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.

Angiogenic protein profiling, phytochemical screening and in silico anti-cancer targets validation of stem, leaves, fruit, and seeds of Calotropis procera in human liver and breast cancer cell lines.

The main focus of anticancer drug discovery is on developing medications that are gentle on normal cells and should have the ability to target multiple anti-cancer pathways. Liver cancer is becoming a worldwide epidemic due to the highest occurring and reoccurring rate in some countries. Calotropis procera is a xerophytic herbal plant growing wildly in Saudi Arabia. Due to its anti-angiogenic and anticancer capabilities, "C. procera" is a viable option for developing innovative anticancer medicines. However, no study has been done previously, to discover angiogenic and anti-cancer targets which are regulated by C. procera in liver cancer. In this study, leaves, stems, flowers, and seeds of C. procera were used to prepare crude extracts and were fractionated into four solvents of diverse polarities. These bioactivity-guided solvent fractions helped to identify useful compounds with minimal side effects. The phytoconstituents present in the leaves and stem were identified by GC-MS. In silico studies were done to predict the anti-cancer targets by major bioactive constituents present in leaves and stem extracts. A human angiogenesis antibody array was performed to profile novel angiogenic targets. The results from this study showed that C. procera extracts are an ideal anti-cancer remedy with minimum toxicity to normal cells as revealed by zebrafish in vivo toxicity screening assays. The novel antiangiogenic and anticancer targets identified in this study could be explored to design medication against liver cancer.

Identification of steroidal cardenolides from Calotropis procera as novel HIV-1 PR inhibitors: A molecular docking & molecular dynamics simulation study.

Background & objectives Despite advancements in antiretroviral therapy, drug-resistant strains of HIV (human immunodeficiency virus) remain a global health concern. Natural compounds from medicinal plants offer a promising avenue for developing new HIV-1 PR (protease) inhibitors. This study aimed to explore the potential of compounds derived from Calotropis procera, a medicinal plant, as inhibitors of HIV-1 PR. Methods This in silico study utilized natural compound information and the crystal structure of HIV-1 PR. Molecular docking of 17 steroidal cardenolides from Calotropis procera against HIV-1 PR was performed using AutoDock 4.2 to identify compounds with higher antiviral potential. A dynamic simulation study was performed to provide insights into the stability, binding dynamics, and potential efficacy of the top potential antiviral compound as an HIV-1 therapeutic. Results We found that all tested cardenolides had higher binding affinities than Amprenavir, indicating their potential as potent HIV-1 PR inhibitors. Voruscharin and uscharidin displayed the strongest interactions, forming hydrogen bonds and hydrophobic interactions with HIV-1 PR. Voruscharin showed improved stability with lower RMSD (Root Mean Square Deviation) values and reduced fluctuations in binding site residues but increased flexibility in certain regions. The radius of gyration analysis confirmed a stable binding pose between HIV-1 PR and Voruscharin. Interpretation & conclusions These findings suggest that Calotropis procera could potentially be a source of compounds for developing novel HIV-1 PR inhibitors, contributing to the efforts to combat HIV. Further studies and clinical trials are needed to evaluate the safety and efficacy of these compounds as potential drug candidates for the treatment of HIV-1 infection.

Network Pharmacology Approach to Identify the Calotropis Phytoconstituents' Potential Epileptic Targets and Evaluation of Molecular Docking, MD Simulation, and MM-PBSA Performance.

Epilepsy originates from unusual electrical rhythm within brain cells, causes seizures. Calotropis species have been utilized to treat a wide spectrum of ailments since antiquity. Despite chemical and biological investigations, there have been minimal studies on their anticonvulsant activity, and the molecular targets of this plant constituents are unexplored. This study aimed to investigate the plausible epileptic targets of Calotropis phytoconstituents through network pharmacology, and to evaluate their binding strength and stability with the identified targets. In detail, 125 phytoconstituents of the Calotropis plant (C. procera and C. gigantea) were assessed for their drug-likeness (DL), blood-brain-barrier (BBB) permeability and oral bioavailability (OB). Network analysis revealed that targets PTGS2 and PPAR-γ were ranked first and fourth, respectively, among the top ten hub genes significantly linked with antiepileptic drug targets. Additionally, docking, molecular dynamic (MD) simulation, and Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) were employed to validate the compound-gene interactions. Docking studies suggested ergost-5-en-3-ol, stigmasterol and ß-sitosterol exhibit stronger binding affinity and favorable interactions than co-crystallized ligands with both the targets. Furthermore, both MD simulations and MM-PBSA calculations substantiated the docking results. Combined data revealed that Calotropis phytoconstituents ergost-5-en-3-ol, stigmasterol, and ß-sitosterol might be the best inhibitors of both PTGS2 and PPAR-γ.

