Roseus Roots Research Articles

Catharanthus roseus (L.) G. Don counteracts the ampicillin resistance in multiple antibiotic-resistant Staphylococcus aureus by downregulation of PBP2a synthesis.

It is essential to revisit the global biodiversity, search for ethnopharmacologically relevant plants, and unveil their untapped potential to overcome the complications associated while treating infections triggered by multiple antibiotic-resistant Staphylococcus aureus. Catharanthus roseus (L.) G. Don of the Apocynaceae family is a medicinal plant used for remedial purposes against infectious diseases from ancient times. In this study, we intended to evaluate the mechanism by which the ethanolic extract of C. roseus root (EECRR) causes the reversal of ampicillin resistance in S. aureus. To achieve this goal, we have stained EECRR-treated S. aureus with acridine orange, analysed DNA damage by comet assay, and studied the alteration of plasmid band pattern and expression of penicillin-binding protein 2a (PBP2a) protein. Experiments revealed better S. aureus killing efficiency of EECRR at its minimum inhibitory concentration (MIC) doses due to DNA damage and reducing plasmid band intensities along with a decline in the expression of PBP2a in EECRR-treated cells at half-MIC dose. EECRR proved to be an efficient growth inhibitor of S. aureus that reduces the expression of PBP2a. Therefore, EECRR can also render ampicillin-resistant S. aureus susceptible to the antibiotic.

A Comparison of Antibacterial Effects of Catharanthus roseus and Camellia sinensis (Black Tea) and Their Synergistic Effect along with Antibiotic against Multiple Antibiotic Resistant Strains of Staphylococcus aureus

ABSTRACT Community associated strains of Staphylococcus aureus were isolated and treated with extracts of Catharanthus roseus and Camellia sinensis (black tea) to examine their antibacterial potential, either alone or in combination with an antibiotic, ampicillin. Results of MIC values (Minimum Inhibitory Concentration), growth inhibitory patterns, SEM images of the clinical and standard strains in the presence of their ethanolic extracts indicated that C. roseus root had greater antibacterial potential than black tea. The FIC (Fractional Inhibitory Concentration) values suggested that the combination of C. roseus root extract and ampicillin had a greater antibacterial activity due to a synergistic effect, than that of the combination of black tea and ampicillin, which exerted an additive effect.

Phytochemical, antioxidant, anti-proliferative and antimicrobial properties of Catharanthus roseus root extract, saponin-enriched and aqueous fractions.

Catharanthus roseus (L.) G. Don (C. roseus) is a well-known medicinal plant for its source of alkaloids solely found in the leaves. Other parts including the root are usually discarded after the alkaloid extraction. This study sought to investigate phytochemical profiles, antioxidant, antimicrobial and cytotoxic properties ofthe C. roseus root extract (RE) and its two sub-fractions including saponin-enriched (SE) and aqueous (AQ) fractions. The results showed that the RE was a rich source of saponins (1744.44mgESE/g) and phenolics (51.27mgGAE/g), which comprised of gallic acid (25.74mg/g), apigenin (1.45mg/g) and kaempferol (1.58mg/g). The SE fraction was enriched with 31% of saponins and 63% of phenolics higher than those ofthe RE; whereas the concentrations of saponins and phenolics of the AQ fraction were lower than those of theRE by 40% and 74%, respectively. The content of gallic acid in the SE fraction was 1.4-fold and 1.5-fold higher than those of the RE or AQ fraction, respectively. The SE fraction demonstrated potent antioxidant capacity, which was significantly higher than the RE or AQ fraction, and also exhibited strong anti-proliferative activity against 11 cancer cell lines including A2780 (ovarian), H460 (lung), A431 (skin), MIA PaCa-2 (pancreas), Du145 (prostate), HT29 (colon), MCF-7 (breast), BE2-C (neuroblastoma), SJ-G2, U87 and SMA (glioblastoma) with low GI50 values (≤ 2.00µg/mL). The SE fraction was also shown to effectively inhibit the growth of both bacteria (Escherichia coli, Enterobacter aerogenes and Staphylococccus lugdunensis) and fungi (Candida albicans and Aspergillus niger). These findings warrant further investigation to isolate major compounds from theSE fraction and further test their antioxidant, anticancer and antimicrobial activities.

