Tropical lichen, Dirinaria consimilis, induces ROS-mediated activation of MAPKs and triggers caspase cascade mediated apoptosis in brain and cervical cancer cells.

Lichens are the symbiotic association between photobiont algae and mycobiont fungi having diverse phytochemicals. However, they are still an underexplored natural resource for biological activities. In the present report, we have evaluated the anti-brain and anti-cervical cancer activity of tropical lichen, Dirinaria consimilis (DCME) through the cell viability assay, cell cycle analysis, annexin V-FLUOS staining, morphological analysis, ROS-induction mechanism, evaluation of antioxidant levels, and western blotting study. The WST-1-based cell viability assay showed the cytotoxic nature of DCME towards U87 (IC50-52.65 ± 1.04 µg/ml) and HeLa (IC50-77.60 ± 2.23 µg/ml) cells. Interestingly, DCME does not showed any toxicity towards non-malignant fibroblast cell line WI-38 (IC50-685.80 ± 19.51 µg/ml). Furthermore, the cell cycle analysis showed sub-G1 arrest (apoptosis), and annexin V-FLUOS staining showed an increase in early apoptosis population dose-dependently. Confocal-based morphological data confirmed the DNA condensation and fragmentation upon treatment. Furthermore, DCME treatment induces ROS and regulates the levels of antioxidant enzymes (SOD, Catalase, GST, and GSH) in both U87 and HeLa cells. Finally, the western blotting data revealed the increase in Bax/Bcl-2 ratio, activation of Bid, Caspase-8, -9 and -3 along with degradation of PARP. Moreover, regulation of MAP kinases and activation of p53 was also observed upon DCME treatment. Herein, we first reported the anticancer activity of D. consimilis against brain and cervical cancer cells. Performed in-depth anticancer study revealed the ROS-mediated regulation of MAP kinases and activation of caspase cascade in U87 and HeLa cells upon DCME treatment.

The natural flavones, acacetin and apigenin, induce Cdk-Cyclin mediated G2/M phase arrest and trigger ROS-mediated apoptosis in glioblastoma cells.

Brain and CNS-related cancers are rare; however, 0.3 million incidences and 0.24 million deaths in 2018 demonstrates the unrelenting associated dangers. Glioblastoma is a brain cancer of star-shaped glial cells. It is almost universally fatal within 2 years of diagnosis despite maximal medical therapies. This study aims to evaluate the in-depth anticancer activity of acacetin and apigenin on glioblastoma cells (U87). In the present report, we have isolated two flavonoids, acacetin and apigenin; and studied the in-depth anticancer activity on U87 cells. Selective cytotoxicity of acacetin and apigenin was observed towards the U87 cells (IC50: 43.73 ± 1.19 and 48.18 ± 1.37 µM, respectively). The flow cytometer-based result revealed the induction of G2/M phase arrest along with the increase in sub G1 population upon compound treatment. Annexin-V-FLUOS and DAPI staining also confirmed the apoptosis-inducing effects of compounds. Flow cytometer and confocal microscopy-based DCFH-DA staining showed ROS-inducing effect of the compounds. The up-regulation of p21 and down-regulation of Cyclin-A1, Cyclin-B1, and Cdk-1 revealed the G2/M phase arrest mechanism of acacetin and apigenin. Furthermore, western blotting result confirmed the activation of intrinsic pathway of apoptosis upon acacetin treatment and activation of both extrinsic and intrinsic pathways of apoptosis upon apigenin treatment through the regulation of Bax, t-Bid, caspase 8, caspase 9, caspase 3, and PARP. The obtained result showed a significant effect (P < 0.05) of acacetin and apigenin on U87 cells. Acacetin and apigenin-induced ROS is responsible for the induction of cell cycle arrest and activation of caspase-cascade pathways in U87 cells.

A natural antioxidant, tannic acid mitigates iron-overload induced hepatotoxicity in Swiss albino mice through ROS regulation.