Antimicrobial, Antigenotoxicity, and Characterization of Calotropis procera and Its Rhizosphere-Inhabiting Actinobacteria: In Vitro and In Vivo Studies.

Calotropis procera (C. procera) is a wild shrub that is a medicinal plant found in abundance throughout Saudi Arabia. In this study, we investigated the phytochemical composition and antigenotoxic properties of the ethanolic extract of C. procera, in addition to the antimicrobial activity of the plant and its rhizospheric actinobacteria effects against pathogenic microorganisms. Soil-extract medium supplemented with glycerol as a carbon source and starch-casein agar medium was used for isolation of actinobacteria from rhizosphere. From the plant, a total of 31 compounds were identified using gas chromatography/mass spectrometry (GC-MS). The main components were α-amyrin (39.36%), lupeol acetate (17.94%), phytol (13.32%), hexadecanoic acid (5.55%), stigmasterol (3.16%), linolenic acid (3.04%), and gombasterol A (2.14%). C. procera plant extract's antimicrobial activity was investigated using an agar well-diffusion assay and minimum inhibitory concentration (MIC) against six pathogenic microbial strains. The plant extract of C. procera was considered significantly active against Staphylococcus aureus, Klebsiella pneumonia, and Escherichia coli, with inhibition zones of 18.66 mm, 21.26 mm, and 21.93 mm, respectively. The plant extract was considered to be a moderate inhibitor against Bacillus subtilis, with MIC ranging from 0.60-1.50 mg/mL. On the other hand, the isolated actinobacteria were considered to be a moderate inhibitor against S. aureus (MIC of 86 µg/mL), and a potent inhibitor, strain CALT_2, against Candida albicans (MIC of 35 µg/mL). The 16S rRNA gene sequence analysis showed that the potential strains belonged to the genus Streptomyces. The effect of C. procera extract against cyclophosphamide (CP)-induced genotoxicity was examined by evaluating chromosome abnormalities in mouse somatic cells and DNA fragmentation assays. The current study revealed that oral pretreatment of C. procera (50, 100, and 200 mg/kg b.w.) for 1, 7, and 14 days to cyclophosphamide-treated animals significantly reduced chromosomal abnormalities as well as DNA fragmentation in a dose-dependent manner. Moreover, C. procera extract had antimicrobial and antigenotoxic effects against CP-induced genotoxicity.

Non-classical cardenolides from Calotropis gigantea exhibit anticancer effect as HIF-1 inhibitors.

Six new non-classical cardenolides (1-6), and seventeen known ones (7-23) were isolated from Calotropis gigantea. All cardenolides showed inhibitory effect on hypoxia inducible factor-1 (HIF-1) transcriptional activity with IC50 of 8.85 nM-16.69 µM except 5 and 7. The novel 19-dihydrocalotoxin (1) exhibited a comparable HIF-1 inhibitory activity (IC50 of 139.57 nM) to digoxin (IC50 of 145.77 nM), a well-studied HIF-1 inhibitor, and 11, 12, 14, 16 and 19 presented 1.4-15.4 folds stronger HIF-1 inhibition than digoxin. 1 and 11 showed a dose-dependent inhibition on HIF-1α protein, which led to their HIF-1 suppressing effects. Compared with LO2 and H9c2 normal cell lines, both 1 and 11 showed selective cytotoxicity against various cancer cell lines including HCT116, HeLa, HepG2, A549, MCF-7, A2780 and MDA-MB-231. Moreover, a comprehensive structure-activity relationship was concluded for these non-classical cardenolides as HIF-1 inhibitors, which may shed some light on the rational design and development of cardenolide-based anticancer drugs.

The Anticonvulsant Effect of Hydroethanolic Leaf Extract of Calotropis procera (Ait) R. Br. (Apocynaceae).

