Combination of ethyl acetate fraction from Calotropis gigantea stem bark and sorafenib induces apoptosis in HepG2 cells.

The cytotoxicity of the ethyl acetate fraction of the Calotropis gigantea (L.) Dryand. (C. gigantea) stem bark extract (CGEtOAc) has been demonstrated in many types of cancers. This study examined the improved cancer therapeutic activity of sorafenib when combined with CGEtOAc in HepG2 cells. The cell viability and cell migration assays were applied in HepG2 cells treated with varying concentrations of CGEtOAc, sorafenib, and their combination. Flow cytometry was used to determine apoptosis, which corresponded with a decline in mitochondrial membrane potential and activation of DNA fragmentation. Reactive oxygen species (ROS) levels were assessed in combination with the expression of the phosphatidylinositol-3-kinase (PI3K)/ protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) pathway, which was suggested for association with ROS-induced apoptosis. Combining CGEtOAc at 400 µg/mL with sorafenib at 4 µM, which were their respective half-IC50 concentrations, significantly inhibited HepG2 viability upon 24 h of exposure in comparison with the vehicle and each single treatment. Consequently, CGEtOAc when combined with sorafenib significantly diminished HepG2 migration and induced apoptosis through a mitochondrial-correlation mechanism. ROS production was speculated to be the primary mechanism of stimulating apoptosis in HepG2 cells after exposure to a combination of CGEtOAc and sorafenib, in association with PI3K/Akt/mTOR pathway suppression. Our results present valuable knowledge to support the development of anticancer regimens derived from the CGEtOAc with the chemotherapeutic agent sorafenib, both of which were administered at half-IC50, which may minimize the toxic implications of cancer treatments while improving the therapeutic effectiveness toward future medical applications.

Calotropis procera extract inhibits prostate cancer through regulation of autophagy.

Current treatment options available for prostate cancer (PCa) patients have many adverse side effects and hence, new alternative therapies need to be explored. Anticancer potential of various phytochemicals derived from Calotropis procera has been studied in many cancers but no study has investigated the effect of leaf extract of C. procera on PCa cells. Hence, we investigated the effect of C. procera leaf extract (CPE) on cellular properties of androgen-independent PC-3 and androgen-sensitive 22Rv1 cells. A hydroalcoholic extract of C. procera was prepared and MTT assay was performed to study the effect of CPE on viability of PCa cells. The effect of CPE on cell division ability, migration capability and reactive oxygen species (ROS) production was studied using colony formation assay, wound-healing assay and 2',7'-dichlorodihydrofluorescein diacetate assay, respectively. Caspase activity assay and LDH assay were performed to study the involvement of apoptosis and necrosis in CPE-mediated cell death. Protein levels of cell cycle, antioxidant, autophagy and apoptosis markers were measured by western blot. The composition of CPE was identified using untargeted LC-MS analysis. Results showed that CPE decreased the viability of both the PCa cells, PC-3 and 22Rv1, in a dose- and time-dependent manner. Also, CPE significantly inhibited the colony-forming ability, migration and endogenous ROS production in both the cell lines. Furthermore, CPE significantly decreased NF-κB protein levels and increased the protein levels of the cell cycle inhibitor p27. A significant increase in expression of autophagy markers was observed in CPE-treated PC-3 cells while autophagy markers were downregulated in 22Rv1 cells after CPE exposure. Hence, it can be concluded that CPE inhibits PCa cell viability possibly by regulating the autophagy pathway and/or altering the ROS levels. Thus, CPE can be explored as a possible alternative therapeutic agent for PCa.

Calotropis procera latex protein reduces inflammation and bone loss in ligature-induced period ontitis in male rats.

