Biomedical subjects
Publications and source records attributed to R Verpoorte.
The aerial part of Leptadenia arborea has been shown to contain pinoresinol (1), syringaresinol (2), leucanthemitol (3) and E-ferulaldehyde (4). These known compounds are being reported for the first time from this plant. Among them, syringaresinol has shown an inhibitory effect against acetylcholinesterase. The IC(50) (the concentration of 50% enzyme inhibition) value of this compound was 200 microg/ml.
Studies on carrageenin-induced rat paw edema, yeast-induced hyperthermia in rat and writhing response induced by acetic acid in mice showed that the alcoholic extract of stems of Tabernaemontana pandacaqui (T. pandacaqui) has significant anti-inflammatory, antipyretic and antinociceptive activities. These activities are due to alkaloidal components since they were also observed when the crude alkaloidal (CA) fraction separated from alcoholic extract was tested in the same models.
Recent achievements have been made in the metabolic engineering of plant secondary metabolism. Various pathways have been altered using genes encoding biosynthetic enzymes or genes encoding regulatory proteins. In addition, antisense genes have been used to block competitive pathways, thereby increasing the flux towards the desired secondary metabolites.
Cell cultures of Mandevilla velutina have proved to be an interesting production system for biomass and secondary metabolites able to inhibit the hypotensive activity of bradykinin, a nonapeptide generated in plasma during tissue trauma. The crude ethyl acetate extract of cultured cells contains about 31- to 79-fold more potent anti-bradykinin compounds (e.g., velutinol A) than that obtained with equivalent extracts of tubers. Somaclonal variation may be an explanation for the wide range of inhibitor activity found in the cell cultures. The heterogeneity concerning morphology, differentiation, carbon dissimilation, and velutinol A production in M. velutina cell cultures is reported. Cell cultures showed an asynchronous growth and cells in distinct developmental stages. Meristematic cells were found as the major type, with several morphological variations. Cell aggregates consisting only of meristematic cells, differentiated cells containing specialized cell structures such as functional chloroplasts (cytodifferentiation) and cells with embryogenetic characteristics were observed. The time course for sucrose metabolism indicated cell populations with significant differences in growth and metabolic rates, with the highest biomass-producing cell line showing a cell cycle 60% shorter and a metabolic rate 33.6% higher than the control (F2 cell population). MALDI-TOF mass spectrometric analysis of velutinol A in selected cell lines demonstrated the existence of velutinol A producing and nonproducing somaclones. These results point to a high genetic heterogeneity in general and also in terms of secondary metabolite content.
Geraniol 10-hydroxylase (G10H) is a cytochrome P450 monooxygenase involved in the biosynthesis of iridoid monoterpenoids and several classes of monoterpenoid alkaloids found in a diverse range of plant species. Catharanthus roseus (Madagascar periwinkle) contains monoterpenoid indole alkaloids, several of which are pharmaceutically important. Vinblastine and vincristine, for example, find widespread use as anti-cancer drugs. G10H is thought to play a key regulatory role in terpenoid indole alkaloid biosynthesis. We purified G10H from C. roseus cells. Using degenerate PCR primers based on amino acid sequence information we cloned the corresponding cDNA. The encoded CYP76B6 protein has G10H activity when expressed in C. roseus and yeast cells. The stress hormone methyljasmonate strongly induced G10h gene expression coordinately with other terpenoid indole alkaloid biosynthesis genes in a C. roseus cell culture.
Salicylic acid and its putative biosynthetic precursors were assayed isocratically by RP-HPLC with UV detection at 280 nm. Optimum resolution was provided by an HPLC mobile phase consisting of MeOH-1% aqueous HOAc (40:60, v/v), at pH 4. Furthermore, for the analysis of 2,3-dihydroxybenzoic acid (2,3-DHBA) in Catharanthus roseus cell cultures after elicitation, a mobile phase consisting of acetonitrile-1% aqueous HCOOH containing 0.25% trichloroacetic acid (1:5, v/v), at pH 2, was used. The recovery for the free form of 2,3-DHBA was about 80% after a one-step extraction of the cells. The detection limit of 2,3-DHBA was 3 microg by using saligenin as an internal standard.
