Green-synthesized CuO and ZnO nanoparticles derived from Calotropis gigantea (Apple of Sodom): enhancing plant growth, efficient dye removal, and potent antibacterial applications.

Nanoparticles, owing to their unique physicochemical properties, have garnered significant attention in various scientific disciplines, including materials science, chemistry, biology, and environmental engineering. In recent years, the synthesis of metal oxide nanoparticles, such as NiO, Fe2O3, ZnO, SnO2, and CuO via green routes, has gained attraction due to their diverse applications in fields ranging from catalysis and electronics to medicine and environmental remediation. This study focuses on the green synthesis of copper oxide (CuO) and zinc oxide (ZnO) nanoparticles using Calotropis gigantea (Apple of Sodom) leaf extract as a reducing agent and stabilizer, with zinc nitrate (ZnNO3.6H2O) and copper nitrate (CuNO3.3H2O) as precursors. The hexagonal phase of ZnO and monoclinic plan structure of CuO with high crystallinity was confirmed by XRD and elemental composition by EDX analysis. With the help of an SEM image, particle size measured for CuO and ZnO using ImageJ software was found to be 56.08 nm and 46.49 nm, respectively. This study investigates the efficacy of nanoparticles in wastewater treatment, particularly focusing on methylene blue dye decolorization using the statistical processing of response surface methodology (RSM) using the Box-Behnken method. Additionally, it explores the impact of synthesized nanoparticles on seed growth enhancement, using Vigna radiata (green gram) seeds immersed in various doses of nanoparticles (0, 0.5, 1, 1.5, 2 mg/30 mL). Furthermore, the antibacterial activity of the nanoparticles against both gram-positive and gram-negative bacteria is evaluated. The results confirm the effectiveness of the materials for methylene blue dye removal, achieving 80.53% with CuO and 78.25% with ZnO. Significant seed growth was observed with a low nanoparticle dosage of 1.5 mg/30 mL, resulting in the highest seedling vigour index and germination percentage. This reduces the need for fertilizers and lessens environmental impact.

The dichloromethane fraction from Calotropis gigantea (L.) dryand. Stem bark extract prevents liver cancer in SDT rats with insulin-independent diabetes mellitus.

ETHNOPHARMACOLOGICAL RELEVANCE: Calotropis gigantea (L.) Dryand. (C. gigantea) is a traditional medicinal plant, recognized for its effectiveness in managing diabetes, along with its notable antioxidant, anti-inflammatory, and anticancer properties. Type II diabetes mellitus (T2DM) is characterized by chronic metabolic disorders associated with an elevated risk of hepatocellular carcinoma (HCC) due to hyperglycemia and impaired insulin response. The scientific validation of C. gigantea's ethnopharmacological efficacy offers advantages in alleviating cancer progression in T2DM complications, enriching existing knowledge and potentially aiding future clinical cancer treatments. AIM: This study aimed to investigate the preventive potential of the dichloromethane fraction of C. gigantea stem bark extract (CGDCM) against diethylnitrosamine (DEN)-induced HCC in T2DM rats, aiming to reduce cancer incidence associated with diabetes while validating C. gigantea's ethnopharmacological efficacy. MATERIALS AND METHODS: Spontaneously Diabetic Torii (SDT) rats were administered DEN to induce HCC (SDT-DEN-VEH), followed by treatment with CGDCM. Metformin was used as a positive control (SDT-DEN-MET). All the treatments were administered for 10 weeks after the initial DEN injection. Diabetes-related parameters, including serum levels of glucose, insulin, and glycosylated hemoglobin (HbA1c), as well as liver function enzymes (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase), were quantified. Serum inflammation biomarkers interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were evaluated. Liver tissue samples were analyzed for inflammation protein expression (IL-6, TNF-α, transforming growth factor-ß1 (TGF-ß1), and α-smooth muscle actin (α-SMA)). Histopathological evaluation was performed to assess hepatic necrosis, inflammation, and fibrosis. Liver cell proliferation was determined using immunohistochemistry for Ki-67 expression. RESULTS: Rats with SDT-DEN-induced HCC treated with CGDCM exhibited reduced serum glucose levels, elevated insulin levels, and decreased HbA1c levels. CGDCM treatment also reduced elevated hepatic IL-6, TNF-α, TGF-ß1, and α-SMA levels in SDT-DEN-VEH rats. Additionally, CGDCM treatment prevented hepatocyte damage, fibrosis, and cell proliferation. No adverse effects on normal organs were observed with CGDCM treatment, suggesting its safety for the treatment of HCC complications associated with diabetes. Additionally, the absence of adverse effects in SD rats treated with CGDCM at 2.5 mg/kg further supports the notion of its safe usage. CONCLUSIONS: These findings suggest that C. gigantea stem bark extract exerts preventive effects against the development of HCC complications in patients with T2DM, expanding the potential benefits of its ethnopharmacological advantages.

