Tropane Alkaloid Levels in the Leaves of Micropropagated Datura innoxia Plants

Three androgenic DATURA INNOXIA Mill. (Solanaceae) plants which had been characterised in regard to their tropane alkaloid levels were micropropagated through axillary buds. The alkaloid contents in the leaves of more than 300 micropropagated plants were investigated by ELISA and HPLC, after one and two culture cycles and compared to those of one of seedlings obtained from the same original line from which the androgenic plants had been produced. The plant with the higher alkaloid level yielded the population with the most alkaloids. After a second cycle of micropropagation, all the populations contained more alkaloids than those they had been derived from. All the micropropagated plant populations contained higher levels of alkaloids than plants grown from seeds.     

Variation in the Tropane Alkaloid Content of Hyoscyamus muticus Plants and Cell Culture Clones

Systematic studies were carried out on two different strains (Gatersleben and Cairo) of HYOSCYAMUS MUTICUS L. (Solananaceae) in order to analyse the variation in the contents of the two main tropa-alkaloids in individual plants and protoplast-derived cell culture clones. The hyoscyamine content was determined by radioimmunoassay, the scopolamine content by radio- and enzymeimmunoassay and the total tropane alkaloid content by quinuclidinyl benzilate assay. The development stage of the plant is important for alkaloid production. Clear maximal foliar scopolamine and hyoscyamine contents were reached at the onset of flowering and during the full blooming stage, respectively. In the roots the changes in the production of these alkaloids were not considerable. Hyoscyamine is a major alkaloid in the plants as well as in the cell culture clones. In a few exceptions the scopolamine content was greater than that of hyoscyamine, but this phenomenon was not inherited by the cultured clones derived from these plants. High and low tropane alkaloid production was inherited by the selfed F (1), generation plants. Great variability in these alkaloids was observed among individual plants or clones in the same plant or clone population. There was a significant difference between the Cairo strain and the Gatersleben strain as regards their ability to produce tropane alkaloids. Haploid plants of both strains contained more hyoscyamine and scopolamine than the diploid ones. Hyoscyamine and scopolamine were the main alkaloids in the clones and in the plants. Via QNB-assay an interesting clone was found which contains remarkable amounts of unknown tropane-group alkaloids.     

Long-Term Cultivation of Digitalis lanata Clones Propagated in Vitro: Cardenolide Content of the Regenerated Plants1

Shoot cultures of DIGITALIS LANATA Ehrh. (Scrophulariaceae) were established by inoculating meristem explants from axillary buds in a halfstrength liquid Murashige and Skoog medium. After short- and long-term cultivation IN VITRO, the shoots were rooted on a solid medium, transferred into soil, and grown in the greenhouse. The cardenolide content of the regenerated plants was determined by HPLC. The vegetatively propagated plants showed good homogeneity and are identical with the parent plant in terms of their digoxigenin-glycoside content. Long-term cultivation IN VITRO had no negative influence on the homogeneity and digoxigenin-glycoside content of the propagated plants. With this method, valuable genotypes can be preserved for a longer time than the natural vegetation period.     

In vitro amoebicidal testing of natural products; part I. Methodology.

An IN VITRO test procedure utilising ENTAMOEBA HISTOLYTICA is described for the evaluation of crude extracts of plants and of isolated compounds. The test has been used to evaluate a number of standard antiamoebic drugs (emetine, 2, 3-dehydroemetine, metronidazole, 5-chloro-8-hydroxyquinoline), antimalarial drugs (amodiaquine, mepacrine, primaquine, chloroquine, quinine), CINCHONA alkaloids (quinidine, quinidinone, cinchonamine, 10-methoxycinchonamine, 3-epiquinamine, aricine, crude extracts), quassinoids (bruceantin, bruceine C, quassin, BRUCEA crude extract) and the alkaloid canthin-6-one. The applicability of the test to screening plant extracts is discussed and the results are compared with IN VITRO cytoxicity tests to guinea-pig ear keratinocytes.     

