Secondary metabolic profiling of Serratia marcescens NP10 reveals new stephensiolides and glucosamine derivatives with bacterial membrane activity.

Secondary metabolic profiling, using UPLC-MSE and molecular networking, revealed the secondary metabolites produced by Serratia marcescens NP10. The NP10 strain co-produced cyclic and open-ring stephensiolides (i.e., fatty acyl chain linked to Thr-Ser-Ser-Ile/Leu-Ile/Leu/Val) and glucosamine derivatives (i.e., fatty acyl chain linked to Val-glucose-butyric/oxo-hexanoic acid), with the structures of sixteen new stephensiolides (L-Y) and three new glucosamine derivatives (L-N) proposed. Genome mining identified sphA (stephensiolides) and gcd (glucosamine derivatives) gene clusters within Serratia genomes available on NBCI using antiSMASH, revealing specificity scores of the adenylation-domains within each module that corroborates MSE data. Of the nine RP-HPLC fractions, two stephensiolides and two glucosamine derivatives exhibited activity against Staphylococcus aureus (IC50 of 25-79 µg/mL). 1H NMR analysis confirmed the structure of the four active compounds as stephensiolide K, a novel analogue stephensiolide U, and glucosamine derivatives A and C. Stephensiolides K and U were found to cause membrane depolarisation and affect the membrane permeability of S. aureus, while glucosamine derivatives A and C primarily caused membrane depolarisation. New members of the stephensiolide and glucosamine derivative families were thus identified, and results obtained shed light on their antibacterial properties and mode of membrane activity.

Essential Oil Composition and Traditional Uses of Salvia dentata, a Poorly Known Medicinal Plant from Namaqualand, South Africa.

South Africa has a rich history of medicinal plant species and their documented uses as traditional medicines, and is also home to three well-known, blue-flowered sage species of ethnobotanical importance. The Namaqualand bloublomsalie (Salvia dentata) has so far remained unstudied and apparently overlooked. Our study is the first to report on the essential oil chemistry of this medicinally relevant species and provide a comparison with the other two (well-studied) closely related Cape bloublomsalies (Salvia africana and S. chamelaeagnea). The data, generated from three geographically isolated populations comprised of 13 individual plants of S. dentata, revealed diagnostically high levels of camphor (14.37%), α-pinene (11.43%), camphene (10.18%), 1,8-cineole (eucalyptol) (9.42%) and bornyl acetate (8.56%) which provide a distinct chemical profile from the other two species.

Corrigendum: Mass spectrometry metabolomics and feature-based molecular networking reveals population-specific chemistry in some species of the Sceletium genus.

[This corrects the article DOI: 10.3389/fnut.2022.819753.].

Mass Spectrometry Metabolomics and Feature-Based Molecular Networking Reveals Population-Specific Chemistry in Some Species of the Sceletium Genus.

The Sceletium genus has been of medicinal importance in southern Africa for millennia and Sceletium tortuosum (Aizoaceae), one of eight species in the genus has gained pharmaceutical importance as an anxiolytic and anti-depressant due to the presence of mesembrine alkaloids. S. tortuosum is used for the manufacture of herbal teas, dietary supplements and other phytopharmaceutical products. This study aimed to provide a metabolomic characterization of S. tortuosum and its sister species as these are not easy to distinguish using morphology alone. Plant samples were thus collected from various locations in the succulent Karoo (South Africa) and analyzed through liquid chromatography-mass spectrometry (LC-MS), using MSE fragmentation as a putative tool for chemical identities. Metabolomics-based analyses in combination with molecular networking were able to distinguish between the four species of Sceletium based on the presence of 4-(3,4-dimethyoxyphenyl)-4-[2-acetylmethlamino)ethyl]cyclohexanone (m/z 334.2020; RT 6.60 min), mesembrine (m/z 290.1757; RT 5.10 min) and 4'-O-demethylmesembrenol (m/z 276.1597; RT 4.17 min). Metabolomic profiles varied according to the different localities and metabolites occurred at variable quantitative levels in Sceletium ecotypes. Molecular networking provided the added advantage of being able to observe mesembrine alkaloid isomers and coeluting metabolites (from the joubertiamine group) that were difficult to discern without this application. By combining high-throughput metabolomics together with global and feature based-molecular networking, a powerful metabolite profiling platform that is able to discern chemical patterns within and between populations was established. These techniques were able to reveal chemotaxonomic relationships and allowed for the discovery of chemical markers that may be used as part of monitoring protocols during the manufacture of phytopharmaceutical and dietary products based on Sceletium.

