Naturally occurring benzodiazepines: current status of research and clinical implications.

Naturally occurring benzodiazepines (BZDs) were first detected in mammalian tissues in 1986. They comprise a variety of 1,4-benzodiazepines corresponding to drugs commercially available for the treatment of anxiety disorders, sleep disturbances and epileptic seizures. Several biosynthetic pathways leading to the formation of BZDs are currently being discussed and have led to the proposition of possible precursor molecules. For years, the identification of naturally occurring BZDs in mammalian organisms was mostly confined to post mortem CNS material for sensitivity reasons. While radioimmunoassay and radioreceptor assay techniques have been tentatively applied to quantitations of genuine BZDs from human milk and cerebrospinal fluid, accurate measurements in peripheral blood have only recently become accessible, e. g., by gas chromatography/selected ion monitoring-mass spectrometry (GC/SIM-MS). This review summarizes existing evidence of benzodiazepines' occurrence in nature and discusses implications for neuropsychiatric disorders.

[Enantiomeric monoterpenes in ether oil from Achillea millefolium s. I.--a taxonomically useful marker?]. / Enantiomere Monoterpene im ätherischen Ol von Achillea millefolium s. l.--eine zusätzliche taxonomische Bestimmungshilfe?

The Achillea millefolium complex is a group of taxonomically hardly separable species. Yarrow has the tendency to hybridize and to vary in phenotype. An obvious characterization of the species or hybrids is not just important for the taxonomical distinction but also for a reliable assessment of herbal drug quality. Most of the Achillea plants are still gathered from natural populations. According to the variation in phenotype, mixtures with Achillea species, which contain allergy setting compounds, often cannot be determined. Morphometric investigations exclusively do not replace a further chemical characterization. Therefore we tried to assess the composition of the chiral monoterpenes alpha-pinene, beta-pinene and sabinene. They were selected because of their frequency in the essential oil of Achillea species. By method of M.C.S.S. (Moving Capillary Stream Switching) the differentiation of stereoisomeres succeeded directly from the essential oil, which was distilled or extracted by headspace trapping at room temperature. The enantiomeric distribution neither depends on the method of extraction, nor on the habitat or the developmental stage of Yarrow. Since the compositions of enantiomeres from several Achillea species and their hybrides are of different pattern, it seemed to represent an additional marker. Though investigations of all species within the Achillea millefolium group and possibly of further chiral compounds are necessary to ensure these results.

Identification of benzodiazepines in Artemisia dracunculus and Solanum tuberosum rationalizing their endogenous formation in plant tissue.

Sterile cultivated plant cell tissues and cell regenerates of several species were tested for their binding affinity to the central human benzodiazepine receptor. Binding activity was found in extracts of Artemisia dracunculus cell tissue (IC(50) = 7 microg/ml) and, to a lesser extent, in plant regenerates of potato herb (Solanum tuberosum). Preparative HPLC led to the isolation of fractions with a significant displacing potency in the benzodiazepine receptor binding assay. Using on-line HPLC-electrospray-tandem mass spectrometry (HPLC-ESI-MS/MS) in the "selected reaction monitoring" (SRM) mode, delorazepam and temazepam were found in amounts of about 100 to 200 ng/g cell tissue of Artemisia dracunculus, whereas sterile potato herb contained temazepam and diazepam ranging approximately from 70 to 450 ng/g cell tissue. It is the first report on the endogenous formation of benzodiazepines by plant cells, as any interaction of microorganisms and environmental factors was excluded.

Identification of Melissa officinalis Subspecies by DNA Fingerprinting.

The random amplified polymorphic DNA analysis (RAPD) is a method to study genetic variability within and between populations and species on the basis of the amplification of anonymous fragments from genomic DNA templates by means of polymerase chain reaction (PCR). We applied RAPD analysis in order to distinguish medicinal plant subspecies at the level of their genomes. In this study we investigated various samples of two MELISSA subspecies and showed that RAPD analysis is a fast and reliable method to distinguish subspecies on the pharmaceutical market that have been previously classified according to the distribution pattern of compounds present in the lemon balm oil.

