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.

Antiangiogenic activity of sophorolipids extracted from refined bleached deodorized palm olein.

OBJECTIVES: Sophorolipids (SLs) are a group of surface-active glycolipids produced by a type of nonpathogenic yeast Candida bombicola in the presence of vegetable oil through fermentation technology. SLs have shown antitumor activity; however, the mechanism of action underlying the anticancer activity of SLs is poorly understood. This work evaluated the anticancer activity of SLs fermented from palm oil by exploring its antiangiogenic activity. MATERIALS AND METHODS: The SLs that were fermented and further characterized for their biochemical activities. Cytotoxicity study was performed to assess cytostatic properties. A series of in vitro and ex vivo angiogenesis assay was also carried out. The relative fold change in the expression of p53 mRNA by SLs was also studied. RESULTS: Altogether, the data show that SLs derived from palm oil fermentation process inhibited neovascularization in the ex vivo tissue segments and also the endothelial cell proliferation between 50% and 65% inhibition as a whole. The palm oil derived SLs also caused downregulation of the suppression level of vascular endothelial growth factor and also upregulate the p53 mRNA level. The analytical studies revealed the presence of high amount of phenolic compounds but with relatively weak antioxidant activity. The gas chromatography-mass spectrometry studies revealed abundant amount of palmitic and oleic acid, the latter an established antiangiogenic agent, and the former being proangiogenic. CONCLUSION: Therefore, it can be concluded from this study that SLs derived from fermented palm oil have potent antiangiogenic activity which may be attributed by its oleic acid component.

Establishment of in vitro and in vivo anti-colon cancer efficacy of essential oils containing oleo-gum resin extract of Mesua ferrea.

Proven the great potential of essential oils as anticancer agents, the current study intended to explore molecular mechanisms responsible for in vitro and in vivo anti-colon cancer efficacy of essential oil containing oleo-gum resin extract (RH) of Mesua ferrea. MTT cell viability studies showed that RH had broad spectrum cytotoxic activities. However, it induced more profound growth inhibitory effects towards two human colon cancer cell lines i.e., HCT 116 and LIM1215 with an IC50 values of 17.38 ± 0.92 and 18.86 ± 0.80 µg/mL respectively. RH induced relatively less toxicity in normal human colon fibroblasts i.e., CCD-18co. Cell death studies conducted, revealed that RH induced characteristic morphological and biochemical changes in HCT 116. At protein level it down-regulated expression of multiple pro-survival proteins i.e., survivin, xIAP, HSP27, HSP60 and HSP70 and up-regulated expression of ROS, caspase-3/7 and TRAIL-R2 in HCT 116. Furthermore, significant reduction in invasion, migration and colony formation potential was observed in HCT 116 treated with RH. Chemical characterization by GC-MS and HPLC methods revealed isoledene and elemene as one the major compounds. RH showed potent antitumor activity in xenograft model. Overall, these findings suggest that RH holds a promise to be further studied for cheap anti-colon cancer naturaceutical development.

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

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.

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-ß 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.

Flavonoids-Rich Orthosiphon stamineus Extract as New Candidate for Angiotensin I-Converting Enzyme Inhibition: A Molecular Docking Study.

This study aims to evaluate the in vitro angiotensin-converting enzyme (ACE) inhibition activity of different extracts of Orthosiphon stamineus (OS) leaves and their main flavonoids, namely rosmarinic acid (RA), sinensetin (SIN), eupatorin (EUP) and 3'-hydroxy-5,6,7,4'-tetramethoxyflavone (TMF). Furthermore, to identify possible mechanisms of action based on structure-activity relationships and molecular docking. The in vitro ACE inhibition activity relied on determining hippuric acid (HA) formation from ACE-specific substrate (hippuryl-histidyl-leucine (HHL)) by the action of ACE enzyme. A High Performance Liquid Chromatography method combined with UV detection was developed and validated for measurement the concentration of produced HA. The chelation ability of OS extract and its reference compounds was evaluated by tetramethylmurexide reagent. Furthermore, molecular docking study was performed by LeadIT-FlexX: BioSolveIT's LeadIT program. OS ethanolic extract (OS-E) exhibited highest inhibition and lowest IC50 value (45.77 ± 1.17 µg/mL) against ACE compared to the other extracts. Among the tested reference compounds, EUP with IC50 15.35 ± 4.49 µg/mL had highest inhibition against ACE and binding ability with Zn (II) (56.03% ± 1.26%) compared to RA, TMF and SIN. Molecular docking studies also confirmed that flavonoids inhibit ACE via interaction with the zinc ion and this interaction is stabilized by other interactions with amino acids in the active site. In this study, we have demonstrated that changes in flavonoids active core affect their capacity to inhibit ACE. Moreover, we showed that ACE inhibition activity of flavonoids compounds is directly related to their ability to bind with zinc ion in the active site of ACE enzyme. It was also revealed that OS extract contained high amount of flavonoids other than RA, TMF, SIN and EUP. As such, application of OS extract is useful as inhibitors of ACE.

