mRNA transcriptome profiling of human hepatocellular carcinoma cells HepG2 treated with Catharanthus roseus-silver nanoparticles

BACKGROUND The demand for the development of cancer nanomedicine has increased due to its great therapeutic value that can overcome the limitations of conventional cancer therapy.However,the presence of various bioactive compounds in crude plant extracts used for the synthesis of silver nanoparticles(AgNPs)makes its precise mechanisms of action unclear.AIM To assessed the mRNA transcriptome profiling of human HepG2 cells exposed to Catharanthus roseus G.Don(C.roseus)-AgNPs.METHODS The proliferative activity of hepatocellular carcinoma(HepG2)and normal human liver(THLE3)cells treated with C.roseusAgNPs were measured using MTT assay.The RNA samples were extracted and sequenced using BGIseq500 platform.This is followed by data filtering,mapping,gene expression analysis,differentially expression genes analysis,Gene Ontology analysis,and pathway analysis.RESULTS The mean IC 50 values of C.roseusAgNPs on HepG2 was 4.38±1.59μg/mL while on THLE3 cells was 800±1.55μg/mL.Transcriptome profiling revealed an alteration of 296 genes.C.roseusAgNPs induced the expression of stress-associated genes such as MT,HSP and HMOX-1.Cellular signalling pathways were potentially activated through MAPK,TNF and TGF pathways that are responsible for apoptosis and cell cycle arrest.The alteration of ARF6,EHD2,FGFR3,RhoA,EEA1,VPS28,VPS25,and TSG101 indicated the uptake of C.roseus-AgNPs via both clathrin-dependent and clathrinindependent endocytosis.CONCLUSION This study provides new insights into gene expression study of biosynthesised AgNPs on cancer cells.The cytotoxicity effect is mediated by the aberrant gene alteration,and more interestingly the unique selective antiproliferative properties indicate the C.roseusAgNPs as an ideal anticancer candidate.

Synthesis of silver nanoparticles using leaves of Catharanthus roseus Linn.G.Don and their antiplasmodial activities

Objective:To develop a novel approach for the green synthesis of silver nanoparticles using aqueous leaves extracts of Catharanthus roseus(C.roseus) Linn.G.Don which has been proven aclive against malaria parasite Plasmodium falciparum(P.falciparum).Methods:Characterizations were determined by using ultraviolet-visible(UV-Vis) spectrophotometry,scanning electron microscopy(SEM).energy dispersive X-ray and X-ray diffraction.Results:SLM showed the formation of silver nanoparticles with an average size of 35-55 nm.X-ray diffraction analysts showed that the particles were crystalline in nature with face centred cubic structure of the hulk silver with the broad peaks at 32.4.46.4 and 28.0.Conclusions:It can be concluded that the leaves of C.roseus can he good source for synthesis of silver nanoparticle which shows aiitiplasmodial activity against P.falciparum.The important outcome of the study will he the development of value added products from medicinal plants C.roseus lor hionicdical and nanotechnology based industries.

Engineering Terpenoid Indole Alkaloids Biosynthetic Pathway in Catharanthus roseus Hairy Root Cultures by Overexpressing the Geraniol 10-hydroxylase Gene

Catharanthus roseus contains important anti-tumor terpenoid indole alkaloids (TIAs) such as vinblastine and vincristine. Cytochrome P450 enzyme geraniol 10-hydroxylase (G10H) is a putative rate-limiting enzyme involved in the TIAs biosynthetic pathway in C. roseus. In this study the g10h gene driven by the cauliflower mosaic virus 35S (CaMV 35S) promoter was introduced into C. roseus through Agrobacterium-mediated transformation. The integration and overexpression of the target gene (g10h) in hairy root lines were confirmed by polymerase chain reaction and RT-QPCR analysis respectively. Overexpression of g10h in transgenic hairy root lines significantly enhanced the accumulations of monomeric alkaloid ajmalicine and dimeric alkaloids, vincristine and vinblastine. Total TIAs production in hairy roots reached (9.51) mg/g DW, over 3-fold higher than that in the untransformed root lines. This is the first report that engineering of g10h into TIAs-producing plant species results in significant enhancement of TIAs accumulation in cultured hairy roots. This study demonstrates that the putative rate-limiting step catalyzed by G10H is indeed the real rate-limiting step involved in the TIAs biosynthetic pathway in C. roseus, which is one of the key targets for promoting TIAs production by genetic engineering.

