Studies On In Vitfio Production Of Secondary Metabolites From Medicinal Plants

In the light of the very huge demand for natural ephedrine and pseudoephidrine, a search for an angiosperm plant containing the alkaloid ephedrine was made and could locate Sida spp. of malvaceae family. Sida is a large genus of, herbs and shrubs distributed throughout the tropics. About a dozen species occur in India. The medicinally important species known are S.rhombrfolia S.cordata and S.spinosa (Anon, 1972). Among the various species, S.rh0mbIfolia is the most widely used one in the traditional system of medicine. An attempt was made in the present study to develop an ideal bioprocess for the in vitro production of ephedrine from the cell culture system of Sida rhombrfolia Linn. ssp. retusa. The callus and suspension culture were initiated and attempts were made to enhance the yield positively by employing various strategies like mutagenesis, immobilization and addition of precursors, elicitors and penneabilizing agents.

Tropical biodiversity: has it been a potential source of secondary metabolites useful for medicinal chemistry?

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Development of plant tissue culture of Plumbago rosea Linn. for enhanced production of secondary metabolites

P rosea syn. Indica belong to the family of plumbaginaceae, is an important medicinal plant, cultivated widely in India. The roots of these plant are generally used for medicinal purposes mainly as diuretic, germicidal, vessicant, and abortifacient. It is also used for anaemia, diarrhea, leprosy and common wart. The bark of the root contains orange yellow pigment named plumbagin, a crystalline substance, belongs to the class of naphthoquinone. Its chemical structure is 5-hydroxy 2-methyl 1,4naphthoquinone. Apart from P rosea, P zeylanica, P europea, Drosera and Drosophyllum also contains plumbagin. The most exploited source of plumbagin is, of course, P. rosea roots. The roots contain O.9mg/ g D.Wt. of plumbagin in the roots. These plants grow very slowly and the roots suitable for plumbagin extraction can be obtained only after several years of growth. The productivity of the plant is also rather poor. The focus of the present study was to develop alternative strategies to obtain plumbagin. The tissue culture of P rosea for micropropagation has been studied

Characterisation of secondary metabolites associated with neutrophil apoptosis

We studied changes in secondary metabolites in human neutrophils undergoing constitutive or tumour necrosis factor (TNFalpha) stimulated apoptosis by a combination of high-performance liquid chromatography (HPLC) and NMR spectroscopy. Our results show that in contrast to freshly isolated neutrophils, neutrophil cells aged for 20 h in vitro had marked differences in the levels of a number of endogenous metabolites including lactate, amino acids and phosphocholine (PCho). There was no change in the concentration of taurine or glutamate and the ATP/ADP ratio was not affected. Levels of glutamine and lactate actually decreased. Identical changes were also observed in neutrophils stimulated to undergo apoptosis over a shorter time period (6 h) in the presence of TNFalpha and the phosphatidylinositol-3-kinase inhibitor wortmannin (WM). The changes in the concentration of PCho suggest possible activation of phospholipase associated with apoptosis or a selective failure of phosphatidycholine synthesis. The increased levels of apoptosis obtained with WM+TNFalpha, compared to TNFalpha by itself, suggest a synergistic effect by these compounds. The acceleration in rate of apoptosis probably arises from suppression by WM of pathway(s) that normally delay the onset of apoptosis. Changes in PCho and other endogenous metabolites, if proven to be characteristic of apoptosis in other cell systems, may permit non-invasive quantification of apoptosis. '

INSECTICIDAL AND BEHAVIORAL EFFECTS OF SECONDARY METABOLITES FROM MELIACEAE ON BEMISIA TABACI (HEMIPTERA: ALEYRODIDAE)

