Secondary Alkaloids Research Articles

Chiral liquid chromatography-tandem mass spectrometry for enantiomeric profiling of ten alkaloids in tobacco: Assessing interconversion in precursor materials and aerosol

Multiple chiral enantiomer separation poses challenges in analyzing complex matrices like natural plant materials. Currently, there is no straightforward method for simultaneous separation and analysis of ten alkaloids and enantiomers, mainly due to most of their low levels. Here, we present an improved chiral liquid chromatography-tandem mass spectrometry method for accurate quantification of ten alkaloids and enantiomers. This method exhibits recoveries of 92.3 to 118.1 %, intra-day precisions of 0.6 to 4.7 %, and inter-day precisions of 1.2 to 4.5 %, with detection limits ranging from 0.03 to 3.0 ng/mL. Successfully applied to the ten alkaloids and enantiomers quantification in precursor tobacco materials and aerosols from traditional cigarettes and heated tobacco products, our results show higher concentrations of ten alkaloids in traditional cigarette aerosol compared to heated tobacco products aerosol. During aerosol formation, (R)-cotinine, N-methylanabasine, and β-nornicotyrine were produced. Notably, high temperatures during aerosol formation favored the (R)-enantiomer of several chiral alkaloids, and lead to the formation of new secondary alkaloids. This proposed mechanism represents the first attempt to elucidate the interconversion of alkaloids during aerosol formation.

Impact of nicotine pathway downregulation on polyamine biosynthesis and leaf ripening in tobacco.

Traditional breeding and molecular approaches have been used to develop tobacco varieties with reduced nicotine and secondary alkaloid levels. However, available low‐alkaloid tobacco varieties have impaired leaf quality likely due to the metabolic consequences of nicotine biosynthesis downregulation. Recently, we found evidence that the unbalanced crosstalk between nicotine and polyamine pathways is involved in impaired leaf ripening of a low‐alkaloid (LA) Burley 21 line having a mutation at the Nic1 and Nic2 loci, key biosynthetic regulators of nicotine biosynthesis. Since the Nic1 and Nic2 loci are comprised of several genes, all phenotypic changes seen in LA Burley 21 could be due to a mixture of genetics‐based responses. Here, we investigated the commercial burley variety TN90 LC and its transgenic versions with only one downregulated gene, either putrescine methyl transferase (PMT‐RNAi) or PR50‐protein (PR50‐RNAi). Nicotine levels of cured lamina of TN90 LC, TN90 PMT‐RNAi and TN90 PR50‐RNAi, were 70.5 ± 3.8, 2.4 ± 0.5, and 6.0 ± 1.1 mg/g dry weight, respectively. Low‐alkaloid transgenic lines showed delayed leaf maturation and impaired leaf quality. We analyzed polyamine contents and ripening markers in wild‐type TN90 control plants (WT) and the two transgenic lines. The ripening markers revealed that the PMT‐RNAi line showed the most pronounced impaired leaf maturation phenotype at harvest, characterized by higher chlorophyll (19%) and glucose (173%) contents and more leaf mesophyll cells per area (25%), while the ripening markers revealed that maturation of PR50‐RNAi plants was intermediate between PMT‐RNAi and WT lines. Comparative polyamine analyses showed an increase in free and conjugated polyamines in roots of both transgenic lines, this being most pronounced in the PMT‐RNAi plants. For PMT‐RNAi plants, there were further perturbations of polyamine content in the leaves, which mirrored the general phenotype, as PR50‐RNAi transgenic plants looked more similar to the WT than PMT‐RNAi transgenic plants. Activity of ornithine decarboxylase, the enzyme that catalyzes the committing step of polyamine biosynthesis, was significantly higher in roots and mature leaves of PMT‐RNAi plants in comparison to WT, while there was no increase observed for arginine decarboxylase. Treatment of both transgenic lines with polyamine biosynthesis inhibitors decreased the polyamine content and ameliorated the phenotype, confirming the intricate interplay of polyamine and nicotine biosynthesis in tobacco and the influence of this interplay on leaf ripening.

The Impact of Alkaloid-Producing Epichloë Endophyte on Forage Ryegrass Breeding: A New Zealand Perspective.

