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.

钴(Ⅱ)和镍(Ⅱ)对长春花C.roseus细胞悬浮生长的影响

研究了pH5.5条件下,钴(Ⅱ)和镍(Ⅱ)对长春花C.roseus细胞悬浮培养生长的影响。当介质中钴的起始浓度为0.50 2.0μg/Ll时,能轻微刺激C.roseus细胞的生长,[Co(Ⅱ)]>4.0μg/ml,[Ni(Ⅱ)]>2.0μg/ml时,阻碍细胞生长,其抑制程度与介质中钴(Ⅱ)。镍(Ⅱ)的浓度相关;它们的最小致死浓度分别为50.0μg/ml Co(Ⅱ)和10.0μg/ml Ni(Ⅱ)。钴(Ⅱ)较容易被C.roseus所吸收,而镍(Ⅱ)的毒性相对较大。

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.

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.

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.

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.

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 .

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,

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

A self made gas-liquid jet bioreactor in spray flow mode is used in the two-stage multiple shoot culture of C. roseus. The experimental results show that the gas-liquid jet bioreactor is suitable for the multiple shoot culture of C. roseus in two stages, and the multiple shoots of C. roseus reach as high as 35g(dry wt.) · L-1 of biomass content and as high as 1.18% (dry wt.) of total indole alkaloids.

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.