The influence of phytohormones on growth, organ differentiation and fructan production in callus of Symphytum officinale L.

Callus derived from Symphytum officinale L. regenerants was cultured in the presence of various phytohormones. The growth rate of callus was stimulated by all phytohormones at various concentrations. With 1-naphthaleneacetic acid no organ differentiation could be observed. With indole-3-butyric acid at low concentrations only roots were formed, whereas 6-benzylaminopurine, kinetin and zeatin at various concentrations induced either root or shoot formation or the simultaneous regeneration of both. Minor amounts of fructans were formed at high 6-benzylaminopurine-, zeatin- and at all indole-3-acetic acid-concentrations. The concentration of 1-naphthaleneacetic acid had no influence on the fructan content. Highest rates of fructan synthesis occurred at low zeatin-concentrations up to 1.5 mg/l. Only zeatin at all concentrations induced the synthesis of polyfructans, whereas appreciable amounts of oligofructans were formed under the influence of all other phytohormones.

Inhibition of zeaxanthin formation and of rapid changes in radiationless energy dissipation by dithiothreitol in spinach leaves and chloroplasts.

Dithiothreitol, which completely inhibits the de-epoxidation of violaxanthin to zeaxanthin, was used to obtain evidence for a causal relationship between zeaxanthin and the dissipation of excess excitation energy in the photochemical apparatus in Spinicia oleracea L. In both leaves and chloroplasts, inhibition of zeaxanthin formation by dithiothreitol was accompanied by inhibition of a component of nonphotochemical fluorescence quenching. This component was characterized by a quenching of instantaneous fluorescence (F(o)) and a linear relationship between the calculated rate constant for radiationless energy dissipation in the antenna chlorophyll and the zeaxanthin content. In leaves, this zeaxanthin-associated quenching, which relaxed within a few minutes upon darkening, was the major component of nonphotochemical fluorescence quenching determined in the light, i.e. it represented the ;high-energy-state' quenching. In isolated chloroplasts, the zeaxanthin-associated quenching was a smaller component of total nonphotochemical quenching and there was a second, rapidly reversible high-energy-state component of fluorescence quenching which occurred in the absence of zeaxanthin and was not accompanied by F(o) quenching. Leaves, but not chloroplasts, were capable of maintaining the electron acceptor, Q, of photosystem II in a low reduction state up to high degrees of excessive light and thus high degrees of nonphotochemical fluorescence quenching. When ascorbate, which serves as the reductant for violaxanthin de-epoxidation, was added to chloroplast suspensions, zeaxanthin formation at low photon flux densities was stimulated and the relationship between nonphotochemical fluorescence quenching and the reduction state in chloroplasts then became more similar to that found in leaves. We conclude that the inhibition of zeaxanthin-associated fluorescence quenching by dithiothreitol provides further evidence that there exists a close relationship between zeaxanthin and potentially photoprotective dissipation of excess excitation energy in the antenna chlorophyll.

Differences in the capacity for radiationless energy dissipation in the photochemical apparatus of green and blue-green algal lichens associated with differences in carotenoid composition.

Green algal lichens, which were able to form zeaxanthin rapidly via the de-epoxidation of violaxanthin, exhibited a high capacity to dissipate excess excitation energy nonradiatively in the antenna chlorophyll as indicated by the development of strong nonphotochemical quenching of chlorophyll fluorescence (FM, the maximum yield of fluorescence induced by pulses of saturating light) and, to a lesser extent, FO (the yield of instantaneous fluorescence). Blue-green algal lichens which did not contain any zeaxanthin were incapable of such radiationless energy dissipation and were unable to maintain the acceptor of photosystem II in a low reduction state upon exposure to excessive photon flux densities (PFD). Furthermore, following treatment of the thalli with an inhibitor of the violaxanthin de-epoxidase, dithiothreitol, the response of green algal lichens to light became very similar to that of the blue-green algal lichens. Conversely, blue-green algal lichens which had accumulated some zeaxanthin following long-term exposure to higher PFDs exhibited a response to light which was intermediate between that of zeaxanthin-free blue-green algal lichens and zeaxanthin-containing green algal lichens. Zeaxanthin can apparently be formed in blue-green algal lichens (which lack the xanthophyll epoxides, i.e. violaxanthin and antheraxanthin) as part of the normal biosynthetic pathway which leads to a variety of oxygenated derivatives of ß-carotene during exposure to high light over several days. We conclude that the pronounced difference in the capacity for photoprotective energy dissipation in the antenna chlorophyll between (zeaxanthin-containing0 green algal lichens and (zeaxanthin-free) blue-green algal lichens is related to the presence or absence of zeaxanthin, and that this difference can explain the greater susceptibility to high-light stress in lichens with blue-green phycobionts.

