Butylated hydroxyl-toluene, 2,4-Di-tert-butylphenol, and phytol of Chlorella sp. protect the PC12 cell line against H2O2-induced neurotoxicity.

Oxidative stress is considered the main cause of cellular damage in a number of neurodegenerative disorders. One suitable ways to prevent cell damage is the use of the exogenous antioxidant capacity of natural products, such as microalgae. In the present study, four microalgae extracts, isolated from the Persian Gulf, were screened to analyze their potential antioxidant activity and free radical scavenging using ABTS, DPPH, and FRAP methods. The methanolic extracts (D1M) of green microalgae derived from Chlorella sp. exhibited potent free radical scavenging activity. In order to characterize microalgae species, microscopic observations and analysis of the expression of 18S rRNA were performed. The antioxidant and neuroprotective effects of D1M on H2O2-induced toxicity in PC12 cells were investigated. The results demonstrated that D1M significantly decreased the release of nitric oxide (NO), formation of intracellular reactive oxygen species (ROS), and the level of malondialdehyde (MDA), whereas it enhanced the content of glutathione (GSH), and activity of heme oxygenase 1 (HO-1), NAD(P)H: quinone oxidoreductase 1 (NQO1), and catalase (CAT) in PC12 cells exposed to H2O2. The pretreatment of D1M improved cell viability as measured by the MTT assay and invert microscopy, reduced cell apoptosis as examined by flow cytometry analysis, increased mitochondrial membrane potential (MMP), and diminished caspase-3 activity. The GC/MS analysis revealed that D1M ingredients have powerful antioxidant and anti-inflammatory compounds, such as butylated hydroxytoluene (BHT), 2,4-di-tert-butyl-phenol (2,4-DTBP), and phytol. These results suggested that Chlorella sp. extracts have strong potential to be applied as neuroprotective agents, for the treatment of neurodegenerative disorders.

Toxic metabolite profiling of Inocybe virosa.

Wild mushroom foraging involves a high risk of unintentional consumption of poisonous mushrooms which is a serious health concern. This problem arises due to the close morphological resemblances of toxic mushrooms with edible ones. The genus Inocybe comprises both edible and poisonous species and it is therefore important to differentiate them. Knowledge about their chemical nature will unambiguously determine their edibility and aid in an effective treatment in case of poisonings. In the present study, the presence of volatile toxic metabolites was verified in Inocybe virosa by gas chromatography. Methyl palmitate, phenol, 3,5-bis (1,1-dimethyl ethyl) and phytol were the identified compounds with suspected toxicity. The presence of the toxin muscarine was confirmed by liquid chromatography. The in vitro study showed that there was negligible effect of the digestion process on muscarine content or its toxicity. Therefore, the role of muscarine in the toxicity of Inocybe virosa was studied using a bioassay wherein metameters such as hypersalivation, immobility, excessive defecation, heart rate and micturition were measured. Administration of muscarine resulted in an earlier onset of symptoms and the extract showed a slightly stronger muscarinic effect in comparison to an equivalent dose of muscarine estimated in it. Further, the biological fate of muscarine was studied by pharmacokinetics and gamma scintigraphy in New Zealand white rabbits. Significant amount of the toxin was rapidly and effectively concentrated in the thorax and head region. This study closely explains the early muscarinic response such as miosis and salivation in mice. By the end of 24 h, a relatively major proportion of muscarine administered was accumulated in the liver which stands as an explanation to the hepatotoxicity of Inocybe virosa. This is one of the rare studies that has attempted to understand the toxic potential of muscarine which has previously been explored extensively for its pharmaceutical applications.

In vivo and in vitro antimicrobial activity of phytol, a diterpene molecule, isolated and characterized from Adhatoda vasica Nees. (Acanthaceae), to control severe bacterial disease of ornamental fish, Carassius auratus, caused by Bacillus licheniformis PKBMS16.

