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.     

Enhancement of anthraquinone production in Morinda citrifolia cell suspension cultures after stimulation of the proline cycle with two proline analogs

Synthesis of anthraquinones (AQs) involves the shikimate and 2-C-methyl-D-erythritol 4-phosphate pathways. The proline cycle is linked to the pentose phosphate pathway (PPP) to generate NADPH needed in the first steps of this pathway. The effect of two proline analogs, azetidine-2-carboxylic acid (A2C) and thiazolidine-4-carboxylic acid (T4C), were evaluated in Morinda citrifolia suspension cultures. Both analogs gave higher proline accumulation after 6 and 10 days (68 and 179% after 6 days with A2C at 25 and 50 μM, respectively, and 111% with T4C added at 100 μM). Induction of the proline cycle increased the AQ content after 6 days (~40% for 50 μM A2C and 100 μM T4C). Whereas A2C (50 μM) increased only AQ production, T4C also enhanced total phenolics. However, no induction of the PPP was observed with any of the treatments. This pathway therefore does not limit the supply of carbon skeletons to secondary metabolic pathways.     

Synergistic effect of methyl jasmonate and cyclodextrins on anthraquinone accumulation in cell suspension cultures of Morinda citrifolia and Rubia tinctorum

Plant in vitro culture is a platform for producing secondary metabolites that combines safety, quality and low environmental impact. Besides, it is possible to increase the accumulation of these compounds by different strategies, such as elicitation. In this work, we analyzed the effects of the combination of methyl jasmonate (MeJ) and two cyclodextrins (CDs) on the production of anthraquinones (AQs) in cell cultures of Rubiaceae (Morinda citrifolia and Rubia tinctorum). These secondary metabolites have been traditionally used as dyes and have interesting therapeutic applications. The experiments were designed according to a full factorial design of two factors (MeJ and a CD) in two levels (0 and 0.1 mM for MeJ, and 0 and 20 mM of the CD). MeJ and (2-hydroxypropyl)-β-cyclodextrin (HPCD) synergistically increased intracellular AQ content in suspension cultures of R. tinctorum, and, to a lesser extent, in suspension cultures of M. citrifolia. Combination of MeJ with another CD, 2-methyl-β-cyclodextrin, led to a more intense and later increase in AQ content in cell cultures of R. tinctorum when compared to MeJ–HPCD treatment. However, the combination of CD and MeJ failed to induce a drastic AQ release to the culture media. Nevertheless, our results show that combination of strategies (using a CD and MeJ) was successful to increase secondary metabolite accumulation in suspension cultures. To our knowledge, this is the first report of synergistic effect of MeJ and CD on AQ accumulation in plant in vitro cultures.

     

Anthraquinones in cell suspension cultures of Morinda citrifolia

From cell suspension cultures of Morinda citrifolia five known anthraquinones, rubiadin, lucidin, morindone, lucidin-3-β-primeveroside and morindone-6-β-primeveroside, and seven new anthraquinones were isolated. Six of the seven new quinones were characterized as 2-methyl-3,5,6-trihydroxyanthraquinone, 3-hydroxymorindone, 5,6-dihydroxylucidin, 2-methyl-3,5,6-trihydroxyanthraquinone-6-β-primeveroside, 3-hydroxymorindone-6-β-primeveroside and 5,6-dihydroxylucidin-3-β-primeveroside, respectively.     

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     

海巴戟的组织培养及快速繁殖

1植物名称海巴戟（MorindacitrifoliaL．）。 2材料类别从美国加里福尼亚叶启恩先生处得到大溪种海巴戟种子,擦伤外壳后播种于砂盆中,可比未用粗砂擦伤外壳的提前4个月发芽（后者为6个月）,植株长出2片完全叶时,取茎段和顶芽作实验材料。     

Phytochemical composition and in vitro biological activities of Morinda citrifolia fruit juice