Differences in the susceptibility to light stress in two lichens forming a phycosymbiodeme, one partner possessing and one lacking the xanthophyll cycle.

The effect of high light levels on the two partners of a Pseudocyphellaria phycosymbiodeme (Pseudocyphellaria rufovirescens, with a green phycobiont, and P. murrayi with a blue-green phycobiont), which naturally occurs in deep shade, was examined and found to differ between the partners. Green algae can rapidly accumulate zeaxanthin, which we suggest is involved in photoprotection, through the xanthophyll cycle. Blue-green algae lack this cycle, and P. murrayi did not contain or form any zeaxanthin under our experimental conditions. Upon illumination, the thallus lobes with green algae exhibited strong nonphotochemical fluorescence quenching indicative of the radiationless dissipation of excess excitation energy, whereas thallus lobes with blue-green algae did not possess this capacity. The reduction state of photosystem II was higher by approximately 30% at each PFD beyond the light-limiting range in the blue-green algal partner compared with the green algal partner. Furthermore, a 2-h exposure to high light levels resulted in large reductions in the efficiency of photosynthetic energy conversion which were rapidly reversible in the lichen with green algae, but were long-lasting in the lichen with blue-green algae. Changes in fluorescence characteristics indicated that the cause of the depression in photosynthetic energy conversion was a reversible increase in radiationless dissipation in the green algal partner and "photoinhibitory damage" in the blue-green algal partner. These findings represent further evidence that zeaxanthin is involved in the photoprotective dissipation of excessive excitation energy in photosynthetic membranes. The difference in the capacity for rapid zeaxanthin formation between the two partners of the Pseudocyphellaria phycosymbiodeme may be important in the habitat selection of the two species when living separate from one another.

Light Response of CO(2) Assimilation, Dissipation of Excess Excitation Energy, and Zeaxanthin Content of Sun and Shade Leaves.

Intact attached sun leaves of Helianthus annuus and shade leaves of Monstera deliciosa and Hedera helix were used to obtain light response curves of CO(2) uptake, the content of the carotenoid zeaxanthin (formed by violaxanthin de-epoxidation), as well as nonphotochemical quenching (q(NP)), and the rate constant of radiationless energy dissipation (k(D)). The latter two parameters were calculated from the decrease of chlorophyll a fluorescence at closed photosystem II traps in saturating pulses in the light. Among the three species, the light-saturated capacity of CO(2) uptake differed widely and light saturation of CO(2) uptake occurred at very different photon flux densities. Fluorescence quenching and zeaxanthin content exhibited features which were common to all three species: below light-saturation of CO(2) uptake nonphotochemical quenching occurred in the absence of zeaxanthin and was not accompanied by a decrease in the yield of instantaneous fluorescence. Nonphotochemical quenching, q(NP), increased up to values which ranged between 0.35 and 0.5 when based on a control value of the yield of variable fluorescence determined after 12 hours of darkness. As light saturation of CO(2) uptake was approached, q(NP) showed a secondary increase and the zeaxanthin content of the leaves began to rise. This was also the point from which the yield of instantaneous fluorescence began to decrease. The increase in zeaxanthin was paralleled by an increase in the rate constant for radiationless energy dissipation k(D), which opens the possibility that zeaxanthin is related to the rapidly relaxing "high-energy-state quenching" in leaves.