Effects of Near-Ultraviolet Light on Alkaloid Production in Catharanthus roseus Plants

Artificial near-ultraviolet light with a peak at 370 nm and the light of natural radiation with wavelengths between 290 and 380 nm stimulated the synthesis of dimeric indole alkaloids in intact plants of CATHARANTHUS ROSEUS. The artificial light also specifically stimulated an IN VITRO FMN-mediated, non-enzymatic coupling of vindoline and catharanthine to synthesize an iminium intermediate, the IN VIVO precursor of dimeric alkaloid synthesis. These results suggest that near-ultraviolet light is necessary for catharanthine oxidation as a trigger reaction of dimer synthesis in the plants.     

Antihepatotoxic Principles of Artemisia capillaris Buds1

Since an extract of the crude drug "inchinko", the buds of ARTEMISIA CAPILLARIS, showed significant antihepatotoxic activity by means of carbon tetrachloride-induced liver lesion IN VIVO and IN VITRO, the extract was fractionated by monitoring the activity to yield a number of flavonoids and a coumarin (6,7-dimethylesculetin). Antihepatotoxic effects of these constituents were determined by IN VITRO assay methods using carbon tetrachloride- and galactosamine-induced cytotoxicity in primary cultured rat hepatocytes. Some analogues of dimethylesculetin were also assayed for liver-protective activity. Both the antihepatotoxic activity and the dimethylesculetin content in this plant were found to vary markedly with the date of harvesting, which was assumed to be a reason for remarkable variations of the antihepatotoxic activity in commercially available preparations of this crude drug.     

Blockade of adrenergic and cholinergic transmissions by tetramethylpyrazine.

The pharmacological actions of an amide alkaloid, tetramethylpyrazine (TMPZ), isolated from the stem of JATROPHA PODAGRICA H OOK (Euphorbiaceae) have been investigated on the electrically-evoked contractions, relaxations and twitches of some cholinergically- and adrenergically-innervated muscle preparations IN VITRO and IN VIVO. The amide alkaloid depressed or abolished the electrically-evoked contractions of the chick oesophagus, rabbit duodenum and guinea-pig vas deferens IN VITRO. Tetramethylpyrazine also inhibited or abolished the indirect electrically-induced twitches of the rat isolated hemidiaphragm, or the contractions of the cat nictitating membrane evoked by pre-ganglionic cervial sympathetic nerve electrical stimulation IN VIVO. Moreover, TMPZ prevented or reversed the high frequency electrical stimulation-induced relaxations of the rabbit isolated duodenum. These results were taken to imply blocking actions of the amide alkaloid at the cholinergic and adrenergic neuro-effector junctions, the neuro-muscular junction, and at the ganglia (presumably indicating blockade of peripheral cholinergic and adrenergic transmissions). The inhibitory effects of TMPZ on the electrically-provoked contractions (or relaxations) of the cholinergically-innervated and adrenergically-innervated muscle preparations set up are thought to be possibly linked with the non-specific spasmolytic action of the amide alkaloid, or, probably mediated through its local anaesthetic (membrane-stabilizing) activity; since its inhibitory effects were not modified by any specific antagonist. The pharmacological implications of the above findings are discussed.     

Cardenolides in Digitalis lanata Cells Transformed with Ti-Plasmids

Crown galls were induced by transformation of leaves, leaf discs, and shoots of the plant DIGITALIS LANATA with the AGROBACTERIUM TUMEFACIENS strains C58 pTi C58, B6S3 pTi B6S3, and A136 pTi A6NCtmr-338::Tn5. Integration of plasmid DNA in the genome of D. LANATA was demonstrated by hybridization experiments. The transformed cells synthesized opines and showed hormone-autotrophic growth. The crown galls formed on leaves of D. LANATA plants contained digitoxigenin derivatives (up to 0.8 muimol digitoxin equivalents g (-1) dry weight). Transformed cell lines derived from the crown galls built cardenolides IN VITRO (ca. 0.03 mumol digitoxin equivalents g (-1) dry weight). The rate of cardenolide biosynthesis IN VITRO did not decrease during a cultivation period of 12 months.     