A link between urease and polyamine metabolism in Cryptococcus neoformans.

The urease enzyme of Cryptococcus neoformans is linked to different metabolic pathways within the yeast cell, several of which are involved in polyamine metabolism. Cryptococcal biogenic amine production is, however, largely unexplored and is yet to be investigated in relation to urease. The aim of this study was therefore to explore and compare polyamine metabolism in wild-type, urease-negative and urease-reconstituted strains of C. neoformans. Mass spectrometry analysis showed that agmatine and spermidine were the major extra- and intracellular polyamines of C. neoformans and significant differences were observed between 26 and 37 °C. In addition, compared to the wild-type, the relative percentages of extracellular putrescine and spermidine were found to be lower and agmatine higher in cultures of the urease-deficient mutant. The inverse was true for intracellular spermidine and agmatine. Cyclohexylamine was a more potent polyamine inhibitor compared to DL-α-difluoromethylornithine and inhibitory effects were more pronounced at 37 °C than at 26 °C. At both temperatures, the urease-deficient mutant was less susceptible to cyclohexylamine treatment compared to the wild-type. For both inhibitors, growth inhibition was alleviated with polyamine supplementation. This study has provided novel insight into the polyamine metabolism of C. neoformans, highlighting the involvement of urease in biogenic amine production.

Profiling the Production of Antimicrobial Secondary Metabolites by Xenorhabdus khoisanae J194 Under Different Culturing Conditions.

Species from the genus Xenorhabdus, endosymbiotic bacteria of Steinernema nematodes, produce several antibacterial and antifungal compounds, some of which are anti-parasitic. In this study, we report on the effect growth conditions have on the production of antimicrobial compounds produced by Xenorhabdus khoisanae J194. The strain was cultured in aerated and non-aerated broth, respectively, and on solid media. Production of antimicrobial compounds was detected after 24 h of growth in liquid media, with highest levels recorded after 96 h. Highest antimicrobial activity was obtained from cells cultured on solid media. By using ultraperformance liquid chromatography linked to mass spectrometry and HPLC, a plethora of known Xenorhabdus compounds were identified. These compounds are the PAX lipopeptides (PAX 1', PAX 3', PAX 5, and PAX 7E), xenocoumacins and xenoamicins. Differences observed in the MS-MS fractionation patterns collected in this study, when compared to previous studies indicated that this strain produces novel xenoamicins. Three novel antimicrobial compounds, khoicin, xenopep and rhabdin, were identified and structurally characterized based on MS-MS fractionation patterns, amino acid analysis and whole genome analysis. The various compounds produced under the three different conditions indicates that the secondary metabolism of X. khoisanae J194 may be regulated by oxygen, water activity or both. Based on these findings X. khoisanae J194 produce a variety of antimicrobial compounds that may have application in disease control.

Oligomerisation of tryptocidine C, a Trp-rich cyclodecapeptide from the antimicrobial tyrothricin complex.

Tryptocidine C (TpcC, cyclo[D-Phe1-Pro2-Trp3-D-Trp4-Asn5-Gln6-Trp7-Val8-Orn9-Leu10]) is a broad-spectrum antimicrobial peptide in the tyrothricin complex produced by a soil bacterium, Brevibacillus parabrevis. Electrospray mass spectrometric studies reveal the oligomerisation of TpcC into dimers and higher oligomers, analogous to tyrocidine C (TrcC, Trp7 replaced by Tyr7). Ion mobility mass spectrometry (IMMS) further confirms the formation of stable peptide dimers and tetramers with diameters of 2.7 nm and 3.3 nm, respectively, calculated from collisional cross section (CCS). Molecular dynamic simulations and docking studies support the formation of amphipathic dimers, with a diameter of 2.5 ± 0.07 nm calculated from low energy model CCS. Circular dichroism and IMMS studies point towards dynamic hydrogen-bonded conformational changes up to 28-33 µM after which the structures become more static (or in equilibrium). Fluorescence studies indicate aromatic stacking of Trp residues with a CMC of 18 µM in aqueous solutions. The concentration and time dependent interaction of Trp in oligomers indicate cooperativity in the TpcC oligomerisation that leads to the formation of higher order microscopic structures. Scanning electron microscopy studies unequivocally shows that TpcC forms nanospheres with a mean diameter of 25 nm. Repeated smaller oligomeric units, possibly dimers and tetramers, self-assemble to form these nanospheres.