OBJECTIVE: Calotropis procera latex protein (CpLP) is a popular anti-inflammatory and therefore we aimed to study its effects on inflammatory bone loss. DESIGN: Male Wistar rats were subjected to a ligature of molars. Groups of rats received intraperitoneally CpLP (0.3 mg/kg, 1 mg/kg, or 3 mg/kg) or saline (0.9% NaCl) one hour before ligature and then daily up to 11 days, compared to naïve. Gingiva was evaluated by myeloperoxidase activity and interleukin-1 beta (IL-1ß) expression by ELISA. Bone resorption was evaluated in the region between the cement-enamel junction and the alveolar bone crest. The histology considered alveolar bone resorption and cementum integrity, leukocyte infiltration, and attachment level, followed by immunohistochemistry bone markers between 1st and 2nd molars. Systemically, the weight of the body and organs, and a leukogram were performed. RESULTS: The periodontitis significantly increased myeloperoxidase activity and the IL-1ß level. The increased bone resorption was histologically corroborated by periodontal destruction, leukocyte influx, and attachment loss, as well as the increasing receptor activator of the nuclear factor-kappa B ligand (RANKL)/osteoprotegerin (OPG) ratio, and Tartrate-resistant acid phosphatase (TRAP)+ cells when compared to naïve. CpLP significantly reduced myeloperoxidase activity, level of IL-1ß, alveolar bone resorption, periodontal destruction, leukocyte influx, and attachment loss. The CpLp also reduced the RANKL/OPG ratio and TRAP+ cells, when compared with the saline group, and did not affect the systemic parameters. CONCLUSIONS: CpLP exhibited a periodontal protective effect by reducing inflammation and restricting osteoclastic alveolar bone resorption in this rat model.

Anastomosing laticifer in the primary and secondary structures of Calotropis procera (Aiton) W.T.Aiton (Apocynaceae) stems.

An in-depth understanding of the development and distribution of laticifer (latex secretory structure) will be important for the production of both rubber and medicines and will support studies on plant adaptations to their environments. We characterize here and describe the ontogenesis of the laticifer sytem in Calotropis procera (Apocynaceae), an invasive subshrub species in arid landscapes. Anatomical and histochemical evaluations of the primary and secondary structures of the stem were carried out on a monthly basis during a full year, with ultrastructural evaluations of laticifer on the stem apex during the rainy season. In the primary structure, laticifer differentiate early from procambium and ground meristem cells of the cortex and medulla and become concentrated adjacent to the external and internal phloem of the bicollateral bundles. In the secondary structure, laticifer differentiates from fusiform derivative cells of the phloem close to the sieve-tube elements. The laticifer is of the articulated, anastomosing, branched type, and it originates from precursor cells that loose the transversal and longitudinal walls by dissolution. Latex is a mixture of terpenes, alkaloids, flavonoids, mucilage, and proteins. The apical meristem and vascular cambium where the laticifer system begins its development are active throughout the year, including during the dry season. The vascular cambium produces phloem with laticifer precursor cells during the rainy season, with high temperatures and long days. The ability of C. procera to grow under water deficit conditions and produce laticifer throughout the year contribute to its wide distribution in arid environments.

Calotropis Procera Induced Caspase-Dependent Apoptosis and Impaired Akt/mTOR Signaling in 4T1 Breast Cancer Cells.

INTRODUCTION: Calotropis procera (Aiton) Dryand (Apocynaceae) is an herb that has been commonly used in folk medicine to treat various diseases for more than 1500 years. AIMS: Our goal was to investigate the anti-metastatic effects of phenolics extracted from C. procera (CphE) against 4T1 breast cancer cells and in BALB/c mice. METHODS: 4T1 cells were treated with CphE and quercetin (positive control) at concentrations that inhibited cell viability by 50% (IC50). Levels of reactive oxygen species (ROS), wound healing, and protein expressions were determined following standard protocols. For the in vivo pilot study, the syngeneic BALB/c mouse model was used. 4T1 cells were injected into mammary fat pads. Tumors were allowed to grow for 9 days before gavage treatment with CphE (150 mg GAE/kg/day) or PBS (controls) for one week. Excised tumors, liver, and lungs were analyzed for gene and protein expression and histology. RESULTS: In vitro results showed that CphE suppressed cell viability through apoptosis induction, via caspase-3 cleavage and total PARP reduction. CphE also scavenged ROS and suppressed Akt, mTOR, ERK1/2, CREB, and Src activation contributing to cell motility inhibition. CphE reduced IR, PTEN, TSC2, p70S6, and RPS6, protein levels, which are proteins involved in the PI3K/Akt/mTOR pathway, suggesting this pathway as CphE primary target. In vivo results showed downregulation of ERK1/2 activation by phosphorylation in tumor tissues, accompanied by angiogenesis reduction in tumor and lung tissues. A reduction of Cenpf mRNA levels in liver and lung tissues strongly suggested anti-invasive cancer activity of CphE. CONCLUSION: CphE inhibited 4T1 cell signal pathways that play a key role in cell growth and invasion. The potential for in vitro results to be translated in vivo was confirmed. A complete animal study is a guarantee to confirm the CphE anticancer and antimetastatic activity in vivo.