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.

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.

Swarna Bhasma Induces Antigen-Presenting Abilities of Macrophages and Helps Antigen Experienced CD4+ T Cells to Acquire Th1 Phenotypes Against Leishmania donovani Antigens.

In leishmaniasis, the protective immunity is largely mediated by proinflammatory cytokine producing abilities of T cells and an efficient parasite killing by phagocytic cells. Notwithstanding a substantial progress that has been made during last decades, the mechanisms or factors involved in establishing protective immunity against Leishmania are not identified. In ancient Indian literature, metallic "bhasma," particularly that of "swarna" or gold (fine gold particles), is indicated as one of the most prominent metal-based therapeutic medicine, which is known to impart protective and curative properties in various health issues. In this work, we elucidated the potential of swarna bhasma (SB) on the effector properties of phagocytes and antigen-activated CD4+ T cells in augmenting the immunogenicity of L. donovani antigens. The characterization of SB revealing its shape, size, composition, and measurement of cytotoxicity established the physiochemical potential for its utilization as an immunomodulator. The activation of macrophages with SB enhanced their capacity to produce nitric oxide and proinflammatory cytokines, which eventually resulted in reduced uptake of parasites and their proliferation in infected cells. Further, in Leishmania-infected animals, SB administration reduced the generation of IL-10, an anti-inflammatory cytokine, and enhanced pro-inflammatory cytokine generation by antigen activated CD4+ T cells with increased frequency of double (IFNγ+/TNFα+) and triple (IFNγ+TNFα+IL-2+) positive cells and abrogated disease pathogeneses at the early days of infection. Our results also suggested that cow-ghee (A2) emulsified preparation of SB, either alone or with yashtimadhu, a known natural immune modulator which enhances the SB's potential in enhancing the immunogenicity of parasitic antigens. These findings suggested a definite potential of SB in enhancing the effector functions of phagocytes and CD4+ T cells against L. donovani antigens. Therefore, more studies are needed to elucidate the mechanistic details of SB and its potential in enhancing vaccine-induced immunity.

Identification of phytochemical, antioxidant, anticancer and antimicrobial potential of Calotropis procera leaf aqueous extract.

Since the dawn of civilization, people have turned to plants as a safe and efficient form of treatment for a variety of diseases. It has long been known that Calotropis procera has the potential to treat a number of diseases. In this study, the C. procera leaf aqueous extract was obtained using the maceration method, and p-coumaric was found to be the main compound. The extract was rich in phenols (174.82 mg gallic acid equivalent/g) and flavonoids (1781.7 µg quercetin equivalent/g). The extract had high antioxidant properties, as indicated by the IC50 values obtained for 2,2-diphenyl-1-picrylhydrazyl (DPPH) (366.33 µg/mL) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) (169.04 µg/mL), as well as the ferric ions reducing antioxidant power (FRAP) (1.67 µg ascorbic acid equivalent/g of the extract). The cytotoxicity of the extract was evaluated against the survival of HT 29 cells, and the IC50 was found to be 236.87 µg/mL. The most resistant and sensitive strains to the extract were Escherichia coli and Staphylococcus aureus, respectively. The morphological changes of these strains were demonstrated through scanning electron microscopy and confocal laser scanning microscopy. The C. procera extract could be therefore used as an antioxidant, antimicrobial, and anticancer agent.

Comparative study of microscale and macroscale technique for encapsulation of Calotropis gigantea extract in metal-conjugated nanomatrices for invasive ductal carcinoma.

