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.

Cysteine protease I29 propeptide from Calotropis procera R. Br. As a potent cathepsin L inhibitor and its suppressive activity in breast cancer metastasis.

Breast cancer metastasis is associated with a poor prognosis and a high rate of mortality. Cathepsin L (CTSL) is a lysosomal cysteine protease that promotes tumor metastasis by degrading the extracellular matrix. Gene set enrichment analysis revealed that CTSL expression was higher in tumorous than in non-tumorous tissues of breast cancer patients and that high-level CTSL expression correlated positively with the epithelial-mesenchymal transition. Therefore, we hypothesized that inhibiting CTSL activity in tumor cells would prevent metastasis. In this study, we characterized the inhibitory activity of SnuCalCpI15, the I29 domain of a CTSL-like cysteine protease from Calotropis procera R. Br., and revealed that the propeptide stereoselectively inhibited CTSL in a reversible slow-binding manner, with an inhibitory constant (Ki) value of 1.38 ± 0.71 nM, indicating its potency as an exogenous inhibitor in anti-cancer therapy. SnuCalCpI15 was localized intracellularly in MDA-MB-231 breast cancer cells and suppressed tumor cell migration and invasion. These results demonstrate the potential of SnuCalCpI15 as a novel agent to prevent breast cancer metastasis.

Biogenic synthesis and physicochemical characterization of metal nanoparticles based on Calotropis procera as promising sustainable materials against skin cancer.

The UAE harbors a rich diversity of wild medicinal plants, such as Calotropis procera (CP), that are renowned for their extensive use in traditional medicine due to their abundance of bioactive phytochemicals. Zinc and iron metals possess significant pharmacological effects including antioxidant and anticancer properties. In this study, nanoparticles (NPs) containing zinc and iron were green synthesized utilizing ethanolic and aqueous extracts of CP aerial parts. UV-Vis spectra revealed absorption peaks around 270-275 nm, while FT-IR analysis confirmed successful coating of the NPs with plant's phytochemicals. SEM/EDX analysis indicated a more potent reducing effect of the aqueous extract, whereas the alcoholic extract demonstrated more effective coating of the NPs. DLS showed monodispersed NPs with average sizes of 32.67-202 nm. The alcoholic extract-based zinc and iron NPs exhibited the highest phenolic and flavonoid contents (51.06 ± 2.82 µg of GAE/mg of DW and 66.26 ± 1.12 µg of Qu/mg of DW, respectively) and the strongest antioxidant effect against ABTS and DPPH radicals (IC50 = 52.81 and 148.46 µg/mL, respectively). The aqueous extract-based zinc NPs demonstrated the greatest cytotoxicity against A-431 cell lines (IC50 = 188.97 µg/mL). The findings highlight promising potential of these sustainable materials for therapeutic applications, indicating a need for continued research and development in this area.

Healing of leprosy-associated chronic plantar wounds with a novel biomembrane containing latex proteins from Calotropis procera.

Pre-clinical assays demonstrated that a 1% polyvinyl alcohol biomembrane containing latex proteins (10%) from the medicinal plant Calotropis procera was biocompatible and stimulated healing of incisional and excisional wounds in murine models, and the mechanistic aspects were established. The efficacy of the biomembrane (BioMemCpLP) to promote healing of chronic ulcers in leprosy patients was investigated. The study started with 28 volunteers. Five were excluded later due to different disconformities. Ulcers from 15 patients were continuously treated with BioMemCpLP for 56 days. Five patients were treated only with silver sulfadiazine and three patients received plain hydrocolloid wound dressings with high absorption capacity. In all cases, wound dressings were renewed three times a week for 56 days and ulcers were evaluated weekly for contraction and healing progress. The extent of the healed area in the ulcers treated with BioMemCpLP was greater than in the control groups. Approximately 88% of ulcers treated with BioMemCpLP were fully healed before day 56, against 6% in both control groups. This result was not correlated with age/gender, duration or location of ulcers, deformity or whether or not the patient was cured of leprosy. The results showed that BioMemCpLP was beneficial for treatment of ulcers suffered by leprosy patients without noticeable side effects.

Optimizing antimicrobial synergy: Green synthesis of silver nanoparticles from Calotropis gigantea leaves enhanced by patchouli oil.