Catharanthus roseus:a natural source for the synthesis of silver nanoparticles

Objective:To develop a simple rapid procedure for bioreduction of silver nanoparticles(AgNPs)using aqueous leaves extracts of Catharanthus roseus(C.roseus).Methods:Characterization were determined by using UV-Vis spectrophotometry,scanning electron microscopy(SEM),energy dispersive X-ray and X-ray diffraction.Results:SEM showed the formation of silver nanoparticles with an average size of 67 nm to 48 nm.X-ray diffraction analysis showed that the particles were crystalline in nature with face centered cubic geometry.Conclusions:C.roseus demonstrates strong potential for synthesis of silver nanoparticles by rapid reduction of silver ions(Ag^+ to Ag^0).This study provides evidence for developing large scale commercial production of value-added products for biomedical/nanotechnology-based industries.

Effects of co-administration of methanol leaf extract of Catharanthus roseus on the hypoglycemic activity of metformin and glibenclamide in rats

Objective:To investigate the interacting effects of co-administration of methanol leaf extract of Catharanthus roseus（C.roseus） on the hypoglycemic activity of metformin as well as glibenclamide using experimental rats.Methods:Phytochemical analysis as well as acute toxicity and lethality(LD50) test were carried out on its methanol leaf extract.The alloxan model for experimental induction of diabetes in rats was employed.Six groups comprising five rats each were used.GroupsⅡ,ⅢandⅣreceived 250 mg/kg of extract,100 mg/kg of metformin and 1 mg/kg of glibenclamide respectively,while V and VI were administered metformin-extract and glibenclamide-extract combinations respectively at doses as above.Group I served as negative control and received only distilled water.All administration was done once daily for seven days. Fasting blood glucose was determined at 2,12,24,72 and 168 h using a glucometer.One-way ANOVA with post-hoc tests was used to assess for significant difference due to administration of drug alone and with co-administration of drug and extract.Results:The LD50 was 2 121.32 mg/kg. The phytochemical studies indicated the presence of saponins,tannins,alkaloids,phlotatannins, flavonoids,triterpenoids,reducing sugars,anthraquinones and glycosides.All medicaments significantly reduced blood glucose levels when compared with control alone（P【0.05） with the highest percentage reduction in blood glucose（64.86%） exhibited by metformin-extract combination.Conclusions:The leaf extract of C.roseus significandy increases the hypoglycemic effect of metformin.

Distribution pattern and titer of Candidatus Liberibacter asiaticus in periwinkle(Catharanthus roseus)

Candidatus Liberibacter asiaticus （CaLas）, an uncultured Gram-negative alphaproteobacterium, is the causal agent of Huanglongbing （HLB） in citrus. CaLas resides in phloem sieve tubes and has been shown to be unequally distributed in different tissues. Although HLB is a disease of citrus plants, it has been demonstrated that periwinkle can serve as an experimental host of CaLas, which can be transmitted from citrus to periwinkle via the parasitic plant dodder （Cuscuta spp.）. To investigate the distribution of CaLas in various periwinkle tissues, the bacteria were transmitted from an infected periwinkle plant to healthy periwinkles by top-grafting. The movement of the inoculum and associated titer changes were observed over time in various tissues. CaLas could be detected in the leaves, main stems, and roots of infected periwinkle by conventional PCR, and in all three tissues a clear time-dependent change in CaLas titer was observed, with titer increasing soon after inoculation and then decreasing as disease symptoms became severe. The highest titer was found at 25, 35 and 35 days after inoculation in leaves, main stems and roots, respectively. The titer in leaves was much higher than in the main stems and roots at the same time point, and the spatial distribution of CaLas in the leaves, main stems and roots of infected periwinkle was uneven, similar to what has been shown in citrus. The results provide guidance for selecting the proper periwinkle tissues and sampling times for early detection of CaLas.

