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.

Biotransformation of Gastrodin by Cell Suspension Cultures of Catharanthus roseus

应用长春花 (Catharanthusroseus (L .)G .Don)悬浮细胞培养体系对天麻素进行了生物转化反应研究。经过8d培养形成一个转化产物 ,应用光谱方法鉴定转化产物的结构为对羟基苯甲醇 ,为天麻素水解后形成的甙元。

Cell Growth and Ajmalicine Accumulation in a Full Habituated Catharanthus roseus Cell Line C_ (20)hi

A full habituated cell line C_ 20hi was screened from 2,4_D dependent line (C_ 20D) of Catharanthus roseus (L.) G. Don. The investigation involved the cell growth, ajmalicine production and enzyme activity related to indole alkaloid biosynthesis in both cell lines. These results indicated that C_ 20hi cells grew faster than C_ 20D cells, and average ajmalicine content in C_ 20hi cells was 18.4 times more than that in C_ 20D when cultured in the production medium. In the growth medium, average ajmalicine content in C_ 20hi cells was 31.9 times more than that in C_ 20D cells, while the cell growth has no obvious difference. The comparison of enzyme activities in C_ 20hi and C_ 20D cells indicated that tryptophan decarboxylase (TDC), strictosidine synthase (SSS) and geraniol_10_dehydrogenase (G10H) activities have no close relation to ajmalicine accumulation, although the activities of these enzymes were higher when cells were cultured in the production medium than in the growth medium. The C_ 20hi cells are relatively stable in five years of culture.

Science Letters:Alterations in seedling vigour and antioxidant enzyme activities in Catharanthus roseus under seed priming with native diazotrophs

An experiment was conducted on Catharanthus roseus to study the effect of seed treatments with native diazotrophs on its seedling growth and antioxidant enzyme activities. The treatments had significant influence on various seedling parameters. There is no significant influence on dry matter production with the diazotrophs, Azospirillum and Azotobacter. However, the vital seedling parameters such as germination percentage and vigour index were improved. Azotobacter treatment influenced maximum of 50% germination, whereas Azospirillum and Azotobacter were on par with C. roseus with respect to their vigour index. There was significant difference in the population of total diazotrophs. Azospirillum and Azotobacter between rhizosphere and non-rhizosphere soils of C. roseus had the same trend and were observed at various locations of the study. The activities of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POX) and catalase (CAT) were increased to a significant extent due to the treatment with diazotrophs.

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℃ and 40℃for short-term heat shock experiment and 20℃, 25℃ and 35℃ 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℃ than at 30℃, 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℃, while increased slowly at 30℃ 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℃ than at 25℃ and had a sharp increase under the condition of 35℃. 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℃ 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.

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.

Effects of tip-pruning treatment on source-sink regulation of Catharanthus roseus seedlings

Fifty cultivated Catharanthus roseus seedlings were selected for tip-pruning treatment and the effects of tip-pruning on seedling growth and source-sink regulation were investigated for revealing physiological mechanisms of plants. The results showed that tip-pruning treatment resulted in obvious inhibition of apical dominance and enhancement of branching numbers. The contents of soluble sugars, acid sucrose invertase activity (AI) had a great change in differently positional leaves of the seedling. The sink strength in tip leaves of seedlings dramatically declined after tip-pruning treatment, while that in the leaves at the middle and bottom of seedlings had no obvious changes. The inhibition of apical dominance of tip leaves of seedlings was caused by the diminished sink strength due to tip-pruning treatment,

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.

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.

Simultaneous quantitative determination of five alkaloids in Catharanthus roseus by HPLC-ESI-MS/MS

AIM: To establish a method to simultaneously determine the main five alkaloids of Catharanthus roseus for trace samples, a high-performance liquid chromatography–electrospray ionization-tandem mass spectrometry(HPLC-ESI-MS/MS) analysis method was developed. METHOD: The five Catharanthus alkaloids, vinblastine, vincristine, vinleurosine, vindoline, and catharanthine were chromatographically separated on a C18 HPLC column. The mobile phase was methanol-15 nmol?L–1 ammonium acetate containing 0.02% formic acid(65 : 35, V/V). The quantification of these alkaloids was based on the Multiple Reaction Monitoring(MRM) mode. RESULTS: This method was validated, and the results achieved the aims of the study. The intra- and inter-day precision and accuracy of the five alkaloids were within 1.2%-11.5%(RSD%) and-10.9%-10.5%(RE%). The recovery rates of the five alkaloids of samples were from 79.9% to 91.5%. The five analytes were stable at room temperature for 2 h, at 4 °C for 12 h, and at-20 °C for two weeks. The developed method was applied successfully to determine the content of the five alkaloids in three plant parts of three batches of C. roseus with a minute amount collected from three regions of China. CONCLUSION: The HPLC-ESI-MS/MS method can be used for the simultaneous determination of five important alkaloids in trace C. roseus samples.