Zeaxanthin and the Induction and Relaxation Kinetics of the Dissipation of Excess Excitation Energy in Leaves in 2% O(2), 0% CO(2).

The relationship between the carotenoid zeaxanthin, formed by violaxanthin de-epoxidation, and nonphotochemical fluorescence quenching (q(NP)) in the light was investigated in leaves of Glycine max during a transient from dark to light in 2% O(2), 0% CO(2) at 100 to 200 micromoles of photons per square meter per second. (a) Up to a q(NP) (which can vary between 0 and 1) of about 0.7, the zeaxanthin content of leaves was linearly correlated with q(NP) as well as with the rate constant for radiationless energy dissipation in the antenna chlorophyll (k(D)). Beyond this point, at very high degrees of fluorescence quenching, only k(D) was directly proportional to the zeaxanthin content. (b) The relationship between zeaxanthin and k(D) was quantitatively similar for the rapidly relaxing quenching induced in 2% O(2), 0% CO(2) at 200 micromoles of photons per square meter per second and for the sustained quenching induced by long-term exposure of Nerium oleander to drought in high light (B Demmig, K Winter, A Krüger, F-C Czygan [1988] Plant Physiol 87: 17-24). These findings suggest that the same dissipation process may be induced by very different treatments and that this particular dissipation process can have widely different relaxation kinetics. (c) A rapid induction of strong nonphotochemical fluorescence quenching within about 1 minute was observed exclusively in leaves which already contained a background level of zeaxanthin.

Zeaxanthin Synthesis, Energy Dissipation, and Photoprotection of Photosystem II at Chilling Temperatures.

When leaves of a mangrove, Rhizophora mangle, were exposed to an excess of light at chilling temperatures, synthesis of zeaxanthin through violaxanthin de-epoxidation as well as nonphotochemical fluorescence quenching were markedly reduced. The results suggest a protective role of energy dissipation against the adverse effects of high light and chilling temperatures: leaves of R. mangle that had been preilluminated in 2% O(2), 0% CO(2) at low photon flux density and showed a high level of zeaxanthin, and leaves that had been kept in the dark and contained no zeaxanthin, were both exposed to high light and chilling temperatures (5 degrees C leaf temperature) in air and then held under control conditions in low light in air at 25 degrees C. Measurements of chlorophyll a fluorescence at room temperature showed that the photochemical efficiency of PSII and the yield of maximum fluorescence of the preilluminated leaf recovered completely within 1 to 3 hours under the control conditions. In contrast, the fluorescence responses of the predarkened leaf in high light at 5 degrees C did not recover at all. During a dark/light transient in 2% O(2), 0% CO(2) in low light at 5 degrees C, nonphotochemical fluorescence quenching increased linearly with an increase in the zeaxanthin content in leaves of R. mangle. In soybean (Glycine max) leaves, which contained a background level of zeaxanthin in the dark, a similar treatment with excess light induced a level of nonphotochemical fluorescence quenching that was not paralleled by an increase in the zeaxanthin content.

Photochemical efficiency of photosystem II, photon yield of O2 evolution, photosynthetic capacity, and carotenoid composition during the midday depression of net CO2 uptake in Arbutus unedo growing in Portugal.

During the "midday depression" of net CO2 exchange in the mediterranean sclerophyllous shrub Arbutus unedo, examined in the field in Portugal during August of 1987, several parameters indicative of photosynthetic competence were strongly and reversibly affected. These were the photochemical efficiency of photosystem (PS) II, measured as the ratio of variable to maximum chlorophyll fluorescence, as well as the photon yield and the capacity of photosynthetic O2 evolution at 10% CO2, of which the apparent photon yield of O2 evolution was most depressed. Furthermore, there was a strong and reversible increase in the content of the carotenoid zeaxanthin in the leaves that occurred at the expense of both violaxanthin and ß-carotene. Diurnal changes in fluorescence characteristics were interpreted to indicate three concurrent effects on the photochemical system. First, an increase in the rate of radiationless energy dissipation in the antenna chlorophyll, reflected by changes in 77K fluorescence of PSII and PSI as well as in chlorophyll a fluorescence at ambient temperature. Second, a state shift characterized by an increase in the proportion of energy distributed to PSI as reflected by changes in PSI fluorescence. Third, an effect lowering the photon yield of O2 evolution and PSII fluorescence at ambient temperature without affecting PSII fluorescence at 77K which would be expected from a decrease in the activity of the water splitting enzyme system, i.e. a donor side limitation.

