Studies on Alkaloids of Cephalotaxus sinensis (Rehd et Will.)Li

Seven alkaloids were isolated from Cephalotaxus sinensis (Rehd. et Wils.) Li and were identified by elements analysis, melting point and spectral data. Six of them are known alkaloids: cephalotaxine (Ⅰ), Ⅱ-hydroxyeephalotaxine (Ⅱ), drupaeine (Ⅲ), demethylcephalotaxinone (Ⅳ), c-3epi-witsonine (Ⅴ) and wilsonine (Ⅵ). Cephalo- taxine is the major alkaloid of this plant. (Ⅰ–Ⅳ) are alkaloid of Cephalotaxus type and another two (Ⅴ–Ⅵ) are alkaloid of homoerythrina type. (Ⅶ) seems to be a new alkaloid. Its strueture will be studied further.     

Antitumor alkaloids in callus cultures of Cephalotaxus harringtonia

Leaves and stems of Cephalotaxus harringtonia were induced to callus on Murashige and Skoog medium supplemented with additional vitamins, hypoxanthine, naphthaleneacetic acid and kinetin. Analysis by GC-MS showed cephalotaxine and its antitumor esters (harringtonine, isoharringtonine and homoharringtonine) in both callus and medium, whereas deoxyharringtonine was present (in relatively large amounts) only in the medium. The amount of each alkaloid was 2 to 6 times greater at 6 months than at 3 months except for deoxyharringtonine, which was not increased. At 6 months, the total alkaloid production was 1–3% of levels reported for mature trees, but the pattern was the same: 60% cephalotaxine, 40% esters. The pattern of the parent plant was unique (5% cephalotaxine, 80% homoharringtonine, 15% other esters) and may more accurately represent that of fresh plant material. A new cephalotaxine ester, homodeoxyharringtonine, was also detected in the culture medium.     

Anticarcinogenic Alkaloids in the Cultured Cells of Cephalotaxus fortunei

Calli were induced from the leaves and stems of Cephalotaxus fortunei Hook. f. on MS medium supplemented with 0. 1 mg/L KT and 3 mg/L NAA, and from which the suspension culture cell line of this plant was established for the first time. Factors such as light, pH value of the medium, concentration of plant hormone, carbon resources and addition of substances to the medium, which affect the growth of suspension cells were investigated. The results showed that suspension cells grew appropriately at pH 5.8 with a low concentration of sucrose or glucose, and a low level of NAA. No difference effect on cell growth was seen between sucrose and glucose. Phenylalanine and protein hydrolysate were not suitable for cell growth in suspension cultures, and light inhibited cell growth. A sensitive and rapid high-performance liquid chromatographic method has been developed for detecting the alkaloids in cultured cells. The results revealed the following contents of cephalotaxine and its anticancer esters in cultured cells: harringtonine, isoharringtonine and homoharringtonine. The total alkaloid production in cell suspension cultures was doubled as that in solid cultures. The relative amounts of cephalotaxine, drupacine, harringtonine, homoharringtonine and isoharringtonine in suspension cells was 22%, 6%, 8%, 23% and 41% respectively. In addition, other alkaloid as deoxyharringtonine and some steroids, including ergdst-5-en-3-ol. stigmasta-5, 22-dien-3-ol, β-sitosterin and 2-naphthalenamine have also been detected in cell cultures using GC/MS combined technique.     

Elicitor-stimulation of Monoterpene Indole Alkaloid Formation in Suspension Cultures of Catharanthus roseus

Upon treatment of 5 cell lines of Catharanthus roseus with homogenates of various fungi, as well as with chemically defined phytoalexin elicitors, all except one (non-alkaloid producing #916) responded with browing and accumulation of tryptamine within 6 – 24 h. Cells of line #615 responded with not only accumulating tryptamine, but also N-acetyl tryptamine, strictosidine lactam, ajmalicine, tabersonine, lochnericine, and catharanthine. Based on amounts of alkaloids accumulated, cells of line #615 performed best when treated with homogenates of Alternaria zinnae, Pythium apbanidermatum, Verticillium dabliae, and Rhodotorula rubs. A Pythium homogenate concentration of 5 % and a Rhodotorula homogenate concentration of 0.5 % effected maximum alkaloid yields, and, thus, were used in subsequent studies. These revealed a temporary increase of the level of alkaloids in cells and in their medium after 12 – 24 h of treatment. Ten-day-old subcultures responded better than younger and older ones. The elicitor stimulated accumulation of alkaloids and alkaloid composition did not depend on the use of 1-B5 or alkaloid production medium. A 5 l cell suspension of #615 grown in a 7.5 l bioreactor and treated with 5 % Pythium homogenate for 18 h was found to contain strictosidine lactam, ajmalicine, and catharanthine in concentrations of 27, 10, and 13 μg/g DW respectively, the medium contained 42 % of total ajmalicine.     