Thin-layer chromatography (TLC) was used to screen for acetylcholinesterase inhibitors from Amaryllidaceae extracts. The TLC plate was developed and then stained using Ellman's reagent, 5,5'-dithiobis-(2-nitrobenzoic acid), to detect acetylcholinesterase activity. The advantages of this TLC assay method were that we could dereplicate the known inhibitor galanthamine, widely occurring in Amaryllidaceae, at an early stage of the isolation procedure. Moreover, there is no disturbance from sample dissolving solvents as in the microplate assay, and it is a very simple method. The detection limits were 10-200 ng for several known acetylcholinesterase inhibitors tested, and it is thus more sensitive than UV or Dragendorff's reagent detection. Also the minimal detectable amount for an acetylcholinesterase inhibitor tested was much less than that needed for the microplate assay. We screened 15 Amaryllidaceae extracts using this TLC method, and chose candidates for acetylcholinesterase inhibitor isolation.
Sample preparation is still the most critical step in two-dimensional gel electrophoresis (2-DE), and needs to be optimized for each type of sample. To analyze the proteome of the medicinal plant Catharanthus roseus, we developed and evaluated a sequential solubilization procedure for the solubilization of proteins after precipitation in trichloroacetic acid and acetone. The procedure includes solubilization with a conventional urea buffer followed by a stronger solubilizing buffer containing thiourea. The sequential solubilization of the precipitated proteins results in very different spot patterns following 2-DE. The number of protein spots which could be detected in both samples of the sequential solubilization was only about 10% of the total number of spots. Compared to solubilization in a single step, the total number of spots that could be detected in the sequential solubilization procedure was increased by 52%. The method described is simple and is applicable to different types of plant tissue.
Plant secondary metabolism is the source of many natural products with diverse applications, including pharmaceuticals, food colors, dyes and fragrances. Functions in plants include attraction of pollinating insects and protection against pests and pathogens. An important regulatory step in secondary metabolism is transcription of the biosynthetic genes. The aim of this chapter is to discuss results and opportunities concerning modification of secondary metabolism using transcriptional regulators. The transcriptional regulation of two well-studied secondary pathways, the phenylpropanoid pathway and its flavonoid branch, and the terpenoid indole alkaloid biosynthetic pathway, are reviewed. Some examples of successful engineering of these pathways via transcriptional regulators are discussed.
A transgenic Saccharomyces cerevisiae was constructed containing the cDNAs coding for strictosidine synthase (STR) and strictosidine beta-glucosidase (SGD) from the medicinal plant Catharanthus roseus. Both enzymes are involved in the biosynthesis of terpenoid indole alkaloids. The yeast culture was found to express high levels of both enzymes. STR activity was found both inside the cells (13.2 nkatal/g fresh weight) and in the medium (up to 25 nkatal/l medium), whereas SGD activity was present only inside the yeast cells (2.5 mkatal/g fresh weight). Upon feeding of tryptamine and secologanin, this transgenic yeast culture produced high levels of strictosidine in the medium; levels up to 2 g/l were measured. Inside the yeast cells strictosidine was also detected, although in much lower amounts (0.2 mg/g cells). This was due to the low permeability of the cells towards the substrates, secologanin and tryptamine. However, the strictosidine present in the medium was completely hydrolyzed to cathenamine, after permeabilizing the yeast cells. Furthermore, transgenic S. cerevisiae was able to grow on an extract of Symphoricarpus albus berries serving as a source for secologanin and carbohydrates. Under these conditions, the addition of tryptamine was sufficient for the transgenic yeast culture to produce indole alkaloids. Our results show that transgenic yeast cultures are an interesting alternative for the production of plant alkaloids.