Gene transcript profiles of the TIA biosynthetic pathway in response to ethylene and copper reveal their interactive role in modulating TIA biosynthesis in Catharanthus roseus.

Research on transcriptional regulation of terpenoid indole alkaloid (TIA) biosynthesis of the medicinal plant, Catharanthus roseus, has largely been focused on gene function and not clustering analysis of multiple genes at the transcript level. Here, more than ten key genes encoding key enzyme of alkaloid synthesis in TIA biosynthetic pathways were chosen to investigate the integrative responses to exogenous elicitor ethylene and copper (Cu) at both transcriptional and metabolic levels. The ethylene-induced gene transcripts in leaves and roots, respectively, were subjected to principal component analysis (PCA) and the results showed the overall expression of TIA pathway genes indicated as the Q value followed a standard normal distribution after ethylene treatments. Peak gene expression was at 15-30 μM of ethephon, and the pre-mature leaf had a higher Q value than the immature or mature leaf and root. Treatment with elicitor Cu found that Cu up-regulated overall TIA gene expression more in roots than in leaves. The combined effects of Cu and ethephon on TIA gene expression were stronger than their separate effects. It has been documented that TIA gene expression is tightly regulated by the transcriptional factor (TF) ethylene responsive factor (ERF) and mitogen-activated protein kinase (MAPK) cascade. The loading plot combination with correlation analysis for the genes of C. roseus showed that expression of the MPK gene correlated with strictosidine synthase (STR) and strictosidine b-D-glucosidase(SGD). In addition, ERF expression correlated with expression of secologanin synthase (SLS) and tryptophan decarboxylase (TDC), specifically in roots, whereas MPK and myelocytomatosis oncogene (MYC) correlated with STR and SGD genes. In conclusion, the ERF regulates the upstream pathway genes in response to heavy metal Cu mainly in C. roseus roots, while the MPK mainly participates in regulating the STR gene in response to ethylene in pre-mature leaf. Interestingly, the change in TIA accumulation does not correlate with expression of the associated genes. Our previous research found significant accumulation of vinblastine in response to high concentration of ethylene and Cu suggesting the involvement of posttranscriptional and posttranslational mechanisms in a spatial and temporal manner. In this study, meta-analysis reveals ERF and MPK form a positive feedback loop connecting two pathways actively involved in response of TIA pathway genes to ethylene and copper in C. roseus.

Association with arbuscular mycorrhizal fungi influences alkaloid synthesis and accumulation in Catharanthus roseus and Nicotiana tabacum plants

Frequently disregarded, plant associations with arbuscular mycorrhizal fungi (AMF) can influence plant specialized metabolism with important ecological and/or economic implications. In this study, we report on both the influence of mycorrhization on the content of a wide range of alkaloids and differential gene expression of some enzymes involved in alkaloid biosynthetic pathways in the leaves and roots of Catharanthus roseus and Nicotiana tabacum plants. These plants were divided into several treatments: mycorrhizal, inoculated with AMF; non-AMF inoculated plants; and non-AMF inoculated plants with an extra supply of phosphorus. The contents of vindoline, vinblastine, vincristine, catharanthine, ajmalicine and serpentine in C. roseus and of nicotine, anabasine and nornicotine in N. tabacum tobacco plants were determined. Mycorrhizal inoculation increased ajmalicine and serpentine contents in C. roseus roots suggesting that mycorrhization had a greater influence on the accumulation of alkaloids in roots than it did in shoots. The youngest leaves of mycorrhizal C. roseus plants showed lower transcript levels of the genes analysed; however, in older leaves, the expression levels were higher when compared with the leaves of non-mycorrhizal plants. In the case of tobacco, higher leaf to root ratios for nicotine and anabasine were found in plants with a mycorrhizal association. Our results showed that mycorrhization changed the alkaloid content and expression pattern of the genes analysed in both species; however, differences were found between the roots and shoots. In nature, such changes may have a direct influence on the interactions between plants and insects (herbivory) and pathogens. These interactions must be studied further to reveal the ecological influence mycorrhizae may have on chemical defences in a broader sense.