In Vitro Production of Essential Oil from Proliferating Shoots of Rosmarinus officinalis1

The 40-day-old IN VITRO proliferating shoots of ROSMARINUS OFFICINALIS L. var. GENUINA forma ERECTUS produced an appreciable quantity of essential oil, i.e., 1.8% fw, which was similar in its constituents to that obtained from 1-year-old plants, whether naturally grown or IN VITRO-raised potted plants. However, the quantity of various constituents identified so far was generally, but marginally, less in the former case than the latter two kinds of 1-year-old plants with the exception of bornyl acetate and 1,8-cineole, the concentrations of which were higher in the proliferated shoots than the plants. The essential oil content of 1-year-old naturally grown plants was 2.4% fw, while it was 2.38% fw in the IN VITRO-raised potted plants of the same age.     

Anti-tumour effects of the sesquiterpene Lactone parthenin

The anti-tumour effects of the sesquiterpene lactone, parthenin was studied both IN VIVO and IN VITRO. Parthenin showed marked cytotoxic activity to P815 mastocytoma, L1210 leukemia, and M-1 rhabdomyosarcoma cells IN VITRO. IN VIVO studies showed intraperitoneal drug administration could either cure mice or increase their survival time after intraperitoneal of subcutaneous injection with tumour cells.     

In vitro and in vivo assessement of the antimalarial activity of sergeolide

The antimalarial activity of sergeolide (a quassinoid from PICROLEMMA PSEUDOCOFFEA) was investigated both, IN VITRO on PLASMODIUM FALCIPARUM cultures and IN VIVO through a classical test of schizontocidal action against PLASMODIUM BERGHEI in mice. Sergeolide showed a very strong antiplasmodial activity IN VITRO as well as IN VIVO. Low concentrations (0.006 microg/ml) were able to fully inhibit the IN VITRO growth of chloroquine-sensitive and resistant strains of P. FALCIPARUM. Small amounts (0.26 mg/kg/day) markedly reduced the virulence of experimentally induced P. BERGHEI infection in mice. However, sergeolide, because of its high toxicity (LD 50: 1.8 mg/kg), does not seem, in its present form to be useful for malaria curative treatment.     

Influence of Agrobacterium rhizogenes Strains on Biomass and Alkaloid Productivity in Hairy Root Lines of Hyoscyamus muticus and H. albus *

Using leaf explants of IN VITRO grown HYOSCYAMUS ALBUS and H. MUTICUS plantlets, hairy roots were induced following inoculation with AGROBACTERIUM RHIZOGENES strains A (4) and LBA-9402. The transformed roots, appearing after 14 - 17 days incubation on hormone-free MS medium containing 1 g/L cephalexin, were excised and maintained in the same medium. Ten randomly selected hairy root lines from each bacterial treatment of the two plant systems were compared for growth and alkaloid production in half-strength, hormone-free MS medium on 25 (th) day of culture. A. RHIZOGENES strain - A (4) induced hairy root lines of both H. ALBUS and H. MUTICUS were comparatively faster growing than those induced by strain LBA-9402. In contrast to earlier reports, some of the hairy root lines of H. ALBUS induced by A. RHIZOGENES strain A (4) were as fast growing as the hairy root lines of H. MUTICUS. The atropine yields of A (4) induced lines of H. ALBUS were significantly higher (3.5 fold) than the LBA-9402 induced lines. No such relationship between the bacterial strain and alkaloid productivity could, however, be obtained in case of hairy root lines of H. MUTICUS.     