A number of currently used drugs have been obtained from medicinal plants which are a major source of drugs. These drugs are either used in their pure form or modified to a semisynthetic drug. Drug discovery through natural product research has been fruitful over the years. Traditionally, Calotropis procera is used extensively in the management of epilepsy. This study is conducted to explore the anticonvulsant effect of a hydroethanolic leaf extract of Calotropis procera (CPE) in murine models. This effect was evaluated using picrotoxin-induced convulsions, strychnine-induced convulsions, and isoniazid- and pilocarpine-induced status epilepticus in mice of both sexes. The results showed that CPE (100-300 mg/kg) exhibited an anticonvulsant effect against strychnine-induced clonic seizures by significantly reducing the duration (p = 0.0068) and frequency (p = 0.0016) of convulsions. The extract (100-300 mg/kg) caused a profound dose-dependent delay in the onset of clonic convulsions induced by picrotoxin (p < 0.0001) and tonic convulsions (p < 0.0001) in mice. The duration of convulsions was reduced significantly also for both clonic and tonic (p < 0.0001) seizures as well. CPE (100-300 mg/kg), showed a profound anticonvulsant effect and reduced mortality in the pilocarpine-induced convulsions. ED50 (~0.1007) determined demonstrated that the extract was less potent than diazepam in reducing the duration and onset of convulsions but had comparable efficacies. Flumazenil-a GABAA receptor antagonist-did not reverse the onset or duration of convulsions produced by the extract in the picrotoxin-induced seizure model. In isoniazid-induced seizure, CPE (300 mg kg1, p.o.) significantly (p < 0.001) delayed the onset of seizure in mice and prolonged latency to death in animals. Overall, the hydroethanolic leaf extract of Calotropis procera possesses anticonvulsant properties.

Systematic Phytochemical Screening of Different Organs of Calotropis procera and the Ovicidal Effect of Their Extracts to the Foodstuff Pest Cadra cautella.

In developing countries, crop deterioration is mainly caused by inappropriate storage conditions that promote insect infestation. Synthetic pesticides are associated with serious adverse effects on humans and the environment. Thus, finding alternative "green" insecticides is a very pressing need. Calotropis procera (Aiton) Dryand (Apocynaceae) growing in Saudi Arabia was selected for this purpose. LC-MS/MS analysis was applied to investigate the metabolic composition of different C. procera extracts. Particularly, C. procera latex and leaves showed a high presence of cardenolides including calactin, uscharidin, 15ß-hydroxy-calactin, 16ß-hydroxy-calactin, and 12ß-hydroxy-calactin. The ovicidal activity of the extracts from different plant organs (flowers, leaves, branches, roots), and of the latex, against Cadra cautella (Walker) (Lepidoptera, Pyralidae) was assessed. Extracts of C. procera roots displayed the most potent activity with 50% of C. cautella eggs not hatching at 10.000 ppm (1%).

Plant-bacterial endophyte secondary metabolite matching: a case study.

Some studies focused on metabolic relationships between plants and their endophytic bacterial associates, and more research is required to generate critical evidence for these relationships. In the current interest, we tried to confirm the relationship between the traditional medicinal plant, Calotropis procera (Aiton) W.T. Aiton, and its associated endophytes, Bacillus siamensis and Bacillus amyloliquefaciens, as the first matching study regarding the production of bioactive secondary metabolites from the plant vis-a-vis its bacterial endophytes.Secondary metabolites of both the plant and its endophytic bacteria were extracted using different solvents, e.g., water, methanol, and ethyl acetate. All extracts exhibited high quantities of phenolics, flavonoids, tannins, and saponins. In addition, they showed significant antioxidant capacity which was found to be positively correlated with total phenolic contents. The highest total antioxidant capacity (99.28 ± 0.0 mg AA equivalent/g extract) was measured for the aqueous extract of B. siamensis.Antibacterial activity of the different extracts was evaluated against certain pathogenic bacteria, i.e., Escherichia coli, Klebsiella pneumoniae, Streptococcus agalactiae, Salmonella typhi, Serratia marcescens, and Staphylococcus aureus. It was strikingly found that the broadest antibacterial spectrum was revealed by extracts of both C. procera and its endophytic B. siamensis. Interestingly, antibacterial activity was significantly correlated to phenolic and flavonoid contents.