The terpenoid indole alkaloid content of Catharanthus pusillus was investigated during development from young to old plants. Different plant organs were assessed showing that the new leaves were the main repository site with vindoline ( approximately 4.8 mg/g DW) and catharanthine ( approximately 2.2 mg/g DW) being the major metabolites with the highest yields at the second and third sampling time (51 and 70 days, respectively). The other samples analysed, from old, oldest and yellow leaves followed in accumulation levels. The roots and stems were the least accumulative organs, although for the case of tubotaiwine the root was the most important organ. It appeared that the alkaloid content changed coinciding with the different developmental stages of the plants, particularly at flowering and fruiting stages. Moreover, this species seems to constitute a precious source of the monomerics, vindoline and catharanthine, intermediates in the synthesis of the two important antitumor dimerics vincristine and vinblastine, which did not accumulate in this species.
The ethanolic extract of Clerodendrum petasites was tested to evaluate the spasmolytic activity on isolated guinea-pig tracheal smooth muscle. The crude extract (2.25-9.0 mg/ml) dose-dependently caused relaxation of tracheal smooth muscle which was contracted by exposure to histamine. A bioassay-guided fractionation of the crude extract was performed by means of partitioning and centrifugal partition chromatography. Finally the active principle was isolated and identified as the flavonoid hispidulin (EC(50): (3.0+/-0.8)x10(-5) M). These results suggest that hispidulin may be beneficial in the treatment of asthma.
Jasmonic acid is an important plant stress signalling molecule. It induces the biosynthesis of defence proteins and protective secondary metabolites. In alkaloid metabolism, jasmonate acts by coordinate activation of the expression of multiple biosynthesis genes. In terpenoid indole alkaloid metabolism and primary precursor pathways, jasmonate induces gene expression and metabolism via ORCAs, which are members of the AP2/ERF-domain family of plant transcription factors. Other jasmonate-regulated (secondary) metabolic pathways might also be controlled by ORCA-like AP2/ERF-domain transcription factors. If so, such regulators could be used to improve plant fitness or metabolite productivity of plants or cell cultures.
Hypericum is a large genus comprising 200 species, wide spread on temperate region and tropical mountains. Several different species are available in Indian subcontinent. Psychopharmacological profiles of the two different Hypericum species e.g., H. hookerianum and H. patulum available in Nilgiris, India were investigated at two different doses (200 and 400 mg/kg, p.o.) in different animal models viz. Spontaneous motor activity (SMA) test in mice; Exploratory behaviour test by Head dip test in mice and Y-maze test in rats; Effects on pentobarbitone induced sleeping time in mice and study of the effects on body temperature in rats. All the extracts tested showed enhancement in spontaneous motor activity (SMA) in mice and exploratory behavior by head dip test in mice and Y-maze test in rats. The extracts reduce significantly the pentobarbitone induced sleeping time in mice. When tested for their effect on body temperature in rats, the extract of H. hookerianum showed significant reduction in yeast induced pyrexia with no effect on normal body temperature, while H. patulum showed no activity in this experimental model.
Mevalonate kinase was purified to homogeneity from Catharanthus roseus (L.) G. Don suspension-cultured cells. The purified enzyme had an M(r) of 104,600 and a subunit size of about 41,500. Kinetic studies indicated an ordered sequential mechanism of action, in which mevalonate was the first substrate to bind and ADP was the last product to leave the enzyme. True values for the kinetic constants were determined for mevalonate, with K(ma) = 76 microM and K(ia) = 74 microM, and for ATP, with K(mb) = 0.13 mM and K(ib) = 0. 13 mM; the true V(max) was calculated to be 138.7 nkat/mg of protein. Product inhibition was only detectable at rather high concentrations: above 0.7 mM for 5-phosphomevalonate and above 2 mM for ADP, with an ADP/ATP ratio of at least 1. Mevalonate kinase activity was shown to be strongly inhibited by farnesyl diphosphate. Farnesyl diphosphate acted as a competitive inhibitor toward ATP, with a K(i) value of 0.1 microM. Mevalonate kinase activity was dependent on the presence of divalent ions. At a concentration of 2 mM, Mg(2+) and Mn(2+) were best and equally effective in sustaining activity; compared to Mg(2+) and Mn(2+), relative activities of 35, 30, 16, 4.8, and 3.4% were detected at equimolar concentrations of Zn(2+), Fe(2+), Co(2+), Ca(2+), and Ni(2+), respectively. The pH-dependent activity profile of mevalonate kinase showed a broad pH optimum between pH 7 and 10, with a maximum at about pH 8.9.
