochemical and the absorption, distribution, metabolism, excretion and toxicity (ADMET) descriptors identified 28 compounds with favorable ADMET properties. The six compounds-crotofoline A, erythraline, henningsiine, nauclefidine, vinburnine, and voaphylline-were identified as novel potential multi-targeted anti-diabetic compounds, with favorable ADMET properties for further drug development.

Functional annotation of putative QTL associated with black tea quality and drought tolerance traits.

The understanding of black tea quality and percent relative water content (%RWC) traits in tea (Camellia sinensis) by a quantitative trait loci (QTL) approach can be useful in elucidation and identification of candidate genes underlying the QTL which has remained to be difficult. The objective of the study was to identify putative QTL controlling black tea quality and percent relative water traits in two tea populations and their F1 progeny. A total of 1,421 DArTseq markers derived from the linkage map identified 53 DArTseq markers to be linked to black tea quality and %RWC. All 53 DArTseq markers with unique best hits were identified in the tea genome. A total of 5,592 unigenes were assigned gene ontology (GO) terms, 56% comprised biological processes, cellular component (29%) and molecular functions (15%), respectively. A total of 84 unigenes in 15 LGs were assigned to 25 different Kyoto Encyclopedia of Genes and Genomes (KEGG) database pathways based on categories of secondary metabolite biosynthesis. The three major enzymes identified were transferases (38.9%), hydrolases (29%) and oxidoreductases (18.3%). The putative candidate proteins identified were involved in flavonoid biosynthesis, alkaloid biosynthesis, ATPase family proteins related to abiotic/biotic stress response. The functional annotation of putative QTL identified in this current study will shed more light on the proteins associated with caffeine and catechins biosynthesis and % RWC. This study may help breeders in selection of parents with desirable DArTseq markers for development of new tea cultivars with desirable traits.

Anti-aging potential of extracts from Sclerocarya birrea (A. Rich.) Hochst and its chemical profiling by UPLC-Q-TOF-MS.

BACKGROUND: Degradation of components of the extracellular matrix such as elastin and collagen by elastase and collagenase accelerates skin aging. Phytochemicals that inhibit the activity of these enzymes can be developed as anti-aging ingredients. In this study, an investigation of the anti-aging properties of Sclerocarya birrea (A. Rich.) Hochst (Marula) extracts was conducted in vitro with the aim of developing chemically characterized anti-aging ingredients. METHODS: Marula stems, leaves and fruits were extracted using methanol:dichloromethane (DCM) (1:1). The stems were later extracted using acetone, ethanol, methanol:DCM (1:1) and sequentially using hexane, DCM, ethyl acetate and methanol. The stem ethanol extract was defatted and concentrated. Elastase and collagenase inhibition activities of these extracts and Marula oil were determined using spectrophotometric methods. The chemical profile of the ethanolic stem extract was developed using Ultra-performance-liquid chromatography quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS) with MassLynx software. Pure standards were used to confirm the identity of major compounds and were screened for anti-elastase and anti-collagenase activity. RESULTS: Marula stems extracts were the most active as they exhibited anti-elastase activity comparable to that of elafin (> 88%) and anti-collagenase activity as potent as EDTA (> 76%). The leaf extract had moderate anti-elastase activity (54%) but was inactive agains collagenase. Marula fruits and oil exhibited limited activity in both assays. The ethanolic extract of Marula stems was the most suitable based on its acceptability to the cosmetic industry and its anti-collagenase activity (99%). Defatting and concentration improved its antiaging activity and lowered the colour intensity. Six compounds have been tentatively identified in the chemical profile of the ethanolic extract of Marula stems of which four; quinic acid, catechin, epigallocatechin gallate and epicatechin gallate have been confirmed using pure standards. Epigallocatechin gallate and epicatechin gallate were as potent (p < 0.05) as EDTA at 5 µg/ml in the anti-collagenase assay. CONCLUSIONS: The ethanolic extract of Marula stems can be developed into an anti-aging ingredient as it exhibited very good in vitro anti-aging activity and its chemical profile has been developed. Epicatechin gallate and epigallocatechin gallate contribute to the anti-aging activity of Marula stem ethanol extract.

