Effects of nutritional and hormonal factors on growth and production of anthraquinone glucosides in cell suspension cultures of Cinchona succirubra

Cell suspension cultures of Cinchona succirubra were found to produce anthraquinone glucosides. The effects of nutritional and hormonal factors on growth and anthraquinone production were investigated in order to study the enzymecatalyzed glucosylation of these metabolites.Abbreviations AQ Anthraquinone - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid - IAA 3-indole-acetic acid - HPLC High Performance Liquid Chromatography - TLC Thin Layer Chromatography     

Influence of various media constituents on the growth of Cinchona ledgeriana tissue cultures and the production of alkaloids and anthraquinones therein

Using the methods reported by De Fossard et al. (11) the influence of various media constituents on the growth and the alkaloid and anthraquinone production in Cinchona ledgeriana callus cultures was studied. Growth and indole alkaloid production (e.g. cinchonamine) was improved by higher auxin levels. The best growth was observed in the light, although many media resulted in no growth at all in the light. Anthraquinone production was highest at lower auxin levels. Quinoline alkaloid levels (e.g. quinidine) were highest in media with low auxin concentrations. Low and medium cytokinin concentration benefited the quinoline alkaloid production.From the results it was concluded that the pathways leading to the various secondary products, anthraquinones, indole alkaloids and quinoline alkaloids are, at least partly, regulated independently.Abbreviations used IAA indol-acetic acid - IBA indol-butyric acid - NAA -naphtaleneacetic acid - NOA 2-naphtoxy-acetic acid - 2,4-D 2,4-dichlorophenoxy-acetic acid - pCPA parachlorophenoxy-acetic acid - BA benzyladenine     

Purification and some properties of five anthraquinone specific glucosyltransferases from Cinchona succirubra cell suspension culture

Five anthraquinone-specific glucosyltransferases were partially purified from Cinchona succirubra cell suspension culture by fractional precipitation with ammonium sulphate, gel filtration and chromatofocusing on a fast protein liquid chromatography system. Five, distinct glucosylating activities were resolved with apparent pI values of 5.3, 4.8, 4.5, 4.3 and 4.1. They accepted emodin, anthrapurpurin, quinizarin, 2,6-dihydroxy anthraquinone and 1,8- dihydroxy anthraquinone as the best substrates, respectively. These enzymes exhibited similar characteristics as to pH optimum (pH 7) in histidine/HCl buffer, M, 50 000, had no cation requirement and were inhibited by various SH-group reagents. The K_m value of the respective anthraquinones for either of the five enzymes was 10 μM. The physiological role of these novel enzymes is discussed in relation to the biosynthesis of anthraquinone glucosides in this tissue.     

Accumulation of anthraquinones in Morinda citrifolia cell suspensions

Cell suspensions of Morinda citrifolia were cultivated in a B5-medium containing 4% sucrose as the sole carbon source and 1 mg l^-1 naphthyl acetic acid (NAA) or 1 mg l^-1 2,4-dichloro-phenoxyacetic acid (2,4-D). Both auxins were able to support growth but only in the presence of NAA anthraquinone production was observed. 2,4-D inhibited the production in NAA cultures. Anthraquinone synthesis took place in the growth and the stationary phase and amounts of 0.2–0.4 mmol (about 100–200 mg) g^-1 dry weight could be reached.Under both growth conditions sucrose was hydrolyzed extracellularly by invertase. From the resulting monosaccharides, glucose was taken up preferentially and an appreciable uptake of fructose only took place when medium glucose was exhausted. Sugar uptake rates were similar when cells were grown in NAA and in 2,4-D medium but the intracellular sugar contents (expressed on a dry weight basis) differed considerably. The presence of sucrose, glucose and fructose was demonstrated under both growth conditions. The amounts of sucrose and glucose were much lower in the 2,4-D cells than in the NAA-cells especially during the growth phase. Fructose contents were low and comparable, while in NAA cells an unknown sugar (possibly the sugar moiety of the glycosylated anthraquinones) was observed especially at the end of the growth phase and in the stationary phase. The differences in sugar concentrations were even larger due to the lower water contents of the NAA cells.Respiration of 2,4-D cells was much higher than that of NAA cells during the growth phase. A sharp increase in sugar contents (mainly sucrose) occurred in the 2,4-D cells at the end of the growth phase and corresponded with the fall in respiratory activity.A possible correlation between the lack of production of anthraquinones in 2,4-D cells and a less efficient growth metabolism in these cells is discussed.Abbreviations AQ anthraquinones - 2,4-D 2,4-dichloro-phenoxy-acetic acid - DW dry weight - FW fresh weight - NAA naphthyl acetic acid - pCPO p-chloro-phenoxy-acetic acid     