Efficacy of larvicidal and pupicidal activity of Catharanthus roseus aqueous and solvent extracts against Anopheles stephensi Liston and Culex quinquefasciatus Say (Diptera: Culicidae)

Objective: To investigate the larvicidal and pupicidal activities of aqueous, ethyl acetate and methanol extracts of Catharanthus roseus ( C. roseus ) against malaria and filariasis vectors. Methods: The larvicidal and pupicidal activities of C. roseus leaf extracts were tested against the fourth instar larvae and pupae of Anopheles stephensi ( An. stephensi ) and Culex quinquefasciatus ( Cx. quinquefasciatus ). The mortality was observed after 24 and 48 h post the treatment. The data were subjected to probit analysis to determine the lethal concentrations (LC 50 and LC 90 ) at which 50% and 90% of the treated larvae or pupae of the tested species were killed. Results: The larval and pupal mortality were observed after 24 and 48 h of exposure of aqueous, ethyl acetate and methanol extracts of C. roseus ; no mortality was observed in the control group. The LC 50 values against the fourth-instar larvae of An. stephensi were 68.62 and 72.04 mg/mL for the aqueous extract, 82.47 mg/mL for the ethyl acetate extract, and 78.80 and 86.64 mg/mL for the methanol extract, while the aqueous, ethyl acetate and methanol extracts had LC 50 values of 85.21, 76.84 and 94.20 mg/mL against the fourth-instar larvae of Cx. quinquefasciatus . The aqueous, ethyl acetate and methanol extracts had LC 50 values of 118.08, 182.47 and 143.80 mg/mL against the pupae of An. stephensi and 146.20, 226.84 and 156.62 mg/mL against the pupae of Cx. quinquefasciatus , respectively. Conclusions: The aqueous and methanol extracts of C. roseus leaves had an excellent potential to control the malarial vector An. stephensi and filariasis vector Cx. quinquefasciatus .

A BIOREACTOR FOR TWO-STAGE MULTIPLE SHOOT CULTURE OF CATHARANTHUS ROSEUS

1 INTRODUCTIONThe production of commercially important indole alkaloid from tissue culture of C.roseus hasbeen a major objective of research carried out worldwide[1].Of primary importance has beenthe production of catharanthine and vindoline which can be used as precursor for thesynthesis of commercially important antineoplastic agents,vinblastine and vincristine.

Comparative studies of elemental composition in leaves and flowers of Catharanthus roseus growing in Bangladesh

Objective:To investigate the elemental composition of the leaves and flowers of Catharanthus roseus(C.roseus) due to the plant's wide application in the indigenous medicinal system and its chemical constituents' importance.Methods:The atomic absorption spectrophotometer was used for quantitative analysis of various elements.Results:Total 13 important elements were analyzed in leaves and flowers of C.roseus.Results indicated the presence of Na,K,Ca,Mg,Cr,Fe,Zn,Al,Cu,Ni,Pb,Cd and Mn in both leaves and flowers.The most important finding of the work was that,leaves of C.roseus showed high concentration of all elements except K and Zn while flowers of C.roseus showed higher concentration of K and Zn.Conclusions:The elemental composition in both leaves and flowers of C.roseus were found to be different.Therefore,different parts of this medicinal plant are enriched in some micro and macro nutrients like Fe,Ca,Na,K,Zn,which are very important for biological metabolic system as well as human health.

Larvicidal efficacy of Catharanthus roseus Linn.(Family:Apocynaceae)leaf extract and bacterial insecticide Bacillus thuringiensis against Anopheles stephensi Liston.