Inhibition of seed germination by quinolizidine alkaloids

Germination of Lactuca sativa L. was inhibited by mixtures of quinolizidine alkaloids. The alkaloid esters resulted in the strongest inhibition: 6 mM 13-tigloyloxylupanine inhibited germination by 100%, whereas the other lupin alkaloids, such as lupanine and sparteine, gave a 45 and 20% inhibition, respectively. Seedlings of Lupinus albus L., which are not affected by quinolizidine alkaloids, excrete lupanine and 13-tigloyloxylupanine into the surrounding medium by their roots. It is assumed that lupin alkaloids are potential compounds of plant-plant interaction (i.e. allelopathy) besides their role in plant-herbivore interrelations.     

Purine alkaloid producing cell cultures: fundamental aspects and possible applications in biotechnology

Coffea arabica is one of the plant species that has been widely studied with attention largely being given to its secondary products, caffeine and other purine alkaloids. The biosynthesis and significance of these alkaloids for the plant are elucidated and presented. Tissue cell culture and fundamental aspects of cell growth and alkaloid productivity are also discussed. The feasibility of Coffea cultivation in cell suspension has recently attracted the interest of many researchers. Although this cultivation is not of commercial interest, Coffea is especially suitable as a model cell line for reaction engineering studies because the purine alkaloids are well-characterised and readily released in culture medium. The use of free and immobilized coffee cells in various types of bioreactors (stirred tank, expanded bed, and membrane device) is shown.     

Seasonal variation in the secondary chemistry of foliar and reproductive tissues of Delphinium nuttallianum

Plant secondary compounds are critical in affecting interactions between plants and their herbivores. The norditerpene alkaloids are secondary compounds in Delphinium (larkspur) species which are divided into two classes: the N-(methylsuccinimido) anthranoyllycoctonine (MSAL-type) and non MSAL-type, and are known to be toxic to herbivorous insects and livestock. Alkaloid concentrations were measured in a whole plant context in vegetative and reproductive tissues in Delphinium nuttallianum at different stages of plant maturity at two locations to explore how plant maturity affected alkaloid concentrations within a growing season. Alkaloid concentrations differed between vegetative and reproductive tissues, with vegetative tissues having significantly lower alkaloid concentrations than reproductive tissues. However, no systematic differences in alkaloid concentrations were observed at different plant maturity stages across the growing season. Based on the data we suggest that alkaloid allocation in different plant parts of D. nuttallianum is influenced by life history of the plant, consistent with plant defense theory. At one location, as pods mature the qualitative alkaloid composition changed through structural diversification of the alkaloids present. The ecological significance of this structural diversification awaits further exploration.     

Studies on the Alkaloids of Cephalotaxus Isolation of 7-deoxycephalofortuneine,a New Homoerythrina Alkaloid from Cephalotaxus fortuneine Hook. f

Six homoerythrina alkaloids were isolated and identified from the weak base fraction of alkaloids from Cephalotaxus fortunei Hook. f. grown in Anhui Province, China. Five of them were known compounds and identified as cephalofortuneine (2), comosimine (Phelline alkaloid 6) (3), 3-epifortuneine (4), wilsonine (6) and epiwilsonine (7), respectively. One of them is a new alkaloid and proved to be 7-deoxycephalofortuneine (1) on the base of spectral analysis. Its MS shows a molecular ion at m/z 345, 16 mass units less than that of cephalofortuneine (2). Comparison of its 1H-NMR spectrum with that of 2 indicats that 1 lacks 7-hydroxyl group, but it still has 2-hydroxyl group.J3,4ax and J3,4eq are ll.2 Hz and 3.0 Hz, so that 3-Hpresumably is axial. The J1,2 and J2,3 are found to be 4 Hz. These indicat that the 2-H should be equatorial, since J2,3= 4 Hz is small for the trans-axial coupling.     