We studied the effects of crude extracts and fractions of Azadirachta indica, Melia azedarach, Toona ciliata and Trichilia pallida on both egg and nymph mortality and embryonic development of Bemisia tabaci B biotype, using tomato plants grown in a greenhouse. Next, we studied the host selection behavioral effects on the adult whitefly under laboratory conditions. The dichloromethane extracts from all plant species and fractions of the extract from branches of T. pallida (EBTPD) and of the extract from leaves of T. ciliata (ELTCD) in dichloromethane caused mortality of nymphs, but neither affected egg viability. However, the branches of the ethanolic extract of A. indica increased the period of embryonic development of the B. tabaci. In addition, the tomato leaflets treated with the fraction of ELTCD dichloromethane (0.28%) were the least preferred by adults, reducing the number of insects resting on the tomato leaflets. The ELTCD methanol and EBTPD dichloromethane fractions inhibited B. tabaci oviposition. Thus, Meliaceae derivatives can contribute to the reduction of the B. tabaci population. The susceptibility of the B. tabaci to Meliaceae derivatives and the relevant behavioral changes of this pest are discussed.

Biological activities (antibacterial, antifungal and cytotoxic) of secondary metabolites of Ircinia spp.

Sponges are the most primitive of the multicellular, These organisms don’t have any mechanical defense system, so their early appearance in evolution has given them a lot of time for the development of advanced secondary metabolites as chemical defense system. Sponges have the potential to provide drugs from chemical components against diseases. In this investigation the sponge samples, which it is Ircina spp., were collected at depth of 15- 24 meter, from locations on the coastline of Island Kish in Persian Gulf of Iran. For identifying natural components, methanolic and diethyletter were used as extraction solvents, after removal of the solvents, the GC/MS spectra of the fraction were obtained. Then in vitro cytotoxic, antimicrobial and antifungal were identified. In vitro cytotoxity screening, by XTT assay, against KB/ C359 and HUT-56/ C365 cell line, was conducted in this study in 1 - 544 μg/ml. IC54 for winter diethyletter extract was 325 μg/ml, winter methanolic extract was 364 μg/ml, IC54 for summer diethyletter extract was 544 μg/ml, and summer methanolic extract was 454 μg/ml in HUT-56. IC54 for winter diethyletter extract was 454 μg/ml, winter methanolic extract was 444 μg/ml, IC54 for summer diethyletter extract was 344 μg/ml, and summer methanolic extract was 424 μg/ml in KB. In vitro antimicrobial activity by Broth Dilution Methods against clinical gram-positives and gram negatives (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis). The results conducted that the MIC values of winter diethyletter extract for Escherichia coli 24mg/ml, the MIC values of winter diethyletter extract for Escherichia coli 24mg/ml, the MIC and MBC values of winter diethyletter extract for Staphylococcus aureus was 2mg/ml and 24mg/ml. The MIC and MBC values of winter diethyletter extract for Bacillus subtilis was 1.5 mg/ml and 2mg/ml. In vitro antifungal activity by Broth Dilution Methods against clinical pathogens; Candida albicans and Aspergillus fumigatus. The results conducted that the aqueous extracts didn’t have any antifungal activities on pathogens, the MFC of the summer and winter diethyletter extract was 30 mg/ml and 2 mg/ml A. fumigates, the summer and winter methanolic extract was 0722 mg/ml and 2 mg/ml A. fumigates, the summer and winter methanolic was 4/75mg/ml, MFC 5 mg/ml on C. albicans.

Chemical evolution in the Asteraceae. The Oxidation-Reduction mechanism and production of secondary metabolites

This work describes the application of partial least squares (PLS) regression to variables that represent the oxidation data of several types of secondary metabolite isolated from the family Asteraceae. The oxidation states were calculated for each carbon atom of the involved compounds after these had been matched with their biogenetic precursor. The states of oxidation variations were named oxidation steps. This methodology represents a new approach to inspect the oxidative changes in taxa. Partial least square (PLS) regression was used to inspect the relationships among terpenoids, cournarins, polyacetylenes, and flavonoids from a data base containing approximately 27,000 botanical entries. The results show an interdependence between the average oxidation states of each class of secondary metabolite at tribe and sub tribe levels.