Plant Nutritional Elements and Tropane Alkaloid Production in the Roots of Henbane (Hyoscyamus niger)

The effect of calcium on the morphology, growth, tropane alkaloid production (hyoscyamine and scopolamine) and plant nutritional element (calcium, magnesium and potassium) content of roots and root cultures of Hyoscyamus niger L. was examined in this study. It was observed that the tropane alkaloid productivity of root cultures was significantly higher than that of the corresponding field cultures. Murashige-Skoog (MS) medium was found to be optimal for the production of tropane alkaloids of the root cultures of H. niger. Variation in the calcium content of the medium had no substantial effect on the morphology, growth or content of magnesium, potassium and sodium of the root cultures of H. niger. However, increased calcium content in MS medium increased the production of the scopolamine and decreased the production of alkaloid hyoscyamine in the root cultures of H. niger. Collectively, our results are helpful in the optimization of medium composition for the production of highly valuable tropane alkaloid scopolamine using root cultures of H. niger.     

Development of in vitro techniques for the important medicinal plant Veratrum californicum

VERATRUM CALIFORNICUM (Liliaceae) is an important monocotyledonous medicinal plant which is the only source of the anticancer compound cyclopamine. An IN VITRO culture system for somatic embryogenesis and green plant regeneration of VERATRUM CALIFORNICUM was developed. Embryogenic calli were induced from mature embryos on induction medium. Five basal media supplemented with different growth regulators were evaluated for embryogenic callus induction, modified MS medium with 4 mg/L picloram showing the best result for embryogenic callus production. Fine suspension cell lines were established by employing friable embryogenic calli as starting material and AA medium and L2 medium as culture media. The suspension cell lines cultured in AA medium with 4 mg/L NAA appeared to be fresh yellow and fast growing. The suspension cells were cryopreserved successfully and recovered at a high rate. Green plants were regenerated from embryogenic calli maintained on solid medium with 73 % regeneration ability (green plants/100 calli) in 27-month-old culture. The IN VITRO plantlets contained the steroid alkaloids cyclopamine and veratramine. This IN VITRO system will form the basis for metabolic engineering of VERATRUM cells in the context of biotechnological production of pharmaceutically important secondary metabolites. DMSO:dimethyl sulfoxide fw:fresh weight NAA:naphthaleneacetic acid 2,4-D:2,4-dichlorophenoxyacetic acid picloram:4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid dicamba:3,6-dichloro-2-methoxybenzoic acid.     

Assay method for antihepatotoxic activity using carbon tetrachloride induced cytotoxicity in primary cultured hepatocytes*.

Conditions were examined to devise an IN VITRO assay method for antihepatotoxic activity using carbon tetrachloride-induced lesion in primary cultured mouse and rat liver cells. Utilizing 24 hr-preincubated hepatocytes from rats, which give more cells and are more sensitive to the hepatotoxin than mice, a satisfactory assay procedure was established. A good correlation of results obtained by this IN VITRO assay method with those by IN VIVO assay methods was corroborated by screening of some natural products. This assay method offers many advantages: numerous samples disposed at one time at a low expense, small sample sizes required, and little variation and good reproducibility of results.     

Toxicosis by Plant Alkaloids in Humans and Animals in Colombia

Due to its tropical location, chains of mountains, inter-Andean valleys, Amazon basin area, eastern plains and shores on both the Atlantic and Pacific Oceans, Colombia has many ecosystems and the second largest plant biodiversity in the world. Many plant species, both native and naturalized, are currently recognized as toxic for both animals and humans, and some of them are known to cause their toxic effects due to their alkaloid content. Among these, there are plants containing the hepatotoxic pyrrolizidine alkaloids, neurotoxins such as the indolizidine alkaloid swainsonine and the piperidine alkaloids coniine and γ-coniceine and tropane alkaloids. Unfortunately, the research in toxic plants in Colombia is not nearly proportional to its plant biodiversity and the scientific information available is only very scarce. The present review aims at summarizing the scarce information about plant alkaloid toxicosis in animals and humans in Colombia.     