Tannic acid (TA), a water soluble natural polyphenol with 8 gallic acids groups, is abundantly present in various medicinal plants. Previously TA has been investigated for its antimicrobial and antifungal properties. Being a large polyphenol, TA chelates more than 1 metal. Hence TA has been explored for potent antioxidant activities against reactive oxygen species (ROS), reactive nitrogen species (RNS) and as iron chelator in vitro thereby mitigating iron-overload induced hepatotoxicity in vivo. Iron dextran was injected intraperitoneally in Swiss albino mice to induce iron-overload triggered hepatotoxicity, followed by oral administration of TA for remediation. After treatment, liver, spleen, and blood samples were processed from sacrificed animals. The liver iron, serum ferritin, serum markers, ROS, liver antioxidant status, and liver damage parameters were assessed, followed by histopathology and protein expression studies. Our results show that TA is a prominent ROS and RNS scavenger as well as iron chelator in vitro. It also reversed the ROS levels in vivo and restricted the liver damage parameters as compared to the standard drug, desirox. Moreover, this natural polyphenol exclusively ameliorates the histopathological and fibrotic changes in liver sections reducing the iron-overload, along with chelation of liver iron and normalization of serum ferritin. The protective role of TA against iron-overload induced apoptosis in liver was further supported by changed levels of caspase 3, PARP as well as Bax/BCl-2 ratio. Thus, TA can be envisaged as a better orally administrable iron chelator to reduce iron-overload induced hepatotoxicity through ROS regulation.

Glycoside rich fraction from Spondias pinnata bark ameliorate iron overload induced oxidative stress and hepatic damage in Swiss albino mice.

BACKGROUND: Iron in the overloaded condition in liver promotes the overproduction of free radicals that lead to oxidative stress and ultimately hepatic damage. The present study was designed to evaluate the ameliorating potential from iron overloaded hepatotoxicity by the glycosidic fraction from Spondious pinnata bark (SPW1) along with its antioxidant property. METHODS: The fraction was tested for its in vitro antioxidant, free radical scavenging property and iron chelation potential via standard biochemical assays. Iron overload condition was generated by the intraperitoneal administration of iron dextran in mice. The levels of serum enzymes, antioxidant enzymes in liver, markers of hepatic damage, liver iron, and ferritin content were measured in response to the oral treatment of SPW1. Histopathology of the liver sections was performed for visual confirmation of the amelioration potential of SPW1. RESULTS: The fraction exhibited excellent in vitro antioxidant as well as free radical scavenging potential against both reactive oxygen species and reactive nitrogen species. Administration of SPW1 significantly normalized the disturbed levels of antioxidant enzymes, liver iron, lipid peroxidation, liver fibrosis, serum enzyme and ferritin better than standard desirox which were also supported by the morphological study of the liver sections. Phytochemical analysis as well as HPLC study, confirmed that the fraction mainly consisted of glycosidic phenolics and flavonoids that attributed to its biological activities. CONCLUSIONS: The above results suggested that beneficial effects of SPW1 on iron overload induced hepatotoxicity that can be considered as a possible candidate against iron overload diseases.

Evaluation of anticancer activity of Clerodendrum viscosum leaves against breast carcinoma.

INTRODUCTION: The use of natural resources as medicines for cancer therapies has been described throughout history in the form of traditional medicines. However, many resources are still unidentified for their potent biological activities. Clerodendrum viscosum is a hill glory bower reported as a remedy against oxidative stress, skin diseases, and intestinal infections. MATERIALS AND METHODS: We have collected the C. viscosum leaves and used for the preparation of 70% methanolic extract (CVLME). Then, CVLME has been confirmed for anticancer properties on various cancer cell lines by evaluating cytotoxicity, cell cycle analysis, induction of ROS and apoptosis, and nuclear fragmentation. Further, the phytochemical analysis of CVLME was evaluated through high-performance liquid chromatography. RESULTS: Cell proliferation assay revealed the selective cytotoxicity of CVLME against breast cancer cell line (MCF-7). The FACS-based cell cycle analysis showed increased subG1 (apoptosis) population dose dependently. Further, the apoptosis-inducing effect of CVLME was confirmed by annexin staining. Flow cytometry and confocal microscopy revealed the selective ROS generation upon CVLME treatment. The confocal-based morphological study also revealed condensed and fragmented nuclear structure in CVLME-treated MCF-7 cells. Phytochemical investigations further indicated the presence of tannic acid, catechin, rutin, and reserpine which might be the reason for the anticancer activity of CVLME. CONCLUSION: The above-combined results revealed the anticancer effect of CVLME, which may be due to the selective induction of ROS in breast carcinoma.