For 30 years, forage ryegrass breeding has known that the germplasm may contain a maternally inherited symbiotic Epichloë endophyte. These endophytes produce a suite of secondary alkaloid compounds, dependent upon strain. Many produce ergot and other alkaloids, which are associated with both insect deterrence and livestock health issues. The levels of alkaloids and other endophyte characteristics are influenced by strain, host germplasm, and environmental conditions. Some strains in the right host germplasm can confer an advantage over biotic and abiotic stressors, thus acting as a maternally inherited desirable ‘trait’. Through seed production, these mutualistic endophytes do not transmit into 100% of the crop seed and are less vigorous than the grass seed itself. This causes stability and longevity issues for seed production and storage should the ‘trait’ be desired in the germplasm. This makes understanding the precise nature of the relationship vitally important to the plant breeder. These Epichloë endophytes cannot be ‘bred’ in the conventional sense, as they are asexual. Instead, the breeder may modulate endophyte characteristics through selection of host germplasm, a sort of breeding by proxy. This article explores, from a forage seed company perspective, the issues that endophyte characteristics and breeding them by proxy have on ryegrass breeding, and outlines the methods used to assess the ‘trait’, and the application of these through the breeding, production, and deployment processes. Finally, this article investigates opportunities for enhancing the utilisation of alkaloid-producing endophytes within pastures, with a focus on balancing alkaloid levels to further enhance pest deterrence and improving livestock outcomes.

RNA interference (RNAi)‐induced suppression of nicotine demethylase activity reduces levels of a key carcinogen in cured tobacco leaves

Technologies for reducing the levels of tobacco product constituents that may contribute to unwanted health effects are desired. Target compounds include tobacco-specific nitrosamines (TSNAs), a class of compounds generated through the nitrosation of pyridine alkaloids during the curing and processing of tobacco. Studies have reported the TSNA N'-nitrosonornicotine (NNN) to be carcinogenic in laboratory animals. NNN is formed via the nitrosation of nornicotine, a secondary alkaloid produced through enzymatic N-demethylation of nicotine. Strategies to lower nornicotine levels in tobacco (Nicotiana tabacum L.) could lead to a corresponding decrease in NNN accumulation in cured leaves. The major nicotine demethylase gene of tobacco has recently been isolated. In this study, a large-scale field trial was conducted to evaluate transgenic lines of burley tobacco carrying an RNA interference (RNAi) construct designed to inhibit the expression of this gene. Selected transgenic lines exhibited a six-fold decrease in nornicotine content relative to untransformed controls. Analysis of cured leaves revealed a commensurate decrease in NNN and total TSNAs. The inhibition of nicotine demethylase activity is an effective means of decreasing significantly the level of a key defined animal carcinogen present in tobacco products.

Reducing the content of nornicotine in tobacco via targeted mutation breeding

Cultivated tobacco produces secondary alkaloids involved in the formation of nitrosamines with health concerns. The recent identification of target genes in nicotine and nornicotine biosynthetic pathways now allows biotechnological approaches for their control. We demonstrate here that mutation breeding can be used as an alternative to genetically modified (GM) plants for generating nornicotine-free tobacco. Ten alleles of the NtabCYP82E4 gene (nicotine N-demethylase) were identified by screening 1,311 M2 families of tobacco ethylmethane sulphonate (EMS) mutants. Alkaloid analysis indicated that the nornicotine contents of homozygous M2 plants carrying nonsense or missense alleles of NtabCYP82E4 were very low or near-null. Backcrossing with tobacco elite varieties yielded BC1 plants phenotypically undistinguishable from parental lines. This major objective of tobacco breeders in the last few decades could be reached in a period of less than 1.5 years, including the creation of highly mutagenised tobacco mutant collections and the detection of mutated alleles using a simple and versatile detection technology (capillary electrophoresis-single strand conformation polymorphism, CE-SSCP) accessible to most breeding companies and crop species.