The encapsulation of plant extract in nanomatrices has limitations due to its adhesion to walls, size control, high cost and long durations that results in low yield. Macroscale and microscale level techniques for development of micro/nanoparticles may impact the encapsulation of plant extract. This study aimed to evaluate the relative efficiency of microscale and macroscale techniques for encapsulation of plant extract, which is not compared yet. Keeping this in view, encapsulation of Calotropis gigantea leaves extract (CaG) was attained in silver-conjugated poliglusam nanomatrices (POL/Ag) to induce apoptosis in invasive ductal carcinoma (IDC) cells. The ethanolic CaG extract was prepared using percolation method and characterized by chemical tests for its active phytochemical compounds. The droplet-based microfluidic system was utilized as microscale encapsulation technique for CaG in nanomatrices at two different aqueous to oil flow rate ratios 1.0:1.5, and 1.0:3.0. Moreover, conventional batch system was utilized as macroscale encapsulation technique consisted of hot plate magnetic stirrer. The prepared nanomatrices were analysed for antioxidant activity using DPPH test and for cytotoxicity analysis using MCF-7 cells. The characteristic peaks of UV-Vis, FTIR and XRD spectrum confirmed the synthesis of CaG(POL/Ag) by both the encapsulation methods. However, microfluidic system was found to be more expedient because of attaining small and uniform sized silver nanoparticles (92 ± 19 nm) at high flow rate and achieving high encapsulation efficiency (80.25%) as compared to the conventional batch method (52.5%). CaG(POL/Ag) nanomatrices found to have significant antioxidant activity (p = 0.0014) against DPPH radical scavenging activity. The CaG(POL/Ag) of the smallest sized formulated by the microfluidic system has also shown the highest cytotoxicity (90%) as compared to batch method (70%) at 80 µg/mL. Our results indicate that the microscale technique using microfluidic system is a more efficient method to formulate size-controlled CaG(POL/Ag) nanomatrices and achieve high encapsulation of plant extract. Additionally, CaG(Pol/Ag) was found to be an efficient new combination for inducing potent (p < 0.0001) apoptosis in IDC cells. Therefore, CaG(Pol/Ag) can be further tested as an anti-cancer agent for in-vivo experiments.

Calotropis procera (Aiton) Dryand (Apocynaceae): State of the art of its uses and Applications.

Calotropis procera (Aiton) Dryand (Apocynaceae), popularly known as milkweed, has been traditionally used to treat diseases particularly associated with gastric disorders, skin disease and inflammatory processes. The present study aimed to review the current scientific evidence regarding the pharmacological effects of C. procera extracted phytochemicals and possible research opportunities as complementary and alternative medicine. Scientific publications were searched in various electronic databases (PubMed, Scopus, Web of Science, Google Scholar, Springer, Wiley, and Mendeley) using the following search terms: Calotropis procera, medicinal plants, toxicity, phytochemical characterization, and biological effects. Collected data showed that cardenolides, steroid glycoside and flavonoids are the main classes of phytochemicals identified in C. procera latex and leaves. In addition, lignans, terpenes, coumarins, and phenolic acids have been reported. These metabolites have been correlated with their biological activities, including mainly antioxidant, anti-inflammatory, antitumoral, hypoglycemic, gastric protective, anti-microbial, insecticide, anti-fungal, anti-parasitic, among others. However, some of the studies were carried out with only a single dose or with a high dose not achievable under physiological conditions. Therefore, the validity of C. procera biological activity may be questionable. Not less important to highlight are the risks associated with its use and the possibility of accumulation of heavy metals that can be toxic. Furthermore, there are no clinical trials with C. procera to date. In conclusion, the need of bioassayguided isolation of bioactive compounds, bioavailability and efficacy, as well as pharmacological and toxicity studies, are needed using in vivo models and clinical trials in order to support the traditionally claimed health benefits.

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.

Use of Calotropis procera cysteine peptidases (CpCPs) immobilized on glyoxyl-agarose for cheesemaking.

