Aman Shah Abdul Majid

Scopoletin, an active principle of tree tobacco (Nicotiana glauca) inhibits human tumor vascularization in xenograft models and modulates ERK1, VEGF-A, and FGF-2 in computer model

We recently reported the antineovascularization effect of scopoletin on rat aorta and identified its potential anti-angiogenic activity. Scopoletin could be useful as a systemic chemotherapeutic agent against angiogenesis-dependent malignancies if its antitumorigenic activity is investigated and scientifically proven using a suitable human tumor xenograft model. In the present study, bioassay-guided (anti-angiogenesis) phytochemical investigation was conducted on Nicotiana glauca extract which led to the isolation of scopoletin. Further, anti-angiogenic activity of scopoletin was characterized using ex vivo, in vivo and in silico angiogenesis models. Finally, the antitumorigenic efficacy of scopoletin was studied in human colorectal tumor xenograft model using athymic nude mice. For the first time, an in vivo anticancer activity of scopoletin was reported and characterized using xenograft models. Scopoletin caused significant suppression of sprouting of microvessels in rat aortic explants with IC50 (median inhibitory concentration) 0.06μM. Scopoletin (100 and 200mg/kg) strongly inhibited (59.72 and 89.4%, respectively) vascularization in matrigel plugs implanted in nude mice. In the tumor xenograft model, scopoletin showed remarkable inhibition on tumor growth (34.2 and 94.7% at 100 and 200mg/kg, respectively). Tumor histology revealed drastic reduction of the extent of vascularization. Further, immunostaining of CD31 and NG2 receptors in the histological sections confirmed the antivascular effect of scopoletin in tumor vasculature. In computer modeling, scopoletin showed strong ligand affinity and binding energies toward the following angiogenic factors: protein kinase (ERK1), vascular endothelial growth factor A (VEGF-A), and fibroblast growth factor 2 (FGF-2). These results suggest that the antitumor activity of scopoletin may be due to its strong anti-angiogenic effect, which may be mediated by its effective inhibition of ERK1, VEGF-A, and FGF-2.

Isoledene from Mesua ferrea oleo-gum resin induces apoptosis in HCT 116 cells through ROS-mediated modulation of multiple proteins in the apoptotic pathways: A mechanistic study.

Colorectal cancer (CRC) is one of the most common human malignant tumors worldwide. Arising from the transformation of epithelial cells in the colon and/or rectum into malignant cells, the foundation of CRC pathogenesis lies in the progressive accumulation of mutations in oncogenes and tumor-suppressor genes, such as KRAS and APC. Resistance to apoptosis is one of the key mechanisms in the development of CRC as it is for any other kind of cancer. Natural products have been shown to induce the expression of apoptosis regulators that are blocked in cancer cells. In the present study, a series of in vitro assays were employed to study the apoptosis-inducing attributes of Isoledene rich sub-fraction (IR-SF) collected from the oleo-gum resin of M. ferrea. Data obtained, showed that IR-SF inhibited cell proliferation and induced typical apoptotic changes in the overall morphology of all the CRC cell lines tested. Fluorescent staining assays revealed characteristic nuclear condensation, and marked decrease in mitochondrial outer membrane potential in the treated cells. In addition, an increment in the levels of ROS, caspase-8, -9 and -3 was observed. Proteomic analysis revealed that IR-SF up-regulated the expression of pro-apoptotic proteins, i.e., Bid, Bim and cytochrome c. Cytochrome c in turn activated caspases cascade resulting in the induction of apoptosis. Moreover, IR-SF significantly down-regulated Bcl-2, Bcl-w, survivin, xIAP and HSPs pro-survival proteins and induced DNA fragmentation and G0/G1-phase arrest in HCT 116 cells. Chemical characterization of IR-SF by GC-MS and HPLC methods identified Isoledene as one of the major compounds. Altogether, results of the present study demonstrate that IR-SF may induce apoptosis in human colorectal carcinoma cells through activation of ROS-mediated apoptotic pathways.