Calotropis procera (L.) mediated synthesis of AgNPs and their application to control leaf spot of Hibiscus rosa-sinensis (L.).

Nature is gifted with a wide range of ornamental plants, which beautify and clean the nature. Due to its great aesthetic value, there is a need to protect these plants from a variety of biotic and abiotic stresses. Hibiscus rosa-sinensis (L.) is an ornamental plant and it is commonly known as China rose or shoeblack plant. It is affected by several fungal and bacterial pathogens. Current study was designed to isolate leaf spot pathogen of H. rosa-sinensis and its control using silver nanoparticles (AgNPs). Based on molecular and morphological features, the isolated leaf spot pathogen was identified as Aspergillus niger. AgNPs were synthesized in the leaf extract of Calotropis procera and characterized. UV-vis spectral analysis displayed discrete plasmon resonance bands on the surface of synthesized AgNPs, depicting the presence of aromatic amino acids. Fourier transform infrared spectroscopy (FTIR) described the presence of C-O, NH, C-H, and O-H functional groups, which act as stabilizing and reducing molecules. X-ray diffraction (XRD) revealed the average size (~32.43 nm) of AgNPs and scanning electron microscopy (SEM) depicted their spherical nature. In this study, in vitro and in vivo antifungal activity of AgNPs was investigated. In vitro antifungal activity analysis revealed the highest growth inhibition of mycelia (87%) at 1.0 mg/ml concentration of AgNPs. The same concentration of AgNPs tremendously inhibited the spread of disease on infected leaves of H. rosa-sinensis. These results demonstrated significant disease control ability of AgNPs and suggested their use on different ornamental plants.

Synthesis of magnetic nanoparticles and their effects on growth and physiological parameters of Calotropis procera seedlings.

The present study was carried out to elucidate effects of synthesized magnetic nanoparticles (MNPs) on morphological and physiological parameters and main essential oil components of Calotropis procera seedlings. For this purpose, 21-day-old seedlings grown under hydroponic conditions were treated by the different MNP concentrations (0, 50, 100, 150, and 200 mg L-1). The results showed that the growth parameters, chlorophyll pigments, soluble sugars, and total proteins significantly increased in leaf under MNP treatment, except for the root length. As compared to the control, MNPs induced a substantial change in the activities of antioxidant enzymes, H2O2, and malondialdehyde contents. Ascorbate peroxidase activity showed a meaningful increase in leaf treated with 200 mg L-1 MNPs, while superoxide dismutase activity and concentration of H2O2 conspicuously decreased relative to the control. Moreover, MNPs enhanced geranial, 1,8-cineol, a-phellandrene, citronellal, camphor, and terpinen-4-ol contents as major components. These results suggest that MNPs could be a promising method of iron application in agricultural systems. Regarding the effects of MNPs, 200-mg L-1 MNPs were most effective on the production of main essential oils and plant growth that could serve as a favorable elicitor for plant improvement.

Characterization and molecular docking study of cathepsin L inhibitory peptides (SnuCalCpIs) from Calotropis procera R. Br.

Propeptides, released from the autocatalytic activation of its zymogen, are potential inhibitors against proteases involved in cancer cell invasion and migration. Our research team previously obtained novel propeptides (SnuCalCpIs) from transcriptome analysis of the medicinal plant Calotropis procera R. Br. and reported them as promising candidates for cancer therapeutics due to their cathepsin L inhibition activity. In the present study, inhibitory activity among SnuCalCpIs was compared with inhibition efficiency and verified by in silico molecular docking analysis. Only SnuCalCpI03 and SnuCalCpI15, expressed in Escherichia coli, showed inhibitory activity against cathepsin L as competitive inhibitors, and the half-maximal inhibitory concentrations (IC50) values of 2.1 nM and 1.6 nM, respectively. They were stable below 70 °C, maintaining more than 90% inhibitory activity over a wide range of pH (2.0-10.0), except at the isoelectric point (pI). The template-based docking simulation models showed that SnuCalCpI02, SnuCalCpI12, and SnuCalCpI16 could not interact with the substrate-binding cleft of cathepsin L even though they possessed the same conserved domain. In contrast, SnuCalCpI03 and SnuCalCpI15 interacted with cathepsin L along the propeptide binding loop and substrate-binding cleft, resulting in obstruction of substrate access to the active site.