Calotropis procera cysteine peptidases (CpCPs) have presented several potential biotechnological applications. Here, these enzymes were immobilized on glyoxyl-agarose (glyoxyl-CpCPs) with yields of 90-95 % and the recovered activities ranged from 10 % to 15 %, according to enzyme loadings (5, 10, 20, 40, and 50 mgBSAeq/g). Spectrophotometric assays and SDS-PAGE showed that the casein hydrolysis by glyoxyl-CpCPs was similar to soluble CpCPs. In addition, glyoxyl-CpCPs exhibited similar ratio of milk-clotting activity to proteolytic activity in comparison with soluble CpCPs and chymosin. Even after being stored for six months at 8 °C, the residual proteolytic activity of glyoxyl-CpCPs remained close to 100 %. Atomic force microscopy and dynamic light scattering techniques showed that the process of casein micelle aggregation after treatment with glyoxyl-CpCPs was very similar to its soluble form and chymosin. Glyoxyl-CpCPs performed well after five reaction cycles, producing cheeses with yield, moisture, protein, and fat similar to those produced with chymosin.

Insecticidal potential of organic extracts of Calotropis procera to Spodoptera frugiperda

This study evaluated the toxic effects of organic extracts of Calotropis proceraleaves on the survival, development, and reproduction of Spodoptera frugiperda. Solutions of crude methanol extract and hexane and methanol fractions of C. proceraleaves were added at 1.15% and 2.14% concentrations to the artificial diet of S. frugiperda. The mortality and duration of larval and pupal phases, weights of female and male pupae, deformations of pupae and adults, the reduction of adults able to reproduce, pre-oviposition and oviposition periods, the number of postures per female, and the fecundity and fertility of S. frugiperdafemales were also evaluated. The extracts harmed the survival, development, and reproduction of S. frugiperda. The ingestion of extracts and fractions by caterpillars affected adults by decreasing the oviposition period, the number of postures, fecundity, and fertility. The crude MeOH extract at a2.14% concentration harmed the evaluated parameters of the insect, except for pupal mortality, female pupae weight, and pre-oviposition period. The MeOH fraction at 2.14% caused a 50.0% mortality of caterpillars and 16.0% deformation in pupae and 33.0% inadults, reducing by 72.0% the population able to reproduce. The MeOH fraction at the 2.14% concentration caused 25.0% and 38.0% of pupal mortality and deformation, respectively. Calotropis procerahas promising insecticidal properties for a biological insecticide, a convenient and sustainable strategy for protecting plants against S. frugiperda.

Polyphenols-Rich Extract of Calotropis procera Alone and in Combination with Trichoderma Culture Filtrate for Biocontrol of Cantaloupe Wilt and Root Rot Fungi.

Fungal diseases have always been a major problem for cantaloupe crops; however, synthetic fungicides are hazardous to humans and the environment. Consequently, a feasible alternative to fungicides without side effects could be by using bio agents and naturally occurring plants with antibacterial potential. This study has achieved a novel procedure for managing wilt and root rot diseases by potentially using Trichoderma sp. culture filtrates in consortium with plant extract of Calotropis procera, Rhizoctonia solani, Fusarium oxysporum, and Pythium ultimum, which were isolated from infected cantaloupe roots with identified root rot symptoms. The antagonistic activity of four Trichoderma isolates and analysis of antibiotics and filtrate enzymes of the most active Trichoderma isolate were determined as well as phytochemical analysis of C. procera plant extract using HPLC-UV. The obtained results showed that all Trichoderma isolates considerably lowered the radial growth of P. ultimum, R. solani, and F. oxysporum in varying degrees. The scanning electron micrographs illustrate the mycoparasitic nature of Trichoderma sp. on F. oxysporum. The phytochemical analysis of C. procera indicated that phenolic contents were the major compounds found in extracts, such as vanillin (46.79%), chlorogenic acid (30.24%), gallic acid (8.06%), and daidzein (3.45%) but including only a low amount of the flavonoid compounds rutin, naringenin, and hesperetin. The Pot experiment's findings showed that cantaloupe was best protected against wilting and root rot diseases when it was treated with both Trichoderma sp. culture filtrates (10%) and C. procera extract of (15 mg/mL), both alone and in combination. This study demonstrates that the application of bio agent Trichoderma spp. filtrate with C. procera phenol extract appears useful for controlling wilting and root rot disease in cantaloupe. This innovative approach could be used as an alternative to chemical fungicide for the control of wilting and rot root diseases.

Calotropis gigantea stem bark extracts inhibit liver cancer induced by diethylnitrosamine.

