Toxicological and Sedative Effects of Chipilin (Crotalaria longirostrata) Leaf Extracts Obtained by Maceration and Supercritical Fluid Extraction.

Chipilin (Crotalaria longirostrata) is consumed as a vegetable in the preparation of traditional dishes. As a folk medicine, Chipilin extracts are used as a hypnotic and sedative agent; however, there are few reports that support these uses. This study aimed to characterize the compounds present in Chipilin leaf extracts and to investigate their sedative effect using zebrafish as an in vivo model. Extracts were obtained by maceration with water (H2O), ethanol (EtOH), and EtOH-H2O, while oleoresin was obtained by supercritical fluid extraction (SFE). Total phenolic and flavonoid contents were quantified by colorimetric methods. Phytochemical constituents were identified by gas chromatography-mass spectrometry (GC-MS) analysis. The chronic and acute toxicities of Chipilin extracts were tested in zebrafish embryos and larvae, respectively. Chipilin sedative effect was tested by the larvae response to dark-light-dark transitions. EtOH-H2O extracts had the highest value of total phenolics (5345 ± 5.1 µg GAE/g), followed by water and oleoresin (1815 ± 5.1 and 394 ± 5.1 µg GAE/g, respectively). In water extracts were identified the alkaloid trachelanthamidine, 1,2ß-epoxy- and the alkyl ketone 7,9-di-tert-butyl-1-oxaspiro(4,5)deca-6,9-diene-2,8-dione, while oleamide, α-monostearin, and erucamide were detected in all samples except in water extracts. Oleoresin extract had the lowest embryotoxicity (LC50 = 4.99 µg/mL) and the highest sedative effects. SFE is a green alternative to obtain Chipilin extracts rich in erucamide, an endocannabinoid analogue, which plays an important role in the development of the central nervous system and in modulating neurotransmitter release.

Dietary Supplementation with Popped Amaranth Modulates the Gut Microbiota in Low Height-for-Age Children: A Nonrandomized Pilot Trial.

Amaranth has been recognized as a nutraceutical food because it contains high-quality proteins due to its adequate amino acid composition that covers the recommended requirements for children and adults. Since pre-Hispanic times, amaranth has been consumed as popped grain; the popping process improves its nutritive quality and improves its digestibility. Popped amaranth consumption has been associated with the recovery of malnourished children. However, there is no information on the impact that popped amaranth consumption has on gut microbiota composition. A non-randomized pilot trial was conducted to evaluate the changes in composition, structure, and function of the gut microbiota of stunted children who received four grams of popped amaranth daily for three months. Stool and serum were collected at the beginning and at the end of the trial. Short-chain fatty acids (SCFA) were quantified, and gut bacterial composition was analyzed by 16S rRNA gene sequencing. Biometry and hematology results showed that children had no pathology other than low height-for-age. A decrease in the relative abundance of Alistipes putredinis, Bacteroides coprocola, and Bacteroides stercoris bacteria related to inflammation and colitis, and an increase in the relative abundance of Akkermansia muciniphila and Streptococcus thermophiles bacteria associated with health and longevity, was observed. The results demonstrate that popped amaranth is a nutritious food that helps to combat childhood malnutrition through gut microbiota modulation.

Amaranth calcium oxalate crystals are associated with chloroplast structures and proteins.

Calcium oxalate (CaOx) crystals in plants are formed in crystal idioblasts cells and have specific geometric shapes. Their proposed functions include calcium homeostasis and carbon source, among others. Amaranth is a plant that presents high tolerance to abiotic stresses and accumulates considerable amounts of CaOx crystals; however, few studies have focused on characterizing the crystals ultrastructure and none is related to identifying proteins bound to them. This information is of great interest to understand the mechanisms related to CaOx crystal formation and to support their proposed functions. Thus, this work aimed to characterize CaOx crystals in amaranth leaves. Crystals were purified and the proteins bound to them were isolated and identified by nLC-MS/MS. Leaf sections were analyzed by light and electron microscopy. The identified proteins were related to the chloroplast such as ATPb synthase, RuBisCO large subunit, and cell wall-related proteins, which were validated by immunohistochemistry and immunogold labeling. In addition, it was observed that CaOx crystal idioblasts were formed from parenchyma cells associated with mesophyll and veins, in which the thylakoid membranes of degraded chloroplasts turned into crystal chambers. These results significantly advance our understanding of the mechanisms of CaOx crystal formation and the potential function as an alternative carbon source in leaves.