Silver nanoparticles (AgNPs) synthesized from plant extracts have gained attention for their potential applications in biomedicine. Calotropis gigantea has been utilized to synthesize AgNPs, called AgNPs-LCg, and exhibit antibacterial activities against both Gram-positive and Gram-negative bacteria as well as antifungal. However, further enhancement of their antimicrobial properties is needed. The aim of this study was to synthesize AgNPs-LCg and to enhance their antimicrobial and antifungal activities through a hybrid green synthesis reaction using patchouli oil (PO), as well as to characterize the synthesized AgNPs-LCg. Optimization was conducted using the response surface method (RSM) with a central composite design (CCD). AgNPs-LCg were synthesized under optimal conditions and hybridized with different forms of PO-crude, distillation wastewater (hydrolate), and heavy and light fractions-resulting in PO-AgNPs-LCg, PH-AgNPs-LCg, LP-AgNPs-LCg, and HP-AgNPs-LCg, respectively. The samples were then tested for their antibacterial (both Gram-positive and Gram-negative bacteria) and antifungal activities. Our data indicated that all samples, including those with distillation wastewater, had enhanced antimicrobial activity. HP-AgNPs-LCg, however, had the highest efficacy; therefore, only HP-AgNPs-LCg proceeded to the characterization stage for comparison with AgNPs-LCg. UV-Vis spectrophotometry indicated surface plasmon resonance (SPR) peaks at 400 nm for AgNPs-LCg and 360 nm for HP-AgNPs-LCg. The Fourier-transform infrared spectroscopy (FTIR) analysis confirmed the presence of O-H, N-H, and C-H groups in C. gigantea extract and AgNP samples. The smallest AgNPs-LCg were 56 nm, indicating successful RSM optimization. Scanning electron microscopy (SEM) analysis revealed spherical AgNPs-LCg and primarily cubic HP-AgNPs-LCg, with energy-dispersive X-ray spectroscopy (EDX) confirming silver's predominance. This study demonstrated that PO in any form significantly enhances the antimicrobial properties of AgNPs-LCg. The findings pave the way for the exploration of enhanced and environmentally sustainable antimicrobial agents, capitalizing on the natural resources found in Aceh Province, Indonesia.

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.

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.

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.

Efficacy of maturase K and rpL20 protein extracted from C. procera leaves on Anophelesstephensi.

The present work is carried out to protein isolation, purification, and characterization from leaves, stem, and seed of C. procera and to evaluate the larvicidal potential on Anopheles stephensi. The whole protein was isolated using protein extraction buffer and precipitated by ammonium sulphate and larvicidal active protein was purified by the column chromatography. The homogeneity of larvicidal protein was confirmed by the SDS-PAGE. The identification of protein was done by the HPLC and LC-MS/ESI-MS. The crude protein from leaves showed 100% mortality of 3rd instar larvae of An. stephensi at the concentration of 5.5 mg/ml after 24 h of exposure. The crude protein from stem showed 25% mortality and no mortality observed was observed in seed protein. The leaves crude protein was further purified by ion exchange chromatography and eluted fractions were tested for larvicidal potential. The purified single protein fractions L2 and L3 from C. procera leaves showed 100% mortality at concentration of 0.06 mg/ml. The homogeneity of purified protein was confirmed by SDS-PAGE and two bands of 26 kDa and 15 kDa protein were observed. The peptide sequence "R.SQMLENSFLIENVMKR.L" was identified in the trypsin digested homogenous protein fraction L2 and "R.DRGSQKR.N" peptide sequence in L3 fraction by LC-MS/ESI-MS. The CprL2 peptide showed the sequence similarity with the protein maturase K and CprL3 peptide showed the sequence similarity with ribosomal protein L20 of C. procera. The conserved functional domain was also identified in both the CprL2 and CprL3 peptide. The identified proteins showed strong larvicidal efficacy at very low concentration. The identified proteins are novel and natural larvicidal agents against An. stephensi and hence can be used to control the malaria.

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.

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 (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.

Phytochemical analysis, anti-inflammatory, antioxidant activity of Calotropis gigantea and its therapeutic applications.