Cat’s whiskers ( Orthosiphon stamineus ) tea modulates arthritis pathogenesis via the angiogenesis and inflammatory cascade

BackgroundOrthosiphon stamineus is used traditionally to treat gout, arthritis, and inflammatory related conditions. The in vitro anti-inflammatory effects of the plant have been scientifically investigated. The goal of the present study was to evaluate the potential of the 50% ethanol extract of O. stamineus (EOS) to treat rheumatoid arthritis.MethodsAnti-arthritic activity was assessed using the in vitro heat denaturation test and the (FCA)-induced arthritis model. Efficacy was assessed by measurements of paw edema and granulation, X-ray radiography, fluorescence molecular tomography (FMT), and histological evaluation. Levels of (TNF-α), interleukin-1 (IL-1), and (COX-1 and COX-2) were analyzed in vitro in lipopolysaccharide (LPS)-stimulated human macrophage (U937). TNF-α and IL-1 levels in the serum samples of arthritic rats were also measured using an ELISA kit.ResultsTreatment with EOS resulted in dose-dependent inhibition of paw edema in acute and chronic models of inflammation. It also inhibited significantly the production of TNF-α, IL-1 COX-1, and COX-2 in the LPS-stimulated U937 macrophages. EOS significantly suppressed FCA-induced paw edema as well as the serum levels of TNF-α and IL-1. X-rays of the synovial joint of the hind leg showed considerable improvement in joint integrity and recovery of tibia-talus bones from degeneration and osteoporotic lesions. Histology of proximal interphalangeal joints of EOS-treated animals showed obvious protection of cartilage and soft tissue. Finally, FMT analysis strongly supported the anti-arthritic effect of EOS. EOS had high phenolic and total flavonoid content as well as strong antioxidant activity.ConclusionsResults illustrated that the anti-arthritic properties of O. stamineus could be beneficial for prevention and management of rheumatoid arthritis and other chronic inflammatory disorders.Graphical abstractIllustration of the Anti- arthritis efficacy of Orthosiphon Stamineus standardized extract.

Mutagenicity and preclinical safety assessment of the aqueous extract of Clinacanthus nutans leaves

Abstract Context: Clinacanthus nutans (CN) is used traditionally for treating various illnesses. Robust safety data to support its use is lacking. Objective: To evaluate the adverse effects of aqueous extract of CN leaves (AECNL). Materials and methods: The oral toxicity of the AECNL was tested following Organisation for Economic Co-operation and Development (OECD) guidelines. Mutagenicity (Ames test) of AECNL was evaluated using TA98 and TA100 Salmonella typhimurium strains. Results: No mortality or morbidity was found in the animals upon single and repeated dose administration. However, significant body weight loss was observed at 2000 mg/kg during sub-chronic (90 d) exposure. In addition, increased eosinophil at 500 mg/kg and decreased serum alkaline phosphatase levels at 2000 mg/kg were observed in male rats. Variations in glucose and lipid profiles in treated groups were also observed compared to control. Ames test revealed no evidence of mutagenic or carcinogenic effects at 500 μg/well of AECNL. Conclusion: The median lethal dose (LD50) of the AECNL is >5000 mg/kg and the no-observed-adverse-effect level is identified to be greater than 2000 mg/kg/day in 90-d study.

Ischemic optic neuropathy as a model of neurodegenerative disorder: A review of pathogenic mechanism of axonal degeneration and the role of neuroprotection

Optic neuropathy is a neurodegenerative disease which involves optic nerve injury. It is caused by acute or intermittent insults leading to visual dysfunction. There are number of factors, responsible for optic neuropathy, and the optic nerve axon is affected in all type which causes the loss of retinal ganglion cells. In this review we will highlight various mechanisms involved in the cell loss cascades during axonal degeneration as well as ischemic optic neuropathy. These mechanisms include oxidative stress, excitotoxicity, angiogenesis, neuroinflammation and apoptosis following retinal ischemia. We will also discuss the effect of neuroprotective agents in attenuation of the negative effect of factors involve in the disease occurrence and progression.

Evaluation of in vitro and in vivo anti-inflammatory effects of (-)-pseudosemiglabrin, a major phytoconstituent isolated from Tephrosia apollinea (Delile) DC.