Structural Analysis Revealed the Interaction of Cardenolides from Calotropis procera with Na+/K+ ATPases from Herbivores.

BACKGROUND: The herbivores Danaus plexippus (Lepidoptera), Oncopeltus fasciatus, and Aphis nerii (Hemiptera) are special insects that feed on Calotropis procera (Apocynaceae) (Sodom Apple). At least 35 chemically distinct cardenolides have been reported in C. procera. OBJECTIVE: We aimed to evaluate the interaction between cardenolides and Na+/K+ ATPases from herbivores. METHODS: The Na+/K+ ATPases from these insects were modeled, and docking studies were performed involving cardenolides from C. procera. RESULTS: The replacement of serine in sensitive Na+/K+ ATPase by histidine, phenylalanine, and tyrosine in the structures examined suggested spatial impairment caused by interaction, probably making the herbivorous insects resistant against the cardenolides of C. procera. In addition, the ability of the insects to avoid cardenolide toxicity was not correlated with cardenolide polarity. Therefore, the plant fights predation through molecular diversity, and the insects, regardless of their taxonomy, face this molecular diversity through amino acid replacements at key positions of the enzyme targeted by the cardenolides. CONCLUSION: The results show the arsenal of chemically distinct cardenolides synthesized by the C. procera.

Sustainable Development of Chitosan/Calotropis procera-Based Hydrogels to Stimulate Formation of Granulation Tissue and Angiogenesis in Wound Healing Applications.

The formation of new scaffolds to enhance healing magnitude is necessarily required in biomedical applications. Granulation tissue formation is a crucial stage of wound healing in which granulation tissue grows on the surface of a wound by the formation of connective tissue and blood vessels. In the present study, porous hydrogels were synthesized using chitosan incorporating latex of the Calotropis procera plant by using a freeze-thaw cycle to stimulate the formation of granulation tissue and angiogenesis in wound healing applications. Structural analysis through Fourier transform infrared (FTIR) spectroscopy confirmed the interaction between chitosan and Calotropis procera. Latex extract containing hydrogel showed slightly higher absorption than the control during water absorption analysis. Thermogravimetric analysis showed high thermal stability of the 60:40 combination of chitosan (CS) and Calotropis procera as compared to all other treatments and controls. A fabricated scaffold application on a chick chorioallantoic membrane (CAM) showed that all hydrogels containing latex extract resulted in a significant formation of blood vessels and regeneration of cells. Overall, the formation of connective tissues and blood capillaries and healing magnitude decreased in ascending order of concentration of extract.

Allelopathic Effect of Calotropis procera, Hyoscyamus muticus and Pulicaria undulata Extracts on Seed Germination of Portulaca oleracea and Chenopodium murale.

BACKGROUND AND OBJECTIVE: The use of herbicides leads to severe damage, especially to the environment. The aim of this investigation was operated to study the allelopathic effect of Calotropis procera, Hyoscyamus muticus and Pulicaria undulata aqueous extracts on the seed germination of Portulaca oleracea and Chenopodium murale. MATERIALS AND METHODS: The aerial parts of the plants (Calotropis procera, Hyoscyamus muticus and Pulicaria undulata) washed well in distilled water and were dried in room temperature then ground to fine powder. Aqueous extract was prepared using distilled water. RESULTS: In this study, different concentrations of plant extracts (1, 3 and 5%) were used to illustrate which of these concentrations have the potential to inhibit seed growth of Portulaca oleracea and Chenopodium murale. Concentration of 5% for all plant extracts used to inhibited the growth of all seeds of Chenopodium murale while, concentration of 5% for Pulicaria undulata extract only inhibited the growth of all seeds of Portulaca oleracea. CONCLUSION: This work dealt with the use of allelopathy strategy in preventing the growth of some weeds to be one of the safest alternative ways of using herbicides. The results showed the effective effect of plant extracts for both Calotropis procera, Hyoscyamus muticus and Pulicaria undulata on the seed germination of Portulaca oleracea and Chenopodium murale.

Reference genes selection for Calotropis procera under different salt stress conditions.

