Ethanol Extract of Achillea fragrantissima Enhances Angiogenesis through Stimulation of VEGF Production.

OBJECTIVE: Achillea fragrantissima L. (Asteraceae) is a traditionally used medicinal herb in the rural communities of Jordan. METHODS: The present study evaluated the efficacy of the ethanol extract of this species on angiogenesis in both, ex vivo using a rat aortic ring assay and in vivo using a rat excision wound model. RESULTS: In concentrations of 50 and 100 µg/ml, the ethanol extract showed angiogenic stimulatory effect and significantly increased length of capillary protrusions around aorta rings of about 60% in comparison to those of untreated aorta rings. In MCF-7 cells, the ethanol extract of A. fragrantissima stimulated the production of VEGF in a dose-dependent manner. 1% and 5% of ethanol extract of A. fragrantissima containing vaseline based ointment was applied on rat excision wounds for six days and found to be effective in wound healing and maturation of the scar. Both preparations resulted in better wound healing when compared to the untreated control group and vaseline- treated group. This effect was comparable to that induced by MEBO, the positive control. CONCLUSION: The results indicate that A. fragrantissima has a pro-angiogenic effect, which may act through the VEGF signaling pathway.

Correction to: The antiangiogenic activities of ethanolic crude extracts of four Salvia species.

CORRECTION: After the publication [1] it came to the attention of the authors that one of the co-authors was incorrectly included as Hamza Somrain. The correct spelling is as follows: Hamzeh Sumrein.

Correlation of antiangiogenic, antioxidant and cytotoxic activities of some Sudanese medicinal plants with phenolic and flavonoid contents.

BACKGROUND: Consumption of medicinal plants to overcome diseases is traditionally belongs to the characteristics of most cultures on this earth. Sudan has been a host and cradle to various ancient civilizations and developed a vast knowledge on traditional medicinal plants. The present study was undertaken to evaluate the antioxidant, antiangiogenic and cytotoxic activities of six Sudanese medicinal plants which have been traditionally used to treat neoplasia. Further the biological activities were correlated with phytochemical contents of the plant extracts. METHODS: Different parts of the plants were subjected to sequential extraction method. Cytotoxicity of the extracts was determined by dimethylthiazol-2-yl)- 2,5diphenyl tetrazolium bromide (MTT) assay on 2 human cancer (colon and breast) and normal (endothelial and colon fibroblast) cells. Anti-angiogenic potential was tested using ex vivo rat aortic ring assay. DPPH (1,1-diphenyl-2-picrylhydrazyl) assay was conducted to screen the antioxidant capabilities of the extracts. Finally, total phenolic and flavonoid contents were estimated in the extracts using colorimetric assays. RESULTS: The results indicated that out of 6 plants tested, 4 plants (Nicotiana glauca, Tephrosia apollinea, Combretum hartmannianum and Tamarix nilotica) exhibited remarkable anti-angiogenic activity by inhibiting the sprouting of microvessels more than 60%. However, the most potent antiangiogenic effect was recorded by ethanol extract of T. apollinea (94.62%). In addition, the plants exhibited significant antiproliferative effects against human breast (MCF-7) and colon (HCT 116) cancer cells while being non-cytotoxic to the tested normal cells. The IC50 values determined for C. hartmannianum, N. gluaca and T. apollinea against MCF-7 cells were 8.48, 10.78 and 29.36 µg/ml, respectively. Whereas, the IC50 values estimated for N. gluaca, T. apollinea and C. hartmannianum against HCT 116 cells were 5.4, 20.2 and 27.2 µg/ml, respectively. These results were more or less equal to the standard reference drugs, tamoxifen (IC50 = 6.67 µg/ml) and 5-fluorouracil (IC50 = 3.9 µg/ml) tested against MCF-7 and HCT 116, respectively. Extracts of C. hartmannianum bark and N. glauca leaves demonstrated potent antioxidant effect with IC50s range from 9.4-22.4 and 13.4-30 µg/ml, respectively. Extracts of N. glauca leaves and T apollinea aerial parts demonstrated high amount of flavonoids range from 57.6-88.1 and 10.7-78 mg quercetin equivalent/g, respectively. CONCLUSIONS: These results are in good agreement with the ethnobotanical uses of the plants (N. glauca, T. apollinea, C. hartmannianum and T. nilotica) to cure the oxidative stress and paraneoplastic symptoms caused by the cancer. These findings endorse further investigations on these plants to determine the active principles and their mode of action.

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.

Increased aqueous solubility and proapoptotic activity of potassium koetjapate against human colorectal cancer cells.