The influence of initial sucrose and nitrate concentrations on the growth of Cinchona ledgeriana cell suspension cultures and the production of alkloids and anthraquinones

The influence of initial concentrations of two of the major medium components, sucrose and nitrate, on the growth and the production of alkaloids and anthraquinones in cell suspension cultures of Cinchona ledgeriana Moens was studied. It was found that maximum growth and maximum alkaloid yield were obtained with a B₅-medium containing the normal level of nitrate and 4% sucrose, whereas the anthraquinone yield was maximum at 8% sucrose at the normal level of nitrate. Maximum contents of secondary metabolites, expressed as g.gDW^-1, were found using a medium containing 2% sucrose, but four times the normal nitrate concentration.     

Analysis of anthraquinones in cell cultures of Cinchona 'Robusta' by HPLC with photodiode array and mass spectrometry detection

An HPLC-PAD-ESI/MS method has been developed for the analysis of anthraquinones in cell cultures of Cinchona 'Robusta'. Using a C18 column and gradient elution with a mobile phase system containing acetonitrile, water and trifluoroacetic acid, a satisfactory separation of both anthraquinone glycosides and aglycones in a crude dichloromethane extract could be obtained. Robustaquinone B was identified as a major anthraquinone in the extracts, and another five anthraquinones were tentatively identified from spectroscopic data. A number of minor unknown compounds were detected and were distinguished from the known anthraquinones. An isocratic system for the quantitative determination of robustaquinone B has also been developed.     

Elicitor-mediated induction of anthraquinone biosynthesis and regulation of isopentenyl diphosphate isomerase and farnesyl diphosphate synthase activities in cell suspension cultures of Cinchona robusta How.

Treatment of a Cinchona robusta How. cell suspension culture with a homogenate of Phytophthora cinnamomi resulted in cessation of growth and a rapid induction of the biosynthesis of anthraquinone-type phytoalexins. The strongest induction of anthraquinone biosynthesis was obtained when the elicitor was added in the early growth phase of the growth cycle. The accumulation of anthraquinones was accompanied by a tri-phasic response in the activity of isopentenyl diphosphate (IPP) isomerase (EC 5.3.3.2): phase I was characterised by a rapid induction of activity, reaching a maximum at 12 h after elicitation. During phase II, IPP isomerase rapidly decreased to levels below those found in untreated cells. At phase III, IPP isomerase activity increased again, reaching a second maximum at about 72 h after elicitation. During phase I, the activity of farnesyl diphosphate synthase (EC 2.5.1.10) was found to be suppressed. Extraction and assay conditions were optimised for IPP isomerase. The presence of Mn²⁺ in the incubation buffer resulted in a marked increase in the activity of the enzymes obtained from cells in phase I. The induction of IPP isomerase in combination with a concomitant inhibition of farnesyl diphosphate synthase might result in an efficient channeling of C₅-precursors into phytoalexin biosynthesis. Received: 23 August 1996 / Accepted: 20 March 1997     

六盘山鸡爪大黄根蒽醌类化合物组织化学定位的研究

采用组织化学方法研究了六盘山鸡爪大黄根蒽醌类化合物的组织化学定位特征及贮藏和积累的规律。结果表明：蒽醌类化合物在根内的贮藏是多位点的,在根周皮的木栓层和栓内层、次生维管组织的维管射线和根中央的部分木薄壁细胞内不同程度地贮藏和积累了一定数量的蒽醌类化合物,次生木质部的木射线和次生韧皮部的韧皮射线是主要贮藏和积累的部位,早期形成的维管射线中蒽醌类化合物的含量较晚期形成的射线含量高。     

