Anticlastogenic, antimutagenic, and cytoprotective properties of Orthosiphon stamineus ethanolic leaves extract.

Orthosiphon stamineus (O.S) is widely consumed for its medidcinal value including anti-inflammatory, anti-infective, and diuretic properties. The present study evaluates the cytoprotective, anti-mutagenic, and anticlastogenic efficacies of standardized extract of Orthosiphon stamineus. Normal liver cell line (WRL68) exposed to hydrogen peroxide and serum-deprived media as insults to evaluate cytoprotective and glutathione activation activities of (Et. O. s). Salmonella typhimurium TA98 and TA100 exposed to different concentrations of (Et. O. s). The influence of Et. O. s on mitotic, replicative indices as well as chromosomal aberration (CA) and sister chromatid exchange (SCE) induced in human peripheral blood lymphocytes by mitomycin C (MMC). The Et. O.s proved to be a potent scavenger for hydrogen peroxide and other free radicals in serum-depraved media, which showed to stimulate glutathione production in liver cells line. Moreover, it did not induce mutations in S. typhimurium subspecies TA98 and TA100. The standardized extract exhibited powerful antimutagenic activities as verified against both 2-nitrofluorene and sodium azide in S. typhimurium TA98 and TA100 cells, respectively. Cytogenetic tests showed high concentrations of Et. O. s to reduce the values of mitotic and replicative indices without any accompanying side effects, such as chromosomal abnormalities or SCE. To ameliorate MMC effects, pretreatment with the extract proofed to be efficient protocol. These data suggests that O. stamineus extract could be useful as cytoprotective, antimutagenic, and anticlastogenic efficacies, which owes to its potent chemoprevention, antioxidant, and glutathione activation properties.

ß-Caryophyllene Induces Apoptosis and Inhibits Angiogenesis in Colorectal Cancer Models.

Beta-Caryophyllene (BCP), a naturally occurring sesquiterpene abundantly found in cloves, hops, and cannabis, is the active candidate of a relatively new group of vascular-inhibiting compounds that aim to block existing tumor blood vessels. Previously, we have reported the anti-cancer properties of BCP by utilizing a series of in-vitro anti-tumor-related assays using human colorectal carcinoma cells. The present study aimed to investigate the effects of BCP on in-vitro, ex-vivo, and in-vivo models of anti-angiogenic assays and evaluate its anti-cancer activity in xenograft tumor (both ectopic and orthotopic) mice models of human colorectal cancer. Computational structural analysis and an apoptosis antibody array were also performed to understand the molecular players underlying this effect. BCP exhibited strong anti-angiogenic activity by blocking the migration of endothelial cells, tube-like network formation, suppression of vascular endothelial growth factor (VEGF) secretion from human umbilical vein endothelial cells and sprouting of rat aorta microvessels. BCP has a probable binding at Site#0 on the surface of VEGFR2. Moreover, BCP significantly deformed the vascularization architecture compared to the negative control in a chick embryo chorioallantoic membrane assay. BCP showed a remarkable reduction in tumor size and fluorescence molecular tomography signal intensity in all the mice treated with BCP, in a dose-dependent relationship, in ectopic and orthotopic tumor xenograft models, respectively. The histological analysis of the tumor from BCP-treated mice revealed a clear reduction of the density of vascularization. In addition, BCP induced apoptosis through downregulation of HSP60, HTRA, survivin, and XIAP, along with the upregulation of p21 expressions. These results suggest that BCP acts at multiple stages of angiogenesis and could be used as a promising therapeutic candidate to halt the growth of colorectal tumor cells.

Cytotoxicity of cucurbitacin E from Citrullus colocynthis against multidrug-resistant cancer cells.

BACKGROUND: Cucurbitacin E (CuE) is an oxygenated tetracyclic triterpenoid isolated from the fruits of Citrullus colocynthis (L.) Schrad. PURPOSE: This study outlines CuE's cytotoxic activity against drug-resistant tumor cell lines. Three members of ABC transporters superfamily, P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and ABCB5 were investigated, whose overexpression in tumors is tightly linked to multidrug resistance. Further factors of drug resistance studied were the tumor suppressor TP53 and the epidermal growth factor receptor (EGFR). METHODS: Cytotoxicity assays (resazurin assays) were used to investigate the activity of Citrullus colocynthis and CuE towards multidrug resistant cancer cells. Molecular docking (In silico) has been carried out to explore the CuE's mode of binding to ABC transporters (P-gp, BCRP and ABCB5). The visualization of doxorubicin uptake was done by a Spinning Disc Confocal Microscope. The assessment of proteins expression was done by western blotting analysis. COMPARE and hierarchical cluster analyses were applied to identify, which genes correlate with sensitivity or resistance to cucurbitacins (CuA, CuB, CuE, CuD, CuI, and CuK). RESULTS: Multidrug-resistant cells overexpressing P-gp or BCRP were cross-resistant to CuE. By contrast, TP53 knock-out cells were sensitive to CuE. Remarkably, resistant cells transfected with oncogenic ΔEGFR or ABCB5 were hypersensitive (collateral sensitive) to CuE. In silico analyses demonstrated that CuE is a substrate for P-gp and BCRP. Immunoblot analyses highlighted that CuE targeted EGFR and silenced its downstream signaling cascades. The most striking result that emerged from the doxorubicin uptake by ABCB5 overexpressing cells is that CuE is an effective inhibitor for ABCB5 transporter when compared with verapamil. The COMPARE analyses of transcriptome-wide expression profiles of tumor cell lines of the NCI identified common genes involved in cell cycle regulation, cellular adhesion and intracellular communication for different cucurbitacins. CONCLUSION: CuE represents a potential therapeutic candidate for the treatment of certain types of refractory tumors. To best of our knowledge, this is the first time to identify CuE and verapamil as inhibitors for ABCB5 transporter.