Objective:To explore the larvicidal activity of Catharanthus roseus(C.roseus) leaf extract and Raeillus thuringiensis(B.thuringiensis) against the malarial vector Anopheles stephensi(An. stephensi),when being used alone or together.Methods:The larvicidal activity was assayed at various concentrations under the laboratory and field conditions.The LC_(50) and LC_(90) values of the C.roseus leaf extract were determined by probit analysis.Results:The plant extract showed larvicidal effects after 24 h of exposure;however,the highest larval mortality was found in the petroleum ether extract of C.roseus against the first to fourth instars larvae with LC_(50)=3.34,4.48, 5.90 and 8.17 g/L,respectively;B.thuringiensis against the first to fourth instars larvae with LC_(50)=1.72.1.93.2.17 and 2.42 g/L.respectively:and the combined treatment with LC_(50)=2.18.2.41. 2.76 and 3.22 g/L,respectively.No mortality was observed in the control.Conclusions:The petroleum ether extract of C.roseus extract and B.thuringiensis have potential to be used as ideal eco-friendly agents for the control of An.stephensi in vector control programs.The combined treatment with this plant crude extract and bacterial toxin has better larvicidal efficacy against An.stephensi.

Antihyperglycemic and antioxidant activities of total alkaloids from Catharanthus roseus in streptozotocin-induced diabetic rats

We evaluated the hypoglycemic and antioxi- dant effects of the total alkaloids of leaves and twigs of Catharanthus roseus Linn. （CTA） in streptozotocin （STZ）- induced diabetic rats. The hypoglycemic effect was mea- sured by blood glucose and plasma insulin level. Oxidative stress was measured in heart, liver and kidney by levels of antioxidant markers, free radical scavengers and lipid peroxides i.e. superoxide dismutase （SOD）, catalase （CAT）, glutathione （GSH） and thiobarbituric acid reactive substances （TBARS）. Biochemical parameters, i.e. aspar- tate aminotransferase （AST）, alanine aminotransferase （ALT）, alkaline phosphate （ALP） were observed in diabetic control and treated rats. Oral administration of CTA for 30 days was followed by a significant （P 〈 0.05） decrease in fasting blood glucose and increase in insulin level as compared with untreated diabetic rats. Also it significantly （P 〈 0.05） reduced ALT, AST and ALP. The treatment also resulted in significant （P 〈 0.05） reductions in GSH, SOD, CAT, and decrease in TBARS in the heart, liver and kidney of diabetic rats. The results suggest that CTA can effectively normalize the impaired antioxidant status in STZ-induced diabetes in a dose-dependent manner.CTA exerted rapid protective effects against lipid peroxi- dation by scavenging of free radicals and reducing the risk of diabetic complications.

Effects of some heavy metals on cell suspension cultures of Catharanthus roseus

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Evaluation of Phytoremediation Potential of <i>Catharanthus roseus</i>with Respect to Chromium Contamination

A major environmental concern due to dispersal of industrial and urban wastes generated by human activities is the contamination of soil. The release of heavy metals into the terrestrial ecosystem is a major problem. Accumulation of heavy metals in environment and particularly in soil is a serious environmental concern, as the accumulated heavy metal ions can find their way into living organisms via contamination of ground water or food chain. This praxis urgently requires and demands governmental regulations in India. Two samples of sludge were collected from Banthar Industrial Pollution Control Company (BIPCC), UP State Industrial Development Corporation (UPSIDC), Leather Technology Park, Banthar, Unnao, India. In the present study, the phytoremediation potential of Catharanthus roseus, a valued medicinal plant, with respect to chromium has been analyzed. C. roseus was shown to absorb up to about 38% of the amount of Cr present in primary and secondary sludge amended soil through roots and accumulate it to about 22% in leaves. Effect of chromium concentration on the status of antioxidant enzyme peroxidase (POD) and detoxification enzyme glutathione-S-transferase (GST) from C. roseus leaves was also observed and determined. Increased expressions of POD and GST were observed on native PAGE under stress conditions as compared to control. C. roseus can well tolerate low amounts of chromium (and accumulate it to about 22% in leaves) and can, thus, prove useful in the reclamation and remediation of chromium contaminated soil and land.