Biosynthesis of anthraquinones in cell cultures of the Rubiaceae

Plants and their derived cell and tissue cultures in the family Rubiaceae accumulate a number of anthraquinones. There are two main biosynthetic pathways leading to anthraquinones in higher plants: the polyketide pathway and the chorismate/o-succinylbenzoic acid pathway. The latter occurs in the Rubiaceae for the biosynthesis of Rubia type anthraquinones. In this pathway, ring A and B of the Rubia type anthraquinones are derived from shikimic acid, -ketoglutarate via o-succinylbenzoate, whereas ring C is derived from isopentenyl diphosphate, a universal building block for all isoprenoids. At present, it is known that isopentenyl diphosphate is formed via the mevalonic acid pathway or the 2-C-methyl-D-erythritol 4-phosphate pathway. Recent findings demonstrate that the 2-C-methyl-D-erythritol 4-phosphate pathway, not the mevalonic acid pathway, is involved in the formation of isopentenyl diphosphate, which constitutes ring C of anthraquinones in the Rubiaceae. This review summarizes the latest results of studies on the biosynthetic pathways, the enzymology and regulation of anthraquinone biosynthesis, as well as aspects of the metabolic engineering. Furthermore, biochemical and molecular approaches in functional genomics, which facilitate elucidation of anthraquinone biosynthetic pathways, are briefly described.     

Increased anthraquinone production in Galium vernum cell cultures induced by polymeric adsorbents

Anthraquinones produced by suspension cultures of Galium vernum are completely retained intracellularly. Surprisingly, in the presence of some polymeric adsorbents anthraquinones are partially released into the culture medium. The secretion and in situ removal stimulates anthraquinone production in cell cultures of Galium vernum. Best results were obtained with Wofatit ES and Amberlite XAD-2.Abbreviations DW dry weight - MS Murashige & Skoog[7]medium - NAA 1-naphthaleneacetic acid     

Effects of nutritional factors on the formation of Anthraquinones in callus cultures of Rheum ribes

Rheum ribes L. (Polygonaceae) is the source of one of the most important crude drugs in Asiatic regions .The medicinal character of rhubarb is due to its anthraquinone content . Different parts of sterile seedlings were cultured on MS medium to study the generation of callus. The explants were cultured with different ranges of plant growth regulators and the best range of plant growth regulators for generation of callus was IBA (1 mg l^–1) and BA (1 mg l^–1). The content of anthraquinones were determined by HPLC . The concentration of sucrose, vitamins and Myo-inositol and ratio of NO₃ to NH₄ in the medium was changed and growth rate and content of 2 anthraquinones was determined . The growth rate of callus declined with increased rate of secondary metabolites production. Myo-inositol 100 mg l^–1 in the medium increased anthraquinone content and in medium that had NO₃/NH₄:1/1 the maximum content of anthraquinone was obtained.     

Regulation of anthraquinone biosynthesis in cell cultures of Morinda citrifolia

Cell cultures of Morinda citrifolia L. are capable of accumulating substantial amounts of anthraquinones. Chorismate formed by the shikimate pathway is an important precursor of these secondary metabolites. Isochorismate synthase (EC 5.4.99.6), the enzyme that channels chorismate into the direction of the anthraquinones, is involved in the regulation of anthraquinone biosynthesis. Other enzymes of the shikimate pathway such as deoxy-D-arabino-heptulosonate 7-phosphate synthase (EC 4.1.2.15) and chorismate mutase (EC 5.4.99.5) do not play a regulatory role in the process. The accumulation of anthraquinones is correlated with isochorismate synthase activity under a variety of conditions, which indicates that under most circumstances the concentration of the branchpoint metabolite chorismate is not a rate-limiting factor. Anthraquinone biosynthesis in Morinda is strongly inhibited by 2,4-D, but much less by NAA. Both auxins inhibit the activity of isochorismate synthase proportionally to the concomitant reduction in the amount of anthraquinone accumulated. However, the correlation between enzyme activity and rate of biosynthesis is less clear when the activity of the enzyme is very high. In this case, a limiting concentration of precursor may determine the extent of anthraquinone accumulation. Partial inhibition of chorismate biosynthesis by glyphosate leads to less anthraquinone accumulation, but also to a reduction in ICS activity. The complexity of the interference of glyphosate with anthraquinone biosynthesis is illustrated by the effect of the inhibitor in cell cultures of the related species Rubia tinctorum L. in these cells, glyphosate leads to an increase in anthraquinone content and a concomitant rise in ICS activity. All data indicate that the main point of regulation in anthraquinone biosynthesis is located at the entrance of the specific secondary route.     

