The Fungus <Emphasis Type="Italic">Penicillium citrinum</Emphasis>, Isolated from Permafrost Sediments,as a Producer of Ergot Alkaloids and New Quinoline Alkaloids Quinocitrinines

Quinocitrinines and ergot alkaloids were synthesized by the strain Penicillium citrinum VKM FW-800 as the culture grews. The major part of these secondary metabolites was secreted into the medium. In the phase of growth deceleration, these metabolites were partly absorbed by the producer cells. Zinc ions stimulated both the primary and secondary metabolic processes. Addition of this microelement into the culture medium stimulated biomass accumulation and the synthesis of clavine alkaloids and quinocitrinines.     

Characterization of prednisone, prednisolone and their metabolites by gas chromatography—mass spectrometry

Human urinary metabolites of the synthetic corticosteroids prednisone and prednisolone were detected in the course of gas chromatographic steroid profiling as methoxime-trimethylsilyl derivatives. Metabolites were provisionaly identified by combined gas chromatography—mass spectrometry. The major metabolites were 11-keto/11-hydroxy conversion products, 20-hydroxy and 4,5-dihydro analogues of the parent drugs. Cortisone, 6-hydroxy and fully saturated A-ring compounds were minor metabolites. Retention indices and mass spectral data are presented.     

Influence of gibberellic acid on <Superscript>14</Superscript>CO<Subscript>2</Subscript> metabolism,growth, and production of alkaloids in <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

Changes in growth parameters, carbon assimilation efficiency, and utilization of ¹⁴CO₂ assimilate into alkaloids in plant parts were investigated at whole plant level by treatment of Catharanthus roseus with gibberellic acid (GA). Application of GA (1 000 g m⁻³) resulted in changes in leaf morphology, increase in stem elongation, leaf and internode length, plant height, and decrease in biomass content. Phenotypic changes were accompanied by decrease in contents of chlorophylls and in photosynthetic capacity. GA application resulted in higher % of total alkaloids accumulated in leaf, stem, and root. GA treatment produced negative phenotypic response in total biomass production but positive response in content of total alkaloids in leaf, stem, and roots. ¹⁴C assimilate partitioning revealed that ¹⁴C distribution in leaf, stem, and root of treated plants was higher than in untreated and variations were observed in contents of metabolites as sugars, amino acids, and organic acids. Capacity to utilize current fixed ¹⁴C derived assimilates for alkaloid production was high in leaves but low in roots of treated plants despite higher content of ¹⁴C metabolites such as sugars, amino acids, and organic acids. In spite of higher availability of metabolites, their utilization into alkaloid production is low in GA-treated roots.     

Urinary metabolites of cannabidiol in dog, rat and man and their identification by gas chromatography—mass spectrometry

Urinary metabolites of cannabidiol (CBD), a non-psychoactive cannabinoid of potential therapeutic interest, were extracted from dog, rat and human urine, concentrated by chromatography on Sephadex LH-20 and examined by gas chromatography—mass spectrometry as trimethylsilyl (TMS), [²H₉]TMS, methyl ester—TMS and methyloxime—TMS derivatives. Fragmentation of the metabolites under electron-impact gave structurally informative fragment ions; computer-generated single-ion plots of these diagnostic ions were used extensively to aid metabolite identification. Over fifty metabolites were identified with considerable species variation. CBD was excreted in substantial concentration in human urine, both in the free state and as its glucuronide. In dog, unusual glucoside conjugates of three metabolites (4″- and 5″-hydroxy- and 6-oxo-CBD), not excreted in the unconjugated state, were found as the major metabolites at early times after drug administration. Other metabolites in all three species were mainly acids. Side-chain hydroxylated derivatives of CBD-7-oic acid were particularly abundant in human urine but much less so in dog. In the latter species the major oxidized metabolites were the products of β-oxidation with further hydroxylation at C-6. A related, but undefined pathway resulted in loss of three carbon atoms from the side-chain of CBD in man with production of 2″-hydroxy-tris,nor-CBD-7-oic acid. Metabolism by the epoxide-diol pathway, resulting in dihydro-diol formation from the Δ-8 double bond, gave metabolites in both dog and human urine. It was concluded that CBD could be used as a probe of the mechanism of several types of biotransformation; particularly those related to carboxylic acid metabolism as intermediates of the type not usually seen with endogenous compounds were excreted in substantial concentration.     