ETHNOPHARMACOLOGICAL RELEVANCE Tephrosia apollinea (Delile) DC (Leguminosae) has been used in folk medicine in Arabian countries to treat inflammatory disorders. The plant has been described to treat swelling, bone fracture, bronchitis, cough, earache and wounds. AIM OF THE STUDY the current study aims to evaluate the anti-inflammatory properties of the major active phytoconstituent of T. apollinea and elucidate the mechanisms by which it inhibits inflammation in vitro and in vivo. MATERIAL AND METHODS The compound, (-)-pseudosemiglabrin (SSG) was isolated as a major component from the aerial parts of T. apollinea using column chromatography techniques. Sub-chronic in vivo anti-inflammatory effect of SSG was assessed using cotton pellet granuloma assay in SD rats and serum levels of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1) and nitric oxide (NO) were measured, whereas, tail flick assay was performed to assess the analgesic effect of SSG. Furthermore, the anti-inflammatory effects of SSG were confirmed by measuring the levels of IL-1, TNF-α, and NO in vitro using human macrophage cell lines (U937). In addition COX inhibition assay was also conducted in cells-free system. In silico study was performed to dock SSG in cyclooxygenase enzymes and opioid receptor to predict its structure-activity and molecular mechanism. RESULTS SSG displayed potential inhibition of granuloma tissue in rats and significantly (P<0.05) lowered the production of cytokines (TNF- α and IL-1) in vivo as well as human macrophages. Further investigation revealed that, SSG selectively inhibited COX-2 by 60% with negligible effect on COX-1. The selectivity of SSG towards COX-2 was confirmed in silico wherein, SSG demonstrated significant binding affinity with binding energy (-9.42kcal/mol). The binding found to be through covalent energy with Ser-530 amino acid residue of the active pocket of COX-2. SSG was found to prolong the flick tail time in mice by two folds. Further computational studies reveal that SSG binds to opioid receptor (µ-OR) through Ile-144 and Thr-218 with affinity two folds compared to the reference compounds, codeine and aspirin. CONCLUSION In the present study the major phytoconstituent (-)-pseudosemiglabrin (SSG) from the aerial parts of T. apollinea demonstrated potent anti-inflammatory effect by inhibiting of granuloma tissue in rats and prolonging the tail flick time in mice. Investigation of levels of pro-inflammatory cytokines in SSG-treated rats and human macrophages demonstrated that SSG significantly inhibited TNF-α and IL-1. Also SSG showed selective inhibitory effect towards COX-2. In silico study exhibited pronounced binding affinity between SSG and µ-opioid receptor better than that of codeine and aspirin. The obtained results justify the use of aerial parts of T. apollinea to treat various inflammatory diseases and indicate that (-)-pseudosemiglabrin has a great potential to be further developed as a promising anti-inflammatory drug.

Chemotherapeutic potentials of the stem bark of Balanite aegyptiaca (L.) Delile: an antiangiogenic, antitumor and antioxidant agent

BackgroundBalanite aegyptiaca (L.) Delile, is a plant with extensive medicinal properties. Its stem bark is traditionally known for its spasmolytic and antiepileptic properties and used to treat yellow fever, jaundice and syphilis. Angiogenesis (sprouting of new blood vessels) is crucial for tumor growth and metastasis. The goal of this study is investigate the antiangiogenic, cytotoxicity and antioxidant activity as well as antitumor in vivo properties of B. aegyptiaca stem bark extracts.MethodThe dried powder of stem bark was extracted sequentially with n-hexane, chloroform, methanol and water. Rat aorta ring assay (RARA) was used as a platform to screen for antiangiogenic affect. The most active extract was subjected to further confirmatory antiangiogenic tests i.e. cell migration, tube formation and VEGF inhibition and finally evaluated for its in vivo antitumor efficacy in nude mice. The cytotoxicity of extracts on four cancer cell lines (HCT-116, K562, U937 and MCF-7) and one normal cells line (HUVEC) was evaluated. To assess the antioxidant activity screening, four methods were used, (DPPH•) and ABTS radical scavenging activity, as well as total flavonoids and phenolic contents.ResultsMethanol extract of B. aegyptiaca stem bark (MBA) showed the highest antiangiogenic, antioxidant and anticancer properties. It was found selectively cytotoxic to leukemia cell lines as well as breast cancer cell line MCF-7. (MBA) thus exhibited antiangiogenic in ex-vivo rat aorta ring model; it was found to excel its antiangiogenic effect via inhibition of the key growth factor (VEGF) as well as to halt HUVEC cell migration and tube formation, furthermore animals bearing colon cancer treated with (MBA) showed significant reduction in tumor growth.ConclusionDifferent extracts of B. aegyptiaca stem bark showed various anticancer and antiangiogenic properties. MBA demonstrated potent antiangiogenic, antioxidant and antitumor in vivo. The outcome of this study suggests the potential of stem bark of the B. aegyptiaca for developing chemotherapeutic agent against solid tumor as well as leukemia.

