Cytotoxic Effects of Some Nepeta Species against Breast Cancer Cell Lines and Their Associated Phytochemical Properties.

Nepeta is one of the largest genera of the Lamiaceae family. Nepeta species are commonly employed in traditional medicine for a variety of ailments, as well as food additives. In addition, they also come to the fore with their rich phytochemical content. In the present study, the quantitative phytochemical content of methanolic extracts and infusions prepared from the aerial parts of 14 Nepeta taxa collected from Turkey and their cytotoxic effects on two breast cancer cell lines, MCF-7 and MDA-MB-231, were investigated by using the MTT (3-(4,5-dimethylthiazol-2-yl))-2,5-diphenyltetrazolium-bromide) test. According to HPLC-PDA analysis, N. racemosa methanolic extract had the highest ursolic acid content with 165.9 mg/g extract. Total sterol, total iridoid, and total triterpenoid content were determined to be greatest in the methanolic extracts of N. meyeri, N. trichocalyx and N. phyllochlamys. The MTT experiment demonstrated that certain Nepeta species suppressed the growth of MCF-7 and MDA-MB-231 cells in a dose-dependent manner. Statistical analysis revealed a strong correlation between the cytotoxic effects of the extracts and their triterpene content. In conclusion, the data obtained from this study are important in terms of forming a basis for advanced anticancer activity studies on breast cancer with Nepeta sp.

Bioassay-guided isolation of cytotoxic compounds from Chrysophthalmum montanum (DC.) Boiss.

Bioassay-guided isolation of the 80% methanol extract of the aerial parts of Chrysophthalmum montanum (DC.) Boiss. (Asteraceae) led to the isolation of four known guaianolide-type sesquiterpene lactones, 6α-acetoxy-4α-hydroxy-1ßH-guaia-9.11(13)-dien-12.8α-olide (1), 6α-acetoxy-4α-hydroxy-9ß.10ß-epoxy-1ßH-guaia-11(13)-en-12.8α-olide (2), 4α,6α-dihydroxy-1ß,5α,7αH-guaia-9(10),11(13)-dien-12,8α-olide (3), and (4α,5α,8ß,10ß)-4,10-dihydroxy-1,11(13)-guaidien-12,8-olide (4), along a steroidal glycoside mixture (5a and 5b). The structures of the compounds were identified on the basis of spectroscopic data. Among them, 2, 4 and a steroidal glycoside mixture were obtained from C. montanum for the first time. All isolates were also first time assayed for in vitro cytotoxicities against four human cancer cell lines, i.e. breast (MCF-7, MDA-MB 231), colon (HT-29), and lung (PC3). Among the isolates, 1-3 showed significant inhibitory effect on the proliferation of cancer cells with viability ranging from 6.86 to 26.51%, while steroidal glycoside mixture showed no cytotoxicity, except against HT-29 (viability 61.99%). Compound 4 exhibited strong and selective cell growth inhibition against HT-29 with viability 20.99% and was identified as a promising compound with high selectivity between cancer cells and normal human lung cells (BEAS-2B), especially against HT-29 (IC50 = 12.2 µg/mL) compared to that of cisplatin. These results suggested that 4 is worthy of further study to determine its cytotoxicity mechanisms.

Phytotoxicity, Toxicity on Brine Shrimp and Insecticidal Effect of Chrysophthalmum gueneri Aytac & Anderb. Growing in Turkey.

OBJECTIVES: The aim of this study was to investigate the probable toxicity on brine shrimp, phytotoxicity, and insecticidal activity of Chrysophthalmum gueneri Aytac & Anderb. MATERIALS AND METHODS: The MeOH (80%) extract obtained from the whole plant of C. gueneri was fractionated through subsequent solvent extractions in increasing polarity with n-hexane, chloroform, and n-butanol. The MeOH (80%) extract and all fractions of C. gueneri were evaluated for their biologic activities using in vitro screening bioassays such as brine shrimp lethality test and phytotoxicity against Lemna minor, as well as insecticidal activity against Rhyzopertha dominica and Tribolium castaneum. RESULTS: The findings showed that the n-hexane and chloroform fractions of the plant had significant phytotoxic activities with 100% growth inhibition (GI) at 1000 µg/mL against L. minor. Moreover, the MeOH (80%) extract (53% GI) and n-butanol fraction (46.6% GI) of the plant had moderate phytotoxic activities at 1000 µg/mL. Otherwise, no samples had toxicity on the brine shrimps. In addition, the remaining water fraction had low insecticidal activity with 20% mortality against T. castaneum. CONCLUSION: Our results show that the n-hexane and chloroform fractions of C. gueneri had potential phytotoxic effects.

Cytotoxic, Phytotoxic and Insecticidal Activities of Chrysophthalmum montanum (DC.) Boiss.