Anti-inflammatory latex proteins of the medicinal plant Calotropis procera: a promising alternative for oral mucositis treatment.

OBJECTIVE AND DESIGN: Oral mucositis (OM) is an intense inflammatory reaction progressing to tissue damage and ulceration. The medicinal uses of Calotropis procera are supported by anti-inflammatory capacity. PII-IAA, a highly homogenous cocktail of laticifer proteins (LP) prepared from the latex of C. procera, with recognized pharmacological properties was tested to treat OM. MATERIALS AND SUBJECTS: Male Golden Sirius hamsters were used in all treatments. TREATMENT: The latex protein samples were injected i.p. (5 mg/Kg) 24 h before mucositis induction (mechanical trauma) and 24 h later. METHODS: Histology, cytokine measurements [ELISA], and macroscopic evaluation [scores] were performed. RESULTS: PII-IAA eliminated OM, accompanied by total disappearance of myeloperoxidase activity and release of IL-1b, as well as reduced TNF-a. Oxidative stress was relieved by PII-IAA treatment, as revealed by MDA and GSH measurements. PII-IAA also reduced the expression of adhesion molecules (ICAM-1) and Iba-1, two important markers of inflammation, indicating modulatory effects. Histological analyses of the cheek epithelium revealed greater deposition of type I collagen fibers in animals given PII-IAA compared with the control group. This performance was only reached when LPPII was treated with iodoacetamide (IAA), an irreversible inhibitor of proteolytic activity of cysteine proteases. The endogenous proteolytic activity of LPPII induced adverse effects in animals. Candidate proteins involved in the phytomodulatory activity are proposed. CONCLUSIONS: Therapy was successful in treating OM with the laticifer protein fraction, containing peptidases and osmotin, from Calotropis procera. The effective candidate from the latex proteins for therapeutic use is PII-IAA.

Calosides A-F, Cardenolides from Calotropis gigantea and Their Cytotoxic Activity.

Phytochemical analysis of the roots of Calotropis gigantea led to the isolation of six new cardenolide glycosides, calosides A-F (1-6), and five known cardenolides (7-11). The structures of 1-6 were elucidated based on NMR and ECD spectroscopic data interpretation. Caloside D (4) is the first naturally occurring example of a cardenolide containing a C-8/C-19 oxygen bridge. In turn, calosides E (5) and F (6) represent the first naturally occurring 3-epi-cannogenol diglycosides having potent cytotoxicity against the PANC-1 cell line (IC50, 0.081 and 0.070 µM, respectively) and HeLa (IC50, both 0.17 µM) cells, under normoglycemic conditions.

Triple-Negative Breast Cancer Cells Exhibit Differential Sensitivity to Cardenolides from Calotropis gigantea.

Triple-negative breast cancers (TNBC) are aggressive and heterogeneous cancers that lack targeted therapies. We implemented a screening program to identify new leads for subgroups of TNBC using diverse cell lines with different molecular drivers. Through this program, we identified an extract from Calotropis gigantea that caused selective cytotoxicity in BT-549 cells as compared to four other TNBC cell lines. Bioassay-guided fractionation of the BT-549 selective extract yielded nine cardenolides responsible for the selective activity. These included eight known cardenolides and a new cardenolide glycoside. Structure-activity relationships among the cardenolides demonstrated a correlation between their relative potencies toward BT-549 cells and Na+/K+ ATPase inhibition. Calotropin, the compound with the highest degree of selectivity for BT-549 cells, increased intracellular Ca2+ in sensitive cells to a greater extent than in the resistant MDA-MB-231 cells. Further studies identified a second TNBC cell line, Hs578T, that is also highly sensitive to the cardenolides, and mechanistic studies were conducted to identify commonalities among the sensitive cell lines. Experiments showed that both cardenolide-sensitive cell lines expressed higher mRNA levels of the Na+/Ca2+ exchanger NCX1 than resistant TNBC cells. This suggests that NCX1 could be a biomarker to identify TNBC patients that might benefit from the clinical administration of a cardiac glycoside for anticancer indications.

Latex proteins downregulate inflammation and restores blood-coagulation homeostasis in acute Salmonella infection.