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.

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

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Progress in NMR-based metabolomics of Catharanthus roseus

Metabolomics has been rapidly developed as an important field in plant sciences and natural products chemistry.As the only natural source for a diversity of monoterpenoid indole alkaloids(MIAs),especially the low-abundance antitumor agents vinblastine and vincristine,Catharanthus roseus is highly valued and has been studied extensively as a model for medicinal plants improvement.Due to multistep enzymatic biosynthesis and complex regulation,genetic modification in the MIA pathway has resulted in complicated changes of both secondary and primary metabolism in C.roseus,affecting not only the MIA pathway but also other pathways.Research at the metabolic level is necessary to increase knowledge on the genetic regulation of the whole metabolic network connected to MIA biosynthesis.Nuclear magnetic resonance(NMR)is a very suitable and powerful complementary technique for the identification and quantification of metabolites in the plant matrix.NMR-based metabolomics has been used in studies of C.roseus for pathway elucidation,understanding stress responses,classification among different cultivars,safety and quality controls of transgenic plants,cross talk between pathways,and diversion of carbon fluxes,with the aim of fully unravelling MIA biosynthesis,its regulation and the function of the alkaloids in the plant from a systems biology point of view.

Influence of native arbuscular mycorrhizal fungi on growth,nutrition and phytochemical constituents of Catharanthus roseus(L.)G.Don

Objective:To study the isolation,identification,mass production and the effect of native arbuscular mycorrhizal fungi(AM fungi)on growth parameters of the Catharanthus roseus(C.roseus).Methods:Acaulospora marrowae,Glomus aggregatum(G.aggregatum),Glomus fasciculatum,Glomusgeosporum,Gigaspora margarita,Gigaspora nigra,Scutellospora heterogama and Scutellospora pellucida were isolated and identified from the root zone soil of C.roseus.Results:A total of nine different AM fungi species such as Acaulospora scrobiculata,The phytochemical analyses showed high concentration of chlorophyll a(0.152±0.0140μg/g),chlorophyll b(0.081±0.006μg/g),total chlorophyll(0.233±0.020μg/g),soluble sugar(0.051±0.004μg/g),reducing sugar(0.060±0,007μg/g),phenols(0.293±0.032μg/g),ortho-dihydroxy phenols(0.275±0.022μg/g),lipids(0.300±0.025μg/g),proteins(0.063±0.003μg/g)and amino acids(1.042±0.056μg/g)in G.aggregatum inoculated C.roseus.G.aggregatum was found to perform better on growth when compared to others and phytochemical constituents of C.roseus.Conclusions:fasciculatum can be used as a potential growth promoters for the C.roseus for better yielding in the agricultural sectors.It is concluded from the present findings that the G.aggregatum and Glomus fasciculatum can be used as a potential growth promoters for the C.roseus for better yielding in the agricultural sectors.

长春花茉莉酸受体CrCOI1的生物信息学分析与原核表达

通过生物信息学方法对长春花茉莉酸受体CrCOI1氨基酸序列、结构域以及二级、三级结构进行分析,利用分子生物学方法构建重组质粒并进行原核表达。结果显示:CrCOI1编码516个氨基酸,为胞质定位,分子量为58.26 k D,等电点pI为5.45,与番薯COI1蛋白的亲缘关系比较近,CrCOI1含有F-box结构域、多个LRR富集亮氨酸重复结构域以及转运抑制响应1结构域;该蛋白二级结构由50.97%α-螺旋、31.59%无规则卷曲、12.79%延伸链以及少部分β-转角(4.65%)组成;经SWISS-MODEL预测其三级结构显示,CrCOI1蛋白由α-螺旋和无规则卷曲组成,其中F-box的3个α-螺旋从蛋白的N端伸出便于结合配体。进一步地成功构建了重组表达质粒pET28a-CrCOI1,并经IPTG诱导后在大肠杆菌BL21中实现异源表达,且发现经16℃、0.4 mmol/L IPTG诱导至12 h后蛋白表达量最高,具有时间依赖性。上述结果表明,我们成功构建了长春花茉莉酸受体CrCOI1的重组表达质粒,实现大肠杆菌中的表达,并对其进行了序列以及结构分析。这为研究CrCOI1的功能以及通过其介导茉莉酸信号调控植物生长发育以及代谢产物合成提供一定依据。