Morinda citrifolia is a plant with broad nutraceutical and therapeutic effects and used in the traditional treatment of several ailments. The objective of this work is to investigate the phytochemistry of the fruit juice of M. citrifolia on one hand and on other hand to evaluate its antiradical and antibacterial activity. The phytochemical investigation was carried out by tube staining tests of the extract of two types of fruit juice of M. citrifolia. The antioxidant activity of these juices was evaluated by reducing the DPPH radical method. Concerning the antibacterial activity, it was tested on the in vitro growth of 10 reference bacterial strains using the well diffusion method. Qualitative phytochemistry of M. citrifolia fruit juices revealed the presence of large groups of secondary metabolites including polyphenols, reducing compounds, mucilage and terpernoids. The antioxidant activity of M. citrifolia fruit juices is dose-dependent and higher than that of ascorbic acid. Antimicrobial activity on other hand revealed that fruit juices inhibit growth inhibitory activity of Staphylococcus aureus, Pseudomonas aeruginosa, Proteus mirabilis, S. epidermidis, Proteus vulgaris, Streptococcus oralis, Enterococcus faecalis and Escherichia coli. This observed difference is significant for each juices on the strains (p < 0.001). These results support the use of M. citrifolia in traditional medicine and are the starting points for the development of a new drug to combat both dietary conditions and chronic conditions associated with oxidative stress.     

海巴戟ACO基因启动子的克隆及功能初步验证

为了进一步明确海巴戟(Morinda citrifolia Linn.)ACO基因(GenBank登录号:ARB05681.1)功能,本研究通过染色体步移法克隆得到2 066 bp启动子.该区域除了含有大量TATA-box、CAAT-box等启动子核心元件外,还含有乙烯等植物激素响应元件、胁迫和防御相关元件、光相关元件...     

Generation of reactive oxygen and antioxidant species by hydrodynamically stressed suspensions of Morinda citrifolia

The generation of reactive oxygen species (ROS) by plant cell suspension cultures, in response to the imposition of both biotic and abiotic stress, is well-documented. This study investigated the generation of hydrogen peroxide by hydrodynamically stressed cultures of Morinda citrifolia, over a 5-h period post-stress imposition. Suspensions were exposed to repeated passages through a syringe, under laminar flow conditions, corresponding to cumulative energy dissipation levels of approximately 3-6 J kg-1. Extracellular hydrogen peroxide was detected using a luminol-based chemiluminescence assay. The addition of exogenous hydrogen peroxide facilitated the detection of low levels of hydrogen peroxide in the presence of antioxidants. Immediately after shear exposure, there was evidence of significant antioxidative capacity in the sheared cell cultures, which potentially masked any oxidative burst (OB), but which decreased over the following 40 min. This antioxidative capacity was determined to derive from the shearing process. Trials in which ground cellular debris was added to control suspensions suggested that some of the antioxidative capacity observed in stressed suspensions was directly associated with debris generated by the shearing process. Using UV-vis spectrophotometry and HPLC, stress-related increases in the levels of phenolic compounds were detected in suspension filtrates. Under the stress conditions investigated, maximum hydrogen peroxide levels of 11.5 muM were observed, 5 h after shear exposure. This study emphasizes the importance of considering both oxidative and antioxidative capacities as part of a holistic approach to the determination of the OB in hydrodynamically stressed plant cell suspension cultures.     

Interfacial inactivation of epoxide hydrolase in a two-liquid-phase system

Enantioselective epoxide hydrolases are useful biocatalysts for the preparation of enantiopure epoxides and diols. The kinetic resolution of racemic epoxides can be carried out in an organic/aqueous biphasic system to allow use of high epoxide concentrations. Enzyme inactivation in such a system, however, may occur by contact with the interface. In this study, we investigated the factors which influence the interfacial inactivation of Agrobacterium radiobacter epoxide hydrolase in an octane/water biphasic system. Rates of interfacial inactivation were measured both in a stirred-cell, which has a planar interface, and in an emulsion reactor. Interfacial inactivation rates measured in the stirred-cell at a fixed interfacial area increased with mixing intensity. Interfacial inactivation rates per unit area were lower in the emulsion reactor than in the stirred-cell and increased with bulk aqueous enzyme concentration. Circular dichroism measurements showed that during biphasic incubation all unadsorbed soluble enzyme existed in the native conformation. Activity assays showed that the dissolved enzyme was also fully active, indicating that inactivated enzyme precipitated from solution. Using an inactive epoxide hydrolase mutant structurally similar to the wild-type enzyme in order to avoid the conversion of the epoxide, it was found that high concentrations of epoxide in the organic phase increased the rate of interfacial inactivation.     