Pharmacological effects of Catharanthus roseus root alkaloids in acetylcholinesterase inhibition and cholinergic neurotransmission

The leaves of Catharanthus roseus constitute the only source of the well known indolomonoterpenic alkaloids vincristine and vinblastine. In this work we studied the biological potential of the roots, which are used in several countries as decocts or hot water extracts for the treatment of a number of conditions. The aqueous extract strongly inhibited acetylcholinesterase (AchE) in an in vitro microassay, an effect ascribable mainly to serpentine (IC(50) = 0.775 microM vs physostigmine IC(50) = 6.45 microM) as assessed with the pure compound. Pure alkaloids were tested for muscarinic and nicotinic antagonism using rat ex-vivo preparations, namely, ileum and diaphragm/phrenic-nerve, respectively. Serpentine competitively blocked muscarinic receptors with a pA(2) of 5.2, whereas the precursor ajmalicine up to 80 microM was undistinguishable from control, and catharanthine exhibited an unsurmountable muscarinic antagonism at greater than 10 microM concentrations. Nicotinic receptor mediated diaphragm contractions were fully inhibited by catharanthine (IC(50) = 59.6 microM) and ajmalicine (IC(50) = 72.3 microM), in a reversible but non-competitive manner, unlike the more potent nicotinic antagonist tubocurarine (IC(50) = 0.35 microM) whose competitive blockade was overcome by a physostigmine-induced increase in acetylcholine. Serpentine up to 100 microM did not change diaphragm contractions suggesting reduced affinity for neuromuscular nicotinic receptors. Despite strong in vitro AchE inhibition, serpentine failed to restore diaphragm contractions upon submaximal tubocurarine blockade, suggesting that poor tissue penetration may prevent serpentine from inhibiting AchE in deep neuromuscular synapses in the ex-vivo preparation. To our knowledge, the present study is the first to assess the effect of C. roseus root extracts, as well as of serpentine, ajmalicine and catharanthine on AchE. The results described herein suggest that the currently overlooked C. roseus roots may constitute a promising source of compounds with pharmaceutical interest. Moreover, given serpentine's potent in vitro AchE inhibitory activity and low cholinergic receptor affinity, it is conceivable that minor structural modifications may yield a potent and selective AchE inhibitor, potentially useful for the pharmacological management of conditions such as Alzheimer's disease and/or myasthenia gravis.

Alkaloid variations in Catharanthus roseus seedlings treated by different temperatures in short term and long term

A study was conducted to investigate the effects of high temperature on variations of alkaloid metabolism in C. roseus seedlings in Key Laboratory of Forest Plant Ecology, Northeast Forestry University, Heilongiang, China. 60-day-old C. roseus seedlings with 3–4 pairs of leaves were incubated in chambers with temperature of 30°C and 40°C for short-term heat shock experiment and 20°C, 25°C and 35°C for long-term experiment. The contents of vindoline, catharanthine, vinblastine and vincristine in C. roseus leaves and root were checked at different temperatures in short term (1–6 h) and long term (1–16 d). Results showed that under short-term heat shock, the contents of vindoline, catharanthine and vinblastine in leaves of the seedlings were higher at 40°C than at 30°C, but after 6 h treatment, the contents of vindoline and catharanthine under the two temperatures came to the same level. Catharanthine was exclusively distributed in C. roseus roots and its content was increased by 40% after two hours incubation at 40°C, while increased slowly at 30°C incubation and reached the highest value at 6 h. In the Long-term experiment, concentrations of monomeric alkaloids catharanthine and vindoline were higher at 20°C than at 25°C and had a sharp increase under the condition of 35°C. While for dimeric alkaloid, it showed that the higher the temperature, the earlier the peak value of vinblastine content appears. Vincristine had a continuous enhancement and attained 0.027 mg·g−1 at 16th day under 35°C condition which was higher than those in the other conditions. It was concluded that high temperature could promote the accumulation of different alkaloids in C. roseus and the accumulation characteristic is highly related to treatment time.