Lippia graveolens HBK oleoresins, extracted by supercritical fluids, showed bactericidal activity against multidrug resistance Enterococcus faecalis and Staphylococcus aureus strains.

OBJECTIVE: The aim of this work was to characterize Lippia graveolens oleoresins, obtained by Supercritical Fluid Extraction (SFE), from crops collected at different locations in Mexico. The antimicrobial effect of oleoresins was tested in reference strains and clinical isolates of susceptible and multidrug-resistant (MDR) strains of Enterococcus faecalis and Staphylococcus aureus. SIGNIFICANCE: The increasing of MDR strains is becoming a global public health problem that has led to the search for new treatments, and essential oils have resurged as a source of compounds with bactericidal functions. Oregano essential oil has attracted attention recently, however, this oil is mainly obtained by hydro-distillation (uses large amounts of water) or solvents extraction (potential contaminant). SFE has gained popularity as it represents an environmentally friendly technology. METHODS: L. graveolens oleoresins were obtained by SFE, total phenol contents were quantified by Folin-Ciocalteu method, the identification of compounds and thymol and carvacrol quantification was carried out by GC-MS. The antimicrobial activity was tested by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). RESULTS: SFE showed higher yields compared with the hydro-distillation process. L. graveolens grown in different Mexican locations showed differences in oleoresin composition and a slightly different antimicrobial capacity against clinical isolates. CONCLUSIONS: It was demonstrated that SFE is an efficient technology for extracting L. graveolens oleoresins. Additionally, the solvent-free extraction method and the observed antimicrobial effect increase the applications of these oleoresins in fields, such as cosmetics, food industry, medicine, amongst others.

Biomass, chlorophyll fluorescence, and osmoregulation traits let differentiation of wild and cultivated Amaranthus under water stress.

Amaranths are recognized by their high nutritive value and their natural tolerance to environmental stresses. In this study, physiological differences in response to water stress were compared between A. hybridus, a wild species considered as weed, and A. hypochondriacus, the most cultivated species for grain production, under the hypothesis that wild species have better adaptation to stress. In both species, photosynthetic parameters, pigments, and gene expression of selected genes were assessed. Biomass, effective quantum efficiency (ΦPSII), photochemical quenching (qP), and electron transport rate (ETR) values were reduced only in A. hybridus due to water deficit. Drought stress promoted proline accumulation by twice in A. hybridus but until three times in A. hypochondriacus. In both species, drought stress reduced net assimilation rate (A), transpiration rate (E), stomatal conductance (gs), and the expression of phosphoenol pyruvate carboxylase (PEPC). While, maximum quantum efficiency (Fv/Fm), chlorophyll, betacyanins, and the expression of ribulose1-5, bisphosphate carboxylase/oxygenase large subunit (LSU) did not change when plants were subjected to water stress. Likewise, both species accumulated total phenolic compounds and Oxalyl-CoA gene was up-regulated in response to drought. Our results have shown that A. hypochondriacus, the cultivated species, exhibited better tolerance to drought than A. hybridus, the wild species, probably due to an unconsciously selected trait during the domestication process.

Opuntia cladode powders inhibit adipogenesis in 3 T3-F442A adipocytes and a high-fat-diet rat model by modifying metabolic parameters and favouring faecal fat excretion.

BACKGROUND: Obesity is a major public health concern worldwide. A sedentary life and a nutritional transition to processed foods and high-calorie diets are contributing factors to obesity. The demand for nutraceutical foods, such as herbal weight-loss products, which offer the potential to counteract obesity, has consequently increased. We hypothesised that Opuntia cladodes consumption could assist weight management in an obesity prevention context. METHODS: This study was designed to explore the anti-adipogenic effects of lyophilised Opuntia cladode powders (OCP) in an in vitro cellular model for adipocyte differentiation and an in vivo high-fat-diet (HFD)-induced obesity rat model. Two OCP were tested, one from wild species O. streptacantha and the second from the most known species O. ficus-indica. RESULTS: Pre-adipocytes 3 T3-F442A were treated by OCP during the differentiation process by insulin. OCP treatment impaired the differentiation in adipocytes, as supported by the decreased triglyceride content and a low glucose uptake, which remained comparable to that observed in undifferentiated controls, suggesting that an anti-adipogenic effect was exerted by OCP. Sprague-Dawley rats were fed with a normal or HFD, supplemented or not with OCP for 8 weeks. OCP treatment slightly reduced body weight gain, liver and abdominal fat weights, improved some obesity-related metabolic parameters and increased triglyceride excretion in the faeces. Taken together, these results showed that OCP might contribute to reduce adipogenesis and fat storage in a HFD context, notably by promoting the faecal excretion of fats. CONCLUSIONS: Opuntia cladodes may be used as a dietary supplement or potential therapeutic agent in diet-based therapies for weight management to prevent obesity.