Seasonal variation in toxic steroidal alkaloids of foothill death camas (Zigadenus paniculatus)

Death camas (Zigadenus spp.) is a common poisonous plant in North America with plants occurring in a wide variety of habitats with species of toxic concern occurring primarily in meadows, grasslands, shrublands, and mountains. The toxicity of Zigadenus species has been attributed to a series of steroidal alkaloids. The objective of this study was to evaluate zygacine and total steroidal alkaloid concentrations in different plant tissues of Zigadenus paniculatus as a function of plant maturity. Death camas plants were collected at two locations at different developmental growth stages representing vegetative, flower, seed pod, and shattered seed pod stages. Zygacine represented greater than 50% of the total steroidal alkaloids at all developmental stages. In bulbs, total steroidal alkaloid and zygacine concentrations did not change significantly as a function of plant phenology, and concentrations were lower than what were observed in above ground plant parts. Total steroidal alkaloid and zygacine concentrations in above ground parts were highest at early vegetative growth stages and decreased over the growing season. In plant reproductive parts, total steroidal alkaloid and zygacine concentrations increased until maturity and then decreased as the plant senesced. The concentrations of steroidal alkaloids reported here suggest that the toxic risk associated with death camas is greatest in the early vegetative growth stages followed by the flower and pod stages. There is a toxic risk to livestock as long as the plant is present, and caution should be taken when grazing livestock in areas with death camas until the plant senesces.     

Alkaloids of some south american erythroxylum species

Fourteen South American species of Erythroxylum representing four sections of the genus were examined for tropane and related alkaloids. The alkaloid content of the dried material ranged from 0.002 to 0.20 %. Commonly, the alkaloids involved were esters of various tropanols with benzoic and phenylacetic acids. A new alkaloid, nortropacocaine, was isolated from E. mamacoca. Mass spectrometry indicated the existence of other new bases, cuscohygrine and, in some species dihydrocuscohygrine. Chemotaxonomic implications are discussed.     

The Only African Wild Tobacco,Nicotiana africana: Alkaloid Content and the Effect of Herbivory

Herbivory in some Nicotiana species is known to induce alkaloid production. This study examined herbivore-induced defenses in the nornicotine-rich African tobacco N. africana, the only Nicotiana species indigenous to Africa. We tested the predictions that: 1) N. africana will have high constitutive levels of leaf, flower and nectar alkaloids; 2) leaf herbivory by the African bollworm Helicoverpa armigera will induce increased alkaloid levels in leaves, flowers and nectar; and 3) increased alkaloid concentrations in herbivore-damaged plants will negatively affect larval growth. We grew N. africana in large pots in a greenhouse and exposed flowering plants to densities of one, three and six fourth-instar larvae of H. armigera, for four days. Leaves, flowers and nectar were analyzed for nicotine, nornicotine and anabasine. The principal leaf alkaloid was nornicotine (mean: 28 µg/g dry mass) followed by anabasine (4.9 µg/g) and nicotine (0.6 µg/g). Nornicotine was found in low quantities in the flowers, but no nicotine or anabasine were recorded. The nectar contained none of the alkaloids measured. Larval growth was reduced when leaves of flowering plants were exposed to six larvae. As predicted by the optimal defense theory, herbivory had a localized effect and caused an increase in nornicotine concentrations in both undamaged top leaves of herbivore damaged plants and herbivore damaged leaves exposed to one and three larvae. The nicotine concentration increased in damaged compared to undamaged middle leaves. The nornicotine concentration was lower in damaged leaves of plants exposed to six compared to three larvae, suggesting that N. africana rather invests in new growth as opposed to protecting older leaves under severe attack. The results indicate that the nornicotine-rich N. africana will be unattractive to herbivores and more so when damaged, but that potential pollinators will be unaffected because the nectar remains alkaloid-free even after herbivory.     