Responses of transformed Catharanthus roseus roots to femtomolar concentrations of salicylic acid

Catharanthus roseus transformed roots were cultured in the presence of salicylic acid (SA) at concentrations between 0.1 fM and 100 pM and the effect on root growth was evaluated. Significant morphological changes in the lateral roots were recorded on day two in the SA treatment. Presence of SA increased root cap size and caused the appearance of lateral roots closer to the root tip. The bioassay was sensitive enough to allow testing of low concentrations of other growth regulators that may affect root morphology and physiology.

Transcription factor Agamous-like 12 from Arabidopsis promotes tissue-like organization and alkaloid biosynthesis in Catharanthus roseus suspension cells

In Catharanthus roseus, monomeric terpenoid indole alkaloids (TIAs) are biosynthesized in specific tissues, particularly in roots, but failed to be produced by in vitro undifferentiated suspension cells. In this paper, we describe the impact of the root-specific MADS-box transcription factor Agamous-like 12 ( Agl12) from Arabidopsis thaliana on the differentiation of suspension cells from C. roseus. The expression of Agl12 is sufficient to promote an organization of suspension cells into globular parenchyma-like aggregates but is insufficient by itself to induce complete morphological root differentiation. Agl12 expression selectively increases the expression of genes encoding enzymes involved in the early biosynthesis steps of the terpenic precursor of alkaloids. The transgenic cell lines expressing Agl12 produced significant amounts of ajmalicine, an antihypertensive TIA that normally accumulates in C. roseus roots. The present paper indicates that transcription factors involved in tissue or organ differentiation may constitute new metabolic engineering tools that could help to design in vitro cultured cells able to produce specific valuable secondary metabolites.

Tryptophan Decarboxylase from Transformed Roots of Catharanthus roseus

Tryptophan decarboxylase (TDC, EC 4.1.1.28) from Catharanthus roseus hairy roots was purified 80-fold. Antibodies against TDC were obtained and they recognized only one protein of 55 kDa in crude extracts from hairy root cultures. Elicitation of transformed root cultures with macerozyme yielded a marked increase in TDC activity, which was accompanied by a similar increase in the amount of immunoreactive TDC protein. These results suggest that the alkaloid accumulation, produced by elicitation, requires the synthesis of new TDC polypeptide in C. roseus root cultures and establishes important differences in the regulatory control of this enzyme in root cultures compared to developing seedlings, where the posttranslational regulation apparently plays a major role.

Multiple forms of NADPH-cytochrome P450 oxidoreductases in the madagascar periwinkle Catharanthus roseus

NADPH-cytochrome P450 oxidoreductase (CPR, EC 1.6.2.4) is the redox partner of classical P450-monooxygenases, which have crucial roles in the metabolism of terpenes, alkaloids, flavonoids, phytoalexins, etc. It becomes evident that, contrary to animals and yeast, various CPR isoforms occur in some plants, although their specific physiological functions are largely unknown. C. roseus-CPR has been reported as encoded by a single gene and early papers concerning the C. roseus-CPR protein also reported a single CPR polypeptide. The observation of diverse CPRs during purification or by immunoblot were attributed to proteolytic degradation. We obtained CPR immunotype of C. roseus roots using two heterologous antisera directed against the CPR from Sorghum bicolor and Helianthus tuberosus, respectively. Both antisera developed the same immunogenic profile with two cross-reactive polypeptides. Further evaluation of anti-H. tuberosus CPR serum excluded non-specific binding of antiserum with C. roseus microsomal proteins. The two immuno-reactive polypeptides are probably not the result of proteolytic degradation, since increasing protease inhibitor concentration during the extraction and manipulation of the samples did not affect the occurrence of these two CPR forms. Roots from plants growing in the field showed identical CPR immunotypes with those seen in vitro, indicating that this immunoprofile actually belongs to C. roseus roots. The lectin concanavalin A was able to inhibit the CPR activity from C. roseus hairy roots; therefore, the immuno-reactive polypeptides probably result from post-translational glycosylation of the original polypeptide. Not only the roots, but also the flowers, leaves and the stem showed more than a single CPR form. The different tissues of the plant showed different immuno-reactive bands, which were reproducible even though they came from tissues of plants growing in the field. This opens the possibility of the occurrence of diverse tissue-specific CPRs.