Genetic Engineering of Nicotiana tabacum for Reduced Nornicotine Content

Nornicotine is an undesirable secondary alkaloid in cultivated tobacco, because it serves as a precursor to N'-nitrosonornicotine (NNN), a tobacco-specific nitrosamine with suspected carcinogenic properties. Nornicotine is produced through the oxidative N-demethylation of nicotine by a nicotine N-demethylase enzyme during the senescence and curing of tobacco leaves. While the nornicotine content of most commercial burley tobacco is low, a process termed "conversion" can bestow considerably increased nornicotine levels in a portion of the plants within the population. Previously, we isolated a nicotine N-demethylase gene, designated CYP82E4, and demonstrated that RNAi-induced silencing of CYP82E4 and its close homologues is an effective means for suppressing nicotine to nornicotine conversion. In this study, we used real-time polymerase chain reaction to confirm the central role of CYP82E4 in nicotine N-demethylation by demonstrating that the transcript accumulation of CYP82E4 is enhanced as much as 80-fold in converter vs nonconverter tobacco. We also show the design of an optimized RNAi construct (82E4Ri298) that suppressed nicotine to nornicotine conversion from 98% to as low as 0.8% in a strong converter tobacco line, a rate of nornicotine production that is about 3.6-fold lower than typically detected in commercial varieties. Southern blot analysis showed that a single copy of the RNAi transgene was as effective in suppressing nornicotine accumulation as multiple copies. Greenhouse-grown transgenic plants transformed with the RNAi construct were morphologically indistinguishable from the empty vector or wild-type controls. These results demonstrate that the genetic transformation of tobacco with the 82E4Ri298 construct is an effective strategy for reducing nornicotine and ultimately NNN levels in tobacco. Alkaloid; cytochrome P450; gene silencing; nicotine N-demethylase; N'-nitrosonornicotine; plant genetic engineering; metabolic engineering; Nicotiana tabacum L.; real-time PCR; RNA interference; tobacco-specific nitrosamines.

Stimulation of nicotine demethylation by NaHCO(3) treatment using greenhouse-grown burley tobacco.

Experiments were conducted in tobacco (Nicotiana tabacum L.) to investigate the effect of sodium bicarbonate (NaHCO(3)) on the conversion of nicotine to nornicotine, a secondary alkaloid that can form the tobacco-specific nitrosamine N-nitrosonornicotine (NNN). The results showed that, under optimum conditions, NaHCO(3) stimulated nicotine conversion in converter plants to the maximum level predetermined by the genetic background. The conversion level in NaHCO(3)-treated leaves was 2-3 times higher than that in control leaves. For young seedlings the optimum concentration of sodium bicarbonate was 0.8% aqueous solution, and for adult plants the optimum concentration was 1%. Lower concentrations resulted in partial stimulation, whereas higher concentration damaged leaf tissue and resulted in a lower conversion level. Studies with different temperatures (from 22 to 43 degrees C) showed that 37 degrees C was optimal. This temperature allowed the least amount of time, 2-3 days for mature leaves and 4-6 days for green leaves, for the major converters to reach >95% of nicotine conversion. An examination of leaves from different growth stages and stalk positions showed that the amount of time needed for conversion was longer for young leaves and shorter for mature leaves. Treatment of leaves with NaHCO(3) affords a rapid and convenient means of identifying and removing nornicotine converter plants during growth in the greenhouse or field.

Potential nitrite scavengers as inhibitors of the formation of N-nitrosamines in solution and tobacco matrix systems.

The ability of 20 compounds, all but one tobacco constituents, to inhibit the formation of tobacco-specific N-nitrosamines (TSNA) was investigated in buffer and detergent solution and in tobacco midrib and lamina systems. In solution at pH 5.5, ascorbic acid and the phenolic acids caffeic and ferulic acid were the most potent inhibitors of the reaction between nornicotine and nitrite, with nearly complete inhibition at molar ratios test compound/nitrite > 1:1. Also, cysteine > dihydrocaffeic acid > protocatechuic acid approximately catechin acted as strong inhibitors with >90% inhibition at a ratio of 3:1. Lower inhibitions were observed with chlorogenic acid > p-coumaric acid > sclareol > serine. Rutin showed an inhibition of 34% at a ratio of 0.1:1. Sclareol, alanine, proline, and serine did not significantly affect the N-nitrosonornicotine (NNN) formation. alpha-Tocopherol and glutathione enhanced NNN formation at pH 5.5 but were inhibitors at pH 3. Cinnamic acid, vanillic acid, eugenol, and esculin enhanced NNN formation. Increased NNN formation was also observed for dihydrocaffeic acid, chlorogenic acid, protocatechuic acid, and catechin at a less-than-equimolar ratio of test compound to nitrite. The tobacco matrix experiments were performed with air-cured, ground tobacco midrib and lamina. Caffeic acid, ferulic acid, dihydrocaffeic acid and catechin were potent inhibitors of the formation of TSNA in the midrib as well as in the lamina. Also protocatechuic acid, glutathione, ascorbic acid, p-coumaric acid, chlorogenic acid and cysteine were inhibitors, while alpha-tocopherol and rutin inhibited the reaction in the midrib but not in the lamina. Cinnamic acid, vanillic acid, eugenol, alanine, proline and serine showed small effects only. The molar ratio of secondary alkaloid(s)/nitrite in the test systems were 0.1:1 (solution), approximately 0.25:1 (midrib), and approximately 1:1 (lamina) and is most likely the major contributor to the observed order of inhibition potency (solution > midrib > lamina) of the test compounds. The vicinal phenolic hydroxyl groups of polyphenols and the simultaneous presence of a phenol group and an olefinic bond in hydroxycinnamic acids were the most characteristic structural elements of the potent inhibitors.