Bacillus licheniformis, a pathogenic new strain of bacteria is considered as the main cause of high mortalities and economic losses among the ornamental fish farms of India. The present study aimed to investigate the anti-bacterial and Immunostimulant activity of three selected Indian medicinal plants, Allium sativum, Adhatoda vasica and Centella asiatica for treating Bacillus licheniformis PKBMS16 by subsequent experimental and clinical trials using different organic polar and non-polar solvents. The antimicrobial and Immunostimulant activity of methanolic crude extracts of Adhatoda vasica was fractions and active constituents was further characterized by chromatography and mass spectroscopy studies using FTIR, 1HNMR and 13c NMR to identify as well as to determine the nature of the pure compound which is phytol (C20H40O), a diterpene alcohol with a molecular weight of m/z 297. In order to study the in vivo anti-pathogenic influence of the biologically active compounds, phytol were incorporated to the artificial diets at the concentration of 2, 5 and 8 mg/kg and fed to the1.0 × 105 CFU/ml of Bacillus licheniformis PKBMS16 injected experimentally challenged ornamental goldfish Carassius auratus for twenty days. Phytol treated group significantly (P < 0.01 and P < 0.05) reduced the rate of fish mortality. After the termination of survivability assay the estimation of hemato-biochemical parameters have been performed and revealed the significant recovery of health condition on 20th days post treatment. Therefore, the present study concluded that the low toxicity along with high bioactivity and tolerance by lower vertebrate supports the potential of phytol as a new compound for inducing fish immunity.

One-Step Preparative Separation of Phytosterols from Edible Brown Seaweed Sargassum horneri by High-Speed Countercurrent Chromatography.

Sargassum horneri, a sargassaceae brown alga, is one of the main species in the subtidal seaweeds flora extensively distributed in the Yellow and East China Sea. It has been proven that the phytosterols are an important class of bioactive substances in S. horneri. In this work, a counter-current chromatography approach is proposed for preparative separation of phytol and two analogue sterols from a crude extract of S. horneri. A two-phase solvent system composed of n-hexane-acetonitrile-methanol (5:5:6, v/v) was selected and optimized. The effects of rotary speed and flow rate on the retention of the stationary phase were carefully studied. Under the optimum conditions, phytol and two analogue sterols, fucosterol and saringosterol, were baseline separated, producing 19.8 mg phytol, 23.7 mg fucosterol, and 3.1 mg saringosterol from 300 mg of crude S. horneri extract in one-step separation. The purities of three target compounds were all above 85%. The structures of phytol and two sterols were identified by nuclear magnetic resonance spectroscopy.

Chemical Compositions and Antioxidant Activities of Essential Oils Extracted from the Petals of Three Wild Tree Peony Species and Eleven Cultivars.

The aim of this study was to investigate the essential oil (EO) compositions and antioxidant activities from petals of three wild tree peony species (Paeonia delavayi, P. lutea, and P. rockii) and eleven P. suffruticosa cultivars from different cultivar groups. The EOs yields varied from 0.63% to 1.25% (v/v) among samples when using supercritical CO2 extraction. One hundred and sixty-three components were detected by GC/MS; and among them, linalool oxide, (Z)-5-dodecen-1-yl acetate, nonadecane, (Z)-5-nonadecene, heneicosane, phytol, and linoleic acid ethyl ester were dominant. According to hierarchical cluster analysis, principal component analysis and correspondence analysis, P. lutea, P. delavayi, and 'High Noon' were clustered in a group described as having a refreshing herbal-like note due to high rates of phytol and linalool oxide. Notably, P. lutea and P. delavayi also had strong DPPH and ABTS radical scavenging activities. These results suggest that P. lutea and P. delavayi are the most promising candidates as useful sources of fragrances and natural antioxidants.