Calotropis procera is a perennial Asian shrub with significant adaptation to adverse climate conditions and poor soils. Given its increased salt and drought stress tolerance, C. procera stands out as a powerful candidate to provide alternative genetic resources for biotechnological approaches. The qPCR (real-time quantitative polymerase chain reaction), widely recognized among the most accurate methods for quantifying gene expression, demands suitable reference genes (RGs) to avoid over- or underestimations of the relative expression and incorrect interpretation. This study aimed at evaluating the stability of ten RGs for normalization of gene expression of root and leaf of C. procera under different salt stress conditions and different collection times. The selected RGs were used on expression analysis of three target genes. Three independent experiments were carried out in greenhouse with young plants: i) Leaf100 = leaf samples collected 30 min, 2 h, 8 h and 45 days after NaCl-stress (100 mM NaCl); ii) Root50 and iii) Root200 = root samples collected 30 min, 2 h, 8 h and 1day after NaCl-stress (50 and 200 mM NaCl, respectively). Stability rank among the three algorithms used showed high agreement for the four most stable RGs. The four most stable RGs showed high congruence among all combination of collection time, for each software studied, with minor disagreements. CYP23 was the best RG (rank of top four) for all experimental conditions (Leaf100, Root50, and Root200). Using appropriated RGs, we validated the relative expression level of three differentially expressed target genes (NAC78, CNBL4, and ND1) in Leaf100 and Root200 samples. This study provides the first selection of stable reference genes for C. procera under salinity. Our results emphasize the need for caution when evaluating the stability RGs under different amplitude of variable factors.

Control of ß-sitosterol biosynthesis under light and watering in desert plant Calotropis procera.

Most scientific studies on Calotropis procera refer to the plant as an important source of pharmaceutical compounds and its valuable benefits in medicine. One of the most important substances in this plant is the potential immunostimulant ß-sitosterol (BS) that acts in improving human health. This study focused on the effects of lighting before and after irrigation on the BS accumulation pathway namely steroid biosynthesis. Studying the enzymes in BS biosynthetic pathway indicated the upregulation at dawn and predusk of the SMT2 and SMO2 genes encoding sterol methyltransferase 2 and methylsterol monooxygenase, two key enzymes in BS accumulation in C. procera. The results almost indicated no regulation at the different time points of the CYP710A gene encoding sterol 22-desaturase, an enzyme that acts in depleting ß-sitosterol towards the biosynthesis of stigmasterol. RNA-Seq data was validated via quantitative RT-PCR and results were positive. The data of ultra-performance liquid chromatography-tandem mass spectrometry analysis with regard to BS accumulation also aligned with those of RNA-Seq analysis. We focused on the effects of light before and after watering on BS accumulation in C. procera. Our results show that BS accumulation is high at dawn in both dehydrated and well-watered condition. While, the BS was dramatically decrease at midday in well-watered plants. This increase/decrease in BS content is correlated with rates of expression of SMT 2 gene. This gene is a key convertor between the different branches in the cardiac glycoside biosynthesis. Accordingly, it could be suggested that BS (or one of the descendent product) may play an important role in C. procera tolerance to drought/light intensity conditions.

Coroglaucigenin induces senescence and autophagy in colorectal cancer cells.

OBJECTIVES: Coroglaucigenin (CGN), a natural product isolated from Calotropis gigantean by our research group, has been identified as a potential anti-cancer agent. However, the molecular mechanisms involved remain poorly understood. MATERIALS AND METHODS: Cell viability and cell proliferation were detected by MTT and BrdU assays. Flow cytometry, SA-ß-gal assay, western blotting and immunofluorescence were performed to determine CGN-induced apoptosis, senescence and autophagy. Western blotting, siRNA transfection and coimmunoprecipitation were carried out to investigate the mechanisms of CGN-induced senescence and autophagy. The anti-tumour activities of combination therapy with CGN and chloroquine were observed in mice tumour models. RESULTS: We demonstrated that CGN inhibits the proliferation of colorectal cancer cells both in vitro and in vivo. We showed that the inhibition of cell proliferation by CGN is independent of apoptosis, but is associated with cell-cycle arrest and senescence in colorectal cancer cells. Notably, CGN induces protective autophagy that attenuates CGN-mediated cell proliferation. Functional studies revealed that CGN disrupts the association of Hsp90 with both CDK4 and Akt, leading to CDK4 degradation and Akt dephosphorylation, eventually resulting in senescence and autophagy, respectively. Combination therapy with CGN and chloroquine resulted in enhanced anti-tumour effects in vivo. CONCLUSIONS: Our results demonstrate that CGN induces senescence and autophagy in colorectal cancer cells and indicate that combining it with an autophagy inhibitor may be a novel strategy suitable for CGN-mediated anti-cancer therapy.