Avaliação toxicológica aguda e screening hipocrático da epiisopilosina, alcalóide secundário de Pilocarpus microphyllus Stapf.

A espécie Pilocarpus microphyllus Stapf. recebe a designação geral de jaborandi, fonte industrial de pilocarpina, utilizada no tratamento do glaucoma. O jaborandi contém vários alcalóides secundários. A epiisopilosina foi submetida a testes farmacológicos para determinação da DL50’ screening hipocrático e determinação do peso corporal dos animais sobreviventes da DL50. A análise dos resultados revelou que a epiisopilosina apresentou DL50 duas vezes maior que a pilocarpina. Os animais que sobreviveram à determinação da DL50 ganharam peso no período de 14 dias de observação. A eplisopilosina demonstrou ser um estimulante periférico do sistema nervoso parassimpático, semelhante à pilocarpina, somente em altas doses.

Determination of Mainstream and Sidestream Cigarette Smoke Components for Cigarettes of Different Tobacco Types and a Set of Reference Cigarettes

Abstract The yield of selected components in the mainstream and sidestream smoke for cigarettes of different tobacco types and a set of Kentucky Reference cigarettes have been measured. The BAT ‘fishtail’ chimney apparatus was used in conjunction with standard FTC Cambridge pad collection apparatus for the simultaneous collection of both sidestream (SS) and mainstream (MS) cigarette smoke, respectively. Comparison of MS FTC yields and MS yields from the BAT MS/SS smoke collection device showed that the smoke yields for the products of this study were similar for the two methods. Sidestream to mainstream smoke ratios (SS/MS) for TPM, nicotine, HO, ‘tar’, CO and COwere similar to those reported in the literature, despite the variety of collection devices employed in literature reports. The total nicotine accountabilities for the single tobacco cigarettes and the set of Reference cigarettes of this study were relatively constant (~53 %). Enantiomeric separation of the alkaloids in the different smoke streams was accomplished by chiral gas chromatography-mass selective detection. The levels and the enantiomeric ratios of nicotine and nornicotine varied in the smoke streams of the different cigarettes. The level and enantiomeric ratios of anaba-sine and anatabine in sidestream smoke were below detection limits of the method employed. The enantio-meric ratios of these secondary alkaloids could not be calculated. The fraction ofd -nicotine transferred to side-stream smoke did not change significantly when compared to that yielded in mainstream smoke even though the temperatures experienced during the side-stream smoke formation process are quite different (lower) from those occurring in the mainstream smoke formation process. Relatively low concentrations of the secondary alkaloids provided only a preliminary evaluation of their enantiomeric distribution in cigarette smoke.