Pharmacokinetics of Para-Aminosalicylic Acid and Its 2 Major Metabolites: A Potential Relationship to the Development of Gastrointestinal Intolerance.

Para-aminosalicylic acid (PAS), often the last drug remaining for treatment of drug-resistant tuberculosis, is notorious for causing gastrointestinal intolerance; however, the cause of PAS intolerance is uncertain. The objective of this study was to assess relationships between peak concentrations of PAS administered as a granular slow-release enteric coated formulation, and its metabolites acetyl-PAS and glycine-PAS, and intolerance. PAS and its metabolites were measured in 29 adult patients with drug-resistant tuberculosis at Brooklyn Hospital, Cape Town, randomized to receive granular slow-release enteric-coated PAS 4 g twice daily or 8 g once daily for 1 week, followed by the alternative regimen. Concentrations of PAS and its metabolites were determined by liquid chromatography and tandem mass spectrometry, and a visual analogue scale evaluated intolerance. Spearman's correlation test assessed the relationship between maximum plasma concentrations (Cmax ) and intolerance scores. A large interindividual variability was observed for the PAS Cmax (40.42-68.55 mg/L) following 4 g twice daily; (62.69-102.41 mg/L) for 8 g once daily and a similar wide Cmax range found for the metabolites acetyl-PAS and glycine-PAS. Twenty-six patients reported at least 1 intolerance episode, but most visual analogue scale scores clustered around 0. Significant inverse associations were found between acetyl-PAS Cmax and bloating (rho = -0.448; P = .025) and diarrhea (rho = -0.407; P = .044) for the twice-daily regimen and a similar inverse association found for glycine-PAS and diarrhea (rho = -0.412; P = .041). Plasma concentrations of the metabolites did not correlate with the occurrence of gastrointestinal symptoms, but higher metabolite concentrations correlated with lower intolerance scores; slow metabolism of PAS and its continued presence in the intestinal tract may be the main cause of intolerance.

Polyphenolic and Physicochemical Properties of Simple-Spined Num-Num (Carissa edulis) Fruit Harvested at Ripe Stage of Maturation.

Wildly grown in most regions of the world, Carissa edulis is a highly underutilised fruit with significant antioxidant characteristics. The phyto and physicochemical properties of C. edulis berries at different stages of ripening are evaluated in this work. Total flavonoids (TF), total phenolic content (TPC) and antioxidant activity were determined spectrophotometrically, while concentration of polyphenols was determined using liquid chromatography coupled to diode array detection and electrospray ionization mass spectrometry. Results showed that antioxidant activity was lowest (18.36 ± 0.12 mg TE/g) in RS3 and decreased with TPC upon increased ripening. Conversely, TF increased with ripening progression with TF found to be highest in RS3 (5.92 ± 0.03 mg CE/g). Identified phenolic acids in C. edulis were quinic acid, protocatechuoyl-hexose, neochlorogenic acid, chlorogenic acid, cryptochlorogenic acid and dicaffeoylquinic acid. Identified flavonoids included rutin, catechin, procyanidin dimer, procyanidin trimer, quercetin-3-O-glucosyl-xyloside, quercetin-3-O-robinobioside, quercetin-3-O-glucoside and quercetin-3-OH-3-methylglutaryl-glucoside. Physicochemical properties of C. edulis varied among samples with sugar/acid ratio of C. edulis ranging from 25.70 for RS1 to 50.36 for RS3. Ripening stage of C. edulis undoubtedly affects the phyto and physicochemical properties of C. edulis.