Microbial secondary metabolites ameliorate growth, <Emphasis Type="Italic">in planta</Emphasis> contents and lignification in <Emphasis Type="Italic">Withania somnifera</Emphasis> (L.) Dunal

In the present investigation, metabolites of Streptomyces sp. MTN14 and Trichoderma harzianum ThU significantly enhanced biomass yield (3.58 and 3.48 fold respectively) in comparison to the control plants. The secondary metabolites treatments also showed significant augmentation (0.75–2.25 fold) in withanolide A, a plant secondary metabolite. Lignin deposition, total phenolic and flavonoid content in W. somnifera were maximally induced in treatment having T. harzianum metabolites. Also, Trichoderma and Streptomyces metabolites were found much better in invoking in planta contents and antioxidants compared with their live culture treatments. Therefore, identification of new molecular effectors from metabolites of efficient microbes may be used as biopesticide and biofertilizer for commercial production of W. somnifera globally.     

Simultaneous assay of cocaine, heroin and metabolites in hair, plasma, saliva and urine by gas chromatography—mass spectrometry

As part of an ongoing research program on the development of drug detection methodology, we developed an assay for the simultaneous measurement of cocaine, heroin and metabolites in plasma, saliva, urine and hair by solid-phase extraction (SPE) and gas chromatography—mass spectrometry (GC—MS). The analytes that could be measured by this assay were the following: anhydroecgonine methyl ester; ecgonine methyl ester; ecgonine ethyl ester; cocaine; cocaethylene; benzoylecgonine; cocaethylene; norcocaethylene; benzoylnorecgonine; codeine; morphine; norcodeine; 6-acetylmorphine; normorphine; and heroin. Liquid specimens were diluted, filtered and then extracted by SPE. Additional handling steps were necessary for the analysis of hair samples. An initial wash procedure was utilized to remove surface contaminants. Washed hair samples were extracted with methanol overnight at 40°C. Both wash and extract fractions were collected, evaporated and purified by SPE. All extracts were evaporated, derivatized with N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) with 1% trimethylchlorosilane (TMCS) and analyzed by GC—MS. The limit of detection (LOD) for cocaine, heroin and metabolites in biological specimens was approximately 1 ng/ml with the exception of norcodeine, normorphine and benzoylnorecgonine (LOD = 5 ng/ml). The LOD for cocaine, heroin and metabolites in hair was approximately 0.1 ng/mg of hair with the exception of norcodeine (LOD = 0.3 ng/mg) and normorphine and benzoylnorecgonine (LOD = 0.5 ng/mg). Coefficients of variation ranged from 3 to 26.5% in the hair assay. This assay has been successfully utilized in research on the disposition of cocaine, heroin and metabolites in hair, plasma, saliva and urine and in treatment studies.     

Characterisation of two phenotypes of <Emphasis Type="Italic">Centella asiatica</Emphasis> in Southern Africa through the composition of four triterpenoids in callus,cell suspensions and leaves

Two morphologically distinct phenotypes of Centella asiatica (Type-1 and Type-2) in South Africa were compared in relation to the levels of triterpenoid saponins with the aim of assessing their potential for biotechnological manipulation of triterpenoid synthesis. The metabolites investigated included madecassoside and asiaticoside and their sapogenins madecassic—and asiatic acid; produced in cultured undifferentiated cells (cell suspensions and calli) and leaves. Weight determination in plant cell suspensions and the accumulation of secondary metabolites after 16 days for Type-1 and 20 days for Type-2 were investigated since these secondary metabolites accumulate during the period that follows the active growth phase. The four triterpenoids of interest were analysed and quantified by HPLC in crude ethanolic extracts. A difference in bioactive triterpenoids was exhibited that was tissue specific and varied between the two phenotypes. The triterpenoids from leaf tissue were more easily quantifiable in each phenotype than in the case of the undifferentiated cells (callus and cell suspensions), which had lower, but still quantifiable, levels of these targeted secondary metabolites. Leaves contained the highest triterpenoid levels (ranging from 1.8 to 5% dry weight for the triterpenoid acids and their glycosides, respectively), with the free acids occurring in a ratio of approximately 1:2.5 in relation to the glycoside content.     