Chiral-Gas Chromatograpy-Selected Ion Monitoring-Mass Selective Detection Analysis of Secondary Alkaloids in Tobacco and Tobacco Smoke

Abstract Chiral gas chromatography-mass selective detection has been successfully employed in the analysis of secondary alkaloids in selected tobacco materials and cigarette smoke condensate. No extensive sample preparation is involved. A lower detection limit of ~2 % d-nornicotine, d-anabasine and d-anatabine in a mixture of l and d-isomers was achieved. The levels and the enantiomeric ratios of nicotine, nornicotine, anabasine and anatabine varied in different tobacco types. The enantiomeric ratios of nicotine, anabasine and anatabine in mainstream cigarette condensate also varied but were generally representative of the enantiomeric ratios for the alkaloids found in the leaf. The enantiomeric ratio for nornicotine in mainstream cigarette condensate also varied and was not representative of the enantiomeric ratios for the alkaloids found in the leaf. Preferential decomposition or racemization may account for the differences seen in the yields of isomers of nornicotine transferred to the mainstream smoke condensate. An experiment was conducted to determine if the d-nornicotine present in tobacco contributed to the yield of d-nicotine in mainstream smoke condensate. The results of that experiment indicated that the yield of d-nicotine transferred to mainstream smoke did not change significantly when either large levels of endogenous nornicotine were present in the leaf or when large levels of exogenous levels of d-, l-nornicotine were applied to the tobacco rod prior to smoking. The limiting factor in the production of d-nicotine in mainstream cigarette smoke condensate may be the concentration of methyl radicals present to react. Further work must be conducted to unravel the mechanism involved in the production of d-nicotine in cigarette smoke condensate.

Nachweis von Nicotin im Kopfhaar von natürlich mumifizierten Körpern aus dem christlichen Sayala (Ägyptisch-Nubien)

The investigations of hair samples obtained from human skeleton from the Christian cemetery of Sayala demonstrate the presence of nicotine. 12 of 39 individual hair samples were positive. These results indicate the use of plants containing nicotine as principal or secondary alkaloid.

Stability of Alkaloid Production in Flue‐Cured Tobacco

Alkaloid production in flue‐cared tobacco (Nicotiana tabacum L.) genotypes must be within an established acceptable level for the genotypes to be released as cultivars. The objectives of this study were to examine the stability of alkaloid production in cultivars and advanced breeding lines relative to standards set in the Regional Minimum Standards Program. Stability of alkaloid production was examined using data from the Regional Farm Test entries (1986– 1988) and the North Carolina Official Variety Tests (1979–1983). Averaged across 55 environments, the two check cultivars did not differ in nicotine or nornicotine production; however, ‘NC 95’ produced higher levels of anabasine and anatabine and had a higher ratio of total secondary alkaloids to total alkaloids (TSA/TA) than ‘NC 2326’. As indicated by the coefficients of variation, there were stability differences for nornicotine, anabasine, and anatabine production as well as TSA/TA in the Regional Farm Tests but none for nicotine production. In the North Carolina Official Variety Tests, there was a significant cultivar ✕ year interaction for nicotine production and a significant location effect for nornicotine production. The unstable production of secondary alkaloids suggests that TSA/TA should be examined across both years in which the entries are in the program before rejection or acceptance.

Organic and inorganic constituents of salt tolerant taro ( Colocasia esculenta var Antiquorum) tissues cultured in saline media

Salinity tolerant taro tissues were selected and cultured in vitro on saline media (50–350 mOsm). Salt tolerance in these tissues was associated with increased levels of calcium oxalate, chlorophyll, protein and some secondary alkaloids. The concentration of other secondary alkaloids as well as the levels of quaternary alkaloids decreased. Calcium, potassium, magnesium and sodium content of the tissues decreased with increased salinity. These findings are discussed in relation to salinity tolerance in taro.

Totaquina

IN a recent paper (Asiatic Res.,35, 777; 1939), M. Ciuca states that in its search for an anti-malarial preparation cheaper than quinine but equally efficacious, the Malaria Commission of the League of Nations instituted research into the efficacy, compared with that of quinine, of a certain number of secondary alkaloid mixtures, such as kinetum, chineto, cinchona febrifuge, etc., used in the treatment of malaria in various countries. Research carried out in more than 4,000 patients in malarious countries proved that the efficacy of preparations containing 60-80 per cent crystallizable alkaloids including 15 per cent quinine was equal to that of quinine alone. The Commission has given the name of ‘Totaquina’ to a new preparation which is a mixture of cinchona bark alkaloids containing at least 75 per cent crystallizable alkaloids and not less than 15 per cent quinine. The advantage of the new” preparation is a distinctly lower price, which is mainly due to the method of extraction, while its efficacy is equal to or only slightly less than that of quinine.