Esomeprazole to treat women with preterm preeclampsia: a randomized placebo controlled trial.

BACKGROUND: Preterm preeclampsia has a high rate of fetal death or disability. There is no treatment to slow the disease, except delivery. Preclinical studies have identified proton pump inhibitors as a possible treatment. OBJECTIVE: The purpose of this study was to examine whether esomeprazole could prolong pregnancy in women who have received a diagnosis of preterm preeclampsia. STUDY DESIGN: We performed a double-blind, randomized controlled trial at Tygerberg Hospital in South Africa. Women with preterm preeclampsia (gestational age 26 weeks+0 days to 31 weeks+6 days) were assigned randomly to 40-mg daily esomeprazole or placebo. The primary outcome was a prolongation of gestation of 5 days. Secondary outcomes were maternal and neonatal outcomes. We compared circulating markers of endothelial dysfunction that was associated with preeclampsia and performed pharmacokinetic studies. RESULTS: Between January 2016 and April 2017, we recruited 120 participants. One participant was excluded because of incorrect randomization, which left 59 participants in the esomeprazole and 60 participants in the placebo group. Median gestational age at enrolment was 29+4 weeks gestation. There were no between-group differences in median time from randomization to delivery: 11.4 days (interquartile range, 3.6-19.7 days) in the esomeprazole group and 8.3 days (interquartile range, 3.8-19.6 days) in the placebo group (3 days longer in the esomeprazole arm; 95% confidence interval, -2.9-8.8; P=.31). There were no placental abruptions in the esomeprazole group and 6 (10%) in the placebo group (P=.01, P=.14 adjusted). There were no differences in other maternal or neonatal outcomes or markers of endothelial dysfunction. Esomeprazole and its metabolites were detected in maternal blood among those treated with esomeprazole, but only trace amounts in the umbilical cord blood. CONCLUSION: Daily esomeprazole (40 mg) did not prolong gestation in pregnancies with preterm preeclampsia or decrease circulating soluble fms-like tyrosine kinase 1 concentrations. Higher levels in the maternal circulation may be needed for clinical effect.

An Electrospray Ionization Mass Spectrometry Study on the "In Vacuo" Hetero-Oligomers Formed by the Antimicrobial Peptides, Surfactin and Gramicidin S.

It was previously observed that the lipopeptide surfactants in surfactin (Srf) have an antagonistic action towards the highly potent antimicrobial cyclodecapeptide, gramicidin S (GS). This study reports on some of the molecular aspects of the antagonism as investigated through complementary electrospray ionization mass spectrometry techniques. We were able to detect stable 1:1 and 2:1 hetero-oligomers in a mixture of surfactin and gramicidin S. The noncovalent interaction between GS and Srf, with the proposed equilibrium: GS~Srf↔GS+Srf correlated to apparent K d values of 6-9 µM in gas-phase and 1 µM in aqueous solution. The apparent K d values decreased with a longer incubation time and indicated a slow oligomerization equilibrium. Furthermore, the low µM K dapp values of GS~Srf↔GS+Srf fell within the biological concentration range and related to the 2- to 3-fold increase in [GS] needed for bacterial growth inhibition in the presence of Srf. Competition studies indicated that neither Na+ nor Ca2+ had a major effect on the stability of preformed heterodimers and that GS in fact out-competed Ca2+ and Na+ from Srf. Traveling wave ion mobility mass spectrometry revealed near symmetrical peaks of the heterodimers correlating to a compact dimer conformation that depend on specific interactions. Collision-induced dissociation studies indicated that the peptide interaction is most probably between one Orn residue in GS and the Asp residue, but not the Glu residue in Srf. We propose that flanking hydrophobic residues in both peptides stabilize the antagonistic and inactive peptide hetero-oligomers and shield the specific polar interactions in an aqueous environment. Graphical Abstract ᅟ.

High-throughput analysis of 19 endogenous androgenic steroids by ultra-performance convergence chromatography tandem mass spectrometry.