Protein analysis reveals differential accumulation of late embryogenesis abundant and storage proteins in seeds of wild and cultivated amaranth species.

BACKGROUND: Amaranth is a plant naturally resistant to various types of stresses that produces seeds of excellent nutritional quality, so amaranth is a promising system for food production. Amaranth wild relatives have survived climate changes and grow under harsh conditions, however no studies about morphological and molecular characteristics of their seeds are known. Therefore, we carried out a detailed morphological and molecular characterization of wild species A. powellii and A. hybridus, and compared them with the cultivated amaranth species A. hypochondriacus (waxy and non-waxy seeds) and A. cruentus. RESULTS: Seed proteins were fractionated according to their polarity properties and were analysed in one-dimensional gel electrophoresis (1-DE) followed by nano-liquid chromatography coupled to tandem mass spectrometry (nLC-MS/MS). A total of 34 differentially accumulated protein bands were detected and 105 proteins were successfully identified. Late embryogenesis abundant proteins were detected as species-specific. Oleosins and oil bodies associated proteins were observed preferentially in A. cruentus. Different isoforms of the granule-bound starch synthase I, and several paralogs of 7S and 11S globulins were also identified. The in silico structural analysis from different isoforms of 11S globulins was carried out, including new types of 11S globulin not reported so far. CONCLUSIONS: The results provide novel information about 11S globulins and proteins related in seed protection, which could play important roles in the nutritional value and adaptive tolerance to stress in amaranth species.

Microbial contamination in methanol biofilters inoculated with a pure strain of Pichia pastoris: A potential limitation for waste revalorization.

Novel biotechnologies to valorize waste emissions are based on the use of specialized microbial groups that produce different compounds of industrial interest. On this scenario, the retention of such specific microorganisms in the system is of critical interest; however, the potential limitations of working with simplified cultures in a competitive open environment are neither fully explored nor well understood. In this work, a series of biofilters treating methanol vapors coupled with heterologous endochitinase production were used to evaluate the performance of a specialized microbial population during a typical open-to-environment operation. The biofilters were inoculated with a transformed strain of Pichia pastoris and were operated identically for about 90 days. The results showed that the biofiltration performance became diverse with time in terms of the elimination capacity (EC) shifting from a variation coefficient of 1.5% (EC = 274 ± 24, 279 ± 5, and 281.9 ± 25 g/[m3 h]) at the beginning of the operation to 33% (EC = 297 ± 9, 338 ± 7, and 341 ± 2 g/[m3 h]) at the end of operation. Epifluorescence analysis and cloning-sequencing suggested that P. pastoris remained as the dominant microorganism of methanol degradation, whereas diverse airborne bacteria, including Ochrobactrum spp. and Klebsiella oxytoca, played a secondary role possibly associated with the consumption of intermediates. Overall, this study found that low diversity systems operated under non-sterile conditions could be susceptible to contamination with external microorganisms causing a diversifying behavior at the performance and microbial community levels. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2715, 2019.

Opuntia spp.: Characterization and Benefits in Chronic Diseases.