A Transient Study of Formation of Conjugates during TNT Metabolism by Plant Tissues

ABSTRACT The formation of TNT-derived conjugates was investigated in hairy root tissue cultures of Catharanthus roseus and in aquatic plant systems of Myriophyllum aquaticum. The temporal profiles of four TNT-derived conjugates, TNT-1, 2A-1, TNT-2 and 4A-1, were determined over 3 to 16-day exposure durations. When axenic C. roseus roots were exposed separately to 2,4,6 trinitrotoluene, 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene, the array and levels of conjugates varied. Exposure of axenic roots to either 4-amino-2,6-dinitrotoluene or 2-amino-4,6-dinitrotoluene resulted in the formation of only 4A-1 and 2A-1, respectively, and not TNT-1 and TNT-2. However, amendment of previously unexposed roots with TNT produced all four conjugates. The conjugates were preferentially accumulated within the biomass phase of root cultures. Significantly, conjugates TNT-1 and TNT-2 were observed in the biomass phase of intact M. aquaticum plants exposed to TNT. The results clearly indicate the presence of common TNT transformation products in two diverse plants species and tissue type. The distribution of conjugates formed via monoamine derivatives of TNT, however, may be a function of several factors, including the starting xenobiotic type and/or level. Initial bulk rate constants for disappearance of 2,4,6 trinitrotoluene, 2-amino-4,6-dinitrotoluene, and 4-amino-2,6-dinitrotoluene were also determined. Their magnitude followed the order: TNT >> 4-A-2,6-DNT > 2-A-4,6-DNT.

Effect of the medium pH on the release of secondary metabolites from roots ofDatura stramonium, Catharanthus roseus, andTagetes patula cultured in vitro

The release of alkaloids from root culturesDatura stramonium andCatharanthus roseus and thiophenes from root cultures ofTagetes patula was found to increase when the pH of the culture media (ranging from 4.8 to 7.0) was reduced to 3.5. The extent of the effect was different in each type of culture. Increases ranged from 4- to 20-fold, which in some cases accounted for 75% of the total secondary metabolite pool produced per flask. When the release of individual metabolites was measured, even larger increases, were observed (nearly 400-fold for ajmalicine). Increased release of alkaloids fromC. roseus roots were also observed in cultures growing in a 14-L fermentor, when the medium pH was reduced. Reduction of the pH of the media did not affect growth of the root cultures in subsequent subcultures. The importance of this treatment as a stategy to improve the recovery of secondary metabolites from producing cultures is discussed.

Economic assessment of plant cell culture for the production of ajmalicine

Cost estimates have been prepared for commercial-scale production of ajmalicine-rich Catnaranthus roseus biomass using plant cell culture. At the current state of the technology the cost would be approximately $7.30/lb dry biomass ($3215/kg ajmalicine). Naturally-grown C. roseus roots have a 50% lower ajmalicine concentration but would cost only ca. $0.70/lb ($619/kg ajmalicine). The principal reason for the high cost of the plant cell culture route is not the slow specific growth rate (0.35 day(-1)), but rather the slow specific product accumulation rate (0.26 mg/g day). This rate will have to be increased by a factor of 40 to make the process competitive.