Antioxidant and anti-inflammatory properties of Ilex guayusa tea preparations: a comparison to Camellia sinensis teas.

Ilex guayusa tea preparations are now commercially available as Runa tea. Little is known regarding the antioxidant and anti-inflammatory bioactivities of this tea. The I. guayusa teas had a total polyphenolic content between 54.39 and 67.23 mg GAE per g dry mass and peroxyl radical scavenging capacities between 1773.41 and 2019 µmol TE per g dry mass, nearly half of that for the Camellia sinensis teas. The I. guayusa teas afforded 60-80% protection from oxidative stress in the Caco-2 cellular antioxidant assay, comparable to the C. sinensis teas. The anti-inflammatory activity in lipopolysaccharide-stimulated RAW 264.7 cells of I. guayusa teas was similarly comparable to the C. sinensis teas with nitric oxide production reduced by 10-30%. Major compounds identified by mass spectrometry were the phenolic mono- and dicaffeoylquinic acid derivatives. I. guayusa teas are a good source of dietary phenolic compounds with cellular antioxidant and anti-inflammatory properties.

Proteomic and metabolomic analysis reveals rapid and extensive nicotine detoxification ability in honey bee larvae.

Despite potential links between pesticides and bee declines, toxicology information on honey bee larvae (Apis mellifera) is scarce and detoxification mechanisms in this development stage are virtually unknown. Larvae are exposed to natural and synthetic toxins present in pollen and nectar through consumption of brood food. Due to the characteristic intensive brood care displayed by honey bees, which includes progressive feeding throughout larval development, it is generally assumed that larvae rely on adults to detoxify for them and exhibit a diminished detoxification ability. We found the opposite. We examined the proteomic and metabolomic responses of in vitro reared larvae fed nicotine (an alkaloid found in nectar and pollen) to understand how larvae cope on a metabolic level with dietary toxins. Larvae were able to effectively detoxify nicotine through an inducible detoxification mechanism. A coordinated stress response complemented the detoxification processes, and we detected significant enrichment of proteins functioning in energy and carbohydrate metabolism, as well as in development pathways, suggesting that nicotine may promote larval growth. Further exploration of the metabolic fate of nicotine using targeted mass spectrometry analysis demonstrated that, as in adult bees, formation of 4-hydroxy-4-(3-pyridyl) butanoic acid, the result of 2'C-oxidation of nicotine, is quantitatively the most significant pathway of nicotine metabolism. We provide conclusive evidence that larvae are capable of effectively catabolising a dietary toxin, suggesting that increased larval sensitivity to specific toxins is not due to diminished detoxification abilities. These findings broaden the current understanding of detoxification biochemistry at different organizational levels in the colony, bringing us closer to understanding the capacity of the colony as a superorganism to tolerate and resist toxic compounds, including pesticides, in the environment.

The metabolic fate of nectar nicotine in worker honey bees.

Honey bees (Apis mellifera) are generalist pollinators that forage for nectar and pollen of a very large variety of plant species, exposing them to a diverse range of secondary metabolites produced as chemical defences against herbivory. Honey bees can tolerate high levels of many of these toxic compounds, including the alkaloid nicotine, in their diet without incurring apparent fitness costs. Very little is known about the underlying detoxification processes mediating this tolerance. We examined the metabolic fate of nicotine in newly emerged worker bees using radiolabeled nicotine and LC-MS/MS analysis to determine the kinetic distribution profile of nicotine as well as the absence or presence and identity of any nicotine-derived metabolites. Nicotine metabolism was extensive; virtually no unmetabolised nicotine were recovered from the rectum. The major metabolite found was 4-hydroxy-4-(3-pyridyl) butanoic acid, the end product of 2'C-oxidation of nicotine. It is the first time that 4-hydroxy-4-(3-pyridyl) butanoic acid has been identified in an insect as a catabolite of nicotine. Lower levels of cotinine, cotinine N-oxide, 3'hydroxy-cotinine, nicotine N-oxide and norcotinine were also detected. Our results demonstrated that formation of 4-hydroxy-4-(3-pyridyl) butanoic acid is quantitatively the most significant pathway of nicotine metabolism in honey bees and that the rapid excretion of unmetabolised nicotine does not contribute significantly to nicotine tolerance in honey bees. In nicotine-tolerant insects that do not rely on the rapid excretion of nicotine like the Lepidoptera, it is possible that the 2'C-oxidation of nicotine is the conserved metabolic pathway instead of the generally assumed 5'C-oxidation pathway.