Biological Validations of Fecal Glucocorticoid,Testosterone, and Progesterone Metabolite Measurements in Captive Stumptail Macaques (<Emphasis Type="Italic">Macaca arctoides</Emphasis>)

Measuring hormone metabolites in fecal samples allows the noninvasive assessment of some steroid hormones in primates. However, noninvasive hormone assays need analytical and biological validation owing to variation in hormone metabolism and excretion between the sexes and across species. We aimed to validate the measurement of fecal glucocorticoid (fGC), testosterone (fT), and progesterone (fP) metabolites in 15 captive stumptail macaques (Macaca arctoides). We collected fecal samples before and after we induced a stress response by restraining and injecting the subjects with saline solution. We then measured hormone metabolites using a methanol extraction technique and ¹²⁵I radioimmunoassay kits. We analyzed the change in glucocorticoid production before and after the stressor, as well as sexual and social rank differences. For fT metabolite levels we investigated variation with sex, age, and social rank, and for fP metabolite levels, we tested for sexual and cycle phase differences. We found a significant increase in fGC metabolite levels 22–25 h poststressor in both sexes. The increase was greater in high-ranking than in low-ranking individuals. Levels of fT metabolites were higher in males than in females, correlated positively with rank only in males, and correlated negatively with age in both sexes. fP metabolite levels were higher in females than in males, and were higher during the luteal phase than in the follicular phase. These findings indicate that our assays reliably detected hormonal changes related to stress (fGC) and detected differences between social and sexual categories (fT, fP) in stumptail macaques.     

The Plant-Stress Metabolites,Hexanoic Aacid and Melatonin,Are Potential “Vaccines” for Plant Health Promotion

A plethora of compounds stimulate protective mechanisms in plants against microbial pathogens and abiotic stresses. Some defense activators are synthetic compounds and trigger responses only in certain protective pathways, such as activation of defenses under regulation by the plant regulator, salicylic acid (SA). This review discusses the potential of naturally occurring plant metabolites as primers for defense responses in the plant. The production of the metabolites, hexanoic acid and melatonin, in plants means they are consumed when plants are eaten as foods. Both metabolites prime stronger and more rapid activation of plant defense upon subsequent stress. Because these metabolites trigger protective measures in the plant they can be considered as “vaccines” to promote plant vigor. Hexanoic acid and melatonin instigate systemic changes in plant metabolism associated with both of the major defense pathways, those regulated by SA- and jasmonic acid (JA). These two pathways are well studied because of their induction by different microbial triggers: necrosis-causing microbial pathogens induce the SA pathway whereas colonization by beneficial microbes stimulates the JA pathway. The plant’s responses to the two metabolites, however, are not identical with a major difference being a characterized growth response with melatonin but not hexanoic acid. As primers for plant defense, hexanoic acid and melatonin have the potential to be successfully integrated into vaccination-like strategies to protect plants against diseases and abiotic stresses that do not involve man-made chemicals.     

Actinotetraoses I–K: Tetrasaccharide Metabolites Produced by an Insect‐Derived Actinobacteria,Amycolatopsis sp. HCa1

An isolate of rare actinobacteria strain Amycolatopsis sp. HCa1 obtained from the gut of grasshopper produced seven different metabolites in vitro. The metabolites isolated from its mycelia cakes were characterized by NMR and MS analyses. Actinotetraose hexatiglate (or tigloside; 1 ) with nonreducing glucotetraose skeleton was isolated as a major constituent; three new tetrasaccharide derivatives actinotetraoses I–K ( 2 – 4 , resp.) and three known actinotetraoses A–C ( 5 – 7 , resp.) were also isolated.     