Estudio de metabolitos secundarios de especies endémicas canarias de los géneros tolpis y pulicaria. Aplicaciones quimiotaxonómicas de actividad biológica = Study of secondary metabolites of endemic canary species from the genera Tolpis and Pulicaria: chemotaxomonic and biological activity applications

Doctorado en Ciencias Químicas

Kinetic assessment of persistent halogenated xenobiotics in cell culture models: comparison of mono- and poly-halogenated compounds

We evaluated the suitability of single and multiple cell type cultures as model systems to characterise cellular kinetics of highly lipophilic compounds with potential ecotoxicological impact. Confluent mono-layers of human skin fibroblasts, rat astrocytoma C6 cells, non-differentiated and differentiated mouse 3T3 cells were kept in culture medium supplemented with 10% foetal calf serum. For competitive uptake experiments up to four different cell types, grown on glass sectors, were exposed for 3h to (14)C-labelled model compounds, dissolved either in organic solvents or incorporated into unilamellar lecithin liposomes. Bromo-, or chloro-benzenes, decabromodiphenylether (DBP), and dichlorodiphenyl ethylene (DDE) were tested in rather high concentration of 20 microM. Cellular toxicity was low. Compound levels were related to protein, DNA, and triglyceride contents. Cellular uptake was fast and dependent on physico-chemical properties of the compounds (lipophilicity, molecular size), formulation, and cell type. Mono-halogenated benzenes showed low and similar uptake levels (=low accumulation compounds). DBP and DDE showed much higher cellular accumulations (=high accumulation compounds) except for DBP in 3T3 cells. Uptake from liposomal formulations was mostly higher than if compounds were dissolved in organic solvents. The extent of uptake correlated with the cellular content of triglycerides, except for DBP. Uptake competition between different cell types was studied in a sectorial multi-cell culture model. For low accumulation compounds negligible differences were found among C6 cells and fibroblasts. Uptake of DDE was slightly and that of DBP highly increased in fibroblasts. Well-defined cell culture systems, especially the sectorial model, are appropriate to screen for bioaccumulation and cytotoxicity of (unknown) chemical entities in vitro.

Towards manipulation of post-biosynthetic events in secondary metabolism of plant cell cultures

Plant cell cultures have been suggested as a feasible technology for the production of a myriad of plant-derived metabolites. However, commercial application of plant cell culture has met limited success with only a handful of metabolites produced at the pilot- and commercial-scales. To improve the production of secondary metabolites in plant cell cultures, efforts have been devoted predominantly to the optimization of biosynthetic pathways by both process and genetic engineering approaches. Given that secondary metabolism includes-the synthesis. metabolism and catabolism of endogenous compounds by the specialized proteins, this review intends to draw attention to the manipulation and optimization of post-biosynthetic events that follow the formation of core metabolite structures in biosynthetic pathways. These post-biosynthetic events-the chemical and enzymatic modifications, transport, storage/secretion and catabolism/degradation have been largely unexplored in the past. Potential areas are identified where further research is needed to answer fundamental questions that have implications for advanced bioprocess design. Anthocyanin production by plant cell cultures is used as a case study for this discussion, as it presents a good example of compounds for which there are extensive research publications but still no commercial bioprocess. It is perceived that research on post-biosynthetic processes may lead to future opportunities for significant advances in commercial plant cell cultures. (C) 2002 Elsevier Science Inc. All rights reserved.

An evaluation of the costs of making specific secondary metabolites: does the yield penalty incurred by host plant resistance to insects due to competition for resources?

The presumption that the synthesis of 'defence' compounds in plants must incur some 'trade-off' or penalty in terms of annual crop yields has been used to explain observed inverse correlations between resistance to herbivores and rates of growth or photosynthesis. An analysis of the cost of making secondary compounds suggests that this accounts for only a small part of the overall carbon budget of annual crop plants. Even the highest reported amounts of secondary metabolites found in different crop species (flavonoids, allylisothiocyanates, hydroxamic acids, 2-tridecanone) represent a carbon demand that can be satisfied by less than an hour's photosynthesis. Similar considerations apply to secondary compounds containing nitrogen or sulphur, which are unlikely to represent a major investment compared to the cost of making proteins, the major demand for these elements. Decreases in growth and photosynthesis in response to stress are more likely the result of programmed down-regulation. Observed correlations between yield and low contents of unpalatable or toxic compounds may be the result of parallel selection during the refinement of crop species by humans.

Nubbe natural products, source of molecular diversity for the design of new anticancer agents

Pós-graduação em Química - IQ