BACKGROUND: Calotropis procera latex protein fraction (LP) was previously shown to protect animals from septic shock. Further investigations showed that LP modulate nitric oxide and cytokines levels. OBJECTIVES: To evaluate whether the protective effects of LP, against lethal bacterial infection, is observed in its subfractions (LPPII and LPPIII). METHODS: Subfractions (5 and 10 mg/kg) were tested by i.p. administration, 24 h before challenging with lethal injection (i.p.) of Salmonella Typhimurium. LPPIII (5 mg/kg) which showed higher survival rate was assayed to evaluate bacterial clearance, histopathology, leukocyte recruitment, plasma coagulation time, cytokines and NO levels. FINDINGS: LPPIII protected 70% of animals of death. The animals given LPPIII exhibited reduced bacterial load in blood and peritoneal fluid after 24 h compared to the control. LPPIII promoted macrophage infiltration in spleen and liver. LPPIII restored the coagulation time of infected animals, increased IL-10 and reduced NO in blood. MAIN CONCLUSIONS: LPPIII recruited macrophages to the target organs of bacterial infection. This addressed inflammatory stimulus seems to reduce bacterial colonisation in spleen and liver, down regulate bacterial spread and contribute to avoid septic shock.

Essential Oil of Calotropis procera: Comparative Chemical Profiles, Antimicrobial Activity, and Allelopathic Potential on Weeds.

Plants are considered green resources for thousands of bioactive compounds. Essential oils (EOs) are an important class of secondary compounds with various biological activities, including allelopathic and antimicrobial activities. Herein, the present study aimed to compare the chemical profiles of the EOs of the widely distributed medicinal plant Calotropis procera collected from Saudi Arabia and Egypt. In addition, this study also aimed to assess their allelopathic and antimicrobial activities. The EOs from Egyptian and Saudi ecospecies were extracted by hydrodistillation and analyzed via GC-MS. The correlation between the analyzed EOs and those published from Egypt, India, and Nigeria was assessed by principal component analysis (PCA) and agglomerative hierarchical clustering (AHC). The allelopathic activity of the extracted EOs was tested against two weeds (Bidens pilosa and Dactyloctenium aegyptium). Moreover, the EOs were tested for antimicrobial activity against seven bacterial and two fungal strains. Ninety compounds were identified from both ecospecies, where 76 compounds were recorded in Saudi ecospecies and 33 in the Egyptian one. Terpenes were recorded as the main components along with hydrocarbons, aromatics, and carotenoids. The sesquiterpenes (54.07%) were the most abundant component of EO of the Saudi sample, while the diterpenes (44.82%) represented the mains of the Egyptian one. Hinesol (13.50%), trans-chrysanthenyl acetate (12.33%), 1,4-trans-1,7-cis-acorenone (7.62%), phytol (8.73%), and myristicin (6.13%) were found as the major constituents of EO of the Saudi sample, while phytol (38.02%), n-docosane (6.86%), linoleic acid (6.36%), n-pentacosane (6.31%), and bicyclogermacrene (4.37%) represented the main compounds of the Egyptian one. It was evident that the EOs of both ecospecies had potent phytotoxic activity against the two tested weeds, while the EO of the Egyptian ecospecies was more effective, particularly on the weed D. aegyptium. Moreover, the EOs showed substantial antibacterial and antifungal activities. The present study revealed that the EOs of Egyptian and Saudi ecospecies were different in quality and quantity, which could be attributed to the variant environmental and climatic conditions. The EOs of both ecospecies showed significant allelopathic and antimicrobial activity; therefore, these EOs could be considered as potential green eco-friendly resources for weed and microbe control, considering that this plant is widely grown in arid habitats.

Plant latex thrombin-like cysteine proteases alleviates bleeding by bypassing factor VIII in murine model.