11-Oxygenated steroids such as 11-ketotestosterone and 11-ketodihydrotestosterone have recently been shown to play a putative role in the development and progression of castration resistant prostate cancer. In this study we report on the development of a high throughput ultra-performance convergence chromatography tandem mass spectrometry (UPC(2)-MS/MS) method for the analysis of thirteen 11-oxygenated and six canonical C19 steroids isolated from a cell culture matrix. Using an Acquity UPC(2) BEH 2-EP column we found that UPC(2) resulted in superior selectivity, increased chromatographic efficiency and a scattered elution order when compared to conventional reverse phase ultra-performance liquid chromatography (UPLC). Furthermore, there was a significant improvement in sensitivity (5-50 times). The lower limits of quantification ranged between 0.01-10ngmL(-1), while the upper limit of quantification was 100ngmL(-1) for all steroids. Accuracy, precision, intra-day variation, recovery, matrix effects and process efficiency were all evaluated and found to be within acceptable limits. Taken together we show that the increased power of UPC(2)-MS/MS allows the analyst to complete in vitro assays at biologically relevant concentrations for the first time and in so doing determine the routes of steroid metabolism which is vital for studies of androgen responsive cancers, such as prostate cancer, and could highlight new mechanisms of disease progression and new targets for cancer therapy.

Determination of sotolon content in South African white wines by two novel HPLC-UV and UPLC-MS methods.

Sotolon has been reported to play an important role in the atypical ageing and aroma character of many wines. A number of analytical techniques for sotolon analysis in wine have been reported, but these often require extensive sample preparation. In this work we report a HPLC-UV method and a novel UPLC-MS method to determine sotolon concentrations in white wines with little sample preparation applied for the first time for the evaluation of sotolon levels in South African wines. The validation showed that the instrumental methods had good accuracy, repeatability and linearity, but the UPLC-MS method proved more sensitive. For both methods, quantification limits were lower than the sotolon odour threshold in wine (10µg/L), 0.86µg/L and 0.013µg/L, for HPLC-UV and UPLC-MS methods, respectively. Sotolon levels in 65 South African white wines were often found to be lower than the reported odour threshold, with the highest concentration being 9.11µg/L. However, for low levels (<1µg/L), unknown interferences in certain wines led to sotolon not being quantified with the HPLC-UV method, which made the UPLC-MS method more suitable.

Speciation of [Pt(IV) Cl6-n Brn]2- (n = 0-6) and some of their mono-aquated [Pt(IV) Cl5-n Brn (H2O)]- (n = 0-5) anions in solution at low concentrations by means of ion-pairing reversed-phase ultra-high-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry.

RATIONALE: The speciation of the purely inorganic [PtCl6-n Brn](2-) (n = 0-6) anions and their corresponding mono-aquated [PtCl5-n Brn (H2O)](-) (n = 0-5) anions is of considerable importance to the precious metal refining and recycling industry, to ensure optimum recovery and separation efficiencies. Speciation of platinum complexes present in precursor solutions used for the preparation of precious metal nano-crystals of defined size and morphology appears also to be important. The various possible Pt(IV) complex anions in dilute aqueous can be characterized using ion-pairing reversed-phase high-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOFMS). METHODS: Ion-pairing reversed-phase ultra-high-performance LC separation of the Pt(IV) complex anions present in aqueous solutions prior to detection by means of high-resolution ESI-Q-TOFMS using a low ESI source cone voltage (5 V) allows for the clear identification of all the platinum complexes from the characteristic pattern of fragment ions (m/z), presumably generated by 'reductive conversion' in the ESI source of the mass spectrometer. Sufficient chromatographic resolution for the series of Pt(IV) complexes is achieved using the (n-butyl)3 NH(+) ion generated in a formic acid/water/methanol (pH ~3.5) mobile phase. This mobile phase composition facilitates a low-background for optimal ESI-Q-TOFMS detection with enhanced sensitivity. RESULTS: Direct-infusion mass spectrometry of the inorganic platinum complexes in aqueous solution is impractical due to their low volatility, but more importantly as a result of the very extensive series of fragment ions generated in the ESI source, which leads to virtually uninterpretable mass spectra. However, with prior separation, and by using low ESI cone voltages (5 V), the mass spectra of the separated analyte ions show simpler and systematic fragmentation patterns [Pt(IV) X5](-) → [Pt(III) X4 ](-) → [Pt(II) X3](-) → [Pt(I)X2 ](-) (X = Cl(-) and Br(-)), resulting in clear assignments. This methodology facilitates the characterization of the partially aquated [PtCl5-n Brn (H2O)](-) (n = 0-5) anions derived from the homo- and heteroleptic [PtCl6-n Brn](2-) (n = 0-6) anions, in equilibrated solutions at low concentrations. CONCLUSIONS: Speciation of homo- and heteroleptic [PtCl6-n Brn](2-) (n = 0-6) anions, together with some of their partially aquated [PtCl5-n Brn (H2O)](-) (n = 0-5) species in dilute solution, can successfully be carried out by means of prior ion-pairing reversed-phase LC separation coupled to high-resolution ESI-Q-TOFMS at low ESI cone-voltage settings.