Several fractions of Calotropis gigantea extracts have been proposed to have potential anticancer activity in many cancer models. The present study evaluated the anticancer activity of C. gigantea stem bark extracts in liver cancer HepG2 cells and diethylnitrosamine (DEN)-induced primary liver cancer in rats. The carcinogenesis model induced by DEN administration has been widely used to study pathophysiological features and responses in rats that are comparable to those seen in cancer patients. The dichloromethane (CGDCM), ethyl acetate, and water fractions obtained from partitioning crude ethanolic extract were quantitatively analyzed for several groups of secondary metabolites and calactin contents. A combination of C. gigantea stem bark extracts with doxorubicin (DOX) was assessed in this study to demonstrate the enhanced cytotoxic effect to cancer compared to the single administration. The combination of DOX and CGDCM, which had the most potential cytotoxic effect in HepG2 cells when compared to the other three fractions, significantly increased cytotoxicity through the apoptotic effect with increased caspase-3 expression. This combination treatment also reduced ATP levels, implying a correlation between ATP and apoptosis induction. In a rat model of DEN-induced liver cancer, treatment with DOX, C. gigantea at low (CGDCM-L) and high (CGDCM-H) doses, and DOX + CGDCM-H for 4 weeks decreased the progression of liver cancer by lowering the liver weight/body weight ratio and the occurrence of liver hyperplastic nodules, fibrosis, and proliferative cells. The therapeutic applications lowered TNF-α, IL-6, TGF-ß, and α-SMA inflammatory cytokines in a similar way, implying that CGDCM had a curative effect against the inflammation-induced liver carcinogenesis produced by DEN exposure. Furthermore, CGDCM and DOX therapy decreased ATP and fatty acid synthesis in rat liver cancer, which was correlated with apoptosis inhibition. CGDCM reduced cleaved caspase-3 expression in liver cancer rats when used alone or in combination with DOX, implying that apoptosis-inducing hepatic carcinogenesis was suppressed. Our results also verified the low toxicity of CGDCM injection on the internal organs of rats. Thus, this research clearly demonstrated a promising, novel anticancer approach that could be applied in future clinical studies of CGDCM and combination therapy.

Successful Pre-Clinical Management of Irinotecan-Debilitated Animals: A Protein- Based Accessory Phytomedicine.

BACKGROUND: Calotropis procera is a laticiferous plant (Apocynaceae) found in tropical regions all over the world. The ultrastructural characteristics of laticifers, their restricted distribution among different taxonomic groups, and in some species in each clade, as peptidases from latex, make them very attractive for biological analysis. OBJECTIVE: The study aims to investigate the effects of LP-PII-IAA (laticifer protein (LP) sub-fraction II (PII) of C. procera presenting an iodoacetamide-inhibited cysteine proteinase activity) on irinotecan-induced intestinal mucositis, a serious adverse effect of this medicine for the treatment of cancer. METHODS: LP-PII-IAA is composed of closely related isoforms (90%) of peptidases derived from catalysis and an osmotin protein (5%). Animals receiving co-administration of LP-PII-IAA presented a significant decrease in mortality, absence of diarrhea, histological preservation, and normalization of intestinal functions. RESULTS: Clinical homeostasis was accompanied by a reduction in MPO activity and declined levels of IL-1ß, IL-6 and KC, while the IL-10 level increased in LP-PII-IAA-treated animals. COX-2 and NF-kB immunostaining was reduced and the levels of oxidative markers (GSH, MDA) were normalized in animals that received LP-PII-IAA. CONCLUSION: We suggest that peptidases from the latex of Calotropis procera were instrumental in the suppression of the adverse clinical and physiological effects of irinotecan.