Correlation between Polyamines and Pyrrolidine Alkaloids in Developing Tobacco Callus

Since the diamine putrescine can be metabolized into the pyrrolidine ring of tobacco alkaloids as well as into the higher polyamines, we have investigated the quantitative relationship between putrescine and these metabolites in tobacco callus cultured in vitro. We measured levels of free and conjugated putrescine and spermidine, and pyrrolidine alkaloids, as well as activities of the putrescine-biosynthetic enzymes arginine and ornithine decarboxylase. In callus grown on high (11.5 micromolar) α-naphthalene acetic acid, suboptimal for alkaloid biosynthesis, putrescine and spermidine conjugates were the main putrescine derivatives, while in callus grown on low (1.5 micromolar) α-naphthalene acetic acid, optimal for alkaloid formation, nornicotine and nicotine were the main putrescine derivatives. During callus development, a significant negative correlation was found between levels of perchloric acid-soluble putrescine conjugates and pyrrolidine alkaloids. The results suggest that bound putrescine can act as a pool for pyrrolidine alkaloid formation in systems where alkaloid biosynthesis is active. In addition, changes in arginine decarboxylase activity corresponding to increased alkaloid levels suggest a role for this enzyme in the overall biosynthesis of pyrrolidine alkaloids.     

The production of alkaloids in Annona cacans seedlings is affected by the application of GA4+7 + 6-Benzyladenine

The Annonaceae family presents alkaloids with ecological functions and pharmacological interest. This is the first study to evaluate if the application of plant growth regulators to seeds of this family alters the production of alkaloids over time from germination. This study was carried out in four sequential stages of Annona cacans Warm. development from the resumption of embryo during seed imbibition with the application of plant regulator GA₄₊₇ + 6-Benzyladenine. The concentration of total alkaloids was quantified using the oxoaporphine alkaloid liriodenine as reference standard. In addition, the liriodenine concentration was measured and the profile evaluated by ultra-high-performance liquid chromatography (UHPLC). Results have shown that alkaloids are present in all phases and in all tissues, at higher concentrations in roots (up to 100 times). The proportion of total alkaloids and liriodenine was modified in response to the application of plant regulators. Roots doubled the content of alkaloids and liriodenine. In cotyledonary leaves, the amount of total alkaloids decreased; however, liriodenine remained unchanged. Our results have shown that the use of plant growth regulators based on gibberellins and cytokinins modified the production of alkaloids in tissues in a specific way.     

Concomitant occurrence of pyrrolizidine and quinolizidine alkaloids in the hemiparasite Osyris alba L. (Santalaceae)

The investigation of the alkaloid extracts of the hemiparasitic plant Osyris alba, collected from three different localities in southern France, revealed the concomitant presence of both pyrrolizidine (PA) and quinolizidine (QA) alkaloids in the samples from two of these localities. The sample from the third locality contained only PAs. The eight QAs identified were sparteine, N-methylcytisine, cytisine, methyl-12-cytisine acetate, hydroxy-N-methylcytisine, N-acetylcytisine, lupanine, and anagyrine. Of the eleven detected PAs, eight were identified as chysin A, chysin B, 1-carboxypyrrolizidine-7-olide, senecionine, integerrimine, retrorsine, senecivernine and a new alkaloid janfestine (7R-hydroxychysin A or 1R-carbomethoxy-7R-hydroxypyrrolizidine). PAs were mainly present as their N-oxides This is, to our knowledge, the first report demonstrating the simultaneous presence of two classes of alkaloids, quinolizidine and pyrrolizidine alkaloids, in a single parasitic plant. As these alkaloids do not occur in the same host plant, the results indicate that Osyris must have tapped more than one host plant concomitantly. Since both quinolizidine and pyrrolizidine alkaloids serve as defence compounds against herbivores, affecting different molecular targets, the simultaneous acquisition of the two types of alkaloids by a single plant could provide a novel mode of defence of hemiparasites against herbivores.     

Variability in tissue cultures of Choisy a ternata originating from a single tree

The extent of the variability has been studied in a population of variant strains of Choisya ternata obtained from two cell lines. The cultures differed from each other in morphological, physiological and biochemical traits. In particular, some of them yielded one of the two dihydrofuroquinoline alkaloids (platydesminiuro) in greater amounts than the whole plant while the other alkaloid (balfourodinium) was always produced in much lower quantities than in the whole plant or in cultures of rootless foliated stems. The variants obtained from the first line biotransformed ellipticine and gave rise to protoplast-derived clones more easily than the variants originating from the second line. Neither photoautotrophy, nor habituation, nor growth rate were correlated with alkaloid accumulation: these characters cannot be used for the selection of high-producing cultures. On the other hand, a photoautotrophic strain accumulated three free sterols not detected in the others or in the plant.     