Piper betle and its bioactive metabolite phytol mitigates quorum sensing mediated virulence factors and biofilm of nosocomial pathogen Serratia marcescens in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper betle, a tropical creeper plant belongs to the family Piperaceae. The leaves of this plant have been well known for their therapeutic, religious and ceremonial value in South and Southeast Asia. It has also been reported to possess several biological activities including antimicrobial, antioxidant, antinociceptive, antidiabetic, insecticidal and gastroprotective activities and used as a common ingredient in indigenous medicines. In Indian system of ayurvedic medicine, P. betle has been well recognized for its antiseptic properties and is commonly applied on wounds and lesions for its healing effects. AIM OF THE STUDY: To evaluate the anti-quorum sensing (anti-QS) and antibiofilm efficacy of P. betle and its bioactive metabolite phytol against Serratia marcescens. MATERIALS AND METHODS: The P. betle ethyl acetate extract (PBE) was evaluated for its anti-QS efficacy against S. marcescens by assessing the prodigiosin and lipase production at 400 and 500µgml-1 concentrations. In addition, the biofilm biomass quantification assay was performed to evaluate the antibiofilm activity of PBE against S. marcescens. Besides, the influence of PBE on bacterial biofilm formation was assessed through microscopic techniques. The biofilm related phenomenons like exopolysaccharides (EPS) production, hydrophobicity and swarming motility were also examined to support the antibiofilm activity of PBE. Transcriptional analysis of QS regulated genes in S. marcescens was also done. Characterization of PBE was done by separation through column chromatography and identification of active metabolites by gas chromatography -mass spectrometry. The major compounds of active fractions such as hexadecanoic acid, eugenol and phytol were assessed for their anti-QS activity against S. marcescens. Further, the in vitro bioassays such as protease, biofilm and HI quantification were also carried out to confirm the anti-QS and antibiofilm potential of phytol in PBE. RESULTS: PBE inhibits QS mediated prodigiosin pigment production in S. marcescens, which confirmed its anti-QS potential against S. marcescens. At 500µgml-1 concentration, PBE significantly inhibited the production of protease, lipase, biofilm and EPS to the level of 71%, 68%, 65% and 43% in S. marcescens, respectively. Further, their antibiofilm efficacy was confirmed through microscopic techniques. In addition, PBE effectively inhibited the hydrophobicity and swarming motility. Additionally, the results of qPCR analysis validated the downregulation of QS genes. Chromatographic techniques the presence of hexadecanoic acid, eugenol and phytol in PBE and the potential bioactive compound with anti-QS activity was identified as phytol. In vitro assays with phytol evidenced the potent inhibition of QS-controlled prodigiosin, protease, biofilm and hydrophobicity in S. marcescens, without exerting any deleterious effect on its growth. CONCLUSION: This study demonstrates the promising anti-QS and antibiofilm activities of PBE and its active metabolite phytol, and confirms the ethnopharmacological applications of these leaves against S. marcescens infections.

Isolation and Identification of Mosquito (Aedes aegypti) Biting-Deterrent Compounds from the Native American Ethnobotanical Remedy Plant Hierochloë odorata (Sweetgrass).

Hierochloë odorata (L.) P. Beauv. (Poaceae), commonly known as sweetgrass, has documented use as an insect repellent by the Flatheads of Montana and Blackfoot of Alberta. Both the Flatheads of Montana and Blackfoot of Alberta would use braided plant material in a sachet in clothing or burn them from one end as incense, air/clothing freshener, and insect repellent. This study evaluated the insect-repellent properties of this plant using an in vitro mosquito Aedes aegypti feeding bioassay-directed approach to identify the compound(s) responsible for the observed activities. Evaluation of crude extracts produced from H. odorata revealed that the hydrodistillate had the highest level of mosquito biting deterrence. Fractionation of this extract, followed by re-evaluation for mosquito biting deterrence, produced many active fractions, which were evaluated by spectroscopic techniques and determined to contain phytol, coumarin, and 2-methoxy-4-vinylphenol. Phytol and coumarin were both determined to be responsible for the Ae. aegypti biting deterrency. Scientific evidence reported here validates its traditional use as a biting-insect deterrent.

A New Succinate Derivative from Ajuga decumbens.

A new succinate derivative, ethyl (5-formylfuran-2-yl)methyl succinate (1), along with three known compounds (2-4) have been isolated from the whole plants of Ajuga decumbens Thunb. Their structures were elucidated by extensive spectroscopic (1D and 2D NMR) and HR-ESI-MS data analysis, and literature values. Compound 1 was isolated as a new succinate derivative, and compounds 2 and 3 were for the first time separated from A. decumbens.

Neuroprotective effect of the marine macroalga Gelidiella acerosa: identification of active compounds through bioactivity-guided fractionation.