Ameliorating effects of white mulberry on iron-overload-induced oxidative stress and liver fibrosis in Swiss albino mice.

Excess iron causes oxidative damage of biomolecules, leading to tissue injury primarily liver failure. In this study, we explored the remediating effects of Morus alba L. (MAME) on iron-overload-induced oxidative stress and liver injury in mice. The In vitro study revealed the antioxidant and free radical scavenging properties of MAME. Intraperitoneal injection of iron-dextran was administered in Swiss albino mice to induce iron-overload condition and the mice were further treated with MAME. MAME treatment significantly decreased liver iron, serum ferritin level, oxidative stress, and restored serum parameters and liver antioxidants. Moreover, biochemical and histopathological analyses confirmed the alleviated liver damage and fibrosis upon MAME treatment. The protective effect of MAME against iron-overload-induced apoptosis was confirmed by upregulation of protein levels of Bax, Caspase-3, and PARP. The treatment also affected the expression of MAPKs (ERK, JNK, and p38). GC-MS analysis revealed the presence of various bioactive phytochemicals in MAME that may be responsible for ameliorating effects of excess iron. Thus MAME can be envisaged as an effective iron chelator in the treatment of iron-overload-induced liver injury and fibrosis.

Potent anti-inflammatory Terminalia chebula fruit showed in vitro anticancer activity on lung and breast carcinoma cells through the regulation of Bax/Bcl-2 and caspase-cascade pathways.

The present study was aimed to investigate the anticancer and anti-inflammatory activities of Terminalia chebula fruit (TCME). The TCME was evaluated for in vitro anticancer activity on A549 and MCF-7 cells. TCME showed cytotoxicity toward A549 (IC50 - 359.06 ± 20.04 µg/ml), and MCF-7 (IC50 - 61.02 ± 5.55 µg/ml) cells. The flow-cytometer analysis revealed increase in sub G1 population and apoptotic population, which were observed through cell cycle analysis and annexin-V-FLUOS staining. Confocal microscopy showed DNA fragmentation in both the cell lines upon TCME treatment. Moreover, TCME treatment induces activation of apoptosis-related caspase-cascade pathways in both the cell lines. TCME treatment on RAW 264.7 cells revealed the anti-inflammatory properties by regulating nitrite and TNF-α production; iNOS, COX-2 levels, and translocation of NF-κB protein. Finally, HPLC analysis revealed the bioactive phytocompounds present in TCME. In conclusion, the combined results showed the potent anticancer and anti-inflammatory properties of T. chebula fruit. PRACTICAL APPLICATIONS: Lung cancer is a leading cause of death in men with 35.5% incidences and 30.8% mortality rate worldwide. On the contrary, breast cancer possesses 55.2% incidences and 16.6% mortality rate among the female worldwide. The present findings revealed the anti-lung and -breast cancer activity along with the potent anti-inflammatory potentials of Terminalia chebula fruit. These findings will helpful to isolate the active drug molecules from the Terminalia chebula fruit and mark them as an anticancer and anti-inflammatory agent.

An ellagic acid isolated from Clerodendrum viscosum leaves ameliorates iron-overload induced hepatotoxicity in Swiss albino mice through inhibition of oxidative stress and the apoptotic pathway.