Opuntia species have been used for centuries as food resources and in traditional folk medicine for their nutritional properties and their benefit in chronic diseases, particularly diabetes, obesity, cardiovascular diseases, and cancer. These plants are largely distributed in America, Africa, and the Mediterranean basin. Opuntia spp. have great economic potential because they grow in arid and desert areas, and O. ficus-indica, the domesticated O. species, is used as a nutritional and pharmaceutical agent in various dietary and value-added products. Though differences in the phytochemical composition exist between wild and domesticated (O. ficus-indica) Opuntia spp., all Opuntia vegetatives (pear, roots, cladodes, seeds, and juice) exhibit beneficial properties mainly resulting from their high content in antioxidants (flavonoids, ascorbate), pigments (carotenoids, betalains), and phenolic acids. Other phytochemical components (biopeptides, soluble fibers) have been characterized and contribute to the medicinal properties of Opuntia spp. The biological properties of Opuntia spp. have been investigated on cellular and animal models and in clinical trials in humans, allowing characterization and clarification of the protective effect of Opuntia-enriched diets in chronic diseases. This review is an update on the phytochemical composition and biological properties of Opuntia spp. and their potential interest in medicine.

Comparative shotgun proteomic analysis of wild and domesticated Opuntia spp. species shows a metabolic adaptation through domestication.

UNLABELLED: The Opuntia genus is widely distributed in America, but the highest richness of wild species are found in Mexico, as well as the most domesticated Opuntia ficus-indica, which is the most domesticated species and an important crop in agricultural economies of arid and semiarid areas worldwide. During domestication process, the Opuntia morphological characteristics were favored, such as less and smaller spines in cladodes and less seeds in fruits, but changes at molecular level are almost unknown. To obtain more insights about the Opuntia molecular changes through domestication, a shotgun proteomic analysis and database-dependent searches by homology was carried out. >1000 protein species were identified and by using a label-free quantitation method, the Opuntia proteomes were compared in order to identify differentially accumulated proteins among wild and domesticated species. Most of the changes were observed in glucose, secondary, and 1C metabolism, which correlate with the observed protein, fiber and phenolic compounds accumulation in Opuntia cladodes. Regulatory proteins, ribosomal proteins, and proteins related with response to stress were also observed in differential accumulation. These results provide new valuable data that will help to the understanding of the molecular changes of Opuntia species through domestication. BIOLOGICAL SIGNIFICANCE: Opuntia species are well adapted to dry and warm conditions in arid and semiarid regions worldwide, and they are highly productive plants showing considerable promises as an alternative food source. However, there is a gap regarding Opuntia molecular mechanisms that enable them to grow in extreme environmental conditions and how the domestication processes has changed them. In the present study, a shotgun analysis was carried out to characterize the proteomes of five Opuntia species selected by its domestication degree. Our results will help to a better understanding of proteomic features underlying the selection and specialization under evolution and domestication of Opuntia and will provide a platform for basic biology research and gene discovery.

Dietary cladode powder from wild type and domesticated Opuntia species reduces atherogenesis in apoE knock-out mice

Dietary intake of Opuntia species may prevent the development of cardiovascular diseases. The present study was designed to characterize the biological antioxidant and anti-inflammatory properties of Opuntia species and to investigate whether Opuntia cladodes prevent the development of atherosclerosis in vivo, in apoE−KO mice. The effects of the two Opuntia species, the wild Opuntia streptacantha and the domesticated Opuntia ficus-indica, were tested on the generation of intra- and extracellular reactive oxygen species (ROS) production and kinetics of the LDL oxidation by murine CRL2181 endothelial cells and on the subsequent inflammatory signaling leading to the adhesion of monocytes on the activated endothelium and the formation of foam cells. Opuntia species blocked the extracellular ROS (superoxide anion) generation and LDL oxidation by CRL2181, as well as the intracellular ROS rise and signaling evoked by the oxidized LDL, including the nuclear translocation of the transcription factor NFκB, the expression of ICAM-1 and VCAM-1 adhesion molecules, and the adhesion of monocytes to CRL2181. In vivo, Opuntia significantly reduced the formation of atherosclerotic lesions and the accumulation of 4-hydroxynonenal adducts in the vascular wall of apoE-KO mice, indicating that Opuntia cladodes prevent lipid oxidation in the vascular wall. In conclusion, wild and domesticated Opuntia species exhibit antioxidant, anti-inflammatory, and antiatherogenic properties which emphasize their nutritional benefit for preventing cardiovascular diseases (AU)

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Antiatherogenic and antitumoral properties of Opuntia cladodes: inhibition of low density lipoprotein oxidation by vascular cells, and protection against the cytotoxicity of lipid oxidation product 4-hydroxynonenal in a colorectal cancer cellular model