Growth and alkaloid production of callus culture induced from Securinega suffruticosa

The growth of, and production of alkaloids by, callus derived from budding stem explants of the germinated seeds of Securinega suffruticosa has been studied. The major alkaloids produced were securinine and allosecurinine with the latter being present in the greatest amount. The effects of pH, growth hormones, sucrose concentration and light and dark on callus growth and alkaloid production have been examined in detail. The pattern of alkaloid production in the callus culture appeared to be similar to that in the root of the securinega plant.     

The sequential appearance and metabolism of alkaloids in Heimia salicifolia

The seeds of Heimia salicifolia do not contain alkaloids. Two unidentified alkaloids were detected in 1-week-old seedlings; these alkaloids were absent from older plant samples. Lyfoline, cryogenine, and lythrine were first detected in 2-week-old plants. Sinicuichine was first observed in 3-week-old plants and nesodine in 2-month-old plants. The maximum rates of synthesis for most of these alkaloids occurred in 1- to 2-month-old plants. Following administration of ¹⁴CO₂ to H. salicifolia plants, small quantities of alkaloids were purified to constant specific activity without alkaloid dilution; 95.6% of the administered ¹⁴CO₂ was assimilated and up to 0.16% of this activity was incorporated into known alkaloids. Sinicuichine and lyfoline were shown to undergo catabolism, while cryogenine was degraded very slowly, if at all. Evidence is presented for the conversion of lyfoline to lythrine.     

Norditerpene alkaloid concentrations in tissues and floral rewards of larkspurs and impacts on pollinators

Plant secondary compounds mediate interactions with insects and other animals. The norditerpene alkaloids are significant secondary compounds in Delphinium (larkspur) species which are divided into two classes: the 7, 8-methylenedioxylycoctonine (MDL-type) and N-(methylsuccinimido) anthranoyllycoctonine (MSAL-type), and are known to be toxic to herbivorous insects and livestock. Alkaloid concentrations were measured in a whole plant context in vegetative and floral tissues as well as rewards (pollen and nectar) in Delphinium barbeyi and Delphinium nuttallianum. Alkaloid concentrations differed between vegetative tissues, floral tissues and floral rewards. Alkaloid concentrations in floral parts were consistent with optimal defense theory, with tissues more closely tied to plant fitness, such as fruits, being more heavily defended than foliage. However, alkaloid concentrations were significantly lower in nectar compared to other tissues. The norditerpene alkaloids influenced the activity of bumble bees, the dominant pollinator of larkspur, but the effects were concentration dependent. Alkaloids in nectar are found at concentrations that have no effect on bee activity; however, if alkaloid concentrations in nectar were similar to those in foliage bee activity would be reduced significantly. These results suggest that nectar with low alkaloid concentrations may be beneficial to plant fitness by limiting adverse effects on pollinator activity.     

Iridoids and alkaloids from Castilleja host plants for platyptilia pica. Rhexifoline content of P. pica

Iridoid and alkaloid analyses were conducted on Castilleja sulphurea, C. occidentalis, C. rhexifolia and C. hispida (Scrophulariaceae). Pyrrolizidine alkaloids were found in C. rhexifolia and some C. sulphurea populations, but other C. sulphurea populations had quinolizidine alkaloids or none at all. No C. occidentalis populations contained alkaloids. C. hispida was found to contain lamprolobine and the quinolizidine alkaloid anagyrine. All taxa contained the pyridinemonoterpene rhexifoline. The iridoid content of the Castilleja species were all qualitatively similar. Major iridoids were aucubin, catalpol, penstemonoside and shanzhiside methyl ester, with traces of 8-epiloganin and gardoside methyl ester. Larvae of Platyptilia pica (Pterophoridae) hosted by Castilleja were found to excrete and not sequester iridoids. The adult moths contained rhexifoline alkaloid, but at a low concentration level. Systematic implications of the results for Castilleja are discussed.