Preventive effect of Catharanthus roseus (Linn.) against high-fructose diet-induced insulin resistance and oxida-tive stress in male Wistar rats

High intake of dietary fructose has been shown to exert a number of adverse metabolic effects in humans and experimental animals. The aim of the present study was to investigate whether Catharanthus roseus (L.) G. Don (Apocynaceae) leaf powder alleviates high-fructose diet-induced insulin resistance and oxidative stress in rats. Adult male Wistar rats of body weight around 200 g were divided into four groups, two of which were fed with standard pellet diet and the other two with high fructose (66%) diet. C. roseus leaf powder suspension in water (100 mg/kg body weight/day) was administered orally to each group of the standard pellet diet fed rats and the high fructose diet fed rats for 60 days. Fructose fed rats (F-group) showed hyperglycemia, hyperinsulinemia, insulin resistance and hyperlipidemia (P < 0.05). C. roseus treatment in fructose fed rats (F+CR-group) prevented the rise in glucose levels by 32.2%, insulin by 46.4%, and impaired insulin sensitivity by 63.6%. C. roseus treatment completely prevented the fructose induced plasma lipid alterations. Regarding liver antioxidant status, oxidative stress was observed in fructose fed rats (F-group), while C. roseus treatment in F+CR-group prevented the fructose induced oxidative stress. Our data indicate the preventive role of C. roseus against fructose-induced insulin resistance and oxidative stress;hence this plant can be used as an adjuvant for the prevention and/or management of insulin resistance and disorders related to it.

Effect of Catharanthus roseus (Vince rosea) and Turmeric (Curcuma longa) Extracts as Green Corrosion Inhibitors for Mild Steel in 1 M HCl

The corrosion inhibition of mild steel by Catharanthus roseus (Vince rosea) and Turmeric (Curcuma longa) extracts as green inhibitors in 1 M HCl solutions has been investigated by using potentiodynamic polarization, electrochemical impedance spectroscopy (EIS), hydrogen evolution measurements and scanning electron microscope (SEM) techniques. Results obtained show that these investigated compounds are good inhibitors and act as mixed type inhibitors. The inhibition efficiencies increase with increasing inhibitor concentration. The SEM results show the formation of protective film on the mild steel surface in the presence of Catharanthus roseus and Turmeric extracts. The results obtained from different tested techniques are in good agreement.

Biosynthetic pathway of terpenoid indole alkaloids in Catharanthus roseus

Catharanthus roseus is one of the most extensively investigated medicinal plants, which can produce more than 130 alkaloids, including the powerful antitumor drugs vinblastine and vincristine. Here we review the recent advances in the biosynthetic pathway of terpenoid indole alkaloids （TIAs） in C. roseus, and the identification and characterization of the corresponding enzymes involved in this pathway. Strictosidine is the central intermediate in the biosynthesis of different TIAs, which is formed by the condensation of secologanin and tryptamine. Secologanin is derived from terpenoid （isoprenoid） biosynthetic pathway, while tryptamine is derived from indole biosynthetic pathway. Then various specific end products are produced by different routes during downstream process. Although many genes and corresponding enzymes have been characterized in this pathway, our knowledge on the whole TIA biosynthetic pathway still remains largely unknown up to date. Full elucidation of TIA biosynthetic pathway is an important prerequisite to understand the regulation of the TIA biosynthesis in the medicinal plant and to produce valuable TIAs by synthetic biological technology.

Terpenoid Indole Alkaloids Biosynthesis and Metabolic Engineering in Catharanthus roseus

Catharanthus roseus L. （Madagascar periwinkle） biosynthesizes a diverse array of secondary metabolites including anticancer dimeric alkaloids （vinblastine and vincristine） and antihypertensive alkaloids （ajmalicine and serpentine）. The multi-step terpenoid indole alkaloids （TIAs） biosynthetic pathway in C. roseus is complex and is under strict molecular regulation. Many enzymes and genes involved in the TIAs biosynthesis have been studied in recent decades. Moreover, some regulatory proteins were found recently to control the production of TIAs in C. roseus. Based on mastering the rough scheme of the pathway and cloning the related genes, metabolic engineering of TIAs biosynthesis has been studied in C. roseus aiming at increasing the desired secondary metabolites in the past few years. The present article summarizes recent advances in isolation and characterization of TIAs biosynthesis genes and transcriptional regulators involved in the second metabolic control in C. roseus. Metabolic engineering applications in TIAs pathway via overexpression of these genes and regulators in C. roseus are also discussed.