Toxicological studies of Orthosiphon stamineus (Misai Kucing) standardized ethanol extract in combination with gemcitabine in athymic nude mice model.

Pancreatic cancer has the highest mortality rate among cancers due to its aggressive biology and lack of effective treatment. Gemcitabine, the first line anticancer drug has reduced efficacy due to acquired resistance. The current study evaluates the toxicological effects of Orthosiphon stamineus (O.s) and its marker compound (rosmarinic acid) in combination with gemcitabine. O.s (200 or 400 mg/kg/day) and rosmarinic acid (32 mg/kg/day) were administered orally and gemcitabine (10 mg/kg/3 days) intraperitoneally either alone or in combination treatment for fourteen days. Parameters including blood serum biochemistry, hematology, myeloid-erythroid ratio, incident of lethality, and histopathological analysis of liver, kidney, and spleen tissues were studied. Neither, individual drugs/extract nor chemo-herbal combinations at tested doses induced any toxicity and damage to organs in nude mice when compared to control group. Toxicological data obtained from this study will help to select the best doses of chemo-herbal combination for future pancreatic xenograft tumor studies.

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

BACKGROUND: Orthosiphon 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. METHODS: Anti-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. RESULTS: Treatment 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. CONCLUSIONS: Results illustrated that the anti-arthritic properties of O. stamineus could be beneficial for prevention and management of rheumatoid arthritis and other chronic inflammatory disorders. Illustration of the Anti- arthritis efficacy of Orthosiphon Stamineus standardized extract.

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

BACKGROUND: Balanite 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. METHOD: The 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. RESULTS: Methanol 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. CONCLUSION: Different 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.

In vivo toxicity and antitumor activity of essential oils extract from agarwood (Aquilaria crassna).

BACKGROUND: Aquilaria crassna has been used in traditional Asian medicine to treat vomiting, rheumatism, asthma, and cough. Furthermore, earlier studies from our laboratory have revealed that the essential oil extract from agarwood inhibited colorectal carcinoma cells. Despite of the wide range of ethno-pharmacological uses of agarwood, its toxicity has not been previously evaluated through systematic toxicological studies. Therefore, the potential safety of essential oil extract and its in vivo anti-tumor activity had been investigated. METHODS: In the acute toxicity study, Swiss female mice were given a single dose of the essential oil extract at 2000 mg/kg/day orally and screened for two weeks after administration. Meanwhile, in the sub-chronic study, two different doses of the extract were administered for 28 days. Mortality, clinical signs, body weight changes, hematological and biochemical parameters, gross findings, organ weights, and histological parameters were monitored during the study. Other than that, in vivo anti-tumor study was assessed by using subcutaneous tumors model established in nude mice. RESULTS: The acute toxicity study showed that the LD50 of the extract was greater than 2000 mg/kg. In the repeated dose for 28-day oral toxicity study, the administration of 100 mg/kg and 500 mg/kg of essential oil per body weight revealed insignificant difference in food and water intakes, bodyweight change, hematological and biochemical parameters, relative organ weights, gross findings or histopathology compared to the control group. Nevertheless, the essential oil extract, when supplemented to nude mice, caused significant growth inhibition of the subcutaneous tumor of HCT 116 colorectal carcinoma cells. CONCLUSION: Collectively, the data obtained indicated that essential oil extract from agarwood might be a safe material, and this essential oil is suggested as a potential anti-colon cancer candidate.

Optimization of Cat's Whiskers Tea (Orthosiphon stamineus) Using Supercritical Carbon Dioxide and Selective Chemotherapeutic Potential against Prostate Cancer Cells.

Cat's whiskers (Orthosiphon stamineus) leaves extracts were prepared using supercritical CO2 (SC-CO2) with full factorial design to determine the optimum extraction parameters. Nine extracts were obtained by varying pressure, temperature, and time. The extracts were analysed using FTIR, UV-Vis, and GC-MS. Cytotoxicity of the extracts was evaluated on human (colorectal, breast, and prostate) cancer and normal fibroblast cells. Moderate pressure (31.1 MPa) and temperature (60°C) were recorded as optimum extraction conditions with high yield (1.74%) of the extract (B2) at 60 min extraction time. The optimized extract (B2) displayed selective cytotoxicity against prostate cancer (PC3) cells (IC50 28 µg/mL) and significant antioxidant activity (IC50 42.8 µg/mL). Elevated levels of caspases 3/7 and 9 in B2-treated PC3 cells suggest the induction of apoptosis through nuclear and mitochondrial pathways. Hoechst and rhodamine assays confirmed the nuclear condensation and disruption of mitochondrial membrane potential in the cells. B2 also demonstrated inhibitory effects on motility and colonies of PC3 cells at its subcytotoxic concentrations. It is noteworthy that B2 displayed negligible toxicity against the normal cells. Chemometric analysis revealed high content of essential oils, hydrocarbon, fatty acids, esters, and aromatic sesquiterpenes in B2. This study highlights the therapeutic potentials of SC-CO2 extract of cat's whiskers in targeting prostate carcinoma.