Manipulation of the tyrothricin production profile of Bacillus aneurinolyticus.

A group of non-ribosomally produced antimicrobial peptides, the tyrocidines from the tyrothricin complex, have potential as antimicrobial agents in both medicine and industry. Previous work by our group illustrated that the more polar tyrocidines rich in Trp residues in their structure were more active toward Gram-positive bacteria, while the more non-polar tyrocidines rich in Phe residues had greater activity toward Plasmodium falciparum, one of the major causative pathogens of malaria in humans. Our group also found that the tyrocidines have pronounced antifungal activity, dictated by the primary sequence of the tyrocidine. By simply manipulating the Phe or Trp concentration in the culture medium of the tyrothricin producer, Bacillus aneurinolyticus ATCC 10068, we were able to modulate the production of subsets of tyrocidines, thereby tailoring the tyrothricin complex to target specific pathogens. We optimized the tailored tyrothricin production using a novel, small-scale, high-throughput deep 96-well plate culturing method followed by analyses of the peptide mixtures using ultra-performance liquid chromatography linked to mass spectrometry. We were able to gradually shift the production profile of the tyrocidines and analogues, as well as the gramicidins between two extremes in terms of peptide subsets and peptide hydrophobicity. This study demonstrated that tyrothricin peptide subsets with targeted activity can be efficiently produced by simple manipulation of the aromatic amino acid profile of the culture medium.

Survey of South African fruit juices using a fast screening HILIC-MS method.

Adulteration of fruit juices--by the addition of sugar or other less expensive fruit juices as well as preservatives, artificial sweeteners and colours--was tested for by using a developed screening method. The method employs hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) using electrospray ionisation in the negative mode and ultraviolet light detection. Different fruit juices can be differentiated by the content of marker compounds like sorbitol, certain phenolic molecules and their saccharide profile. This method was used to test 46 fruit juice samples from the retail market as well as 12 control samples. The study focused on the main types of fruit juices consumed on the South African market including apple, orange, grape and blends of these juices with other fruits like mango, pear and guava. Overall, the 46 samples tested mostly agreed with label claims. One grape juice sample was adulterated, probably with apple juice. Natamycin above the legal limits was found in two samples. In addition, two samples contained natamycin and one sample benzoate without it being indicated on the label. The method is well suited as a quick screening method for fruit juice adulteration and if used routinely would reduce fruit juice adulteration without the cost of the current array of tests needed for authenticity testing.

Direct surfactin-gramicidin S antagonism supports detoxification in mixed producer cultures of Bacillus subtilis and Aneurinibacillus migulanus.