OBJECTIVES: To investigate the in vitro cytotoxic, phytotoxic, and insecticidal activity of Chrysophthalmum montanum (DC.) Boiss. MATERIALS AND METHODS: The crude methanol (80%) extract of the aerial parts of C. montanum was fractionated to obtain n-hexane, chloroform, n-butanol, and remaining water fractions. The crude extract and subsequent solvent fractions of the plant were evaluated for their biological activities using screening bioassays such as cytotoxicity on brine shrimp lethality, phytotoxicity against Lemna minor L., and insecticidal activity against Rhyzopertha dominica and Tribolium castaneum. RESULTS: The cytotoxicity assay revealed that the crude extract, n-hexane, and chloroform fractions of the plant had positive lethality with LD50 values of 71.51, 126.62, and 75.95 µg/mL, respectively. The extract and its fractions, except for the remaining water fraction, showed phytotoxic activity, which was expressed as percentage growth regulation in a concentration-dependent manner. n-hexane and chloroform fractions in particular had 100% growth inhibition (GI) at 1000 µg/mL, followed by the n-butanol fraction (62.6% GI) and crude extract (40.0% GI) of the plant at the same concentration. Otherwise, all samples had no insecticidal activity against R. dominica and T. castaneum. CONCLUSION: This study demonstrates that C. montanum contains bioactive compounds related to potential biological activities such as cytotoxic and phytotoxic.

Olive leaf extracts protect cardiomyocytes against 4-hydroxynonenal-induced toxicity in vitro: comparison with oleuropein, hydroxytyrosol, and quercetin.

Olive (Olea europaea) leaf, an important traditional herbal medicine, displays cardioprotection that may be related to the cellular redox modulating effects of its polyphenolic constituents. This study was undertaken to investigate the protective effect of the ethanolic and methanolic extracts of olive leaves compared to the effects of oleuropein, hydroxytyrosol, and quercetin as a positive standard in a carbonyl compound (4-hydroxynonenal)-induced model of oxidative damage to rat cardiomyocytes (H9c2). Cell viability was detected by the MTT assay; reactive oxygen species production was assessed by the 2',7'-dichlorodihydrofluorescein diacetate method, and the mitochondrial membrane potential was determined using a JC-1 dye kit. Phospho-Hsp27 (Ser82), phospho-MAPKAPK-2 (Thr334), phospho-c-Jun (Ser73), cleaved-caspase-3 (cl-CASP3) (Asp175), and phospho-SAPK/JNK (Thr183/Tyr185) were measured by Western blotting. The ethanolic and methanolic extracts of olive leaves inhibited 4-hydroxynonenal-induced apoptosis, characterized by increased reactive oxygen species production, impaired viability (LD50: 25 µM), mitochondrial dysfunction, and activation of pro-apoptotic cl-CASP3. The ethanolic and methanolic extracts of olive leaves also inhibited 4-hydroxynonenal-induced phosphorylation of stress-activated transcription factors, and the effects of extracts on p-SAPK/JNK, p-Hsp27, and p-MAPKAPK-2 were found to be concentration-dependent and comparable with oleuropein, hydroxytyrosol, and quercetin. While the methanolic extract downregulated 4-hydroxynonenal-induced p-MAPKAPK-2 and p-c-Jun more than the ethanolic extract, it exerted a less inhibitory effect than the ethanolic extract on 4-hydroxynonenal-induced p-SAPK/JNK and p-Hsp27. cl-CASP3 and p-Hsp27 were attenuated, especially by quercetin. Experiments showed a predominant reactive oxygen species inhibitory and mitochondrial protecting ability at a concentration of 1-10 µg/mL of each extract, oleuropein, hydroxytyrosol, and quercetin. The ethanolic extract of olive leaves, which contains larger amounts of oleuropein, hydroxytyrosol, verbascoside, luteolin, and quercetin (by HPLC) than the methanolic one, has more protecting ability on cardiomyocyte viability than the methanolic extract or each phenolic compound against 4-hydroxynonenal-induced carbonyl stress and toxicity.

Fatty acid composition of Juniperus species (Juniperus section) native to Turkey.

Fatty acid compositions of seeds of five taxa of the Juniperus section of the genus Juniperus L. (Cupressaceae), i. e. J. drupacea Lab., J. communis L. var. communis, J. communis var. saxatilis Pall., J. oxycedrus L. subsp. oxycedrus, and J. oxycedrus subsp. macrocarpa (Sibth. & Sm.) Ball, were investigated. Methyl ester derivatized fatty acids of the lipophylic extracts of the five species were comparatively analyzed by capillary gas chromatography-mass spectrometry (GC-MS). Juniperus taxa showed uniform fatty acid patterns, among which linoleic (25.8 - 32.5%), pinolenic (11.9 - 24.1%) and oleic acids (12.4 - 17.2%) were determined to be the main fractions in the seed oils. Juniperonic acid was found to be remarkably high in J. communis var. saxatilis (11.4%), J. oxycedrus subsp. oxycedrus (10.4%), and J. communis var. communis (10.1%). To the best of our knowledge, the present work discloses the first report on the fatty acid compositions of seeds of this Juniperus section grown in Turkey.

Isolation of active constituents from cherry laurel (Laurocerasus officinalis Roem.) leaves through bioassay-guided procedures.