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.

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

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.

Diuretic activity of aqueous extract of Nigella sativa in albino rats.

The study aims to evaluate the diuretic effect and acute toxicity of a crude aqueous extract of Nigella sativa using animal models. To evaluate the diuretic activity of the plant, Albino rats were divided into five groups. The control group received normal saline (10 mL/kg), the reference group received furosemide (10 mg/kg) and the test groups were administered different doses (i.e., 10, 30 and 50 mg/kg) of the crude extract by intra-peritoneal route, respectively. Graph Pad Prism was used for the statistical analysis and p-values less than 0.05 were considered statistically significant. We observed significant diuretic, kaliuretic and natriuretic effects in the treated groups in a dose dependent manner. However, urinary pH remained unchanged during the course of the study. The diuretic index values showed good diuretic activity of the crude extract. The Lipschitz values demonstrated that the crude extract, at the dose of 50 mg/kg, showed 46% diuretic activity compared with furosemide. With regard to the acute toxicity study, no lethal effects were observed among Albino mice even at the higher dose of 5000 mg/kg. The extract of Nigella sativa, at the dose of 50 mg/kg, significantly increased the urinary volume and modified the concentration of urinary electrolytes, and there was observed no signs of acute toxicity associated with the crude extract. Further studies are encouraged to isolate the pure phytochemical responsible for diuresis.

Isolation, Characterization, Crystal Structure Elucidation, and Anticancer Study of Dimethyl Cardamonin, Isolated from Syzygium campanulatum Korth.

Syzygium campanulatum Korth is an equatorial, evergreen, aboriginal shrub of Malaysia. Conventionally it has been used as a stomachic. However, in the currently conducted study dimethyl cardamonin or 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (DMC) was isolated from S. campanulatum Korth, leaf extract. The structural characterization of DMC was carried out by making use of various techniques including UV, IR, NMR spectral followed by LC-MS, and X-ray crystallographic techniques. For determining the purity of compound, highly effective techniques including TLC, HPLC, and melting point were used. The cytotoxicity of DMC and three different extracts of S. campanulatum was evaluated against human colon cancer cell line (HT-29) by three different assays. DMC and ethanolic extract revealed potent and dose-dependent cytotoxic activity on the cancer cell line with IC50 12.6 and 90.1 µg/mL, respectively. Quite astonishingly to our knowledge, this is the very first report on S. campanulatum as being a rich source (3.5%) of DMC, X-ray crystallography, and anticancer activity on human colon cancer cells.

Diuretic activity of Boswellia serrata Roxb. oleo gum extract in albino rats.

The aim of the study was to evaluate the effect of crude aqueous extract of Boswellia serrata Roxb. oleo gum on urinary electrolytes, pH and diuretic activity in normal albino rats. Moreover, acute toxicity of the gum extract was assessed using mice. Albino rats were divided into five groups. Control group received normal saline (10 mg/kg), reference group received furosemide (10 mg/kg) and test groups were given different doses of crude extract (10, 30 and 50 mg/kg) by intra-peritoneal route, respectively. The Graph Pad Prism was used for the statistical analysis and p < 0.05 was considered statistically significant. Significant diuretic, kaliuretic and natriuretic effects were observed in the treated groups in a dose dependent manner. Diuretic index showed good diuretic activity of the crude extract. Lipschitz values indicated that the crude extract, at the dose of 50 mg/kg, showed 44 % diuretic activity compared to the reference drug. No lethal effects were observed among albino mice even at the higher dose of 3000 mg/kg. It is concluded that aqueous extract of Boswellia serrata oleo gum, at the dose of 50 mg/kg showed significant effects on urinary volume and concentration of urinary electrolytes with no signs of toxicity.

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.

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.

Syzygium campanulatum korth methanolic extract inhibits angiogenesis and tumor growth in nude mice.