Opuntia species have been used for thousands of years as a folk medicine in the treatment of diseases. However, the components and protective mechanisms are still unclear. We make the hypothesis that Opuntia species may protect the development of oxidative stress-associated diseases, such as atherosclerosis or colon cancer, via their antioxidant properties. We investigated the protective effect of Opuntia cladode powder against the oxidation of low-density lipoprotein (LDL) evoked by vascular endothelial cells, an important risk factor for atherosclerosis development, and the toxicity of 4-hydroxynonenal (a major lipid peroxidation product) on normal (Apc +/+) and preneoplastic (Apc min/+) immortalized epithelial colon cells. Various Opuntia species classified according to their degree of domestication, from the wildest (Opuntia streptacantha, Opuntia hyptiacantha, Opuntia megacantha), medium (Opuntia albicarpa), to the most domesticated (Opuntia ficus-indica) were tested. Cladode powders prepared from these Opuntia species significantly inhibited LDL oxidation induced by incubation with murine endothelial cells and the subsequent foam cell formation of RAW 264.7 murine macrophages and cytotoxicity on murine endothelial cells. Moreover, Opuntia cladode powder blocked the promotion of colon cancer development on an in vitro model of colonocytes. It may be noted that the phenolic acid and flavonoids content, the antioxidant capacity, and the protective effect were relatively similar in all the cladode powders from wild (O. streptacantha) and domesticated Opuntia. Altogether, these data confirm the therapeutic potential of Opuntia cladodes in diseases associated with oxidative stress

[Preclinical in vitro and in vivo models for the assessment of biological activity in biosimilarity studies]. / Modelos preclínicos in vitro e in vivo para la evaluación de la actividad biológica en estudios de biocomparabilidad.

A drug that contains a recombinant protein as an active principle is called a biotechnological drug or biopharmaceutical.There are currently over 300 biopharmaceuticals worldwide. Many of these contains a similar active principle (biosimilar drug) as other previously registered (innovator drug). It has suggested that due to the complex implications in a formulation containing a protein, the manufacturing process is a key factor for efficacy and safety requirements. In fact, certain variability has been detected of the protein properties in different lots (or batches) of the same manufacturer, which produce changes at a clinical level. For this reason, the evaluation of biosimilar drugs has acquired great relevance, being the preclinical level of one of the more important stages of the development due to its lower cost (with respect to the clinical level) and its high capacity to detect formulation-manufacture problems. However, the demonstration of comparability at physicochemical, preclinical, and clinical levels is required in order to achieve market registration. In this review the in vitro and in vivo models used for the assessment of proposed biosimilars will be discussed.

G protein-coupled receptors mediate coronary flow- and agonist-induced responses via lectin-oligosaccharide interactions.

Blood flow acts parallel to the coronary luminal endothelial surface layer (LESL) and modulates multiple parenchymal functions via the release of paracrine agents. Evidence indicates that the LESL may be a flow-sensing organelle and that perhaps through flow-induced lectin (L)·oligosaccharide (O) complex formation (L·O) participates in this process. LESL integrins and selectins are both lectinic and flow sensitive, but the L properties of flow-sensitive G protein-coupled receptors (GPCRs) are unknown. Therefore, we investigated the presence of L in the LESL and hypothesized that if flow-sensitive GPCRs are L, flow and O will determine their response to receptor activation. The LESL protein fraction isolated from guinea pig hearts was passed through an affinity chromatography column made of three sugars, mannose, galactose, and N-acetylglucosamine, and the lectinic fraction was eluted. Immune dot blot was used to identify L proteins in the LESL fraction. Our results indicate the following. 1) Two-dimensional SDS-PAGE (2D-SDS-PAGE) of the LESL lectinic fraction revealed at least 167 Ls. 2) Among these Ls, we identified three selectins and the GPCRs: angiotensin II, bradykinin (B2-R), adenosine A1 and A2, prolactin, endothelin, α1-adrenergic (α1A-R), thromboxane A2, ß1-adrenergic, ß3-adrenergic, and insulin receptors; the first six GPCRs are known to be flow sensitive. 3) The amplitude of receptor-induced vascular responses by α1A-R and B2-R activation (phenylephrine or bradykinin, respectively) was a function of flow and O (hyaluronidate). Our results support a novel mechanism of GPCR-mediated responses to flow via L·O interaction.

