Composition of the essential oil from cell suspension cultures of Achillea millefolium ssp. millefolium

The composition of the essential oil isolated from Achillea millefolium L. ssp. millefolium cell suspension cultures was analysed by GC and GC-MS. The yield of the oil obtained by hydrodistillation or a simultaneous distillation -extraction of these cultures, harvested at days 8–10 (end of exponential phase), was 0.001 % (w/w). The analysis of the volatiles showed the presence of thirteen components; monoterpenes amounted to 5%, sesquiterpene hydrocarbons attained 40%, while eugenol, demethoxyencecalin and two unidentified compounds amounted to 45% of the total oil. Several methods were tested in an attempt to increase the essential oil production by the cultures: growth on solid medium, growth in light, use of a different culture medium, elicitation with cellulase or yeast extract, and growth in a two-phase system. Of the different methods tested, the growth in B5⁺ medium with Miglyol 812 led to the highest essential oil yield (0.002%, w/w), and resulted in a more diverse oil composition.     

Achillea millefolium (yarrow) cell suspension cultures: Establishment and growth conditions

Summary Friable calli were obtained fromAchillea millefolium L. hypocotyls, in Gamborg B5 medium, supplemented with 1.5mg.1^–1 2,4-D / 0.1mg.1^–1 Kin, and used for the production of cell suspension cultures in the same liquid medium. The growth pattern of the cultures was determined in permanent light or dark conditions and with different inoculum densities, basal media, growth regulators and sucrose concentrations. Different sources and nitrogen amounts were assayed to study the effect on yarrow cell growth. The conditions found to be optimal for growth of yarrow cell suspension cultures were: 70g (f.w.).1^–1 of initial inoculum in Gamborg B5 medium, supplemented with 1.5mg. 1^–1 2,4-D / 0.1mg.1^–1 Kin, NO₃ ^–/NH₄ ⁺ (30/lmM), and 2% sucrose, in darkness. In these culture conditions the cell suspensions showed a doubling time of 35–40h.Abbreviations 2,4-D dichlorophenoxyacetic acid - NAA naphtalenacetic acid - BA benzyladenine - Kin Kinetin     

Ultrastructural Aspects of the Glandular Cells from the Secretory Trichomes and from the Cell Suspension Cultures of Achillea millefolium L. ssp. millefolium

The ultrastructure of the glandular cells of the floret secretorytrichomes from Achillea millefolium L. ssp. millefolium (yarrow)was examined before and after anthesis and compared with theultrastructure of the cells from the cell suspension culturesobtained from the same plant. The profuse tubular structuresobserved in the plastids of the glandular cells of the trichomesduring the pre-secretory stage were much reduced in the secretorystage and showed an osmiophilic content. Some endoplasmic reticulumprofiles could be seen adjacent to the plastids. Later in thesecretory stage, the secretion appeared in the periplasmic spacebetween the cells of the upper tiers and in the sub-cuticularspace. Finally the secretion was released by rupture of thecuticle. At the lag phase, the cells from the cell suspensioncultures of yarrow were characterized by the presence or plastidswith tubular structures, similar to those observed in the plastidsof the trichomes in the pre-secretory stage. By the end of thelag phase accumulations of starch were observed inside the plastids.At the beginning of the exponential phase, the tubular structuresof the plastids started to show an osmiophilic content and theaccumulations of starch were still present. At the end of thisphase starch disappeared from the plastids and only osmiophilictubular structures were observed. Rough endoplasmic reticulumas well as smooth endoplasmic reticulum profiles were frequentlyin close association with plastids and mitochondria. At thestationary phase a very large vacuole filled the cells, andin the remaining cytoplasm some endoplasmic reticulum profilesand osmiophilic droplets were observed.Copyright 1994, 1999Academic Press Achillea millefolium L. spp. millefolium, yarrow, ultrastructure, trichomes, glandular cells, plant cell suspension cultures     

Biotransformation of podophyllotoxin by Hordeum vulgare cell suspension cultures

Hordeum vulgare cell suspension cultures were used to modify podophyllotoxin (1) One major product (1a) and one minor product (1b) were detected in both the culture medium and cells. To optimize the yield of compound 1a, we showed that: (1) the optimal concentration of added podophyllotoxin (1) was 33 mg L^-1; higher concentrations caused cell toxicity; (2) the stage of the cell cycle (lag/log/stationary) at which podophyllotoxin was added only marginally affected the yield of compound 1a; the optimal addition time was after lag phase, in which the yield of compound 1a reached ca. 76%, and (3) biotransformation of podophyllotoxin (1) was relatively slow; podophyllotoxin fed at 4 days after subculture resulted in yields of compound 1a of ca. 56, 64 and 76% after an additional 3, 6 and 10 days of incubation, respectively. Product 1a was purified and identified as isopicropodophyllone (1a) based on MS and NMR data.     