BACKGROUND: Syzygium campanulatum Korth (Myrtaceae) is an evergreen shrub rich in phenolics, flavonoid antioxidants, and betulinic acid. This study sought to investigate antiangiogenic and anti-colon cancer effects of S.C. standardized methanolic extract. METHODS: Betulinic acid was isolated from methanolic extract by crystallization and chromatography techniques. S.C. methanolic extract was analyzed by UV-Vis spectrophotometry, FTIR, LC-MS, and HPLC. Antiangiogenic effect was studied on rat aortic rings, matrigel tube formation, cell proliferation and migration, and expression of vascular endothelial growth factor (VEGF). Antitumor effect was studied using a subcutaneous tumor model of HCT 116 colorectal carcinoma cells established in nude mice. RESULTS: Analysis by HPLC, LC-MS and FTIR confirm presence of betulinic acid in S.C. methanolic extract. Quantitative analysis by HPLC indicates presence of betulinic acid in S.C. extract at 5.42 ± 0.09% (w/w). Antiangiogenesis study showed potent inhibition of microvessels outgrowth in rat aortic rings, and studies on normal and cancer cells did not show any significant cytotoxic effect. Antiangiogenic effect was further confirmed by inhibition of tube formation on matrigel matrix that involves human endothelial cells (IC50 = 17.6 ± 2.9 µg/ml). S.C. extract also inhibited migration of endothelial cells and suppressed expression of VEGF. In vivo antiangiogenic study showed inhibition of new blood vessels in chicken embryo chorioallantoic membrane (CAM), and in vivo antitumor study showed significant inhibition of tumor growth due to reduction of intratumor blood vessels and induction of cell death. CONCLUSION: Collectively, our results indicate S. campanulatum as antiangiogenic and antitumor candidate, and a new source of betulinic acid.

Anti-angiogenic activity of Morinda citrifolia extracts and its chemical constituents.

Morinda citrifolia L. has been used for the treatment of a wide variety of diseases, including cancer. This study was undertaken to evaluate the anti-angiogenic effect of M. citrifolia fruits and leaves. Anti-angiogenic activity was evaluated in vivo using the chick chorioallantoic membrane assay. Bioactivity-guided fractionation and isolation were performed to identify the active constituent, and high-performance liquid chromatography analysis was then used to quantify the amount of this active constituent in the active extracts and fraction. The methanol extracts of fruits and leaves of M. citrifolia and the subsequent chloroform fraction of the fruit methanolic extract were found to have potential anti-angiogenic activity and were more potent compared to suramin. Scopoletin was identified as one of the chemical constituents that may be partly responsible for the anti-angiogenic activity of M. citrifolia fruits. The present findings further support the use of M. citrifolia in cancer or other pathological conditions related to angiogenesis.

Chemical composition, antioxidant and cytotoxicity activities of the essential oils of Myristica fragrans and Morinda citrifolia.

BACKGROUND: In this study the chemical composition, antioxidant activities and cytotoxic effect of the essential oils of Myristica fragrans (nutmeg) and Morinda citrifolia (mengkudu) were determined. RESULTS: Thirty-eight compounds in nutmeg oil and six compounds in mengkudu oil were identified by gas chromatography­mass spectrometry. The free radical scavenging activity of nutmeg oil was superior of that mengkudu oil. The MTT assay of nutmeg oil on human colorectal carcinoma (HCT-116) and human breast carcinoma (MCF-7) cell lines showed IC50 values of 78.61 and 66.45 µg mL⁻¹, respectively. The mengkudu oil exhibited IC50 values of 91.46 and 78.15 µg mL⁻¹ for HCT-116 and MCF-7, respectively. CONCLUSION: The results showed that nutmeg oil can be developed as potent anti-cancer and antioxidant drugs.

Phytochemical analysis, cytotoxic activity and constituents-activity relationships of the leaves of Cinnamomum iners (Reinw. ex Blume-Lauraceae).

The leaves of Cinnamomum iners (Reinw. ex Blume-Lauraceae) have been refluxed successively with chloroform and alcohol to get chloroform extract and alcoholic extract. Both the extracts have been assayed for cytotoxicity against human colorectal tumour cells. The chloroform extract exhibited significant cytotoxicity with IC(50) 31 µg mL(-1) (p < 0.01). However, ethanol extract was found to be much less cytotoxic with IC(50) > 200 µg mL(-1). The chloroform extract has been further proceeded for chemical analysis by GC-TOFMS and 178 components were identified including acids, amines, amides, aldehydes, alcohols, esters, benzene derivatives, bicyclic compounds, terpenes, hydrocarbons, naphthalene derivatives, furan derivatives, azulenes, etc. Nine components representing 51.73% of the total chloroform extract were detected as major components. Caryophyllene (14.41%) and Eicosanoic acid ethyl ester (12.17%) are the most prominent components of the chloroform extract. ß-Caryophyllene (14.41%) as most abundant compound supports potent cytotoxicity as shown by chloroform extract.