ETHNOPHARMACOLOGICAL RELEVANCE: The fresh leaves of Laurocerasus officinalis Roem. (Rosaceae) are externally used against pain and feverish symptoms in Turkish folk medicine. AIM OF THE STUDY: Effects of the extracts, fractions and isolated compounds from the leaves of L. officinalis were investigated using in vivo models of inflammation and pain in mice. METHODS: The crude ethanolic extract from the leaves of plant was sequentially fractionated into five subextracts; explicitly, n-hexane, chloroform, ethyl acetate (EtOAc), n-butanol, and remaining water extracts. Further studies were carried out on the most active EtOAc subextract was further subjected to fractionation through column chromatography. For the anti-inflammatory activity, carrageenan-induced hind paw edema and acetic acid-induced increase in capillary permeability models, and for the antinociceptive activity p-benzoquinone-induced writhing test in mice were employed. RESULTS: Ethanolic extract of the leaves was shown to possess significant inhibitory activity in the assay methods without inducing any gastric damage. Through bioassay-guided fractionation and isolation procedures three phenolic compounds, 2-O-ß-D-glucopyranosyl-2-hydroxyphenyl-acetic acid (1), kaempferol-3-O-ß-D-xylopyranosyl-(1→2)-O-ß-D-glucopyranoside (2) and (+)-catechin (3) were isolated from the active fraction and their structures were elucidated by spectral techniques (1D and 2D NMR, ESIMS). CONCLUSION: The experimental data verified that Laurocerasus officinalis leaves displayed remarkable anti-inflammatory and antinociceptive activity.

New HPLC-chemometric approaches to the analysis of isoflavones in Trifolium lucanicum Gasp.

New HPLC-chemometric approaches were proposed for the simultaneous chromatographic quantification of daidzein, genistein, formononetin, and biochanin A in the samples consisting of the aerial parts of Trifolium lucanicum Gasp. (Leguminosae). Partial least squares and principal component regression algorithms were applied to the multiple chromatographic data set obtained by measuring at 240, 248, 256, and 264 nm to construct HPLC-partial least squares and HPLC-principal component regression calibrations. Chromatographic separation was carried out by using a mobile phase containing methanol, acetate buffer (pH=4.75) and acetonitrile (21:58:21, v/v/v) on the reversed phase column, Supelcosil™ LC-18 (15 cm×4.6 mm id). In addition, conventional HPLC based on the detection at a single wavelength was used for the determination of each compound in the extracts of T. lucanicum. The validity and applicability of the proposed HPLC-chemometric and conventional HPLC methods were performed by analyzing various synthetic plant samples. A good agreement was observed in the application of the proposed HPLC-chemometric tools to the synthetic and extracted samples of T. lucanicum.

Assessment of enzyme inhibitory and antioxidant activities of lignans from Taxus baccata L.

Phytochemical investigations of Taxus baccata L. by successive chromatographic methods resulted in the isolation of the lignans lariciresinol (1), taxiresinol (2), 3'-demethylisolariciresinol-9'-hydroxyisopropylether (3), isolariciresinol (4), and 3-demethylisolariciresinol (5) as well as taxoids. Compounds 1-5 were evaluated for their acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and lipoxygenase (LOX) inhibitory activities, which play a role in the pathogenesis of Alzheimer's disease (AD), by in vitro spectrophotometric methods, while they were also screened for their antioxidant capacity in 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, ferrous ion-chelating effect, and ferric-reducing antioxidant power (FRAP) at 125, 250, 500, and 1000 microg ml(-1). All compounds exhibited a moderate inhibition against both BChE and LOX, whereas they were inactive towards AChE. The compounds displayed a great scavenging activity against DPPH especially at 500 and 1000 microg ml(-1). Besides, they were found to exert noteworthy reducing antioxidant power on ferric ions. In particular, the FRAP of compounds 2 (3.552 +/- 0.02), 4 (3.021 +/- 0.71), and 5 (3.533 +/- 0.01) were as high as that of the reference chlorogenic acid (3.618 +/- 0.01) at 1000 microg ml(-1). None of the compounds exhibited chelating ability against ferrous ions.

Characterization of Szovitsia callicarpa volatile constituents obtained by micro- and hydrodistillation.

The volatile constituents of Szovitsia callicarpa Fisch. & C. A. Mey. were obtained from the fruits by microdistillation, due to the limited plant material availability. Initial GC-MS analyses of the obtained material showed an unidentified major constituent with a molecular ion peak at M+ 280. After hydrodistillation of the plant material, the resulting essential oil was also analyzed by GC and GC-MS, simultaneously. In total, twenty-two compounds, representing 98.6% of the microdistilled sample, were identified, whereas seventeen components were detected in the hydrodistilled oil, representing 98.5% of the total. The GC-MS analyses showed that the samples contained an unidentified major constituent, which was further purified from the hydrodistilled essential oil by micro-column chromatography. The structure was elucidated as alpha-kessyl acetate using 1H- and 13C-NMR spectroscopic and mass spectroscopic techniques. Other major constituents in the analytes were determined as longipinene, longicyclene and kessane. In addition, both samples, as well as the major compound alpha-kessyl acetate, were tested at 1 mg/mL concentration against the pathogen Candida tropicalis and for its free radical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH*) by TLC-bioauthographic techniques. Both preliminary assays at the tested concentration showed no activity.