Photoluminescent reduced graphene oxide quantum dots from latex of Calotropis gigantea for metal sensing, radical scavenging, cytotoxicity, and bioimaging in Artemia salina: A greener route.

In this work, we report the fabrication of green fluorescent reduced graphene oxide quantum dots (rGOQDs) from the latex of Calotropis gigantea by simple one-step microwave assisted greener route. The latex of Calotropis gigantea calcined at 300°C and its ethanolic extract is used for the synthesis of QDs, The rGOQDs showed particle size ranging from 2 to 8nm and it exhibited green fluorescent in longer UV region at 360-520nm. The rGOQDs graphitic nature was confirmed by RAMAN and XRD analysis. The FTIR, XPS demonstrate that presence of functional groups such as CO, COC, -OH, hence it's addressing them as rGOQDs. It is used to design the greener and economically adopted fluorescent probe for the detection of Pb2+ ions. It provides simple and appropriate for the selective and sensitive detection of Pb2+ ions in water purification process. It also trapped the free radicals and neutralized that and act as an excellent radical scavenger in DPPH radical scavenging assessment. These rGOQDs showed excellent biocompatibility on brine shrimp nauplii (Artemia salina) up to 160µg/mL for 24h incubation. Furthermore, rGOQDS were demonstrated as fluorescent bioimaging probe selectively in the inner digestion part of Artemia salina. In summary, stable, economically viable, highly biocompatible, greener method based rGOQDs were prepared for heavy metal ion detecting, radical scavenging, bioimaging applications which can play a vital role in the future nanotechnology-based biomedical field.

Genome Assembly and Annotation of the Medicinal Plant Calotropis gigantea, a Producer of Anticancer and Antimalarial Cardenolides.

Calotropis gigantea produces specialized secondary metabolites known as cardenolides, which have anticancer and antimalarial properties. Although transcriptomic studies have been conducted in other cardenolide-producing species, no nuclear genome assembly for an Asterid cardenolide-producing species has been reported to date. A high-quality de novo assembly was generated for C. gigantea, representing 157,284,427 bp with an N50 scaffold size of 805,959 bp, for which quality assessments indicated a near complete representation of the genic space. Transcriptome data in the form of RNA-sequencing libraries from a developmental tissue series was generated to aid the annotation and construction of a gene expression atlas. Using an ab initio and evidence-driven gene annotation pipeline, 18,197 high-confidence genes were annotated. Homologous and syntenic relationships between C. gigantea and other species within the Apocynaceae family confirmed previously identified evolutionary relationships, and suggest the emergence or loss of the specialized cardenolide metabolites after the divergence of the Apocynaceae subfamilies. The C. gigantea genome assembly, annotation, and RNA-sequencing data provide a novel resource to study the cardenolide biosynthesis pathway, especially for understanding the evolutionary origin of cardenolides and the engineering of cardenolide production in heterologous organisms for existing and novel pharmaceutical applications.

Transcriptomic and metabolic responses of Calotropis procera to salt and drought stress.

BACKGROUND: Calotropis procera is a wild plant species in the family Apocynaceae that is able to grow in harsh, arid and heat stressed conditions. Understanding how this highly adapted plant persists in harsh environments should inform future efforts to improve the hardiness of crop and forage plant species. To study the plant response to droµght and osmotic stress, we treated plants with polyethylene glycol and NaCl and carried out transcriptomic and metabolomics measurements across a time-course of five days. RESULTS: We identified a highly dynamic transcriptional response across the time-course including dramatic changes in inositol signaling, stress response genes and cytokinins. The resulting metabolome changes also involved sharp increases of myo-inositol, a key signaling molecule and elevated amino acid metabolites at later times. CONCLUSIONS: The data generated here provide a first glimpse at the expressed genome of C. procera, a plant that is exceptionally well adapted to arid environments. We demonstrate, through transcriptome and metabolome analysis that myo-inositol signaling is strongly induced in response to drought and salt stress and that there is elevation of amino acid concentrations after prolonged osmotic stress. This work should lay the foundations of future studies in adaptation to arid environments.

