Clove Buds Essential Oil: The Impact of Grinding on the Chemical Composition and Its Biological Activities Involved in Consumer's Health Security.

This study is aimed at identifying the chemical composition of the essential oil extracted from the Syzygium aromaticum seeds, as well as investigating its biological activities, insecticide effect, and allelopathic properties. The extraction yield was about 14.3 and 7.14% for grounded and ungrounded seeds, respectively. The GC-MS analysis allowed the identification of 17 heterogeneous compounds, including eugenol (68.7-87.4%), as major compound, cyperene (20.5-7.2%), phenethyl isovalerate (6.4-3.6%), and cis-thujopsene (1.9-0.8%), respectively, for grounded and ungrounded seeds. Concerning the antibacterial activity, the diameter of the inhibition zone reached 35 mm when the essential oil extracted from grounded seeds was applied against Escherichia coli. Regarding the antioxidant activity via the DPPH radical scavenging test, the IC50 varied from 1.2 ± 0.1 to 2.8 ± 0.5 µg/mL. With respect to reducing power, the efficient concentration EC50 ranged from 32 to 50 µg/mL. The essential oil exhibited also an allelopathic effect against seeds of Hyoscyamus niger, as well as an insecticide effect against Sitophilus oryzae with a DL50 value of 252.4 µL/L air. These findings enhance the use of this spice as a natural food preservative and encourage its use in several fields, including pharmaceutical, cosmetics, agriculture, and therapy, that could be a strategic way to guarantee the consumer's health.

Promoting the accumulation of scopolamine and hyoscyamine in Hyoscyamus niger L. through EMS based mutagenesis.

The overexploitation of medicinal plants is depleting gene pool at an alarming rate. In this scenario inducing the genetic variability through targeted mutations could be beneficial in generating varieties with increased content of active compounds. The present study aimed to develop a reproducible protocol for in vitro multiplication and mutagenesis of Hyoscyamus niger targeting putrescine N-methyltransferase (PMT) and 6ß-hydroxy hyoscyamine (H6H) genes of alkaloid biosynthetic pathway. In vitro raised callus were treated with different concentrations (0.01% - 0.1%) of Ethyl Methane Sulfonate (EMS). Emerging multiple shoots and roots were obtained on the MS media supplemented with cytokinins and auxins. Significant effects on morphological characteristics were observed following exposure to different concentrations of EMS. EMS at a concentration of 0.03% was seen to be effective in enhancing the average shoot and root number from 14.5±0.30 to 22.2 ±0.77 and 7.2±0.12 to 8.8±0.72, respectively. The lethal dose (LD50) dose was calculated at 0.08% EMS. The results depicted that EMS has an intense effect on PMT and H6H gene expression and metabolite accumulation. The transcripts of PMT and H6H were significantly upregulated at 0.03-0.05% EMS compared to control. EMS treated explants showed increased accumulation of scopolamine (0.639 µg/g) and hyoscyamine (0.0344µg/g) compared to untreated.

Sodium nitroprusside stimulated production of tropane alkaloids and antioxidant enzymes activity in hairy root culture of Hyoscyamus reticulatus L.

Hyoscyamus reticulatus L. is a herbaceous biennial belonging to the solanaceae family. Hyoscyamine and scopolamine as main tropane alkaloids accumulated in henbane are widely used in medicine to treat diseases such as parkinson's or to calm schizoid patients. Hairy roots media manipulation which uses elicitors to activate defense mechanisms is one of the main strategies for inducing secondary metabolism as well as increasing the production of valuable metabolites. Cotyledon-derived hairy root cultures were transformed by Agrobacterium rhizogenes. Sodium nitroprusside (SNP), a nitric oxide donor), was used in various concentrations (0, 50, 100, 200 and 300 µM) and exposure times (24 and 48 h). Treatment with SNP led to a significant reduction in fresh and dry weight of hairy roots, compared to control cultures. ANOVA results showed that elicitation of hairy root cultures with SNP at different concentrations and exposure times significantly affected the activity of as antioxidant enzymes such as catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX). The highest hyoscyamine and scopolamine production (about 1.2-fold and 1.5-fold increases over the control) was observed at 50 and 100 µM SNP at 48 and 24 hours of exposure time, respectively. This is the first report of SNP elicitation effects on the production of tropane alkaloids in hairy root cultures.

Mechanisms underlying toxicity and stimulatory role of single-walled carbon nanotubes in Hyoscyamus niger during drought stress simulated by polyethylene glycol.

In this study, seeds of Hyoscyamus niger were exposed to different concentrations (50-800µgmL-1) of single-walled carbon nanotubes (SWCNTs) under different levels of drought stress (0.5-1.5MPa) for 14days. Germinated seeds were subsequently allowed to grow in the same culture media for 7 more days to test the further response of the seedlings in terms of biochemical changes to the employed treatments. Seeds subjected to drought showed reduction in germination percentage, vigor and lengths of roots and shoots. However, inclusion of SWCNTs at the two lowest concentrations significantly alleviated the drought stress (up to moderate levels only)-induced reduction in germination and growth attributes. This happened due to increased water uptake, up-regulation of mechanisms involved in starch hydrolysis, and reduction in oxidative injury indices including H2O2, malondialdehyde contents and electrolyte leakage. The improved plant performance under PEG-induced drought stress was a consequence of changes in the expression of various antioxidant enzymes including SOD, POD, CAT, and APX, and also biosynthesis of proteins, phenolics, and specific metabolites such as proline. Results demonstrate that treatment by low concentrations of SWCNTs can induce tolerance in seedlings against low to moderate levels of drought through enhancing water uptake and activating plant defense system.