A novel metabolic pathway for biodegradation of DDT by the white rot fungi, <Emphasis Type="Italic">Phlebia lindtneri</Emphasis> and <Emphasis Type="Italic">Phlebia brevispora</Emphasis>

1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) was used as the substrate for a degradation experiment with the white rot fungi Phlebia lindtneri GB-1027 and Phlebia brevispora TMIC34596, which are capable of degrading polychlorinated dibenzo-p-dioxin (PCDD) and polychlorinated biphenyls (PCBs). Pure culture of P. lindtneri and P. brevispora with DDT (25 μmol l⁻¹) showed that 70 and 30% of DDT, respectively, disappeared in a low-nitrogen medium after a 21-day incubation period. The metabolites were analyzed using gas chromatography/mass spectrometry (GC/MS). Both fungi metabolized DDT to 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD), 2,2-bis(4-chlorophenyl)acetic acid (DDA) and 4,4-dichlorobenzophenone (DBP). Additionally, DDD was converted to DDA and DBP. DDA was converted to DBP and 4,4-dichlorobenzhydrol (DBH). While DBP was treated as substrate, DBH and three hydroxylated metabolites, including one dihydroxylated DBP and two different isomers of monohydroxylated DBH, were produced from fungal cultures, and these hydroxylated metabolites were efficiently inhibited by the addition of a cytochrome P-450 inhibitor, piperonyl butoxide. These results indicate that the white rot fungi P. lindtneri and P. brevispora can degrade DBP/DBH through hydroxylation of the aromatic ring. Moreover, the single-ring aromatic metabolites, such as 4-chlorobenzaldehyde, 4-chlorobenzyl alcohol and 4-chlorobenzoic acid, were found as metabolic products of all substrate, demonstrating that the cleavage reaction of the aliphatic-aryl carbon bond occurs in the biodegradation process of DDT by white rot fungi.     

Comparison of dynamic responses of cellular metabolites in <Emphasis Type="Italic">Escherichia coli</Emphasis> to pulse addition of substrates

We conducted an integrated study of cell growth parameters, product formation, and the dynamics of intracellular metabolite concentrations using Escherichia coli with genes knocked out in the glycolytic and oxidative pentose phosphate pathway (PPP) for glucose catabolism. We investigated the same characteristics in the wild-type strain, using acetate or pyruvate as the sole carbon source. Dramatic effects on growth parameters and extracellular and intracellular metabolite concentrations were observed after blocking either glycolytic breakdown of glucose by inactivation of phosphoglucose isomerase (disruption of pgi gene) or pentose phosphate breakdown of glucose by inactivation of glucose-6-phosphate dehydrogenase (disruption of zwf gene). Reducing power (NADPH) was mainly produced through PPP when the pgi gene was knocked out, while NADPH was produced through the tricarboxylic acid (TCA) cycle by isocitrate dehydrogenase or NADP-linked malic enzyme when the zwf gene was knocked out. As expected, when the pgi gene was knocked out, intracellular concentrations of PPP metabolites were high and glycolytic and concentrations of TCA cycle pathway metabolites were low. In the zwf gene knockout, concentrations of PPP metabolites were low and concentrations of intracellular glycolytic and TCA cycle metabolites were high.     

In Vitro Metabolism of 20(R)-25-Methoxyl-Dammarane-3, 12, 20-Triol from Panax notoginseng in Human,Monkey, Dog,Rat, and Mouse Liver Microsomes

The present study characterized in vitro metabolites of 20(R)-25-methoxyl-dammarane-3β, 12β, 20-triol (20(R)-25-OCH₃-PPD) in mouse, rat, dog, monkey and human liver microsomes. 20(R)-25-OCH₃-PPD was incubated with liver microsomes in the presence of NADPH. The reaction mixtures and the metabolites were identified on the basis of their mass profiles using LC-Q/TOF and were quantified using triple quadrupole instrument by multiple reaction monitoring. A total of 7 metabolites (M1–M7) of the phase I metabolites were detected in all species. 25(R)-OCH₃-PPD was metabolized by hydroxylation, dehydrogenation, and O-demethylation. Enzyme kinetic of 20(R)-25-OCH₃-PPD metabolism was evaluated in rat and human hepatic microsomes. Incubations studies with selective chemical inhibitors demonstrated that the metabolism of 20(R)-25-OCH₃-PPD was primarily mediated by CYP3A4. We conclude that 20(R)-25-OCH₃-PPD was metabolized extensively in mammalian species of mouse, rat, dog, monkey, and human. CYP3A4-catalyzed oxygenation metabolism played an important role in the disposition of 25(R)-OCH₃-PPD, especially at the C-20 hydroxyl group.     