Zeaxanthin and the Heat Dissipation of Excess Light Energy in Nerium oleander Exposed to a Combination of High Light and Water Stress.

Upon termination of watering of plants of Nerium oleander exposed to high light, photochemical efficiency became reduced as leaf water content decreased. Evidence is presented that this type of photoinhibition reflects to a substantial degree radiationless dissipation of excitation energy, probably mediated by the carotenoid zeaxanthin. During the imposition of water stress, the zeaxanthin content of leaves increased at the expense of violaxanthin and beta-carotene as a water deficit developed over a period of several days. The increase in zeaxanthin content was linearly related to an increase in the rate of radiationless energy dissipation in the antenna chlorophyll as calculated from the characteristics of chlorophyll a fluorescence measured with a pulse amplitude modulated fluorometer at room temperature. The increase in the rate of radiationless dissipation was also linearly related to a decrease in PSII photochemical efficiency as indicated by the ratio of variable to maximum fluorescence. Leaves of well-watered shade plants of N. oleander exposed to strong light showed a similar increase in zeaxanthin content as sun leaves of the same species subjected to drought in strong light. Shade leaves possessed the same capacity as sun leaves to form zeaxanthin at the expense of both violaxanthin and beta-carotene. The resistance of this species to the destructive effects of excess light appears to be related to interconversions between beta-carotene and the three carotenoids of the xanthophyll cycle.

Fructan Synthesis in Tissue Cultures of Symphytum officinale; L. Initiation, Differentiation, and Metabolic Activity.

Tissue cultures originating from different organs i.e. leaves, leaf-stalks, ovaries, anthers, and roots of SYMPHYTUM OFFICINALE were initiated under various growth conditions and subcultured several times to give the first callus generation. From all these calli, whole plants could be regenerated which again were used for the preparation of tissue cultures resulting in the formation of the second callus generation. The different calli and the regenerated plants were analyzed with respect to the fructan-synthesizing capacity. Only calli derived from the leaves of the original plant synthesized fructan whereas calli derived from ovaries, anthers, and roots, which are known to contain large amounts of fructan, were not capable of synthesizing fructan. The regenerated plants obtained from the first callus generation showed ability for fructan synthesis only if the originating callus synthesized fructan. The calli of the second generation, which were prepared from fructan-containing leaves and roots of regenerated plants, showed the capacity for fructan formation. The calli of the second generation obtained from leaves and roots of regenerated, fructan-free plants were not able to synthesize this specific reserve polysaccharide. From these data it can be concluded that the calli of the first generation prepared from roots, ovaries, and anthers have lost their ability for fructan synthesis. Calli initiated from leaves and leaf-stalks preserved the capacity for fructan formation even after many calli generations and regeneration to entire plants. Different phytohormones used in the tissue cultures had only a slight effect upon the fructan formation. An influence of light on fructan synthesis could not be detected.

Photoinhibition and zeaxanthin formation in intact leaves : a possible role of the xanthophyll cycle in the dissipation of excess light energy.