Anti-inflammatory and antioxidant effect of methanol extract of latex of Calotropis procera in rat model of colorectal cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: Preparations derived from the plant Calotropis procera, have been used for medicinal purpose though the plant is known for its toxic effects. The aerial parts of the plant contain latex in plenty and have been found effective in treating disorders of gastrointestinal system and cancer. AIM OF THE STUDY: This study evaluated the efficacy of C. procera dried latex extract prepared in methanol (MeDL) against inflammation and oxidative stress in experimental model of colorectal carcinoma (CRC). MATERIALS AND METHODS: Two subcutaneous injections of chemical carcinogen, 1,2-dimethylhydrazine (DMH; 150 mg/kg) were given at an interval of one week to induce CRC in rats. The MeDL (50 and 150 mg/kg) and aspirin (60 mg/kg) were given daily and their effect was evaluated on markers of oxidative stress and inflammation after completion of 8 weeks following second injection of carcinogen. A comparison was made with normal and experimental control groups. The colon tissue levels of glutathione (GSH), thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), nitrite and myeloperoxidase (MPO) were determined. Enzyme-linked immunosorbent assay was performed to determine the levels of prostaglandin E2 (PGE2) and tumor necrosis factor-alpha (TNF-α) and immunohistochemical analysis was performed for IL-1ß. RESULTS: Induction of cancerous changes in the colon resulted in altered oxidative homeostasis as evident from a reduction in GSH level and SOD activity and rise in TBARS level when compared with normal rats. Elevated levels of nitrite, MPO, TNF-α, PGE2 and immunoreactivity of IL-1ß were also observed in these rats. The levels of these markers were normalized when the rats were treated with MeDL or anti-inflammatory drug, aspirin. CONCLUSION: This study demonstrates that suppression of oxidative stress and inflammation contributes to the beneficial effect of MeDL in rat model of colon carcinogenesis.

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.

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.

Autoproteolysis of Procerain and Procerain B mediated by structural changes.

Procerain (Pc) and Procerain B (PcB) are two latex proteases from Calotropis procera having potential applications in food and other industries. However, autolytic degradation of these proteases limits their potential use in industry. Nevertheless, basic mechanism underlying the autoproteolysis has not been detailed. In order to understand the same, we subjected the enzymes to various denaturing and activating conditions. The results showed that structural changes induced by different denaturing conditions trigger their autoproteolysis. We also observed differential response of Pc, PcB and other papain-like proteases towards autocatalysis in presence of reducing agent in-spite of sharing the same structural fold, including the number of disulfide bonds. The possible reason underlying this intriguing observation is also discussed. Further, present work establishes that structural changes in the proteases lead to autoproteolysis and the enzymes are stable unless they experience structural perturbation. These findings could thus be useful for their practical applications in industries.

Protective effect of methanol extract of latex of Calotropis procera in an experimental model of colorectal cancer.

ETHNOPHARMACOLOGICAL RELEVANCE: The plant, Calotropis procera, has been used for treating various gastrointestinal disorders and cancer. Some of these medicinal properties have been attributed to the latex produced by the plant. AIM OF THE STUDY: To evaluate the efficacy of methanol extract of air-dried latex (MeDL) of C. procera in the rat model of colorectal cancer (CRC). MATERIALS AND METHODS: CRC was induced in the rats by 1,2-dimethylhydrazine (DMH) and the effect of MeDL was evaluated at two doses (50 and 150 mg/kg). MeDL and reference drug aspirin (60 mg/kg) were administered orally starting from 1 h before injecting DMH till 8 weeks after the second dose of DMH. The study also included experimental and normal control groups. Microscopic analysis was carried out to determine the count for aberrant crypt foci (ACF) and histology score whereas enzyme-linked immunosorbent assay and immunohistochemical analyses were performed for markers of carcinogenesis and angiogenesis. Other parameters that were evaluated include deoxyribonucleic acid (DNA) fragmentation, laddering, Bcl2 and Bax immunoreactivity, and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) positivity. RESULTS: Subcutaneous injection of DMH induced pre-neoplastic changes in the colon of rats with the appearance of ACF with multiple crypts (1-3, 4-6 or >6). In the experimental control group, total ACF count was 3.49 ± 0.23/cm of the colon length and the median histology score was 2.0 for architectural abnormalities, 2.0 for dilatation of crypts and 1.5 for hyperplasia/dysplasia against 1.0 for all the characteristics in normal rats. Oral administration of MeDL similar to aspirin, led to a reduction in ACF count and histology score of CRC concomitant with a decrease in the levels of markers of carcinogenesis - ß-catenin and proliferating cell nuclear antigen (PCNA); markers of angiogenesis - matrix metallopeptidase-9 (MMP-9) and vascular endothelial growth factor (VEGF), and an increase in apoptotic DNA fragmentation. CONCLUSION: MeDL confers protection in the rat model of CRC and the study suggests its therapeutic potential in this condition.