Decreased scopolamine yield in field-grown Duboisia plants regenerated from hairy roots

Hairy root cultures were obtained from hybrid clones of Duboisia myoporoides x D. leichhardtii following transformation by Agrobacterium rhizogenes strain A4. Shoots spontaneously regenerating from the hairy root cultures were rooted and transferred to soil. The plants displayed typical morphological alterations known as hairy root syndrome to varying degrees. PCR analysis confirmed that all transformed plants contained the rolA, rolB and rolC genes, irrespective of the degree of morphological alterations. A field test of the transformed regenerated plants revealed that those plants displaying the strongest hairy root syndrome symptoms had the highest content of the tropane alkaloid scopolamine. However, the overall scopolamine and hyoscyamine yield of all transformed plants was clearly reduced compared to untransformed control plants. These results demonstrate that the A. rhizogenes-transformed plants tested in this study do not provide a viable alternative to agricultural farming of hybrid clones of D. myoporoides x D. leichhardtii obtained by conventional breeding.     

Tropane alkaloid production in transformed root cultures of brugmansia Candida

Transformed root cultures of BRUGMANSIA CANDIDA were established by infection with AGROBACTERIUM RHIZOGENES LBA 9402. Several clones with different growth index and tropane alkaloid pattern and content were obtained and two were examined in depth. The alkaloid content and pattern changed during the time course. At 21 days of culture clone 1 revealed an alkaloid spectrum dominated by 3alpha-acetoxytropane (about 50% of the total alkaloid) and hyoscyamine (about 25%), with a ratio of hyoscyamine to scopolamine of 11.2. In clone 40 this ratio was 1.5 and the content of 3alpha-acetoxytropane was low (2%). The maximum concentrations of hyoscyamine were obtained at 3 weeks of culture, and were 700 and 500 microg/g FW in clone 1 and 40, respectively.     

Determination of escin content in androgenic embryos and hairy root culture of Aesculus hippocastanum

Escin, a group of chemically related triterpenic glycosides, is widely used in commercial preparations for the treatment of venous insufficiency. Since the zygotic embryo cotyledons accumulate the highest amount of escin, it is currently extracted from the seeds of horse chestnut, Aesculus hippocastanum L. (Hippocastanaceae), on a large scale. As this material is available during only short period of the year, we studied the possibility of using plant tissue culture to obtain escin. For this purpose, the content of escin in androgenic embryos and hairy root cultures of horse chestnut was studied. Escin content was found to be dependent on the stage of androgenic embryo development and the type of phytoregulator supplemented to the nutritive medium. In the absence of phytoregulators, androgenic embryos at the globular stage of development contained approximately four times less escin than those at the cotyledonary stage. Inclusion of various phytoregulators in the nutritive media stimulated escin production. Among them, 2,4-dichlorophenoxyacetic acid (2,4-D) showed the most pronounced effect, with escin content almost reaching that found in zygotic embryos (6.77% versus 6.96%). Two hairy root clones produced substantial amounts of escin (3.57% and 4.09%), less than zygotic embryos, but higher than cotyledonary embryos on phytoregulator-free medium.     

Haploid plants regenerated from androgenic cell cultures of Digitalis lanata

Androgenic callus was obtained from cold treated anthers and pollen of Digitalis lanata. The callus was mixoploid and contained haploid, diploid and tetraploid cells as shown by impulse cytophotometry. Haploid cell lines were selected by colony cloning. They were unstable and selection had to be repeated every 1-2 months. Mixoploid shoot cultures were derived from embryogenic haploid cell lines via somatic embryos. Haploid shoots were selected by explanting shoot tips. The shoots showed wide variability in cardenolide content and profile. Rooting of the haploid shoots resulted in haploid plants. These plants were smaller in size than diploid plants. Often the flowers were morphologically abnormal and showed male sterility due to crippled anthers.