Comparative studies of chlorophyll a fluorescence, measured with a pulse amplitude modulated fluorometer, and of the pigment composition of leaves, suggest a specific role of zeaxanthin, a carotenoid formed in the xanthophyll cycle, in protecting the photosynthetic apparatus against the adverse effects of excessive light. This conclusion is based on the following findings: (a) exposure of leaves of Populus balsamifera, Hedera helix, and Monstera deliciosa to excess excitation energy (high light, air; weak light, 2% O(2), 0% CO(2)) led to massive formation of zeaxanthin and a decrease in violaxanthin. Over a wide range of conditions, there was a linear relationship between either variable, F(v), or maximum fluorescence, F(m), and the zeaxanthin content of leaves. (b) When exposed to photoinhibitory light levels in air, shade leaves of H. helix had a higher capacity for zeaxanthin formation, at the expense of beta-carotene, than shade leaves of M. deliciosa. Changes in fluorescence characteristics suggested that, in H. helix, the predominant response to high light was an increase in the rate of nonradiative energy dissipation, whereas, in M. deliciosa, photoinhibitory damage to photosystem II reaction centers was the prevailing effect. (c) Exposure of a sun leaf of P. balsamifera to increasing photon flux densities in 2% O(2) and 0% CO(2) resulted initially in increasing levels of zeaxanthin (matched by decreases in violaxanthin) and was accompanied by fluorescence changes indicative of increased nonradiative energy dissipation. Above the light level at which no further increase in zeaxanthin content was observed, fluorescence characteristics indicated photoinhibitory damage. (d) A linear relationship was obtained between the ratio of variable to maximum fluorescence, F(v)/F(m), determined with the modulated fluorescence technique at room temperature, and the photon yield of O(2) evolution, similar to previous findings (O Björkman, B Demmig 1987 Planta 170: 489-504) on chlorophyll fluorescence characteristics at 77 K and the photon yield of photosynthesis.

Photosynthetic capacity, chloroplast pigments, and mineral content of the previous year's spruce needles with and without the new flush: analysis of the forest-decline phenomenon of needle bleaching.

Spruce (Picea abies) damage in the Fichtelgebirge (FRG) occurs as needle bleaching and a depression of CO2 assimilation. Such injury may primarily result from the direct, above-ground effects of air pollution or indirect, below-ground changes in mineral uptake.Typically, the new flush of spruce needles is green and exhibits high photosynthetic capacity. Mies and Zöttl concluded that the older foliage is damaged when nutrients are withdrawn to supply the current year's needles. By removing the terminal buds of single branches in the spring, we produced an experimental set of the previous year's needles with greater mineral reserves than the control needles. During the course of the growing period, the performance of the experimental needles, which lacked competition from the new flush, was compared to that of the control needles of the same age-class on intact branches with the new flush.Throughout the experiment, chloroplast pigments of a healthy control tree were not affected by the elimination of the new flush. However, the chlorophyll and carotenoid content as well as the photosynthetic capacity of the previous year's needles on those branches of a heavily damaged tree where the new flush had been eliminated increased substantially. This increase was associated with an increase in minerals, which seemed to be deficient in the control needles with the new flush. Thus, in contrast to needles of the same age-class on intact branches with undisturbed new growth in the same atmospheric environment, the experimental needles escaped bleaching and a decrease in photosynthesis. It would seem that the bleaching and the loss in photosynthetic capacity typical of trees damaged by forest decline indirectly result from nutrient deficiencies through soil environment changes and/or root damage than directly from atmospheric pollutants.

[Investigations on harpagophytum.]. / Untersuchungen der Gattung Harpagophytum.

Comparing the analytical results of the constituants of naturally growing roots and callus of Harpagophytum procumbens, it could be demonstrated that both, the products of the primary and the secondary metabolism, showed important differences. Harpagosid which is present in significant amounts in the roots and tubers of the fresh plants, was shown to be completely absent in the callus. Stachyose the main reserve carbohydrate again was only produced in minor amounts. Fructose was the predominant sugar in the callus cells.

Carotenoids in the outer cell-wall layer of Scenedesmus (Chlorophyceae).

Scenedesmus obliquus, strain 633, which synthesizes ketocarotenoids and sporopollenin, also forms pink-red-colored cell walls. Both the cell walls left over after autospore liberation and those from homogenates of disrupted green cells have similar carotenoid pigmentation. Canthaxanthin, astaxanthin, an unidentified ketocarotenoid, and lutein were found as integral cell wall components. They are bound to the outer (trilaminar) layer of the complete cell wall which also contains sporopollenin.