Hemostasis is a tightly regulated process which maintains a fluid state of blood within the vasculature and provides thrombotic response upon tissue injury. Various scientific studies have implicated the role of plant latex proteases in hemostasis using in vitro experiments. However, in vivo models substantiate their role in hemostasis. Therefore, in the present study, the effect of plant latex thrombin-like proteases (PTLPs) on hemostasis was investigated systematically using mice tail bleeding as a preclinical model. In this direction, latex protease fractions (LPFs), which showed potent thrombin-like activity, were selected as they act directly on fibrinogen to form clot and quickly stop bleeding. Thrombin-like activity was exhibited mainly by cysteine proteases. Calotropis gigantea, Carica papaya, Jatropha curcas, Oxystelma esculentum, Tabernaemontana divaricata, and Vallaris solanacea LPFs and papain from C. papaya latex significantly reduced bleeding on a topical application in normal and aspirin administered mice. In addition, PTLPs accelerated the clotting of factor VIII deficient plasma, while, papain brought back the clotting time to normal levels acting like a bypassing agent. Further, papain failed to show activity in the presence of specific cysteine protease inhibitor iodoacetic acid; confirming protease role in all the activities exhibited. At the tested dose, PTLPs except C. gigantea did not show toxicity. Further, structural and sequence comparison between PTLPs and human thrombin revealed structural and sequence dissimilarity indicating their unique nature. The findings of the present study may open up a new avenue for considering PTLPs including papain in the treatment of bleeding wounds.

Potent Antagonists of RORγt, Cardenolides from Calotropis gigantea, Exhibit Discrepant Effects on the Differentiation of T Lymphocyte Subsets.

RORγt is the master transcription factor of IL-17 cytokine expression and Th17 lymphocyte differentiation, which are responsible for the induction of many autoimmune diseases. Recently, RORγt has become an attractive target for drug development to treat these types of diseases, and the field of RORγt antagonist research is now extremely competitive. In our current study, molecular docking was applied to demonstrate that cardenolides, including uscharin, calactin, and calotropin derived from Calotropis gigantea, probably directly bind to RORγt. Therefore, the inhibitory effect was further validated using a luciferase reporter assay. Because RORγt is the key transcriptional factor for Th17 differentiation, the effects of these compounds on Th17 differentiation were studied by flow cytometry. The results showed that uscharin, calactin, and calotropin inhibited Th17 differentiation from 100 to 500 nM. Furthermore, uscharin had a better effect than digoxin, a well-known inverse agonist of RORγt, in reducing Th17 polarization. Additionally, the effects of the cardenolides on the differentiation of other Th lineages, including Th1, Th2, and Treg, were investigated. Uscharin suppressed Th1, Th2, and Treg cell differentiation, while calactin suppressed the differentiation of Th1 cells, and calotropin did not influence the other T cell subsets, indicating that calactin suppressed Th1 and Th17 differentiation, and calotropin selectively quenched Th17 polarization. Structural analysis of the three compounds showed that the selectivity of uscharin, calactin, and calotropin on the suppression of the different subsets of T cells is correlated to the minor differences in their chemical structures. Collectively, calactin and calotropin have greater potential to be developed as lead compounds than uscharin to treat autoimmune diseases mediated by Th17 and/or Th1 cells.

Erythrocytes morphology and hemorheology in severe bacterial infection.

BACKGROUND: Severe bacterial infections initiate inadequate inflammation that leads to disseminated intravascular coagulation and death. OBJECTIVES: To evaluate the influence of bacterial infection on blood viscosity and red blood cells (RBCs) morphology, and the ability of Calotropis procera proteins (CpLP) to prevent the patho-hemorheology in infected animals. METHODS: Rheology of blood, atomic force microscopy measurements on specific blood elements and blood count were performed to examine changes in blood viscosity, RBCs morphology, platelets activation, and RBCs indices. FINDINGS: Infected mice hold their blood rheological behaviour as compared to that of the control group. However, they presented hyperactivated platelets, RBCs at different stages of eryptosis, and variation on RBCs indices. CpLP administration in healthy animals altered blood behaviour from pseudoplastic to Bingham-like fluid. Such effect disappeared over time and by inhibiting its proteases. No alterations were observed in RBCs morphology or platelets. Treatment of infected animals with CpLP prevented the changes in RBCs indices and morphology. MAIN CONCLUSIONS: The inflammatory process triggered by bacterial infection induced pathological changes in RBCs and platelets activation. Treatment of infected animals with CpLP prevented the emergence of RBCs abnormal morphology and this may have implications in the protective effect of CpLP, avoiding animal death.

Calotropis gigantea extract induces apoptosis through extrinsic/intrinsic pathways and reactive oxygen species generation in A549 and NCI-H1299 non-small cell lung cancer cells.