OBJECTIVES: Recently, we have isolated koetjapic acid (KA) from Sandoricum koetjape and identified its selective anticancer potentiality against colorectal carcinoma. KA is quite likely to be useful as a systemic anticancer agent against colorectal malignancy. However, with extremely low solubility, KA has to be converted into a biocompatible solubilized form without compromising the bioefficacy. Objective of this study is to enhance solubility of KA and to evaluate anticancer efficacy of potassium koetjapate in human colorectal cancer cells. METHODS: (2-Hydroxypropyl)-ß-cyclodextrin inclusion complex and solid dispersions (carboxymethyl cellulose, polyvinylpyrrolidone and sodium lauryl sulphate) of KA were prepared. In addition, a salt of KA, potassium koetjapate was synthesized. KEY FINDINGS: Potassium koetjapate demonstrated higher solubility than the other tested formulations with enhanced cytotoxicity against HCT 116 cells. The enhanced efficacy of potassium koetjapate is attributed to apoptotic induction of nuclear condensation and disruption of mitochondrial membrane potential in the cells. Interestingly, potassium koetjapate was found to be safe in rats after oral administration (LD50 > 2000 mg/kg). CONCLUSIONS: The salt formulation of KA appears to modulate the capability of the parent compound by enhancing its solubility and improves its bioefficacy against colon cancer cells, suggesting attractive roles for its applications in medicine.

Proapoptotic and antimetastatic properties of supercritical CO2 extract of Nigella sativa Linn. against breast cancer cells.

Nigella sativa, commonly referred as black cumin, is a popular spice that has been used since the ancient Egyptians. It has traditionally been used for treatment of various human ailments ranging from fever to intestinal disturbances to cancer. This study investigated the apoptotic, antimetastatic, and anticancer activities of supercritical carbon dioxide (SC-CO2) extracts of the seeds of N. sativa Linn. against estrogen-dependent human breast cancer cells (MCF-7). Twelve extracts were prepared from N. sativa seeds using the SC-CO2 extraction method by varying pressure and temperature. Extracts were analyzed using FTIR and UV-Vis spectrometry. Cytotoxicity of the extracts was evaluated on various human cancer and normal cell lines. Of the 12 extracts, 1 extract (A3) that was prepared at 60°C and 2500 psi (~17.24 MPa) showed selective antiproliferative activity against MCF-7 cells with an IC50 of 53.34±2.15 µg/mL. Induction of apoptosis was confirmed by evaluating caspases activities and observing the cells under a scanning electron microscope. In vitro antimetastatic properties of A3 were investigated by colony formation, cell migration, and cell invasion assays. The elevated levels of caspases in A3 treated MCF-7 cells suggest that A3 is proapoptotic. Further nuclear condensation and fragmentation studies confirmed that A3 induces cytotoxicity through the apoptosis pathway. A3 also demonstrated remarkable inhibition in migration and invasion assays of MCF-7 cells at subcytotoxic concentrations. Thus, this study highlights the therapeutic potentials of SC-CO2 extract of N. sativa in targeting breast cancer.

The antiangiogenic activities of ethanolic crude extracts of four Salvia species.

BACKGROUND: Angiogenesis is one of cancer hallmarks that are required for both cancer progression and metastasis. In this study we examined the antiangiogenic properties of the ethanolic crude extracts of four Salvia species grown in Jordan. METHODS: The direct antiangiogenic activity was evaluated using various models: ex vivo rat aortic ring assay, in vitro assessment of HUVEC proliferation and migration, and in vivo CAM assay, while we used the changes in the expression of HIF-1α and VEGF in breast cancer cells (MCF 7) as an indicative for the indirect antiangiogenic activity. RESULTS: All four crude extracts showed a potential antiangiogenic activity in the rat aortic assay, however two species were found to be cytotoxic against Fibroblast cell line (PLF); the finding that caused the exclusion of these two extracts from further studies. Of the two remaining extracts, S. triloba showed very promising direct and indirect antiangiogenic activities. S. triloba inhibited the HUVEC proliferation with an IC50 of 90 µg/mL and HUVEC migration by 82% at 150 µg/mL. Furthermore, the in vivo CAM assay also illustrated the high impact of S. triloba against the newly formed vessel in the chicken embryonic membrane. Interestingly, the S. triloba inhibited the expression of VEGF at the mRNA and protein and the HIF-1α mRNA in the MCF 7 breast cancer cells under both normoxic and hypoxic conditions. CONCLUSIONS: Taken together, all these findings of the direct and indirect angiogenic investigations nominated S. triloba as a highly potent antiangiogenic plant that may have chemotherapeutic and/or chemoprevention potentials.

Antiangiogenesis and antioxidant activity of ethanol extracts of Pithecellobium jiringa.