Enhancing terpenoid indole alkaloid production by inducible expression of mammalian Bax in Catharanthus roseus cells

Bax,a mammalian pro-apoptotic member of the Bcl-2 family,triggers hypersensitive reactions when expressed in plants.To investigate the effects of Bax on the biosynthesis of clinically important natural products in plant cells,we generate transgenic Catharanthus roseus cells overexpressing a mouse Bax protein under the β-estradiol-inducible promoter.The expression of Bax in transgenic Catharanthus roseus cells is highly dependent on β-estradiol concentrations applied.Contents of catharanthine and total terpenoid indole alkaloid of the transgenic cells treated with 30 μmol/L β-estradiol are 5.0-and 5.5-fold of the control cells.Northern and Western blotting results show that expression of mammalian Bax induces transcriptional activation of Tdc and Str,two key genes in terpenoid indole alkaloid bio-synthetic pathway of Catharanthus roseus cells,and stimulates the accumulation of defense-related protein PR1 in the cells,showing that the mouse Bax triggers the defense responses of Catharanthus roseus cells and activates the terpenoid indole alkaloid biosynthetic pathway.Thus,our data suggest that the mammalian Bax might be a potential regulatory factor for secondary metabolite biosynthesis in plant cells and imply a new secondary metabolic engineering strategy for enhancing the metabolic flux to natural products by activating the whole biosynthetic pathway rather than by engineering the single structural genes within the pathways.

Involvement of nitric oxide signaling in mammalian Bax-induced terpenoid indole alkaloid production of Catharanthus roseus cells

Bax, a mammalian pro-apoptotic member of the Bcl-2 family, has been demonstrated to be a potential regulatory factor for plant secondary metabolite biosynthesis recently. To investigate the molecular mechanism of Bax-induced secondary metabolite biosynthesis, we determined the contents of nitric oxide (NO) of the transgenic Catharanthus roseus cells overexpressing a mouse Bax protein and checked the effects of NO specific scavenger 2,4-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1- oxyl-3-oxide (cPITO) on Bax-induced terpenoid indole alkaloid (TIA) production of the cells. The data showed that overexpression of the mouse Bax in C. roseus cells triggered NO generation of the cells. Treatment of cPITO not only inhibited the Bax-triggered NO burst but also suppressed the Bax-induced TIA production. The results indicated that the mouse Bax might activate the NO signaling in C. roseus cells and induce TIA production through the NO-dependent signal pathway in the cells. Furthermore, the activities of nitric oxide synthase (NOS) were significantly increased in the transgenic Bax cells as compared to those in the control cells, showing that the mouse Bax may induce NOS of C. roseus cells. Treatment of the transgenic Bax cells with NOS inhibitor PBITU blocked both Bax-induced NO genera- tion and TIA production, which suggested that the mouse Bax might trigger NO generation and TIA production through NOS. However, the NOS-like activities and NO generation in the transgenic Bax cells did not match kinetically and the Bax-induced NOS-like activity was much later and lower than NO production. Moreover, the Bax-induced NO generation and TIA production were only partially inhibited by PBITU. Thus, our results suggested that the Bax-induced NO production and secondary metabolite biosynthesis in C. roseus cells was not entirely dependent on NOS or NOS-like enzymes.

New Sesquiterpene Glycosides from Culture Hairy Roots of Catharanthus roseus

Two new compounds cadin-2-en-1β-ol-1β-D-glucuronopyranoside （1）, guaia-l,7-dien-3β,13-diol-13α-D- glucofuranoside （2） along with three known compounds have been isolated from the culture hairy roots of Catharanthus roseus. Their structures have been elucidated with the help of 500 MHz NMR using 1D and 2D spectral methods： viz： ^1H and ^13C NMR, ^1H-^1H COSY, ^1H-^13C HETCOR and DEPT aided by ELMS, FAB-MS, HR-FABMS and IR spectroscopy.