Antibiotic production as a defence mechanism is a characteristic of a wide variety of organisms. In natural evolutionary adaptation, cellular events such as sporulation, biofilm formation and resistance to antibiotics enable some micro-organisms to survive environmental and antibiotic stress conditions. The two antimicrobial cyclic peptides in this study, gramicidin S (GS) from Aneurinibacillus migulanus and the lipopeptide surfactin (Srf) from Bacillus subtilis, have been shown to affect both membrane and intercellular components of target organisms. Many functions, other than that of antimicrobial activity, have been assigned to Srf. We present evidence that an additional function may exist for Srf, namely that of a detoxifying agent that protects its producer from the lytic activity of GS. We observed that Srf producers were more resistant to GS and could be co-cultured with the GS producer. Furthermore, exogenous Srf antagonized the activity of GS against both Srf-producing and non-producing bacterial strains. A molecular interaction between the anionic Srf and the cationic GS was observed with circular dichroism and electrospray MS. Our results indicate that the formation of an inactive complex between GS and Srf supports resistance towards GS, with the anionic Srf forming a chemical barrier to protect its producer. This direct detoxification combined with the induction of protective stress responses in B. subtilis by Srf confers resistance toward GS from A. migulanus and allows survival in mixed cultures.

Nitrogen supply and abiotic stress influence canavanine synthesis and the productivity of in vitro regenerated Sutherlandia frutescens microshoots.

Environmental stresses can significantly alter the synthesis of both primary and secondary metabolites, resulting in medicinal plants with unpredictable biological activity. Here, in vitro shoot cultures of the medicinal plant Sutherlandia frutescens were used to study the impact of three abiotic stresses (nitrogen availability, drought and salinity), primarily on l-canavanine synthesis. This compound, a non-protein amino acid, is amongst those metabolites linked to the health benefits of Sutherlandia extracts. Nitrogen supplied to microplants positively correlated with canavanine levels, exhibited by a fourfold reduction when nitrates provided were halved. Although the biomass generated was lowered under these conditions, a higher capacity for rooting (52%) in comparison to the controls (37%) became evident. Only a small increase of the canavanine content in microplants growing on 100mM NaCl medium was detected, indicating that salinity stress was not a major limitation on cavanine production, but that it played more of a role in vitro on plantlet morphogenesis. Similarly, PEG as a supplement had little to no effect on canavanine synthesis. We conclude that a deeper understanding of the nutritional requirements for the agricultural crop management of S. frutescens, which serves the herbal products industry, is needed.

Evidence of symbiosis between the soil yeast Cryptococcus laurentii and a sclerophyllous medicinal shrub, Agathosma betulina (Berg.) Pillans.

The interaction between a common soil yeast, Cryptococcus laurentii, and a slow-growing medicinal plant adapted to low-nutrient soils, Agathosma betulina (Berg.) Pillans, was studied. C. laurentii CAB 578 was isolated from the rhizosphere of wild A. betulina, and liquid chromatography-tandem mass spectrometry (LC-MS-MS) analysis revealed that the yeast was capable of producing polyamines, such as cadaverine and spermine, while growing in vitro in a chemically defined medium. Since the exogenous application of polyamines are known to impact on root growth, these findings supported the results obtained when axenic cultures of A. betulina seedlings were inoculated with C. laurentii CAB 578 and cultivated for 5 months under glasshouse conditions. The presence of the yeast increased root growth by 51%. Using soil dilution plates, it was demonstrated that yeast numbers were greater in the vicinity of the roots than in the bulk soil. In addition, fluoromicroscopy, in combination with the fluorescent probes Fungolight and Calcofluor white, revealed the presence of metabolic active yeast colonies on the rhizoplane 5 months after initiation of the experimentation. The study provided evidence for a symbiosis between C. laurentii and A. betulina.

The development of an ultra performance liquid chromatography-coupled atmospheric pressure chemical ionization mass spectrometry assay for seven adrenal steroids.

An ultra performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry (UPLC-APCI-MS) method was developed for the separation and quantification of adrenal steroid metabolites from heterologous expression media. Steroids were extracted by liquid-liquid extraction, separated on a Waters UPLC BEH C18 column, ionized by APCI, and detected using a triple quadrupole mass spectrometer in APCI positive mode with single ion monitoring. The incorporation of UPLC enabled the detection of seven structurally closely related steroids at between 5 and 40 ng/ml using run times of 11 min. The adrenal steroidogenic enzyme cytochrome P450 17-hydroxylase/17,20-lyase (CYP17) was expressed in the yeast Pichia pastoris and in nonsteroidogenic COS-1 cells, and used as a model system to evaluate the detection and quantification of adrenal steroid metabolites by UPLC-APCI-MS.