Context Gelidiella acerosa (Forsskål) Feldmann & G. Hamel (Rhodophyta-Gelidiales) is a marine red macroalga. Our previous work found that a benzene extract of G. acerosa possesses noticeable neuroprotective activity, when evaluated through in vitro and in vivo systems. Objective Bioactive-guided fractionation and identification of active compounds by column chromatography using solvents of varying polarity. Materials and methods Fractionation was done by column chromatography, antioxidant and anticholinesterase activity was assessed by DPPH and cholinesterase inhibition assays (50-200 µg/ml), compound identification was done by LC-MS analysis, the mode of interaction of active compound was analyzed through docking studies and quantification was done by high-performance thin-layer chromatography (HPTLC) analysis. Results The results suggest that fractions F9-F13 exhibited significant (p < 0.05) antioxidant and anticholinesterase activities. Hence, these fractions were pooled together and verified for neuroprotective activity. The pooled fraction was subjected to LC-MS analysis and among all the compounds, phytol was previously reported to possess excellent neuroprotective potential. Hence, the neuroprotective potential of phytol was assessed. The results suggest that phytol showed significant (p < 0.05) antioxidant activities (25-125 µg/ml) with an IC50 value of 95.27 ± 1.65 µg/ml and cholinesterase inhibitory potential (5-25 µg/ml) with IC50 values of 2.704 ± 0.07 and 5.798 ± 0.72 µg/ml for AChE and BuChE, respectively. Molecular docking studies suggest that phytol interacts with cholinesterase through the arginine residue of the enzyme. HPTLC quantification showed that about 6.266 µg of phytol was present per mg of pooled fraction. Conclusion The study suggests that phytol might act as the key compound in contributing to the neuroprotective potential of G. acerosa.

Chemical composition and evaluation of the antimicrobial activity of the essential oil from leaves of Eugenia platysema.

This study describes the qualitative and quantitative chemical composition and evaluates the antibacterial activity of essential oil from Eugenia platysema leaves. Analysis by GC-FID and GC-MS allowed the identification of 22 compounds. Different from the other species of the Eugenia genus, the major compound found in the essential oil was the diterpene phytol (66.05%), being this the first report of the presence of this compound in the essential oils from Eugenia genus. The sesquiterpene elixene was the second most concentrated compound in the studied essential oil (9.16%). The essential oil from E. platysema was tested for its antibacterial activity against cell-walled bacteria and mollicute strains of clinical interest using the microdilution broth assay. The results showed that the essential oil of E. platysema was inactive until 1000 µg mL(-1) against tested bacteria.

Chemical composition and antibacterial activity of the essential oil from Pyrrosia tonkinensis (Giesenhagen) Ching.

The present study aimed to analyse the chemical components of the essential oil from Pyrrosia tonkinensis by GC-MS and evaluate the in vitro antibacterial activity. Twenty-eight compounds, representing 88.1% of the total essential oil, were identified and the major volatile components were trans-2-hexenal (22.1%), followed by nonanal (12.8%), limonene (9.6%), phytol (8.4%), 1-hexanol (3.8%), 2-furancarboxaldehyde (3.5%) and heptanal (3.1%). The antibacterial assays showed that the essential oil of P. tonkinensis had good antibacterial activities against all the tested microorganisms. This paper first reported the chemical composition and antimicrobial activity of the essential oil from P. tonkinensis.

Insecticidal activity of Jatropha curcas extracts against housefly, Musca domestica.

The hexane and ether extracts of leaves, bark and roots of Jatropha curcas were screened for their toxicity against different developmental stages of housefly. The larvicidal, pupicidal and adulticidal activities were analysed at various concentrations (0.78-7.86 mg/cm(2)) of hexane and ether extracts. The lethal concentration values (LC50) of hexane extract of J. curcas leaves were 3.0 and 0.27 mg/cm(2) for adult and larval stages of housefly, respectively, after 48 h. Similarly, the ether extract of leaf showed the LC50 of 2.20 and 4.53 mg/cm(2) for adult and larval stages of housefly. Least toxicity was observed with hexane root extract of J. curcas with LC50 values of 14.18 and 14.26 mg/cm(2) for adult and larvae of housefly, respectively, after 48 h. The variation in LC50 against housefly pupae was found to be 8.88-13.10 mg/cm(2) at various J. curcas extract concentrations. The GC-MS analysis of J. curcas leaf extract revealed the presence of trans-phytol (60.81 %), squalene (28.58 %), phytol (2.52 %) and nonadecanone (1.06 %) as major components that could be attributed for insecticidal activity of J. curcas extracts.

