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Samples of belladonna pilular and powdered extract and tincture from two suppliers were analyzed by GLC as a cooperative effort between two laboratories to develop methodology with substantial improvements in sensitivity, specificity, precision, and working time over the present official method. This goal was achieved, but marked differences in response of the individual extracts to different isolation schemes were noted.
Eight steroidal glycosides of the spirostane type tentatively named atroposides A, B, C, D, E, F, G, and H (according to their increase in polarity) have been isolated from the methanolic extract of Atropa belladonna L. seeds by Sephadex gel filtration and column chromatography on silica gel impregnated with AgNO3. The structure of atroposides A, C, E, G has been elucidated as 3-O-alpha-D-galactopyranoside; 3-O-beta-D-glucopyranosyl(1-->4)-beta-D-galactopyranoside; 3-O-beta-D-glucopyranosyl (1-->2) -beta-D-glucopyranosyl(1-->4)-beta-D-galactopyranoside; and 3-O-alpha-L-rhamnopyranosyl (1-->4)-beta-D-glucopyranosyl (1-->2)-beta-D-glucopyranosyl (1-->4)-beta-D-galactopyranoside, each of (25R)-5 alpha-spirostane-3 beta-ol, respectively. The structure of atroposides B, D, F, and H has been elucidated as 3-O-beta-D-galactopyranoside; 3-O-beta-D-glucopyranosyl (1-->4)beta-D-galactopyranoside; 3-O-beta-D-glucopyranosyl (1-->2)-beta-D-glucopyranosyl (1-->4)-beta-D-galactopyranoside; 3-O-alpha-L-rhamnopyranosyl (1-->4)-B-D-glucopyranosyl (1-->2)-beta-D-glucopyranosyl (1-->4)-beta-D-galactopyranoside, each of (25R)-spirost-5-en-3-b-0l, respectively.
The 3' terminal 1255 nt sequence of Physalis mottle virus (PhMV) genomic RNA has been determined from a set of overlapping cDNA clones. The open reading frame (ORF) at the 3' terminus corresponds to the amino acid sequence of the coat protein (CP) determined earlier except for the absence of the dipeptide, Lys-Leu, at position 110-111. In addition, the sequence upstream of the CP gene contains the message coding for 178 amino acid residues of the C-terminus of the putative replicase protein (RP). The sequence downstream of the CP gene contains an untranslated region whose terminal 80 nucleotides can be folded into a characteristic tRNA-like structure. A phylogenetic tree constructed after aligning separately the sequence of the CP, the replicase protein (RP) and the tRNA-like structure determined in this study with the corresponding sequences of other tymoviruses shows that PhMV wrongly named belladonna mottle virus [BDMV(I)] is a separate tymovirus and not another strain of BDMV(E) as originally envisaged. The phylogenetic tree in all the three cases is identical showing that any subset of genomic sequence of sufficient length can be used for establishing evolutionary relationships among tymoviruses.
Amaryllis belladonna bulbs were examined for constituents inhibitory against the murine P-388 lymphocytic leukemia (PS system). Two in vitro active alkaloids, acetylcaranine (2; 9PS ED50 0.23 microgram/ml) and ambelline (3; 9PS ED50 1.6 micrograms/ml), were isolated accompanied by undulatine. However, the non-chiral anhydrolycorinium chloride (5) was found to be the principal antineoplastic (3 PS, 64-69% life extension at dose levels 10 to 20 mg/kg in vivo, ED50 1.4 micrograms/ml in vitro) component. Quaternary chloride 5 has not been located previously among plant or animal biosynthetic products.
The tropane alkaloid scopolamine is a medicinally important anticholinergic drug present in several solanaceous plants. Hyoscyamine 6 beta-hydroxylase (EC 1.14.11.11) catalyzes the oxidative reactions in the biosynthetic pathway leading from hyoscyamine to scopolamine. We introduced the hydroxylase gene from Hyoscyamus niger under the control of the cauliflower mosaic virus 35S promoter into hyoscyamine-rich Atropa belladonna by the use of an Agrobacterium-mediated transformation system. A transgenic plant that constitutively and strongly expressed the transgene was selected, first by screening for kanamycin resistance and then by immunoscreening leaf samples with an antibody specific for the hydroxylase. In the primary transformant and its selfed progeny that inherited the transgene, the alkaloid contents of the leaf and stem were almost exclusively scopolamine. Such metabolically engineered plants should prove useful as breeding materials for obtaining improved medicinal components.
The nuclear and plastid genomes of the plant cell form a coevolving unit which in interspecific combinations can lead to genetic incompatibility of compartments even between closely related taxa. This phenomenon has been observed for instance in Atropa-Nicotiana cybrids. We have sequenced the plastid chromosome of Atropa belladonna (deadly nightshade), a circular DNA molecule of 156,688 bp, and compared it with the corresponding published sequence of its relative Nicotiana tabacum (tobacco) to understand how divergence at the level of this genome can contribute to nuclear-plastid incompatibilities and to speciation. It appears that (1) regulatory elements, i.e., promoters as well as translational and replicational signal elements, are well conserved between the two species; (2) genes--including introns--are even more highly conserved, with differences residing predominantly in regions of low functional importance; and (3) RNA editotypes differ between the two species, which makes this process an intriguing candidate for causing rapid reproductive isolation of populations.