The osmotin of Calotropis procera latex is not expressed in laticifer-free cultivated callus and under salt stress.

The latex of Calotropis procera has previously been reported to contain osmotin. This protein (CpOsm) inhibited phytopathogens and this was mechanistically characterized. Here, the time-course profile of CpOsm transcripts was examined in the salt-stressed cultivated callus of C. procera in order to better understand its role in the physiology of the plant. Stressed callus (80 mM NaCl) showed an unbalanced content of organic compounds (proline and total soluble sugar) and inorganic ions (Na+, Cl-, and K+). Under salt treatment, the transcripts of CpOsm were detected after 12 h and slightly increased to a maximum at day seven, followed by reduction. Interestingly, CpOsm was not detected in the soluble protein fraction recovered from the salt-stressed callus as probed by electrophoresis, dot/Western blotting and mass spectrometry. The results suggested that (1) CpOsm is not constitutive in cultivated cells (laticifer-free tissues); (2) CpOsm transcripts appear under salt-stressed conditions; (3) the absence of CpOsm in the protein fractions of stressed cultivated cells indicated that salt-induced transcripts were not used for protein synthesis and this accounts to the belief that CpOsm may be a true laticifer protein in C. procera. More effort will be needed to unveil this process. In this study we show evidences that CpOsm gene is responsive to salt stress. However the corresponding protein is not produced in cultivated cells. Therefore, presently the hypothesis that CpOsm is involved in abiotic stress is not fully supported.

Comparative study on cellular entry of incinerated ancient gold particles (Swarna Bhasma) and chemically synthesized gold particles.

Gold nanoparticles (AuNPs) are used for a number of imaging and therapeutic applications in east and western part of the world. For thousands of years, the traditional Indian Ayurvedic approach to healing involves the use of incinerated gold ash, prepared with a variety of plant extracts and minerals depending on the region. Here, we describe the characterization of incinerated gold particles (IAuPs) in HeLa (human cells derived from cervical cancer) and HFF-1 (human foreskin fibroblast cells) in comparison to synthesized citrate-capped gold nanoparticles (AuNPs). We found that while individual IAuP crystallites are around 60 nm in size, they form large aggregates with a mean diameter of 4711.7 nm, some of which can enter cells. Fewer cells appeared to have IAuPs compared to AuNPs, although neither type of particle was toxic to cells. Imaging studies revealed that IAuPs were in vesicles, cytosol, or in the nucleus. We found that their nuclear accumulation likely occurred after nuclear envelope breakdown during cell division. We also found that larger IAuPs entered cells via macropinocytosis, while smaller particles entered via clathrin-dependent receptor-mediated endocytosis.

Photosynthetic limitation and mechanisms of photoprotection under drought and recovery of Calotropis procera, an evergreen C3 from arid regions.

Calotropis procera is a C3 plant native from arid environmental zones. It is an evergreen, shrubby, non-woody plant with intense photosynthetic metabolism during the dry season. We measured photosynthetic parameters and leaf biochemical traits, such as gas exchange, photochemical parameters, A/Ci analysis, organic solutes, and antioxidant enzymes under controlled conditions in potted plants during drought stress, and following recovery conditions to obtain a better insight in the drought stress responses of C. procera. Indeed, different processes contribute to the drought stress resilience of C. procera and to the fast recovery after rehydration. The parameters analyzed showed that C. procera has a high efficiency for energy dissipation. The photosynthetic machinery is protected by a robust antioxidant system and photoprotective mechanisms such as alternative pathways for electrons (photorespiration and day respiration). Under severe drought stress, increased stomatal limitation and decreased biochemical limitation permitted C. procera to maintain maximum rate of Rubisco carboxylation (Vc,max) and photosynthetic rate (Amax). On the other hand, limitation of stomatal or mesophyll CO2 diffusion did not impair fast recovery, maintaining Vc,max, chloroplast CO2 concentration (Cc) and mesophyll conductance (gm) unchanged while electron flow used for RuBP carboxylation (Jc) and Amax increased. The ability to tolerate drought stress and the fast recovery of this evergreen C3 species was also due to leaf anti-oxidative stress enzyme activity, and photosynthetic pigments. Thus, these different drought tolerance mechanisms allowed high performance of photosynthetic metabolism by drought stressed plants during the re-watering period.