Characteristic Chemical Components and Aroma-active Compounds of the Essential Oils from Ranunculus nipponicus var. submersus Used in Japanese Traditional Food.

Ranunculus nipponicus var. submersus is an aquatic macrophyte; it is known as a wild edible plant in Japan for a long time. In this study, the essential oils from the fresh and dried aerial parts of R. nipponicus var. submersus were extracted by hydrodistillation and analyzed by gas chromatography (GC) and GC-mass spectrometry (GC-MS). Moreover, important aroma-active compounds were also detected in the oil using GC-olfactometry (GC-O) and aroma extract dilution analysis (AEDA). Thus, 98 compounds (accounting for 93.86%) of the oil were identified. The major compounds in fresh plant oil were phytol (41.94%), heptadecane (5.92%), and geranyl propionate (5.76%), while those of. Dried plant oil were ß-ionone (23.54%), 2-hexenal (8.75%), and dihydrobovolide (4.81%). The fresh and dried oils had the green-floral and citrus-floral odor, respectively. The GC-O and AEDA results show that phenylacetaldehyde (green, floral odor, FD-factor = 8) and ß-ionone (violet-floral odor, FD-factor = 8) were the most characteristic odor compounds of the fresh oils. ß-Cyclocitral (citrus odor, FD-factor = 64) and ß-ionone (violet-floral odor, FD-factor = 64) were the most characteristic odor compounds of the dried oil. These compounds are thought to contribute to the flavor of R. nipponicus var. submersus.

DPPH scavenging, PRAP activities and essential oil composition of edible Lathyrus ochrus L. (Cyprus Vetch, Luvana) from Cyprus.

The essential oil of the aerial parts of edible Lathyrus ochrus L. was investigated by simultaneous GC, GC/MS analyses under the same conditions. Trace amount of oil (0.01> mL) obtained by hydro distillation of 200 g fresh plants was trapped in 1 mL n-hexane. Twenty components were detected representing 91.55 ± 0.56 % of the oil. The main components were phytol 49.39 ± 0.44 %, hexadecanoic acid 20.64 ± 0.89 % and pentacosane 4.20 ± 0.09 %. Essential oil solution (1% oil: n-hexane) afforded similar DPPH scavenging activity (9.28 ± 1.30 %) when compared with positive controls α-tocopherol (9.74 ± 0.21 %) and BHT (7.79 ± 0.26 %) at the same concentrations. Antioxidant activity of the oil was determined using a new HPTLC-PRAP assay. The oil afforded two fold higher reducing activity of phosphomolybdenum complex (594.85 ± 5.14 AU) when compared with positive controls α- tocopherol (271.10 ± 2.86 AU) and BHT (210.53 ± 1.81 AU) at the same concentration.

A novel ether-linked phytol-containing digalactosylglycerolipid in the marine green alga, Ulva pertusa.

Galactosylglycerolipids (GGLs) and chlorophyll are characteristic components of chloroplast in photosynthetic organisms. Although chlorophyll is anchored to the thylakoid membrane by phytol (tetramethylhexadecenol), this isoprenoid alcohol has never been found as a constituent of GGLs. We here described a novel GGL, in which phytol was linked to the glycerol backbone via an ether linkage. This unique GGL was identified as an Alkaline-resistant and Endogalactosylceramidase (EGALC)-sensitive GlycoLipid (AEGL) in the marine green alga, Ulva pertusa. EGALC is an enzyme that is specific to the R-Galα/ß1-6Galß1-structure of galactolipids. The structure of U. pertusa AEGL was determined following its purification to 1-O-phytyl-3-O-Galα1-6Galß1-sn-glycerol by mass spectrometric and nuclear magnetic resonance analyses. AEGLs were ubiquitously distributed in not only green, but also red and brown marine algae; however, they were rarely detected in terrestrial plants, eukaryotic phytoplankton, or cyanobacteria.

Antiplasmodial activity of the ethnobotanical plant Cassia fistula.