Deadly nightshade (Atropa belladonna) intoxication has been infrequently reported in both children and adults in the literature. In this article, the clinical and laboratory findings of 49 children with acute deadly nightshade intoxication are reviewed. Our purpose was to enlighten the findings of deadly nightshade intoxication in childhood. The most common observed symptoms and signs were meaningless speech, tachycardia, mydriasis, and flushing. None of the children required mechanical ventilation or died in our series. The patients were categorized into two groups, mild/moderate and severe intoxication. Children with and without encephalopathy were accepted as severe and mild/moderate intoxication, respectively. While 43 children were placed in the group of mild/moderate intoxication, six were in severe intoxication group. We found that meaningless speech, lethargy, and coma were more common, but tachycardia was less common in the severe intoxication group (children with encephalopathy) (P < 0.05). In the treatment, neostigmine was used in all children because of no available physostigmine in our country. In conclusion, our findings showed that the initial signs and symptoms of acute deadly nightshade intoxication might be severe in some children, but no permanent sequel and death were seen in children. We also showed that meaningless speech, lethargy, coma, and absence of tachycardia were ominous signs in deadly nightshade intoxication in childhood. Lastly, we suggest that neostigmine may be used in cases of deadly nightshade intoxication if physostigmine cannot be available.
The complete amino acid sequences of [2Fe-2S] ferredoxin from Atropa belladonna and Hyoscyamus niger have been determined by automated Edman degradation of the entire S-carboxymethylcysteinyl proteins and of the peptides obtained by enzymatic digestion. These two ferredoxins exhibited 1-8 differences in their amino acid sequences compared to those of other tropane-alkaloid-containing plants (Scopolia japonica, Datura stramonium, D. metel, and D. arborea), and only 1 or 4 differences compared to S. japonica and D. arborea. In contrast, 9-23 differences were observed among the other solanaceous ferredoxins. This suggests that tropane-alkaloid-containing plants are closely related taxonomically.
Deadly nightshade plants showing severe necrotic lesions on leaves were observed in southern Bohemia. In negatively stained preparations of spontaneously infected deadly nightshade, artificially inoculated host plants and purified preparations two types of virus particles, isometric ones of about 26 nm in diameter and flexuous ones with length of 765 nm were seen by electron microscopy. The virus with isometric particles was identified as belladonna mottle virus (BMV), indistinguishable serologically from the Hungarian isolate of this virus. Identification of the virus with flexuous particles is discussed. Observations of the ultrastructure revealed the presence of filamentous virus particle aggregates and chloroplasts with peripheral vesicles bounded by double membranes, a feature typical for tymoviruses.
For study of the sweat secretion in 27 patients suffered by local hyperhidrosis method of colorimetric determination of functioning sudoriferous glands number and a Minor's tests were used. The confines and intensity of sweat secretion have been determined. Study was carried out before and during the treatment as well as at the moment of clinical recovery. Revealing of vegetative syndromes was provided by Vein's inquirer. In patients with hyperhidrosis vegetative abnormalities were combined with asthenic disorders. Duration of illness had impact on frequency and character of neurasthenic syndrome manifestation. Offered scheme of local hyperhidrosis treatment with staged use of belladonna and antihistaminic preparation "hydroxyzine" (having antimuscarinic action) could be characterized as a well endurable and significantly ameliorative of patient's clinical status.
Serious poisoning with atropine-like substances may produce symptoms which can be confused with acute psychosis. The diagnosis in a case of acute poisoning may be difficult and diagnostic treatment with physostigmine may be considered. A case of serious atropine poisoning caused by consumption of the fruits of deadly nightshade (Atropa belladonna) which commenced with psychosis in a boy of nine years is described. It was known that the child had eaten 20-25 of the berries. He survived after treatment with intravenous administration of physostigmine.
A case is reported of collective intoxication in nine youths through self-brewed belladonna tea. Apart from peripheral anticholinergic symptoms, some of them had delirious states with visual hallucinations. The self-made tea was re-brewed in the laboratory and from the measured concentration of atropine the ingested total dose of atropine was estimated at up to 3 mg or more per person. In delirious states of unclear origin in young people, especially drug abusers, the possibility of intentional intoxication with atropine-containing plants should be considered. Intravenous physostigmine is the antidote of choice.