Phenolic compounds from Achillea millefolium L. and their bioactivity

Since antiquity, Achillea millefolium L. (Asteraceae) has been used in traditional medicine of several cultures, from Europe to Asia. Its richness in bioactive compounds contributes to a wide range of medicinal properties. In this study, we assessed A. millefolium methanolic extract and its isolated components for free radical scavenging activity against 2,2-diphenyl-pycrilhydrazyl, total antioxidant capacity (based on the reduction of Cu(++) to Cu(+)), and ability to inhibit lipid peroxidation. The activity against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum was also tested. Chlorogenic acid, its derivatives and some flavonoids isolated by semipreparative HPLC and identified by NMR and spectrometric techniques were the major bioactive constituents of the methanolic extract. The latter exhibited significant antioxidant properties, as well as its flavonol glycosides and chlorogenic acids. With regard to the antiplasmodial activity, apigenin 7-glucoside was the most effective compound, followed by luteolin 7-glucoside, whereas chlorogenic acids were completely inactive. On the whole, our results confirmed A. millefolium as an important source of bioactive metabolites, justifying its pharmaceutical and ethnobotanical use.     

Biotransformation of eudesmanes by Tessaria absinthioides cell suspension cultures

Tessaria absinthioides callus and cell suspension cultures were established. The most appropriate plant growth regulator combination and culture conditions for cell growth and secondary metabolites were obtained on MS basal media supplemented with 20.0 M IBA/ 18.0 M BA at 22°C and using a photoperiod of 16 h light / 8 h dark. Meanwhile, submerged cultures were initiated by inocula of 5 and 10% (v/ v) and shaken at 120 rpm. The analysis of the presence of the sesquiterpenes in submerged cultures showed that only the eremophilane tessaric acid was accumulated once stationary phase was reached. When ilicic acid was added, only tessaric acid was recovered from biotransformation procedure. However, since no eudesmanes were detected, it is more likely that ilicic acid is not converted to further oxidised eudesmanes. But its disappearance, together with the increase in tessaric acid accumulation, showed that it has been metabolised into the above-mentioned eremophilane. The eudesmanic acid 3-oxo--costic acid was obtained by bioconversion of the precursor -costic acid by cell suspension cultures, together with 3,5-dihydroxycostic and 3,5-dihydroxycostic acids.     

Biotransformation of digitoxigenin by cell suspension cultures of Digitalis purpurea

Digitoxigenin was oxidized to digitoxigenone which was reduced to epidigitoxigenin and then glucosylated to epidigitoxigenin glucoside by cell cultures of Digitalis purpurea. The epidigitoxigenin glucoside, together with digitoxigenone and epidigitoxigenin, was isolated in considerable amounts, whereas digitoxigenin glucoside could only be detected in low concentration. Furthermore, it was confirmed by TLC and HPLC that digitoxigenin was hydroxylated to periplogenin.     

桔梗悬浮细胞对莪二酮的生物转化研究

目的:利用植物悬浮细胞体系对莪二酮进行结构改造研究.方法:采用生物转化技术和天然药物化学手段,分离转化产物单体,并利用波谱学手段对转化产物进行结构鉴定,并利用MTT法对转化产物的抗肿瘤活性进行了评价.结果:分离并鉴定了5个转化产物,分别为1β,10α-环氧基莪二酮(2),3α-羟基-莪二酮(3),3β-羟基-莪二酮(4),1α,10β-环氧基-11-羟基莪二酮(5)和2β-羟基-莪二酮(6).结论:桔梗悬浮细胞对于莪二酮具有良好的转化能力,可以利用其作为植物反应器对莪二酮进行结构改造,以获得水溶性更好或活性更佳的衍生物.     