In our ongoing investigation of new compounds with activity against malaria parasites, we tested the in vitro antiplasmodial activity of fractions and purified compounds from Cassia fistula L., a plant traditionally used by native populations of Tanzania, Zimbabwe, Mozambique and Brazil to treat malaria or symptoms associated with this disease. Crude extracts from leaves, bark and fruits were tested for their antiplasmodial activity against the chloroquine-sensitive strain of Plasmodium falciparum (D10), where leaf extracts showed the highest activity. The chloroform extract of the leaves was further bioassay-guided fractionated using a combination of centrifugal partition chromatography and flash column chromatography. Three main antiplasmodial principles, phytol (1) (IC50 18.9 +/- 0.60 microM), lutein (2) (IC50 12.5 +/- 0.35 microM), and di-lineolylgalactopyranosyl-glycerol (DLGG) (IC50 5.8 +/- 0.27 microM) (3), were isolated and identified using spectroscopic methods. When the three active principles were tested for their cytotoxicity using a Chinese Hamster Ovarian (CHO) cell line, compound 3 showed very weak toxicity (IC50 75.9 +/- 0.28 microM), while the other two compounds were nontoxic, even at the highest concentration tested. The study provides evidence to support the use of Cassia fistula as an antimalarial remedy and describes the antiplasmodial constituents from the leaves.

Distribution of iridiod glucosides and anti-oxidant compounds in Spathodea campanulata parts.

The antioxidant principles isolated from the various parts of the plant are verminoside (leaf, stem bark and flowers; EC(50) = 2.04 µg/ml), Specioside (flowers; EC(50) = 17.44 µg/ml), Kampeferol diglucoside (leaf; EC(50) = 8.87 µg/ml) and Caffeic acid (leaf and fruits). The non anti-oxidant components isolated in the study include ajugol (stem bark and fruits) and phytol (leaf).

Identification of major aroma compounds in the leaf of Morinda citrifolia Linn.

Morinda citrifolia, commonly named noni, has been used as food and as a folk medicine throughout the tropics. The use of the leaves to make hot water beverages is increasing in popularity, especially in Japan and the United States. To better understand the effects of processing on the content of the major aroma compounds, volatile oils were collected from samples of frozen, dried and roasted leaves by steam distillation and then analyzed by GC-MS. Drying of the leaves reduces the quantity of aroma compounds by more than half. Palmitic acid and E-phytol were identified as the major components of the volatile oil. With the exception of E-phytol, all of the known volatile compounds identified in the leaf samples were done so for the first time.

[Studies on chemical constituents of Laurencia tristicha ( II )].

OBJECTIVE: To search for chemical constituents with structural diversity from Laurencia tristicha to supply for biological assay. METHOD: Compounds were isolated by means of column chromatography over normal phase silica gel and Sephadex LH-20, recrystallization and HPLC. Structures were identified by spectroscopic methods including 1D NMR, IR and MS. Cytotoxicities of the purified compounds were evaluated by MTT method. RESULT: Seven compounds were isolated from L. tristicha. Their structures were elucidated as cholesterol (1), cholesta- 5-en-3beta, 7alpha-diol (2), beta-stigmasterol (3), phytol (4), zeaxanthin (5), 4 -hydroxybenzaldehyde (6), indolyl-3-carbaldehyde (7). In the cytotoxic assay compound 2 was active against human cancer cell lines HCT-8, Bel-7402, BGc-823, A549 and HELA with IC50 values of 1.90, 2.02, 1.99, 6.52 and 1.20 microg x mL(-1), respectively. Compound 4 showed cytotoxicity against HCT-8 and HELA with IC50 value of 3.51 and 2.04 microg x mL(-1), and other compounds were inactive ( IC50 > 10 microg x mL(-1)). CONCLUSION: Compounds 1-7 were isolated from L. tristicha for the first time. In additon, compounds 2 and 4 were cytotoxic against several human cancer cell lines.

[Chemical constituents from herb of Alternanthera philoxeroides].

OBJECTIVE: To study the active constituents from Alternanthera philoxeroides. METHOD: The constituents were isolated with silica gel and Toyopearl HW-40C gel column chromatography and purified by HPLC. Their structures were elucidated by spectroscopy. RESULT: Nine compounds were isolated and identified as phaeophytin a (1), pheophytin a' (2), oleanoic acid (3), beta-sitosterol (4), 3beta-hydroxystigmast-5-en-7-one (5), alpha-spinasterol (6), 24-methylenecycloartanol (7), cycloeucalenol (8), phytol (9). CONCLUSION: Compounds 1,2,5,7-9 were isolated from this plant for the first time.