Determination and comparative analysis of major iridoids in different parts and cultivation sources of Morinda citrifolia

Introduction – Noni is a medicinal plant with a long history of use as a folk remedy in many tropical areas, and is attracting more attention worldwide. A comprehensive study on the major phytochemicals in different plant parts (fruit, leaf, seed, root and flower) and sources is of great value for fully understanding their diverse medicinal benefits. Objective – To quantitatively determine the major iridoid components in different parts of noni plants, and compare iridoids in noni fruits collected from different tropical areas worldwide. Methodology – The optimal chromatographic conditions were achieved on a C₁₈ column with gradient elution using 0.1% formic acid aqueous formic acid and acetonitrile at 235 nm. The selective HPLC method was validated for precision, linearity, limit of detection, limit of quantitation and accuracy. Results – Deacetylasperulosidic acid (DAA) was found to be the major iridoid in noni fruit. In order of predominance, DAA concentrations in different parts of the noni plant were dried noni fruit > fruit juice > seed > flower > leaf > root. The order of predominance for asperulosidic acid (AA) concentration was dried noni fruit > leaf > flower > root > fruit juice > seed. DAA and AA contents of methanolic extracts of noni fruits collected from different tropical regions were 13.8–42.9 and 0.7–8.9 mg/g, respectively, with French Polynesia containing the highest total iridoids and the Dominican Republic containing the lowest. Conclusion – Iridoids DAA and AA are found to be present in leaf, root, seed and flower of noni plants, and were identified as the major components in noni fruit. Given the great variation of iridoid contents in noni fruit grown in different tropical areas worldwide, geographical factors appear to have significant effects on fruit composition. The iridoids in noni fruit were stable at the temperatures used during pasteurisation and, therefore, may be useful marker compounds for identity and quality testing of commercial noni products. Copyright © 2010 John Wiley & Sons, Ltd.     

Production of biomass and bioactive compounds by adventitious root suspension cultures of Morinda citrifolia (L.) in a liquid-phase airlift balloon-type bioreactor

To improve root growth and production of bioactive compounds such as anthraquinones (AQ), phenolics, and flavonoids by adventitious root cultures of Morinda citrifolia, the effects of aeration rate, inoculum density, and Murashige and Skoog (MS) medium salt strengths were investigated using a balloon-type bubble bioreactor. The possible mechanisms underlying changes in activities of enzymic (superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase) and nonenzymic (vitamin E) antioxidants, phenylalanine ammonia lyase, and stress levels (accumulation of hydrogen peroxide and proline, peroxidation of lipids) were also studied. Low aeration rate (0.05 vvm [air volume/culture volume/min]) accelerated accumulation of root fresh weight and dry weight (DW). High aeration rates (0.1 to 0.3 vvm) stimulated accumulation of AQ, phenolics, and flavonoids and reduced root growth. Low inoculum densities (5 and 10 g l^–1) increased accumulation of those metabolites but inhibited root growth. Culture of adventitious roots with high concentrations of MS salts (1× and 1.5× MS) resulted in induction of oxidative stress that strongly inhibited root growth. Overall, an aeration rate of 0.05 vvm, 15 g l^–1 inoculum density, and half-strength (0.5×) MS medium were optimal for enhancing accumulation of root dry biomass (4.38 g l^–1), AQ (103.08 mg g^–1 DW), phenolics (54.81 mg g^–1 DW), and flavonoids (49.27 mg g^–1 DW).     

Morinda citrifolia mitigates rotenone-induced striatal neuronal loss in male Sprague-Dawley rats by preventing mitochondrial pathway of intrinsic apoptosis

Objectives: Parkinson disease (PD) is a neurodegenerative disorder affecting mainly the motor system, as a result of death of dopaminergic neurons in the substantia nigra pars compacta. The present scenario of research in PD is directed to identify novel molecules that can be administered individually or co-administered with L-Dopa to prevent the L-Dopa-Induced Dyskinesia (LID) like states that arise during chronic L-Dopa administration. Hence, in this study, we investigated whether Morinda citrifolia has therapeutic effects in rotenone-induced Parkinson’s disease (PD) with special reference to mitochondrial dysfunction mediated intrinsic apoptosis.