SWAPDT: A method for Short-time Withering Assessment of Probability for Drought Tolerance in Camellia sinensis validated by targeted metabolomics.

Climate change is causing droughts affecting crop production on a global scale. Classical breeding and selection strategies for drought-tolerant cultivars will help prevent crop losses. Plant breeders, for all crops, need a simple and reliable method to identify drought-tolerant cultivars, but such a method is missing. Plant metabolism is often disrupted by abiotic stress conditions. To survive drought, plants reconfigure their metabolic pathways. Studies have documented the importance of metabolic regulation, i.e. osmolyte accumulation such as polyols and sugars (mannitol, sorbitol); amino acids (proline) during drought. This study identified and quantified metabolites in drought tolerant and drought susceptible Camellia sinensis cultivars under wet and drought stress conditions. For analyses, GC-MS and LC-MS were employed for metabolomics analysis.%RWC results show how the two drought tolerant and two drought susceptible cultivars differed significantly (p≤0.05) from one another; the drought susceptible exhibited rapid water loss compared to the drought tolerant. There was a significant variation (p<0.05) in metabolite content (amino acid, sugars) between drought tolerant and drought susceptible tea cultivars after short-time withering conditions. These metabolite changes were similar to those seen in other plant species under drought conditions, thus validating this method. The Short-time Withering Assessment of Probability for Drought Tolerance (SWAPDT) method presented here provides an easy method to identify drought tolerant tea cultivars that will mitigate the effects of drought due to climate change on crop losses.

Anti-proliferative properties of commercial Pelargonium sidoides tincture, with cell-cycle G0/G1 arrest and apoptosis in Jurkat leukaemia cells.

Context Pelargonium sidoides DC (Geraniaceae) is an important medicinal plant indigenous to South Africa and Lesotho. Previous studies have shown that root extracts are rich in polyphenolic compounds with antibacterial, antiviral and immunomodulatory activities. Little is known regarding the anticancer properties of Pelargonium sidoides extracts. Objective This study evaluates the anti-proliferative effects of a Pelargonium sidoides radix mother tincture (PST). Materials and methods The PST was characterized by LC-MS/MS. Anti-proliferative activity was evaluated in the pre-screen panel of the National Cancer Institute (NCI-H460, MCF-7 and SF-268) and the Jurkat leukaemia cell line at concentrations of 0-150 µg/mL. The effect on cell growth was determined with sulphorhodamine B and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays after 72 h. The effect on cell cycle and apoptosis induction in Jurkat cells was determined by flow cytometry with propidium iodide and Annexin V: fluorescein isothiocyanate staining. Results Dihydroxycoumarin sulphates, gallic acid as well as gallocatechin dimers and trimers were characterized in PST by mass spectrometry. Moderate anti-proliferative effects with GI50 values between 40 and 80 µg/mL were observed in the NCI-pre-screen panel. Strong activity observed with Jurkat cells with a GI50 value of 6.2 µg/mL, significantly better than positive control 5-fluorouracil (GI50 value of 9.7 µg/mL). The PST arrested Jurkat cells at the G0/G1 phase of the cell cycle and increased the apoptotic cells from 9% to 21%, while the dead cells increased from 4% to 17%. Conclusion We present evidence that P. sidoides has cancer cell type-specific anti-proliferative effects and may be a source of novel anticancer molecules.

Detoxification mechanisms of honey bees (Apis mellifera) resulting in tolerance of dietary nicotine.