Iron is a vital element required for normal cellular physiology in animal systems, but excess iron accumulation in the biological system accelerates oxidative stress, cellular toxicity, tissue injury and organ fibrosis, which ultimately leads to the generation of chronic liver diseases including cancer. A natural antioxidant, ellagic acid (EA) has been previously reported for its pharmacological properties; however, there is no significant evidence available that could illustrate its protective potential against iron-overload induced hepatotoxicity. In the present work, EA was evaluated for its in vitro free radical scavenging and iron chelation potentials. Further, EA was tested in vivo for its protective activity against iron overload-induced hepatotoxicity in Swiss albino mice by evaluating liver iron content, reactive oxygen species (ROS), liver antioxidant enzymes, serum marker levels, liver damage and fibrosis, histopathological study and finally western blotting analysis. EA treatment significantly decreased liver iron and serum ferritin levels. Elevated ROS levels, decreased antioxidant parameters and elevated serum markers were normalized upon treatment with EA. Cellular morphology, iron -overload and liver fibrosis were found to be effectively ameliorated. Finally, the protective effect of EA against iron overload-induced apoptosis was confirmed by western blotting when its treatment upregulated the expressions of caspase-3 and poly(ADP-ribose) polymerase (PARP) proteins. EA revealed hepatoprotective activity against iron overload-induced toxicity through scavenging free radicals, inhibiting excess ROS production, normalizing liver damage parameters and upregulating caspase-3, PARP expression. Collectively, our findings support the possible use of the natural antioxidant EA as a promising candidate against iron-overloaded diseases.

In vitro Antioxidant and Antiproliferative Activities of Various Solvent Fractions from Clerodendrum viscosum Leaves.

BACKGROUND: Free radicals such as reactive oxygen and nitrogen species, generated in the body, play an important role in the fulfillment of various physiological functions but their imbalance in the body lead to cellular injury and various clinical disorders such as cancer, neurodegenaration, and inflammation. OBJECTIVE: The objective of this study is to fight this problem, natural antioxidant from plants can be considered as possible protective agents against various diseases such as cancer which might also modify the redox microenvironment to reduce the genetic instability. This study was designed to evaluate the antioxidant and antiproliferative potential of Clerodendrum viscosum fractions against various carcinomas. MATERIALS AND METHODS: In this present study, 70% methanolic extract of C. viscosum leaves have been fractionated to obtain hexane, chloroform, ethyl acetate, butanol, and water fractions, which were tested for their antioxidant and anticancer properties. RESULTS: It was observed that chloroform and ethyl acetate fractions showed good free radical scavenging properties as well as inhibited the proliferation of human lung cancer (A459), breast (MCF-7), and brain (U87) cells. Moreover, they arrested the cell cycle at G2/M phase of breast and brain cancer. These inhibitory effects were further confirmed by bromodeoxyuridine uptake imaging. Phytochemical investigations further indicate the presence of tannic acid, quercetin, ellagic caid, gallic acid, reserpine, and methyl gallate which might be the reason for these fractions' antioxidant and antiproliferative activities. CONCLUSION: Clerodendrum viscosum leaf chloroform and Clerodendrum viscosum leaf ethyl acetate fractions from C. viscosum showed good reactive oxygen species and reactive nitrogen species scavenging potential. Both the fractions arrested cell cycle at G2/M phase in MCF-7 and U87 cells which lead to induce apoptosis. SUMMARY: Crude extract of Clerodendrum viscosum leaves was fractionated using different solventsAmong them, chloroform and ethyl acetate fractions exhibited excellent free radical scavenging propertiesThe same fractions inhibited the proliferation of human lung cancer (A459), breast (MCF-7), and brain (U87) cellsChloroform and ethyl acetate fractions arrested the cell cycle at G2/M phase of breast and brain cancerPhytochemical investigations further indicate the presence of several bioactive principles present in them. Abbreviations used: CVLME: Clerodendrum viscosum leaf methanolic extract; CVLH: Clerodendrum viscosum leaf hexane; CVLC: Clerodendrum viscosum leaf chloroform; CVLE: Clerodendrum viscosum leaf ethyl acetate; CVLB: Clerodendrum viscosum leaf butanol; CVLW: Clerodendrum viscosum leaf water; BrdU: Bromodeoxyuridine; WST-1: Water soluble tetrazolium salt.