Methods: Male Sprague-Dawley rats were stereotaxically infused with rotenone (3?µg in both SNPc and VTA) and co-treated with the ethyl acetate extract of Morinda citrifolia and levodopa.

Results: The results revealed that rotenone-induced cell death was reduced by MCE treatment as measured by decline in the levels of pro-apoptotic proteins. Moreover, MCE treatment significantly augmented the levels of anti-apoptotic Bcl2 and blocks the release of cytochrome c, thereby alleviating the rotenone-induced dopaminergic neuronal loss, as evidenced by tyrosine hydroxylase (TH) immunostaining in the striatum.

Discussion: Taken together, the results suggest that Morinda citrifolia may be beneficial for the treatment of neurodegenerative diseases like PD.     

Entry into and release of solvents by Escherichia coli in an organic-aqueous two-liquid-phase system and substrate specificity of the AcrAB-TolC solvent-extruding pump

Growth of Escherichia coli is inhibited upon exposure to a large volume of a harmful solvent, and there is an inverse correlation between the degree of inhibition and the log P(OW) of the solvent, where P(OW) is the partition coefficient measured for the partition equilibrium established between the n-octanol and water phases. The AcrAB-TolC efflux pump system is involved in maintaining intrinsic solvent resistance. We inspected the solvent resistance of delta acrAB and/or delta tolC mutants in the presence of a large volume of solvent. Both mutants were hypersensitive to weakly harmful solvents, such as nonane (log P(OW) = 5.5). The delta tolC mutant was more sensitive to nonane than the delta acrAB mutant. The solvent entered the E. coli cells rapidly. Entry of solvents with a log P(OW) higher than 4.4 was retarded in the parent cells, and the intracellular levels of these solvents were maintained at low levels. The delta tolC mutant accumulated n-nonane or decane (log P(OW) = 6. 0) more abundantly than the parent or the delta acrAB mutant. The AcrAB-TolC complex likely extrudes solvents with a log P(OW) in the range of 3.4 to 6.0 through a first-order reaction. The most favorable substrates for the efflux system were considered to be octane, heptane, and n-hexane.     

Effects of juice from Morinda citrifolia (Noni) on gastric emptying in male rats

The effects of juice from Morinda citrifolia (noni) on gastric emptying, gastrointestinal transit, and plasma level of cholecystokinin (CCK) in rats were studied. Male rats were given noni by gavage at levels of 0.25, 1, or 4 ml/kg once per day for one or 7 days. The rats in the control group were given water, while the rats in the experimental group were fasted overnight before measurement of gastrointestinal motility. Gastrointestinal motility was assessed in rats 15 min after intragastric instillation of a test meal containing charcoal (10%) and Na251CrO4 (0.5 microCi/ml). Gastric emptying was determined by measuring the amount of radiolabeled chromium contained in the small intestine as a percentage of the initial amount received. Then, gastrointestinal transit was evaluated by calculating the geometric center of distribution of the radiolabeled marker. Finally, blood samples were collected for measurement of CCK by radioimmunoassay. The administration of noni at 0.25 ml/kg, but not at 1 ml/kg and 4 ml/kg, for 1 day significantly inhibited gastric emptying. In contrast, gastric emptying was significantly inhibited by oral noni (0.25, 1, or 4 ml/kg) for 7 days. Intraperitoneal injection of lorglumide (5 or 10 mg/kg), a selective CCK1 receptor antagonist, effectively attenuated the noni-induced inhibition of gastric emptying. The intestinal transit and body weight, food intake, water intake, urine volume as well as feces weight were not altered by the administration of noni either acutely or chronically, but the administration of oral noni (1 ml/kg) for 7 days increased the level of plasma CCK in male rats. These results suggest that oral noni inhibits gastric emptying in male rats via a mechanism involving stimulation of CCK secretion and CCK1 receptor activation.     