Relation between primary and secondary metabolism in plant cell suspensions

Cell suspensions ofMorinda citrifolia are able to produce large amounts of anthraquinones (AQ) when they are cultivated on a B5-medium containing 1 mg 1^-1 naphtyl acetic acid (NAA); this production is inhibited by addition of 2,4-dichloro-phenoxyacetic acid (2,4-d). Also during cultivation on 1 mg 1^-1 2,4-d AQ-production is absent.It appeared that in the presence of NAA a kind of AQ-production program is switched on: cell division rate is low as well as metabolic activity, while endogenous sugar levels are high. The same properties develop in the presence of auxins like indolyl-acetic acid and p-chloro-phenylacetic acid. With 2,4-d and related auxins (like p-chloro-phenoxyacetic acid) AQ production is absent and emphasis is laid on a developmental program characterized by high cell division rates, high metabolic activity and low endogenous sugar contents. Independent of the type of auxin applied, the cells grow as a suspension consisting of finely dispersed cells. The AQ-producing differentiation program cannot be maintained during a consecutive series of subculturings: with increasing AQ-contents the viability of the cells and the cell division rate decrease.The possible mechanisms of regulation of AQ-production by auxins are discussed as well as the advantages of the use of theMorinda model system in the study of the relation between growth, primary and secondary metabolism.Abbreviations AQ anthraquinones - 2,4-d 2,4-dichloro-phenoxylacetic acid - DW dry weight - EFW extractive free weight - FW fresh weight - IAA indolyl acetic acid - NAA naphtyl acetic acid - pCP p-chloro-phenylacetic acid - pCPO p-chloro-phenoxy-acetic acid     

Uncharacteristic alkaloid synthesis by suspension cultures of Cinchona pubescens fed with L-tryptophan

The growth and alkaloid production of a liquid suspension culture of Cinchona pubescens has been studied, particularly with attention to the effect on the alkaloid spectrum of feeding cultures with L-tryptophan. This treatment did not enhance the production of any of the known alkaloids of Cinchona. Above 2mmM, however, the presence of the amino acid was toxic, causing extreme acidification of the medium and cell death. Under these conditions a number of indole and quinoline derivatives accumulated. The principal component of the alkaloid fraction proved to be norharman; indole-3-aldehyde was also isolated. Both these products probably occur by uncharacteristic metabolism of L-tryptophan. Furthermore, evidence for the degradation of endogenous alkaloids was obtained, as 4-hydroxymethylquinoline was also isolated. None of the known quinoline alkaloids of Cinchona, which were present in untreated cells, could be detected after L-tryptophan treatment, even when large amounts of culture were analysed. It is concluded that, in this instance, Cinchona alkaloid production cannot be improved by feeding with a precursor.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid - BA benzyladenine     

The stimulation of anthraquinone production by Cinchona ledgeriana cultures with polymeric adsorbents

Summary Anthraquinones produced by suspension cultures of Cinchona ledgeriana are released into the medium, which becomes saturated with products late in the growth cycle. When a high affinity polymeric adsorbent, such as the macro-reticular Amberlite XAD-7, is added to the culture the concentration of anthraquinone in the medium is maintained at a low level and production may be stimulated 15-fold, yielding up to 20 mg/1/day. More than 90% of the product is released from the cells. For maximal yields it is shown that both the amount of adsorbent used and the time after sub-culture at which it is added to the system are critical. The value of such a method for product recovery from immobilised cells is discussed.     

QSAR and docking studies of anthraquinone derivatives by similarity cluster prediction

Forty anthraquinone derivatives have been downloaded from PubChem database and investigated in a quantitative structure-activity relationships (QSAR) study. The models describing log P and LD50 of this set were built up on the hypermolecule scheme that mimics the investigated receptor space; the models were validated by the leave-one-out procedure, in the external test set and in a new version of prediction by using similarity clusters. Molecular docking approach using Lamarckian Genetic Algorithm was made on this class of anthraquinones with respect to 3Q3B receptor. The best scored molecules in the docking assay were used as leaders in the similarity clustering procedure. It is demonstrated that the LD50 data of this set of anthraquinones are related to the binding energies of anthraquinone ligands to the 3Q3B receptor.     