BACKGROUND: Angiogenesis plays a critical role in embryonic development and various physiological processes. However, excessive angiogenesis is associated with several pathological conditions including cancer. Pithecellobium jiringa (Jack) Prain is a traditional medicinal plant from the family Leguminosae. It is native to the Southeast Asia, where it has been used traditionally for treatment of various ailments such as hypertension and diabetes. The present work is aimed to study antioxidant and antiangiogenesis activities of P. jiringa ethanol extracts. METHODS: P. jiringa fruit rinds were extracted with ethanol and 50% ethanol. The antioxidant property was analysed using, 1,1-diphenyl-2-picryl-hydrazyl free radical scavenging assay. Phytochemical analysis was performed using thin layer chromatography and colorimetric methods. Then, cell growth inhibition was studied against a panel of human cell lines by MTT test. In vitro inhibition of angiogenesis was studied by the following assays: isolated rat aortic rings cell viability, colony formation, endothelial cell migration, endothelial tube formation on matrigel, and expression of vascular endothelial growth factor by endothelial cells. In vivo antiangiogenesis effect was studied by utilising fertilised chick embryos assay. The results were statistically analysed by analysis of variance. RESULTS: Ethanolic and 50% hydro-ethanolic extracts showed relatively high concentration of total phenolics associated with potent antioxidant activity. The rat aortic rings study conducted showed potent inhibition of the microvessels outgrowth with IC50s 5.27 ± 0.81 µg/ml (ethanolic) and 4.45 ± 0.63 µg/ml (50% hydro-ethanolic). Both extracts arrested the growth of human endothelial cells via down-regulation of VEGF expression, leading to inhibition of other angiogenesis cascades including migration of endothelial cells, and formation of capillary network on matrigel matrix. The extracts also inhibited the neovascularisation of chick embryo chorioallantoic membrane. CONCLUSIONS: P. jiringa extracts inhibit angiogenesis by blocking the VEGF expression thus inhibiting endothelial cells proliferation, migration and differentiation most likely due to presence of the antioxidant phenolics.

Cat's whiskers tea (Orthosiphon stamineus) extract inhibits growth of colon tumor in nude mice and angiogenesis in endothelial cells via suppressing VEGFR phosphorylation.

Cat's whiskers (Orthosiphon stamineus) is commonly used as Java tea to treat kidney stones including a variety of angiogenesis-dependent diseases such as tumorous edema, rheumatism, diabetic blindness, and obesity. In the present study, antitumor potential of standardized 50% ethanol extract of O. stamineus leaves (EOS) was evaluated against colorectal tumor in athymic mice and antiangiogenic efficacy of EOS was investigated in human umbilical vein endothelial cells (HUVEC). EOS at 100 mg/kg caused 47.62 ± 6.4% suppression in tumor growth, while at 200 mg/kg it caused 83.39 ± 4.1% tumor regression. Tumor histology revealed significant reduction in extent of vascularization. Enzyme-linked immunosorbent assay showed EOS (200 mg/kg) significantly reduced the vascular endothelial growth factor (VEGF) level in vitro (211 ± 0.26 pg/ml cell lysate) as well as in vivo (90.9 ± 2 pg/g tissue homogenate) when compared to the control (378 ± 5 and 135.5 ± 4 pg, respectively). However, EOS was found to be noncytotoxic to colon cancer and endothelial cells. In vitro, EOS significantly inhibited the migration and tube formation of human umbilical vein endothelial cells (HUVECs). EOS suppressed VEGF-induced phosphorylation of VEGF receptor-2 in HUVECs. High performance liquid chromatography (HPLC) analysis of EOS showed high rosmarinic acid contents, whereas phytochemical analysis revealed high protein and phenolic contents. These results demonstrated that the antitumor activity of EOS may be due to its VEGF-targeted antiangiogenicity.

Koetjapic acid, a natural triterpenoid, induces apoptosis in colon cancer cells.

Deregulated cell signaling pathways result in cancer development. More than one signal transduction pathway is involved in colorectal cancer pathogenesis and progression. Koetjapic acid (KA) is a naturally occurring seco-A-ring oleanene triterpene isolated from the Sandoricum koetjape stem bark. We report the cellular and molecular mechanisms of anticancer activity of KA towards human colorectal cancer. The results showed that KA induces apoptosis in HCT 116 colorectal carcinoma cells by inducing the activation of extrinsic and intrinsic caspases. We confirmed that KA-induced apoptosis was mediated by DNA fragmentation, nuclear condensation and disruption in the mitochondrial membrane potential. Further studies on the effect of KA on cancer pathways show that the compound causes down-regulation of Wnt, HIF-1α, MAP/ERK/JNK and Myc/Max signaling pathways and up-regulates the NF-κB signaling pathway. The result of this study highlights the anticancer potential of KA against colorectal cancer.