Evaluation of noni (Morinda citrifolia L.) juice antiproliferative activity using the Helianthus tuberosus classical in vitro growth model system

Morinda citrifolia L. (noni) is a tropical plant that has been used for centuries in traditional medicine for numerous purposes and, nowadays, is largely commercialised on the worldwide market as a dietary supplement. Long-term in vitro cultures of Helianthus tuberosus dormant parenchyma explants constitute a classical growth model that can be used to evaluate the proliferative or antiproliferative and/or cytotoxic effects of different mixtures of chemicals present in food or with putative pharmacological applications. Explants of dormant tubers were cultured in vitro with 2%, 10% and 20% (v/v) of noni fruit juice. After four weeks of culture, explant cell growth was reduced 40.5%, 72.5% and 73.9%, respectively, by 2%, 10% and 20% (v/v) of noni juice in comparison to untreated controls. Our results demonstrated that noni fruit juice possesses strong antiproliferative capacity, low cytotoxicity and moderate antioxidant activity.     

库克Noni果汁多糖含量及分子量测定

建立测定库克Noni果汁中多糖含量及分子量的方法。通过醇沉法分离Noni果汁多糖,进一步分离纯化得到均一多糖组分;用精制Noni果汁多糖测得该多糖对葡萄糖的换算因子,对多糖含量进行定量测定;并通过SE-HPLC(Size Exclusion-High Performance Liquid Chromatography)法测定了该多糖的相对分子量。结果表明,多糖含量测定方法简便可行,供试液在4 h内显色稳定,重现性较好,平均回收率为99.3%,RSD为1.93%(n=3);测得该多糖的相对分子量为1140 KDa。     

Antiviral activity of extracts from Morinda citrifolia leaves and chlorophyll catabolites,pheophorbide a and pyropheophorbide a,against hepatitis C virus

The development of complementary and/or alternative drugs for treatment of hepatitis C virus (HCV) infection is still needed. Antiviral compounds in medicinal plants are potentially good targets to study. Morinda citrifolia is a common plant distributed widely in Indo‐Pacific region; its fruits and leaves are food sources and are also used as a treatment in traditional medicine. In this study, using a HCV cell culture system, it was demonstrated that a methanol extract, its n‐hexane, and ethyl acetate fractions from M. citrifolia leaves possess anti‐HCV activities with 50%‐inhibitory concentrations (IC₅₀) of 20.6, 6.1, and 6.6 μg/mL, respectively. Bioactivity‐guided purification and structural analysis led to isolation and identification of pheophorbide a, the major catabolite of chlorophyll a, as an anti‐HCV compound present in the extracts (IC₅₀ = 0.3 μg/mL). It was also found that pyropheophorbide a possesses anti‐HCV activity (IC₅₀ = 0.2 μg/mL). The 50%‐cytotoxic concentrations (CC₅₀) of pheophorbide a and pyropheophorbide a were 10.0 and 7.2 μg/mL, respectively, their selectivity indexes being 33 and 36, respectively. On the other hand, chlorophyll a, sodium copper chlorophyllin, and pheophytin a barely, or only marginally, exhibited anti‐HCV activities. Time‐of‐addition analysis revealed that pheophorbide a and pyropheophorbide a act at both entry and the post‐entry steps. The present results suggest that pheophorbide a and its related compounds would be good candidates for seed compounds for developing antivirals against HCV.     

Steroid conversions by Flavobacterium dehydrogenans in two-liquid-phase systems

The production of 4-androstene-3, 17-dione by Flavobacterium dehydrogenans from androstenolone-acetate in octane/culture (v/v = 1:1) two-liquid phase systems was compared with the conversion rate in a Tween-containing medium commonly used in industry, and in a culture where no organic solvent or detergent was added. Variation of the cell density of the cultures at the moment of steroid, Tween, and/or organic solvent addition showed that the highest conversion rate in the three systems was reached with cells which were in the late exponential growth stage. The rate of 4-androstene-3, 17-dione production at the optimal cell density in the two-liquid-phase system and Tween medium were ca. 6 and 1(1/2) times as high, respectively, as in the aqueous medium without addition. The beneficial effect of octane on the rate of steroid conversion during growth could be explained by viability changes as measured by a new method to determine viability. Furthermore, our results show that a very high degree of conversion can be reached in the two-liquid-phase system [more than 98% (w/w)] which cannot be obtained in an aqueous medium. An easy way to recover the product in high purity is proposed.