Biotransformation of bufadienolides by cell suspension cultures of Saussurea involucrata

The biotransformation of three bioactive bufadienolides, namely, bufotalin (1), telocinobufagin (2), and gamabufotalin (3) by cell suspension cultures of Saussurea involucrata yielded 11 products. Bufotalin yielded 3-epi-bufotalin (1a), 3-epi-desacetylbufotalin (1b), 3-epi-bufotalin 3-O-β-D-glucoside (1c), 1β-hydroxybufotalin (1d), and 5β-hydroxybufotalin (1e); telocinobufagin yielded 3-dehydroscillarenin (2a), 3-dehydrobufalin (2b), and 3-epi-telocinobufagin (2c); and gamabufotalin yielded 3-epi-gamabufotalin (3a), 3-dehydrogamabufotalin (3b), and 3-dehydro-Δ1-gamabufotalin (3c), respectively. Among these 11 products, 1a, 1b, 1c, 1d, 3a and 3c are previously unreported. The structures of these metabolites were elucidated based on NMR spectroscopic analyses and mass spectrometry. Most metabolites showed significant cytotoxic activities against human hepatoma (HepG2) and breast cancer (MCF-7) cell lines. In addition, the time course for the biotransformation of 3 was investigated.     

Biotransformation of 18 beta-glycyrrhetinic acid by cell suspension cultures of Glycyrrhiza glabra.

Two biotransformation products formed from 18 beta-glycyrrhetinic acid by cell suspension cultures of Glycyrrhiza glabra were isolated and their structures determined by chemical and spectral data as 3-O-[alhpa-L-arabinopyranosyl-(1----2)-beta-D-Glucuronopy ranosyl]-24- hydroxy-18 beta-glycyrrhetinic acid and 30-O-beta-D-glycopyrano-syl-18 beta-glycyrrhetinic acid. The formation of glycyrrhizin, the main triterpene glucuronide of the licorice root, was not detected among the biotransformation products. This is the first report of the glucuronylation of an exogenous triterpene in plant cell cultures.     

Biotransformation of thebaine by cell suspension cultures of Papaver somniferum cv. Marianne

Thebaine is biotransformed to neopine by cell suspension cultures of Papaver somniferum cv. Marianne grown in O-B5 medium. Results of precursor stu     

Biotransformation of vinblastine to vincristine by cell suspension cultures ofCatharanthus roseus

Summary Cell suspension cultures ofCatharanthus roseus convert vinblastine to vincristine after 2 days incubation.     

Biotransformation of tabersonine in cell suspension cultures of Catharanthus roseus.

To investigate the reactions involved in the biosynthesis of vindoline from tabersonine, the bioconversion products formed when the latter compound was fed to cell suspension cultures of Catharanthus roseus were isolated and characterized. Two biotransformation products of tabersonine were isolated and shown to be lochnericine, which is formed by epoxidation of tabersonine at positions 14, 15, and lochnerinine, the 11-methoxylation product of lochnericine. The bioconversion ratio of the main biotransformation product, lochnericine, reached a value of 80.6% within three days.     

Germacrane-Type Sesquiterpene Lactones from Achillea millefolium L. and Their Anti-Inflammatory Activity

Six undescribed germacrane-type sesquiterpene lactones, millefoliumons A–F, and two known analogs were isolated from the ethyl acetate fraction of the whole plant of Achillea millefolium L. growing in Xinjiang, China. The structures of these compounds were fully elucidated by their 1D and 2D nuclear magnetic resonance (NMR), and high resolution mass (HR-ESI-MS) spectral data, and comparison with literatures. The absolute configurations of millefoliumons A–F were confirmed by experimental and calculated electronic circular dichroism data (ECD), and ¹³C-NMR calculations and DP4+ probability analysis. All compounds displayed the approximate tendency to inhibit the nitric oxide (NO) release in lipopolysaccharide (LPS)-induced BV2 cells.     