Proteomic analysis of differentially accumulated proteins during ripening and in response to 1-MCP in papaya fruit.

Papaya (Carica papaya L.) is a climacteric fruit susceptible to postharvest losses due to the ethylene-induced ripening. The inhibitor of ethylene action, 1-methylcyclopropene (1-MCP), has been used worldwide as a safe postharvest non-toxic agent, but the physiological and biochemical modifications induced by 1-MCP are not well understood. Using the 2-DE analysis, we report the changes in the protein profiles after 6 and 18 days of postharvest and the effect of the effect of 1-MCP treatment on fruits. Twenty seven protein spots showing differences in abundance during ripening were successfully identified by nano-LC-ESI/MS/MS. Some spots corresponded to the cell wall degrading enzymes related to fruit ripening; others were involved in oxidative damage protection, protein folding, and cell growth and survival that were induced by 1-MCP. This is the first proteomic report analyzing the effect of 1-MCP in papaya ripening. The present data will help to shed light on papaya fruit ripening process.

Identification of differential expressed transcripts in cervical cancer of Mexican patients.

The aim of this study was to identify the gene expression profile in biopsies of patients with cervical intraepithelial neoplasia (CIN) 1, CIN 2, CIN 3, and microinvasive cancer by suppression subtractive hybridization and Southern blotting. After analyzing 1,800 cDNA clones, we found 198 upregulated genes, 166 downregulated, and no significant change of gene expression in 86 clones (p = 0.005). These results were validated by Northern blot analysis (p = 0.0001) in the identification of 28 overexpressed and 7 downregulated transcripts. We observed a set of genes related to the Notch signaling pathway that may be involved in the transformation of cervical cells and in the development to malignancy. The differentially expressed genes may provide useful information about the molecular mechanisms involved in human cervical carcinoma and as diagnostic markers.

Expression, purification and preliminary crystallization of amaranth 11S proglobulin seed storage protein from Amaranthus hypochondriacus L.

11S globulin is one of the major seed storage proteins in amaranth. Recombinant protein was produced as up to approximately 80% of the total bacterial protein using Escherichia coli Rosetta-gami (DE3) containing pET21d with amaranth 11S globulin cDNA. The best expression condition was at 302 K for 20 h using LB medium containing 0.5 M NaCl. The recombinant protein was easily separated from most of the Escherichia coli proteins by precipitation with 0-40% ammonium sulfate solution. It formed aggregates at low temperature and at low salt concentrations. This behaviour may imply that it has a more hydrophobic nature than other 11S seed globulins. The crystals diffracted to 6 A resolution and belonged to space group P6(3), with unit-cell parameters a=b=97.6, c=74.8 A, gamma=120.0 degrees. One subunit of a trimer was estimated to be present in the asymmetric unit, assuming a Vsol of 41%. To obtain the complete structure solution, experiments to improve crystallization and flash-cooling conditions are in progress.

Synthesis and quantitation of six phenolic amides in Amaranthus spp.

Cinnamoylphenethylamines are phenolic amides in which cinnamic acid provides the acid moiety and phenethylamine the amine moiety. Single ion monitoring (SIM) in LC-MS was performed on amaranth leaf extracts. Masses corresponding to sets of regioisomers, including previously reported compounds, were examined. Six peaks were detected and their corresponding standards synthesized for a quantitative LC-MS/MS investigation of cinnamoylphenethylamines in amaranth. Four cinnamoylphenethylamines (caffeoyltyramine, feruloyldopamine, sinapoyltyramine, and p-coumaroyltyramine) are reported in the Amaranthaceae for the first time; also, one rare compound, feruloyl-4-O-methyldopamine, appeared to be quite common in the genus Amaranthus. Feruloyldopamine showed moderate antifungal activity toward an isolate of Fusarium culmorum. Our LC-MS approach, in conjunction with the straightforward synthesis, provides a simple, reliable way of quantitatively investigating cinnamoylphenethylamines in plants. Concentrations of cinnamoylphenethylamines vary widely: feruloyltyramine was present in quantities of 5.26 to 114.31 microg/g and feruloyldopamine in quantities of 0.16 to 10.27 microg/g, depending on the plant sample.