ETHNOPHARMACOLOGICAL RELEVANCE: Herbal remedies can be used to treat a variety of chronic inflammatory illnesses, like rheumatoid arthritis and leprosy. The plant Calotropis gigantea (C. gigantea) belongs to the family Apocynaceae. To treat numerous contagious diseases, C. gigantea is utilized alone or combine with certain medicinal herbs. Traditional Asian and African practitioners employed C. gigantea to treat a variety of inflammatory conditions like boils, rheumatoid arthritis, gout, leprosy and other disorders. AIM OF THE STUDY: The goal of this study is to examine the anti-inflammatory and antioxidant activities of C. gigantea leaf extracts extracted using methanol, petroleum ether, and water. MATERIALS AND METHODS: The leaf extracts of C. gigantea were obtained using the Soxhlet extraction technique. The phytoconstituents present in all three C. gigantea leaf extracts were confirmed by qualitative analysis, and the amounts of the alkaloids, flavonoids, terpenoids and phenols found in the extracts were quantified. C. gigantea crude extracts were subjected to a nitric oxide scavenging experiment to assess their free radical scavenging activities. Protein denaturation and proteinase inhibition assays were used to investigate the effectiveness of extracts to restrict denaturation of protein and to inhibit key enzymes responsible for tissue damage. Further, the membrane stabilization efficacy of plant extracts were examined by the heat-induced hemolysis method. The DPPH and FRAP experiments were performed to determine the antioxidant effectiveness of phytoconstituents extracted using different solvents. The GC-MS study of plant C. gigantea methanolic, aqueous and petroleum ether extracts displayed a broad range of compounds that possess beneficial therapeutic effects. RESULTS: This study reveals that the methanolic extract of C. gigantea provides significantly more anti-inflammatory and antioxidant activity than other extracts. CONCLUSION: Compared to the aqueous and petroleum ether extracts, the methanolic leaf extract of C. gigantea demonstrated greater in vitro anti-inflammatory and antioxidant properties.

Purification, Partial Characterization, and Evaluation of the Antiulcer Activity of Calotropis procera Leaf Lectin.

BACKGROUND: Lectins are proteins with therapeutic and diagnostic potential that can be applied in battling various ailments. AIM AND OBJECTIVE: This study was designed to purify and characterize the hemagglutinating activity derived from the leaves of Calotropis procera and its possible role in protecting the stomach against ethanol-induced lesions. METHODS: The Calotropis procera leaf lectin (ProLec), was isolated by homogenization of the defatted leaf powder in Phosphate-Buffered Saline (PBS) and purified by affinity chromatography on Sephadex G-100. The lectin was eluted from the affinity column by 3% acetic acid and was physicochemically characterized. In a dose-dependent manner, ProLec was administered to rats with ethanol-induced ulcers, and biochemical, histopathological, and toxicological examinations were performed. RESULTS: ProLec is a heterodimer of 75 and 68 kDa. It agglutinated all human RBCs, whereas it showed weak interaction with animal erythrocytes. The protein was optimally active at 25 °C and was labile above this temperature. ProLec exhibited two pH optima and was a metalloprotein requiring Ca, Mn, and Ni. It contains 1.6% tryptophan residues of which about 1% is exposed and critical for lectin activity. The lectin exhibited a potent gastroprotective effect against ethanolinduced gastric lesions with no apparent toxicity to both kidneys and liver. Examination of the pH of the gastric juice of lectin-treated animals indicated a possible role of lectin in maintaining stomach acidity within the normal ranges compared to the gastric juice pH of animals that received ethanol only. CONCLUSION: These results may suggest that ProLec could conceivably be a good future drug for the treatment of gastric ulcers, however, extensive immunological and toxicological research remains to be done.

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.

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.

Recombinant osmotin inclusion bodies from Calotropis procera produced in E. coli BL21(DE3) prevent acute inflammation in a mouse model of listeriosis.

BACKGROUND: The osmotin from the medicinal plant Calotropis procera (CpOsm) has characteristics similar to adiponectin, a human protein with immunoregulatory actions. PURPOSE: This study aimed to investigate whether recombinant osmotin inclusion bodies from C. procera (IB/rCpOsm) produced in E. coli BL21(DE3) can prevent infection-induced inflammation. A virulent strain of Listeria monocytogenes was used as an infection model. METHODS: Cells of E. coli BL21(DE3) carrying the plasmid pET303-CpOsm were used to express the recombinant osmotin, which accumulated at reasonable levels as inclusion bodies (IB/rCpOsm). IB/rCpOsm were purified from induced cells and SDS-polyacrylamide gel electrophoresis followed by mass spectrometry analyses confirmed the identity of the major protein band (23 kDa apparent molecular mass) as CpOsm. Peritoneal macrophages (pMØ) from Swiss mice were cultured with IB/rCpOsm (1 or 10 µg/ml) in 96-well plates and then infected with L. monocytogenes. IB/rCpOsm (0.1, 1 or 10 mg/kg) was also administered intravenously to Swiss mice, which were then infected intraperitoneally with L. monocytogenes. RESULTS: Pretreatment of the pMØ with IB/rCpOsm significantly increased cell viability after infection and reduced the intracellular bacterial load. The infiltration of neutrophils into the peritoneal cavity of mice pretreated with IB/rCpOsm at 10 mg/kg (but not 0.1 and 1 mg/kg) was reduced after infection. In these mice, the bacterial load was high in the peritoneal fluid and the liver, but histological damage was discrete. The treatments with IB/rCpOsm at 10 mg/kg significantly increased the expression of the anti-inflammatory cytokine IL-10. CONCLUSION: This study shows that recombinant osmotin inclusion bodies from C. procera were bioactive and prompted anti-inflammatory actions at therapeutic dosages in the L. monocytogenes infection model.