Mesua ferrea stem bark extract induces apoptosis and inhibits metastasis in human colorectal carcinoma HCT 116 cells, through modulation of multiple cell signalling pathways

Considering the great potential of natural products as anticancer agents, the present study was designed to explore the molecular mechanisms responsible for anticancer activities of Mesua ferrea stem bark extract against human colorectal carcinoma. Based on MTT assay results, bioactive sub-fraction (SF-3) was selected for further studies using HCT 116 cells. Repeated column chromatography resulted in isolation of less active α-amyrin from SF-3, which was identified and characterized by GC-MS and HPLC methods. α-amyrin and betulinic acid contents of SF-3 were measured by HPLC methods. Fluorescent assays revealed characteristic apoptotic features, including cell shrinkage, nuclear condensation, and marked decrease in mitochondrial membrane potential in SF-3 treated cells. In addition, increased levels of caspases-9 and -3/7 levels were also observed in SF-3 treated cells. SF-3 showed promising antimetastatic properties in multiple in vitro assays. Multi-pathway analysis revealed significant down-regulation of WNT, HIF-1α, and EGFR with simultaneous up-regulation of p53, Myc/Max, and TGF-β signalling pathways in SF-3 treated cells. In addition, promising growth inhibitory effects were observed in SF-3 treated HCT 116 tumour spheroids, which give a hint about in vivo antitumor efficacy of SF-3 phytoconstituents. In conclusion, these results demonstrated that anticancer effects of SF-3 towards colon cancer are through modulation of multiple molecular pathways.

50% Ethanol extract of Orthosiphon stamineus modulates genotoxicity and clastogenicity induced by mitomycin C

Abstract Herbal products contain a variety of compounds which may be useful in protecting against cellular damage caused by mutagens. Orthosiphon stamineus (O.s) also known as Cat whiskers. The herb has been shown anti-oxidative properties and can modulate key cellular proteins that have cytoprotective effect. The study aimed to evaluate the effects of different doses (250, 500 and 1000 mg kg−1) of 50% ethanol extract of O.s (Et. O.s) on micro-nucleated polychromatic erythrocytes (MNPCE), Polychromatic to normachromatic erythrocytes ratio (PCE/NCE), Mitotic index (MI), and Chromosomal aberration (CA) in Bab/c mice. Moreover, these parameters were used to evaluate the anti-genotoxic and clastogenic potencies of (Et. O.s) against mitomycin c (MMC) that interact with biological molecules and induce genotoxic and clastogenic disorders in non-tumor cells. MMC (4 mg kg−1) was injected intraperitoneally (i.p.) to the mice before and after treatment with three different doses of (Et. O.s). The results indicated that the extract at different doses did not show significant (p ≥ 0.05) differences in (MNPCE), (PCE/NCE) ratios, and (CA) values. The higher doses sowed high (MI) values compared with untreated control group. MMC showed significant increase (p ≤ 0.001) in (MNPCE), (CA) and reduce (PCE/NCE) and (MI) values compared with untreated control group. Treatment with (Et. O.s) at different doses before and after MMC injection showed to modulate MNPCE, PCE/NCE ratios, CA and MI values in mice bone marrow cells suggesting genoprotective potential of this plant extract.

Preclinical safety assessment and mutagenicity of the hydroethanolic extract of Syzygium campanulatum leaves.

Syzygium campanulatum (Myrtaceae) is a species indigenous to Southeast Asia, a widely consumed medicinal herb and rich in phytochemical content and notable antiangiogenic and anti-colon cancer. Safety reports of administration of S. campanulatum are however lacking. In this study, we investigated the quality of dried leaves, chemical composition analyzed by FTIR and HPLC, phytochemicals content and repeated doses toxicity and mutagenicity effect of the hydroethanolic extract of S. campanulatum leaves (HESCL). The rats were divided into experimental and control groups and fed with 500, 1000, 2000 mg/kg/day of the hydroethanolic extract of S. campanulatum leaves for 28 and 90 days and with a single dose of 5000 mg/kg in acute study. The obtained results showed the dry leaves of S. campanulatum were devoid of any heavy metal and microbial contamination. The major components of HESCL were, respectively betulinic acid (60.43 mg/g.), total glycol saponins, total phenolics, total proteins, total tannins, and total flavonoids. No mutagenicity was detected in S. typhimurium auxotroph and no signs of clinical toxicity and mortality were observed in the experimental groups after 28 and 90-day experiment. However, significant (p l 0.05) statistical deviations were observed in hematological, and biochemical parameters but they were within the normal clinical range for rat, therefore not considered treatment-related. Based on these findings the fifty percent lethal dose was g 5000 mg/kg and the NOAEL was up to 2000 mg/kg for 90 days, as such HESCL is a relatively safe herb.