Shifts in the carbohydrate,polyol, and amino acid pools during rapid cold-hardening and diapause-associated cold-hardening in flesh flies (<Emphasis Type="Italic">Sarcophaga crassipalpis</Emphasis>): a metabolomic comparison

Flesh flies can enhance their cold hardiness by entering a photoperiod-induced pupal diapause or by a temperature-induced rapid cold-hardening process. To determine whether the same or different metabolites are involved in these two responses, derivatized polar extracts from flesh flies subjected to these treatments were examined using gas chromatography–mass spectrophotometry (GC–MS). This metabolomic approach demonstrated that levels of metabolites involved in glycolysis (glycerol, glucose, alanine, pyruvate) were elevated by both treatments. Metabolites elevated uniquely in response to rapid cold-hardening include glutamine, cystathionine, sorbitol, and urea while levels of β-alanine, ornithine, trehalose, and mannose levels were reduced. Rapid cold-hardening also uniquely perturbed the urea cycle. In addition to the elevated metabolites shared with rapid cold-hardening, leucine concentrations were uniquely elevated during diapause while levels of a number of other amino acids were reduced. Pools of two aerobic metabolic intermediates, fumarate and citrate, were reduced during diapause, indicating a reduction of Krebs cycle activity. Principal component analysis demonstrated that rapid cold-hardening and diapause are metabolically distinct from their untreated, non-diapausing counterparts. We discuss the possible contribution of each altered metabolite in enhancing the overall cold hardiness of the organism, as well as the efficacy of GC–MS metabolomics for investigating insect physiological systems.     

Secondary metabolites in taxonomy of the <Emphasis Type="Italic">Penicillium</Emphasis> fungi

A correlation was established between species specificity and synthesis of specific secondary metabolites by the Penicillium fungi. Strains of the subgenus Aspergilloides usually synthesize metabolites of polyketide nature. Most strains of the subgenus Furcatum produce clavine ergot alkaloids and metabolites of diketopiperazine nature. The only clavine ergot alkaloids and diketopiperazine alkaloids produced by strains of the subgenus Biverticillium are rugulovasines and rugulosuvines, respectively. Species designations of the strains of the subgenus Penicillium isolated from permafrost soil, the Mir orbital complex, and sites undergoing anthropogenic load were refined based on the marker secondary metabolites. Changes in the taxonomic position of some strains in the genus Penicillium are suggested.     

Secondary metabolites of fungi of the <Emphasis Type="Italic">Usti</Emphasis> section,genus <Emphasis Type="Italic">Aspergillus</Emphasis> and their application in chemosystematics

Secondary metabolites of 22 fungal strains (genus Aspergillus, section Usti) isolated at diverse geographic regions, including the Arctic permafrost deposits, were studied. The studied strains were found to synthesize a variety of biologically active compounds, structurally identified as drimane sesqueterpenoids, isoquinoline alkaloids (TMC-120 A?C, derivative 1), meroterpenoids (austalides О and J), and anthraquinone pigments (averufin, versicolorin C). Desferritriacetylfusigen production by A. calidoustus isolates is reported for the first time. The individual spectra of secondary metabolites were used for reidentification of 17 strains, of which 15 were identified as A. calidoustus and two, as A. pseudodeflectus.     

<Emphasis Type="Italic">Humulus lupulus</Emphasis> L., a very popular beer ingredient and medicinal plant: overview of its phytochemistry,its bioactivity,and its biotechnology

Humulus lupulus L. (Cannabaceae), commonly named hop, is widely grown around the world for its use in the brewing industry. Its female inflorescences (hops) are particularly prized by brewers because they produce some secondary metabolites that confer bitterness, aromas and antiseptic properties to the beer. These sought-after metabolites include terpenes and sesquiterpenes, found in essential oil, but also prenylated phenolic compounds, mainly acylphloroglucinols (bitter acids) from the series of α-acids (humulone derivatives). These metabolites have shown numerous biological activities, including among others, antimicrobial, sedative and estrogenic properties. This review provides an inventory of hop’s chemistry, with an emphasis on the secondary metabolites and their biological activities. These compounds of biological interest are essentially produced in female inflorescences, while other parts of the plant only synthetize low quantities of them. Lastly, our article provides an overview of the research in plant biotechnology that could bring alternatives for hops metabolites production.     