A new lycorine-type alkaloid, named (-)-amarbellisine, was isolated from the bulbs of Egyptian Amaryllis belladonna L. together with the well known alkaloids (-)-lycorine, (-)-pancracine, (+)-vittatine, (+)-11-hydroxyvittatine, and (+)-hippeastrine. The new alkaloid, containing the pyrrolo[de]phenanthridine ring system, was essentially characterised by spectroscopic and optical methods, and proved to be the 2-methoxy-3a,4,5,7,11b,11c-hexahydro-1H-[1,3]dioxolo[4,5-j]pyrrolo[3,2,1-de]phenanthridinol. By using HPTLC technique we also carried out a comparative study of the relative and total alkaloidal content at two different stages of plant growth. Finally, the antimicrobial activity of the isolated alkaloids was assayed.
Calystegines are norpseudotropine alkaloids accumulating in root cultures of Atropa belladonna, together with tropine derivatives, e.g. hyoscyamine. Both alkaloid groups are derived from the tropane alkaloid pathway. For the investigation of the regulation and individual steps of tropane biosynthesis, methods for the induction of the pathway were tested. Elicitation by chitosan, or defence responses to ABA and methyl jasmonate did not enhance calystegine accumulation, but led to a more or less pronounced decrease. By blocking one arm of the diverged tropane pathway, calystegine accumulation can be increased, but total tropane alkaloid formation does not increase considerably. By elevation of sucrose supply, both, total alkaloids and calystegines in particular were increased approximately threefold. The mechanism of the induction of the biosynthesis by sucrose is not known and needs further experiments.
Raman difference spectroscopy of the belladonna mottle virus (BDMV) and its separated RNA and protein components indicates that molecular interaction occurs between the single-stranded RNA genome and capsid subunits. The molecular interactions that stabilize the virion at pH 5.0 are altered or eliminated at pH 8.0, even though release of the RNA from the capsid is prevented by the addition of divalent metal cations (Ca2+). From the perturbations that occur to Raman lines of cytosine and adenine rings of the encapsidated RNA molecule between pH values of 5.0 and 8.0, it is concluded that cytosines are protonated in significant numbers at the conditions which maintain the native virus structure and that the stacking of adenines is altered by changes in pH. The degree of protonation of RNA bases can be reduced by elevation of the pH to 8.0 for encapsidated RNA or by release of the RNA from the capsid at pH 5.0. Although the protein groups that interact with the viral RNA cannot be identified unambiguously from the Raman spectra, it is apparent that the molecular environments of aromatic amino acid side chains are altered with the same changes in pH (from 5.0 to 8.0) that perturb the cytosine and adenine ring structures. No significant change in secondary structures of the capsid subunit can be detected with changes in pH or with RNA release. On the other hand, the characteristic Raman lines of the phosphate groups of packaged RNA differ from those of naked RNA at all pH values examined, most likely as a result of specific electrostatic binding of divalent cations to RNA phosphates within the virus shell.
Some of the earliest axon pathways to form in the vertebrate forebrain are established as commissural and retinal axons cross the midline of the diencephalon and telencephalon. To better understand axon guidance in the forebrain, we characterized the zebrafish belladonna (bel) mutation, which disrupts commissural and retinal axon guidance in the forebrain. Using a positional cloning strategy, we determined that the bel locus encodes zebrafish Lhx2, a lim-homeodomain transcription factor expressed in the brain, eye and fin buds. We show that bel(Ihx2) function is required for patterning in the ventral forebrain and eye, and that loss of bel function leads to alterations in regulatory gene expression, perturbations in axon guidance factors, and the absence of an optic chiasm and forebrain commissures. Our analysis reveals new roles for Ihx2 in midline axon guidance, forebrain patterning and eye morphogenesis.
A large fraction of homozygous zebrafish mutant belladonna (bel) larvae display a reversed optokinetic response (OKR) that correlates with failure of the retinal ganglion cells to cross the midline and form the optic chiasm. Some of these achiasmatic mutants display strong spontaneous eye oscillations (SOs) in the absence of motion in the surround. The presentation of a stationary grating was necessary and sufficient to evoke SO. Both OKR reversal and SO depend on vision and are contrast sensitive. We built a quantitative model derived from bel fwd (forward) eye behaviors. To mimic the achiasmatic condition, we reversed the sign of the retinal slip velocity in the model, thereby successfully reproducing both reversed OKR and SO. On the basis of the OKR data, and with the support of the quantitative model, we hypothesize that the reversed OKR and the SO can be completely attributed to RGC misrouting. The strong resemblance between the SO and congenital nystagmus (CN) seen in humans with defective retinotectal projections implies that CN, of so far unknown etiology, may be directly caused by a projection defect.
A method for analyzing belladonna alkaloids--hyoscyamine, scopolamine, anisodamine and anisodine, by means of capillary GC and GC-MSD was described. The retention data and characteristic ions of the parent and TMS derivatives of these alkaloids are given and will be very useful for identification of unknown alkaloids. The derivatization of different silylation reagents was compared, and the derivatization with MSTFA was found to be better than with BSA and BSTFA. The method has been used for studying biotransformation of these alkaloids in biosynthesis. Analysis of anisodamine and anisodine by CGC and GC-MS has not been found in the literature. In addition, the advantages of TMS derivatization are also discussed.