BACKGROUND: Calotropis gigantea (CG) is a tall and waxy flower that is used as a traditional remedy for fever, indigestion, rheumatism, leprosy, and leukoderma. However, the precise mechanisms of its anticancer effects have not yet been examined in human non-small cell lung cancer (NSCLC) cells. In this study, we investigated whether CG extract exerted an apoptotic effect in A549 and NCI-H1299 NSCLC cells. METHODS: The ethanol extract of CG was prepared, and its apoptotic effects on A549 and NCI-H1299 NSCLC cells were assessed by using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay, annexin V-fluorescein isothiocyanate/propidium iodide (PI) staining, cell cycle analysis, real-time polymerase chain reaction (RT-PCR), western blotting, JC-1 staining, and ROS detection assay. RESULTS: The CG extract induced apoptosis through the stimulation of intrinsic and extrinsic signaling pathways in A549 and NCI-H1299 lung cancer cells. Cell cycle arrest was induced by the CG extract in both cell lines. Reactive oxygen species (ROS), which can induce cell death, were also generated in the CG-treated A549 and NCI-H1299 cells. CONCLUSIONS: These data confirmed that CG caused apoptosis through the activation of extrinsic and intrinsic pathways, cell cycle arrest, and ROS generation in A549 and NCI-H1299 lung cancer cells. Thus, CG can be suggested as a potential agent for lung cancer therapy.

Acaricidal efficacy of Calotropis procera (Asclepiadaceae) and Taraxacum officinale (Asteraceae) against Rhipicephalus microplus from Mardan, Pakistan.

Medicinal plants are used by traditional folk healers, modern physicians, and veterinarians as an alternative to conventional drugs to treat a wide range of disorders including parasitic diseases. Some compounds from these plants have been shown to have acaricidal activity and repel arthropods. The cattle tick Rhipicephalus microplus is one of the most destructive pests to the livestock industry in tropical and subtropical parts of the world. The potential to develop herbal acaricides to control R. microplus infestations is critical in maintaining cattle herd productivity, reducing economic losses, and curtailing the overuse of synthetic chemical acaricides. Calotropis procera, the apple of Sodom, and Taraxacum officinale, the common dandelion, were evaluated for acaricidal activity against R. microplus larvae and adults in vitro. Both plant species tested are common indigenous species of Pakistan where R. microplus infestations are widespread across livestock species including cattle, sheep, and goats. Whole-plant extracts derived from C. procera and T. officinale significantly reduced the index of egg laying (P < 0.01) and increased the percent inhibition of oviposition of adult female ticks at a concentration of 40 mg/mL when assessed by the adult immersion test (AIT). Calotropis procera and T. officinale treatments at the same concentration also resulted in larval mortality of 96.0% ± 0.57 and 96.7% ± 0.88, respectively, as measured using the larval packet test (LPT). An increasing range of extract concentrations was tested to determine the LD50 and LD90 for C. procera, 3.21 and 21.15 mg/mL, respectively, and T. officinale, 4.04 and 18.92 mg/mL, respectively. These results indicate that further studies are warranted to determine the relative contribution of individual phytochemicals from whole-plant extracts on acaricidal activity. This information will guide the design of further acaricidal efficacy tests using livestock infested with R. microplus.

Molecular cloning and anti-invasive activity of cathepsin L propeptide-like protein from Calotropis procera R. Br. against cancer cells.

Cathepsin L of cancer cells has been shown to play an important role in degradation of extracellular matrix for metastasis. In order to reduce cell invasion, cathepsin L propeptide-like proteins which are classified as the I29 family in the MEROPS peptidase database were characterized from Calotropis procera R. Br., rich in cysteine protease. Of 19 candidates, the cloned and expressed recombinant SnuCalCp03-propeptide (rSnuCalCp03-propeptide) showed a low nanomolar Ki value of 2.3 ± 0.2 nM against cathepsin L. A significant inhibition of tumor cell invasion was observed with H1975, HT29, MDA-BM-231, PANC1, and PC3 with a 76, 67, 67, 63, and 79% reduction, respectively, in invasion observed in the presence of 400 nM of the rSnuCalCp03-propeptide. In addition, thermal and pH study showed rSnuCalCp03-propeptide consisting of secondary structures was stable at a broad range of temperatures (30-70 °C) and pH (2-10, except for 5 which is close to the isoelectric point of 5.2).