Insecticides are thought to be among the major factors contributing to current declines in bee populations. However, detoxification mechanisms in healthy, unstressed honey bees are poorly characterised. Alkaloids are naturally encountered in pollen and nectar, and we used nicotine as a model compound to identify the mechanisms involved in detoxification processes in honey bees. Nicotine and neonicotinoids have similar modes of action in insects. Our metabolomic and proteomic analyses show active detoxification of nicotine in bees, associated with increased energetic investment and also antioxidant and heat shock responses. The increased energetic investment is significant in view of the interactions of pesticides with diseases such as Nosema spp which cause energetic stress and possible malnutrition. Understanding how healthy honey bees process dietary toxins under unstressed conditions will help clarify how pesticides, alone or in synergy with other stress factors, lead to declines in bee vitality.

The antimicrobial effect of colistin methanesulfonate on Mycobacterium tuberculosis in vitro.

Polymyxins have previously been described to have activity against Mycobacterium tuberculosis (MTB), but further research was abandoned due to systemic toxicity concerns to achieve the required MIC. Colistin methanesulfonate (CMS), a polymyxin, is well tolerated when inhaled directly into the lungs, resulting in high local concentrations. We report here for the first time, MIC and MBC data for CMS determined by the microtiter Alamar Blue assay (MABA). We also determined how the MIC would be affected by the presence of pulmonary surfactant (PS) and if any synergy with isoniazid (INH) and rifampicin (RIF) exists. The effect of CMS on the ultrastructure of MTB was also determined. The MIC for CMS was 16 mg/L, while the MBC was 256 mg/L. MIC for CMS in PS was antagonised by eight fold. For synergy, indifference was determined while time-kill assays revealed a greater killing effect when CMS was used together with INH. Ultrastructure analysis suggests that the disruption of the outer polysaccharide layer of MTB by CMS may lead to enhanced uptake of INH. Our findings may provide insight for further investigations of CMS against MTB.

Antioxidant supplementation can reduce the survival costs of excess amino acid intake in honeybees.

Over-consuming amino acids is associated with reduced survival in many species, including honeybees. The mechanisms responsible for this are unclear but one possibility is that excessive intake of amino acids increases oxidative damage. If this is the case, antioxidant supplementation may help reduce the survival costs of high amino acid intake. We tested this hypothesis in African honeybees (Apis mellifera scutellata) using the major antioxidant in green tea, epigallocatechin-3-gallate (EGCG). We first determined the dose-range of EGCG that improved survival of caged honeybees fed sucrose solution. We then provided bees with eight diets that differed in their ratio of essential amino acids (EAA) to carbohydrate (C) (0:1, 1:250, 1:100, 1:75, 1:50, 1:25, 1:10, 1:5 EAA:C) and also in their EGCG dose (0.0 or 0.4 mM). We found that bees fed sucrose only solution survived better than bees fed EAA diets. Despite this, bees preferred a diet that contained intermediate ratios of EAA:C (ca. 1:25), which may represent the high demands for nitrogen of developing nurse bees. EGCG supplementation improved honeybee survival but only at an intermediate dose (0.3-0.5 mM) and in bees fed low EAA diets (1:250, 1:100 EAA:C). That EGCG counteracted the lifespan reducing effects of eating low EAA diets suggests that oxidative damage may be involved in the association between EAAs and lifespan in honeybees. However, that EGCG had no effect on survival in bees fed high EAA diets suggests that there are other physiological costs of over-consuming EAAs in honeybees.

Screening of tea (Camellia sinensis) for trait-associated molecular markers.

This study was done to identify random amplified polymorphic DNA (RAPD) markers that may associate with seven important traits in tea. Sixty RAPD primers were first screened using 18 cultivars under each of the 7 traits, followed by confirmatory screening of 20 promising primers with 32 tea cultivars. Six RAPD primers generated a total of nine specific bands that associated with six desired traits: black tea quality and tolerance to drought, high temperature, low temperature, Phomopsis theae, and high yield. These markers would allow early identification of plant material with the desired traits that can be advanced to the next stage of selection and enhance targeted choice of breeding stocks with the desirable traits. The nine RAPD markers identified in this study could improve precision and efficiency in tea breeding and selection and are an important contribution towards the establishment of marker-assisted selection in tea breeding programmes.