Copper excess promotes propagation and induces proteomic change in root cultures of Hyoscyamus albus L.

Hyoscyamus albus L. seedlings respond positively to copper (Cu) excess. In the present study, to understand how roots cope with Cu excess, propagation and proteome composition in the presence of Cu were examined using a root culture system. When H. albus roots were cultured in a medium without Cu, root growth deteriorated. However, in the presence of Cu, root growth increased in a concentration-dependent manner, and vigorous lateral root development was observed at 200 µM Cu. Cu accumulation in the roots increased with the Cu supply. Subcellular fractionation revealed that the highest amount of Cu was present in the cell wall-containing fraction, followed by the soluble fraction. However, the highest specific incorporation of Cu, in terms of fresh weight, was in the mitochondria-rich fraction. High Cu levels enhanced respiration activity. Comparative proteomic analysis revealed that proteins involved in carbohydrate metabolism, de novo protein synthesis, cell division, and ATP synthesis increased in abundance, whereas the proteasome decreased. These results indicate that Cu promotes propagation of H. albus roots through the activation of the energy supply and anabolism. Newly propagated root tissues and newly generated proteins that bind to Cu may provide space and reservoirs for deposition of additional Cu.

In vitro antibacterial activity and phytochemical analysis of White henbane treated by phytohormones.

This study reported the effect of interaction between cytokinins and auxins to enhance accumulation of alkaloids in White henbane (Hyoscyamus albus L.). Plants of this specie were grown under controlled conditions and treated with plant-hormones: Auxins by: 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 3-Indole Acetic Acid (IAA), Cytokinins by: Kinetin (K) and Benzyl Amino Purine (BAP), at 0-10 and 20 mg L(-1) rates isolated and interacted. The results showed that treatment of 2, 4-D and K at the highest applied rates 20 mg L(-1) increased the accumulation threefold rate estimated to 2.321% in the root plant part and 1.702% in the aerial plant part with the same plant-hormones but dosage of (20x10 mg L(-1)) in order. The TLC for alkaloid extracts shows that H. albus L. contains 6 alkaloids. In this study, it was concluded that the treatment with interaction of (Kx2,4-D) (20x20 mg L(-1)) gives the highest percent of alkaloids in the root and shoot parts compared to plant-hormones separated. The in vitro antibacterial activity was determined on microorganisms: Pseudomonas stutzeri, Staphylococcus aureus, Escherichia coli and Klebsiella pneumoniae. And performed by disc diffusion assay. Respectively, ethanol extracts showed no inhibitory effect on the microorganisms, however alkaloid extracts of H. albus L. of the same treatments with plant-hormones of shoot and root parts showed antibacterial activity against microorganisms that were tested. The results obtained in the present study suggest that alkaloid of H. albus L. can be used in treating diseases caused by the test organisms.

Iron deficiency induces changes in riboflavin secretion and the mitochondrial electron transport chain in hairy roots of Hyoscyamus albus.

Hyoscyamus albus hairy roots secrete riboflavin under Fe-deficient conditions. To determine whether this secretion was linked to an enhancement of respiration, both riboflavin secretion and the reduction of 2,3,5-triphenyltetrazolium chloride (TTC), as a measure of respiration activity, were determined in hairy roots cultured under Fe-deficient and Fe-replete conditions, with or without aeration. Appreciable TTC-reducing activity was detected at the root tips, at the bases of lateral roots and in internal tissues, notably the vascular system. TTC-reducing activity increased under Fe deficiency and this increase occurred in concert with riboflavin secretion and was more apparent under aeration. Riboflavin secretion was not apparent under Fe-replete conditions. In order to examine which elements of the mitochondrial electron transport chain might be involved, the effects of the respiratory inhibitors, barbiturate, dicoumarol, malonic acid, antimycin, KCN and salicylhydroxamic acid (SHAM) were investigated. Under Fe-deficient conditions, malonic acid affected neither root growth, TTC-reducing activity nor riboflavin secretion, whereas barbiturate and SHAM inhibited only root growth and TTC-reducing activity, respectively, and the other compounds variously inhibited growth and TTC-reducing activity. Riboflavin secretion was decreased, in concert with TTC-reducing activity, by dicoumarol, antimycin and KCN, but not by SHAM. In Fe-replete roots, all inhibitors which reduced riboflavin secretion in Fe-deficient roots showed somewhat different effects: notably, antimycin and KCN did not significantly inhibit TTC-reducing activity and the inhibition by dicoumarol was much weaker in Fe-replete roots. Combined treatment with KCN and SHAM also revealed that Fe-deficient and Fe-replete roots reduced TTC in different ways. A decrease in the Fe content of mitochondria in Fe-deficient roots was confirmed. Overall, the results suggest that, under conditions of Fe deficiency in H. albus hairy roots, the alternative NAD(P)H dehydrogenases, complex III and complex IV, but not the alternative oxidase, are actively involved both in respiration and in riboflavin secretion.