Biotransformation of C13-norisoprenoids and monoterpenes by a cell suspension culture of cv. Gamay (Vitis vinifera)

A cell suspension culture of cv. Gamay was studied for its ability to metabolize two different C₁₃-norisoprenoidic volatiles, β-ionone and dehydrovomifoliol, together with monoterpenes, geraniol and linalool, biogenetically common pathways sharing compounds. β-Ionone was totally metabolized leading to fourteen norisoprenoidic volatiles oxygenated mainly at carbons 3 or 4 of the cyclohexane ring or reduced at side chain. The biotransformation of dehydrovomifoliol was at a lesser extent, giving rise to oxygenated and reduced derivatives. The norisoprenoidic metabolites were present both under free and glycosylated forms. Geraniol and linalool were also metabolized, leading to several free and glycosylated compounds. S. Mathieu, J. Wirth contributed equally to the work and should be considered joint first authors. A short part of this paper was published at the proceedings of the 10th Weurman Flavour Research Symposium, Flavour Research at the Dawn of the Twenty-first Century, J.-L.Le Quere, P.-X.Etievant, Editors; Lavoisier,2003/Intercept Ltd, 2003.     

Biotransformation of withanolides by cell suspension cultures of Withania somnifera (Dunal)

The biotransformation potential of cell suspension cultures generated from Withania somnifera leaf was investigated, using withanolides, i.e. withanolide A, withaferin A, and withanone as precursor substrates. Interestingly, the cell suspension cultures showed inter-conversion of withanolides, as well converted to some unknown compounds, released to the culture media. The bio-catalyzed withanolide was detected and quantified by TLC and HPLC, respectively. There is noticeable conversion of withanolide A to withanone, and vice versa though at a lower level. The type of reaction of this biotransformation appears to be substitution of 20-OH group to 17-OH in withanolide A. In this paper, we present for the first time the possibility of biotransformation by inter-conversion of withanolides of pharmacological importance through cell suspension culture of W. somnifera. The possible role of putative cytochrome P₄₅₀ hydroxylases is implicated in the conversion.     

Biotransformation of monoterpenoids by suspension cultures of Lavandula angustifolia

Suspension cultures of Lavandula angustifolia reduced monoterpenoid aldehydes and structurally related compounds to their corresponding primary alcohols. Acyclic primary monoterpenoid alcohols were further metabolized to unidentified compounds. Cyclic, secondary, tertiary and aromatic alcohols were not metabolized.     

Distribution of phenolic compounds in Middleeuropean taxa of the Achillea millefolium L. aggregate

Achillea millefolium L. s.l. is a cytogenetically, morphologically, and chemically polymorphic aggregate. Besides the sesquiterpenes that possess chemotaxonomic relevance and mediate the antiphlogistic activity, the plant contains phenolic compounds such as dicaffeoylquinic acids and flavonoids causing choleretic and spasmolytic effects. To evaluate their contribution to the chemotaxonomy of European taxa of the A. millefolium group, we developed a SPE-HPLC/UV method that allows quantification of the phenolic constituents in the different taxa. The investigated species displayed differences in the quantitative and qualitative composition of phenolic acids and flavonoids. Hence, they seem to be of chemotaxonomic significance, especially for the distinction of the diploid taxa. Combining the obtained results with the data of the sesquiterpene analyses gives a comprehensive insight into the distribution of those pharmacologically relevant plant constituents in the A. millefolium group.     

Biotransformation of cannabinoids by a cell suspension culture of Cannabis sativa L

A cell suspension culture of Cannabis sativa L. is able to convert cannabidiol to bound cannabielsoins and delta-9 tetrahydrocannabinol to cannabicoumaronon. The localization and the mechanism of the bioconversion are discussed.Abbreviations CBD cannabidiol - CBE cannabielsoin - CBon cannabicoumaronon - EHHC hexahydrocannabinol epoxide - EtOAc ethyl acetate - FBS Fast Blue B salt - GLC gas-liquid chromatography - THC delta-9 tetrahydrocannabinol - TLC thin-layer chromatography     

Biotransformation of monoterpenes by Oocystis pusilla

The biotransformation of several monoterpenes by the locally isolated unicellular microalga, Oocystis pusilla was investigated. The metabolites were identified by thin layer chromatography and GC/MS. The results showed that O. pusilla had the ability to reduce the C=C double bond in (+)-carvone to yield trans-dihydrocarvone and traces of cis-dihydrocarvone. O. pusilla also converted (+)-limonene to trans-carveol, as the main product, and yielded carvone and trans-limonene oxide. Furthermore, (−)-linalool was converted to trans-furanoid and trans-pyranoid linalool oxide, thymol was converted to thymoquinone, (−)-carveol was converted to carvone and trans-dihydrocarvone, (−)-menthone and (+)-pulegone were converted to menthol, (L)-citronellal was converted to citronellol, and (+)-β-pinene was converted to trans-pinocarveol.