Anthraquinones in callus cultures of Cinchona ledgeriana

From callus cultures of Cinchona ledgeriana seven known anthraquinones, purpurin, anthragallol-1,2-dimethylether, anthragallol-1,3-dimethylether, rubiadin, 1-hydroxy-2-hydroxymethylanthraquinone, 1-hydroxy-2-methylanthraquinone and morindone-5-methylether (or 1,7-dihydroxy-8-methoxy-2-methylanthraquinone), and eight new anthraquinones, 5,6-dimethoxy-1-(or -4-)hydroxy-2-(or -3-)hydroxymethylanthraquinone, 5-methoxy-2-(or -3-)methyl-1,4,6-trihydroxyanthraquinone, 2-hydroxy-1,3,4-trimethoxyanthraquinone, 4-methoxy-1,3,5-trihydroxyanthraquinone, 1,4-dimethoxy-2,3-methylenedioxyanthraquinone, 1,3-dihydroxy-4-methoxyanthraquinone, 1,3-dihydroxy-2,5-dimethoxyanthraquinone and 2,5-(or 3,5-)dihydroxy-1,3,4-(or -1,2,4-)trimethoxyanthraquinone have been isolated.     

Simultaneous determination of naphthalene and anthraquinone derivatives in Rumex nepalensis Spreng. roots by HPLC: comparison of different extraction methods and validation

Introduction – Rumex nepalensis contains mainly anthraquinone and naphthalene derivatives. Although HPLC methods have been reported for the analysis of anthraquinones, neither a phytochemical analysis of Rumex species nor the simultaneous determination of anthraquinone and naphthalene derivatives in other samples has been reported so far. Objective – To develop and validate a HPLC method for the simultaneous determination of anthraquinone and naphthalene derivatives in R. nepalensis roots. Methodology – Anthraquinones and naphthalenes were extracted from R. nepalensis roots by three methods (reflux, ultrasonication and pressurized liquid extraction) using methanol. Separation was achieved on an RP C₁₈ column with a gradient mobile phase consisting of 0.05% orthophosphoric acid in water (solvent A) and methanol (solvent B) using a UV detector (254 nm). Results – Small differences were observed in the contents of anthraquinone and naphthalene derivatives extracted by the three methods. Chrysophanol‐8‐O‐β‐D‐glucopyranoside and nepodin were detected as major constituents. The method showed a good linearity (r² > 0.9992), high precision (RSD < 5%) and a good recovery (97–105%) of the compounds. The lowest detection limit was found to be 0.97 ng and the method was found to be robust. Conclusion – Reflux and ultrasonication were found to be the best suited methods for the extraction of glycosides and aglycones, respectively. The developed and validated HPLC method is simple, precise and accurate; and can hence be recommended as the method of choice for the analysis of anthraquinones and naphthalenes in R. nepalensis and other Rumex species for both quality control as well as routine analytical purposes. Copyright © 2010 John Wiley & Sons, Ltd.     

Capoamycin,a New Isotetracenone Antibiotic

A new antibiotic was obtained from the cultured broth of an actinomycete identified as Streptomyces capoamus, and has been named capoamycin. The structure of capoamycin was elucidated by NMR spectral analysis and chemical degradation, which revealed that capoamycin was composed of a modified benz[a]anthraquinone chromophore, a β-C-olivoside and (E,E)-2,4- decadienoic acid. Capoamycin inhibited the growth of Gram-positive bacteria, yeasts and fungi, induced differentiation of mouse myeloid leukemia cells (M1) and prolonged the survival periods of mice bearing Ehrlich ascites carcinoma.     

Anthrasesamones from roots of Sesamum indicum

Three anthraquinones, named anthrasesamones A, B and C, were isolated from the roots of Sesamum indicum, and their respective structures were determined to be 1-hydroxy-2-(4-methylpent-3-enyl)anthraquinone, 1,4-dihydroxy-2-(4-methylpent-3-enyl)anthraquinone and 2-chloro-1,4-dihydroxy-3-(4-methylpent-3-enyl)anthraquinone on the basis of spectroscopic evidence. Two known anthraquinones were also isolated for the first time from S. indicum roots and characterized as 2-(4-methylpent-3-enyl)anthraquinone and (E)-2-(4-methylpenta-1,3-dienyl)anthraquinone. Anthrasesamone C is a rare chlorinated anthraquinone in higher plants.