Geranylgeranyl diphosphate (GGPP) synthase, an enzyme that regulates taxane biosynthesis, was purified to homogeneity from cell cultures of Taxus baccata. The molecular weight of the native protein was estimated to be 76+/-2 kDa, resulting from the association of two apparently identical subunits having a molecular weight of 38 kDa. Farnesyl diphosphate (K(m) 2.46 µM) in combination with isopentenyl diphosphate (K(m) 1.5 µM) was the most effective substrate. Dimethyl allyl diphosphate was a poorer substrate (K(m) 12.7 µM). Mn(2+) ion at 4 mM in combination with Mg(2+) of 2 mM gave the greatest stimulation of activity. The pI of the enzyme was lower than 4 and the pH optimum was between 6.9 and 7.2. The enzyme activity was found in the 20000xg (centrifugal force) pellet and a non-ionic detergent was used for its extraction. The inclusion of detergent was not necessary during subsequent chromatographic steps.
A high-performance liquid chromatography (HPLC) method with on-line coupled ultraviolet (UV), mass spectrometry (MS) and biochemical detection for acetylcholinesterase (AChE) inhibitory activity has been developed. By combining the separation power of HPLC, the high selectivity of biochemical detection, and the ability to provide molecular mass and structural information of MS, AChE inhibitors can be rapidly identified. The biochemical detection was based on a colorimetric method using Ellman's reagent. The detection limit of galanthamine, an AChE inhibitor, in the HPLC-biochemical detection is 0.3 nmol. The three detector lines used, i.e., UV, MS and Vis for the biochemical detection were recorded simultaneously and the delay times of the peaks obtained were found to be consistent. This on-line post-column detection technique can be used for the identification of AChE inhibitors in plant extracts and other complex mixtures such as combinatorial libraries.
Strictosidine beta-D-glucosidase (SGD) is an enzyme involved in the biosynthesis of terpenoid indole alkaloids (TIAs) by converting strictosidine to cathenamine. The biosynthetic pathway toward strictosidine is thought to be similar in all TIA-producing plants. Somewhere downstream of strictosidine formation, however, the biosynthesis diverges to give rise to the different TIAs found. SGD may play a role in creating this biosynthetic diversity. We have studied SGD at both the molecular and enzymatic levels. Based on the homology between different plant beta-glucosidases, degenerate polymerase chain reaction primers were designed and used to isolate a cDNA clone from a Catharanthus roseus cDNA library. A full-length clone gave rise to SGD activity when expressed in Saccharomyces cerevisiae. SGD shows approximately 60% homology at the amino acid level to other beta-glucosidases from plants and is encoded by a single-copy gene. Sgd expression is induced by methyl jasmonate with kinetics similar to those of two other genes acting prior to Sgd in TIA biosynthesis. These results show that coordinate induction of the biosynthetic genes forms at least part of the mechanism for the methyl jasmonate-induced increase in TIA production. Using a novel in vivo staining method, subcellular localization studies of SGD were performed. This showed that SGD is most likely associated with the endoplasmic reticulum, which is in accordance with the presence of a putative signal sequence, but in contrast to previous localization studies. This new insight in SGD localization has significant implications for our understanding of the complex intracellular trafficking of metabolic intermediates during TIA biosynthesis.