Rapid assay of cocaine, opiates and metabolites by gas chromatography—mass spectrometry

The simultaneous assay of cocaine, opiates and metabolites in small biological samples continues to be a difficult task. This report focuses upon tabulation of important techniques (extraction, derivatization, chromatographic conditions, detection mode, data acquisition) reported over the last decade that were used in the development of assays for these analytes. The most prevalent procedures for extraction of cocaine, opiates and metabolites were liquid—liquid and solid-phase extraction isolation methods. Following extraction analytes were derivatized and analyzed by gas chromatography—mass spectrometry. The technique most often used for chromatographic separation was fused-silica capillary column gas chromatography. Detection generally was performed by selected ion monitoring in the positive-ion electron-impact ionization mode, although full-scan acquisition and positive- and negative-ion chemical ionization methods have been used. It was apparent from the review that there is a continuing need for greater sensitivity and selectivity in the assay of highly potent opiates and for cocaine and metabolites.     

Determination of isoniazid and its hydrazino metabolites, acetylisoniazid, acetylhydrazine, and diacetylhydrazine in human plasma by gas chromatography—mass spectrometry

A gas chromatographic—mass spectrometric assay for isoniazid and its hydrazino metabolites in human plasma was developed. The trimethylsilyl derivatives of diacetylhydrazine and acetylisoniazid and of the benzaldehyde hydrazones of acetylhydrazine and isoniazid were separated on a 1% OV-17 column and quantitated by single ion monitoring using a LKB 9000 mass spectrometer. Deuterated analogues served as internal standards. The method is well suited for the determination of the hepatotoxic hydrazino metabolites of isoniazid in human plasma following an oral therapeutic dose of isoniazid.     

Regenerative potential,metabolic profile,and genetic stability of <Emphasis Type="Italic">Brachypodium distachyon</Emphasis> embryogenic calli as affected by successive subcultures

Brachypodium distachyon, a model species for forage grasses and cereal crops, has been used in studies seeking improved biomass production and increased crop yield for biofuel production purposes. Somatic embryogenesis (SE) is the morphogenetic pathway that supports in vitro regeneration of such species. However, there are gaps in terms of studies on the metabolic profile and genetic stability along successive subcultures. The physiological variables and the metabolic profile of embryogenic callus (EC) and embryogenic structures (ES) from successive subcultures (30, 60, 90, 120, 150, 180, 210, 240, and 360-day-old subcultures) were analyzed. Canonical discriminant analysis separated EC into three groups: 60, 90, and 120 to 240 days. EC with 60 and 90 days showed the highest regenerative potential. EC grown for 90 days and submitted to SE induction in 2 mg L^?1 of kinetin-supplemented medium was the highest ES producer. The metabolite profiles of non-embryogenic callus (NEC), EC, and ES submitted to principal component analysis (PCA) separated into two groups: 30 to 240- and 360-day-old calli. The most abundant metabolites for these groups were malonic acid, tryptophan, asparagine, and erythrose. PCA of ES also separated ages into groups and ranked 60- and 90-day-old calli as the best for use due to their high levels of various metabolites. The key metabolites that distinguished the ES groups were galactinol, oxaloacetate, tryptophan, and valine. In addition, significant secondary metabolites (e.g., caffeoylquinic, cinnamic, and ferulic acids) were important in the EC phase. Ferulic, cinnamic, and phenylacetic acids marked the decreases in the regenerative capacity of ES in B. distachyon. Decreased accumulations of the amino acids aspartic acid, asparagine, tryptophan, and glycine characterized NEC, suggesting that these metabolites are indispensable for the embryogenic competence in B. distachyon. The genetic stability of the regenerated plants was evaluated by flow cytometry, showing that ploidy instability in regenerated plants from B. distachyon calli is not correlated with callus age. Taken together, our data indicated that the loss of regenerative capacity in B. distachyon EC occurs after 120 days of subcultures, demonstrating that the use of EC can be extended to 90 days.