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.

Anti-migraine activity of freeze-dried latex obtained from Calotropis gigantea Linn.

Migraine which is characterized by a pulsating headache affected an estimated population of 12% worldwide. Herbal products like latex derived from Calotropis gigantea R. Br. (Asclepiadaceae) are a representative intervention to treat migraine traditionally. However, post-harvesting stability issues of latex affect its biological potential. Freeze-drying has been successfully employed for the encapsulation of herbal bioactive compounds resulting in stable dried preparations. Latex derived from Calotropis gigantea (C. gigantea) was microencapsulated using chitosan by freeze-drying (FDCG) method and compared with sun ray-dried latex (ADCG). Current investigation was aimed to improve the shelf life of latex by freeze-drying microencapsulation technique and evaluation of its anti-migraine potential. Dried latex powders (ADCG and FDCG) were evaluated in terms of phenolic content, coloring strength, first-order kinetic, color parameters (L*, a*, b*, C*, and E*), moisture, water activity, solubility, and hygroscopicity. Additionally, apomorphine-induced climbing behavior, L-5-HTP-induced syndrome, and MK-801-induced hyperactivity were used to evaluate the anti-migraine potential of powdered latex. FDCG showed good physicochemical properties due to its higher concentration of phenolic and flavonoid contents. Moreover, FDCG significantly reduced the apomorphine-induced climbing behavior, L-5-HTP-induced syndrome, and MK-801-induced hyperactivity in a dose-dependent manner through an interaction of dopaminergic and serotonergic receptors. In conclusion, the method developed for shelf life improvement of latex offered maximum protection over a period of 10 weeks with retaining its natural biological potential; thus, it can be effectively utilized in the treatment or management of migraine. Anti-migraine effect of Calotropis gigantea freeze-dried latex by inhibition of dopamine and serotonin receptors (D1 and D2: dopamine receptors; 5-HT: serotonin receptors); yellow color represents serotonergic, and blue color indicates dopaminergic neurons.

Adhatoda vasica and Calotropis procera as a resource of novel chemical compounds, their biological bioluminescence assay, and investigation of morphological features of bacterial growth through advanced technologies.

This article reports the three principal groups of compounds for the first time from Adhatoda vasica and Calotropis procera plants species using nuclear magnetic resonance methods in which aliphatic, oxy heterocyclic, and tannins compounds were detected from these plants. The leaves of both species were subjected to testing tyrosinase inhibition and antioxidant activities. ATP bioluminescence use for indirect measurement of the amount of organic residue on the surface of the leaves that provide support to microbial growth. The distinguishing characteristics and intraoperative findings of bacterial diseases involved in treatments were conducted against the positive and negative microbial strains using a scanning electron microscope (SEM). The methanolic extracts of leaves of both species were applied to bacterial strains through broth microdilution method to determine the minimum inhabitation concentrations (MICs) for both species. It was concluded that both plants are a rich resource of bioactive compounds. Their extract may also be used to treat various bacterial diseases and in drug manufacturing. HIGHLIGHTS: New chemical compounds of oxy-heterocyclic, aliphatic, and tannins derivatives are isolated from herbal plants as a source of various drugs. 1 H NMR spectrum and 13 C NMR spectrum of each new derivate were calculated. NMR-spectral analysis of new compound of chemistry class was studied and further applied in various bacterial strains. Tyrosinase inhibition property of bacteria strains by application of active compounds on these strains. Agar overlay bioassays were used to evaluate intercellular morphological features of strains applied on extracts by electron microscope (SEM). a-Glucosidase inhibition assay determined with antioxidants activity through FRAP assay methods.