Chemical Composition and Protective Possibilities of Juglans Nigra Leaves and Green Husks Extracts: DNA Binding and Micronucleus Assay in Human Lymphocytes.

To better understand the mechanism of action of the compounds in the ethanolic extracts of J. nigra leaves and green husks, their binding to CT-DNA was investigated. This study was conducted to elucidate the in vitro protective effect of extracts against chromosomal damage in mitogen-induced human lymphocytes and investigate the possible application of selec+ted extracts as a natural source of polyphenolic compounds. Using HPLC-MS analysis, 103 different compounds were identified as having a higher number of active species, which is consistent with their activity. The frequency of micronuclei (MN) was scored in binucleated cells, and the nuclear proliferation index was calculated. Cyclic voltammetry experiments demonstrate that the nature of the interaction between extracts and CT-DNA is a synergy of electrostatic and intercalative modes, where leaves extracts showed a higher ability to bind to DNA. Extracts showed excellent antioxidant activity. At a concentration of only 4 µg/mL, extract of J. nigra leaves and the green husks reduced the incidence of MN by 58.2% and 64.5%, respectively, compared to control cell cultures.

Beyond Traditional Use of Alchemilla vulgaris: Genoprotective and Antitumor Activity In Vitro.

Alchemilla vulgaris L. (lady's mantle) was used for centuries in Europe and Balkan countries for treatments of numerous conditions and diseases of the reproductive system, yet some of the biological activities of lady's mantle have been poorly studied and neglected. The present study aimed to estimate the potential of A. vulgaris ethanolic extract from Southeast Serbia to prevent and suppress tumor development in vitro, validated by antioxidant, genoprotective, and cytotoxic properties. A total of 45 compounds were detected by UHPLC-HRMS analysis in A. vulgaris ethanolic extract. Measurement of antioxidant activity revealed the significant potential of the tested extract to scavenge free radicals. In addition, the analysis of micronuclei showed an in vitro protective effect on chromosome aberrations in peripheral human lymphocytes. A. vulgaris extract strongly suppressed the growth of human cell lines derived from different types of tumors (MCF-7, A375, A549, and HCT116). The observed antitumor effect is realized through the blockade of cell division, caspase-dependent apoptosis, and autophagic cell death. Our study has shown that Alchemilla vulgaris L. is a valuable source of bioactive compounds able to protect the subcellular structure from damage, thus preventing tumorigenesis as well as suppressing tumor cell growth.

Selected fungi of the genus Lactarius - screening of antioxidant capacity, antimicrobial activity, and genotoxicity.

It is well-known that mushrooms of the genus Lactarius constitute a natural food resource providing health benefits as a nutritient. This genus contains 4 mushrooms identified as L. deliciosus, L. volemus, L. sanguifluus, L. semisanguifluus and L. piperatus which were collected in Serbia. The aim of this study aimed was to identify and characterize the content of phenolic compounds and examine the antioxidant potential of 5 wild edible mushrooms. L. sanguifluus contained the highest content of total phenol compounds (75.25 mg gallic acid equivalents (GAE) per g dry extract weight (GAE/g DE) and exhibited the greatest antioxidant activity through the ability to remove radicals as evidenced by ABTS assay (8.99 mg of trolox equivalents (TE) per g dry extract weight (mg TE/g DE); total reducing power (TRP) assay mg ascorbic acid equivalents per mg of dry extract weight (0.42 mg AAE/g DE) and CUPRAC (14.23 mg TE /g DE). L. deliciosus methanolic extract produced greatest scavenging of the DPPH radical (46%). The methanol mushroom extracts were screened for in vitro antimicrobial activity against a panel of pathogenic bacterial strains using the microdilution method. Of all the extracts tested, L. sanguifluus extract showed the best antibacterial properties. The cytokinesis block micronucleus assay results for the examined mushrooms demonstrated that extracts at a concentration of 3 µg/ml decreased the number of micronuclei (MN) in the range of 19-49% which is significant bearing in mind that radioprotectant amifostine reduced the frequency of MN by only 16.3%. Data thus demonstrate that the 5 wild edible mushrooms of genus Lactarius contain constituents that are beneficial not only as nutrients but also have the potential as antioxidants, antibacterial and antigenotoxic properties.

The effects of Artemisia methanol extracts and ferulic acid, rutin, rosmarinic acid, and quercetin on micronucleus distribution on human lymphocytes.

Methanol extracts of A. absinthium, A. annua, A vulgaris, A. scoparia, and A. alba from Serbia at concentrations of 1.00; 2.00 and 4.00 µg/ml, and as pure compounds identified in examined extracts (ferulic acid, rutin, rosmarinic acid, and quercetin) were examined for their effects on micronucleus distribution in human lymphocytes in vitro. All extracts greatly reduced the number of micronuclei in the concentration of 2.00 µg/ml comparing to the control, whereby A. alba extract exhibited the most prominent effect on decreasing frequency of micronucleus (45.2%), followed by extracts of A. vulgaris, A. absinthium, A. scoparia and A. annua. The same declining trend was observed for the extracts at concentrations of 4.00 µg/ml, and 1.00 µg/ml, respectively. Among pure compounds (concentration 2.00 µg/ml), rutin showed the most pronounced effect on decreasing frequency of micronucleus (41.7%), higher than ferulic acid, quercetin, and rosmarinic acid comparing to the control.

Leaf-surface guaianolides from Amphoricarpos neumaeyri showing protective effect on human lymphocytes DNA.

Analysis of composition of CH2Cl2 surface extract of leaves of Amphoricarpos neumayeri Vis. revealed 16 sesquiterpene lactones with guaianolide skeleton, so called amphoricarpolides, typical for this genus. Four of them, 13-16, were new derivatives and their structures were elucidated by detailed analyses of IR, NMR and MS data. Amphoricarpolide (9), its 15-O-acetyl derivative (5), and two 9ß-hydroxyamphoricarpolides, 3,15-di-O-acetyl- and 3-O-acetyl-15-O-isovaleroyl derivatives (3 and 6, respectively) were tested for in vitro protective effect on chromosome aberrations in peripheral human lymphocytes using cytochalasin-B blocked micronucleus (CBMN) assay. The tested compound exerted a beneficial effect by decreasing DNA damage of human lymphocytes.

Chemical profile and biological activities of Peltigera horizontalis (Hudson) Baumg. thallus and apothecia extracts.

The aim of this study was to determine, for the first time, the chemical composition of Peltigera horizontalis thallus and apothecia extracts (ether, ethyl acetate, dichloromethane and acetone) by HPLC-UV and GC-MS, and evaluate activity of genotoxic, anticholinesterase, antioxidant and antibacterial potential of acetone extracts. Major constituents of thallus extracts were gyrophoric acid, and methyl gyrophorate while dominant component of apothecia extracts was tenuiorin. The predominant volatile compounds in extracts were methyl orsellinate, dodecyl acrylate, orcinol and orcinol monomethyl ether. The thallus acetone extract at concentration of 2.0 µg mL-1 gave the greatest decrease in the micronuclei frequency (22.4%) of all tested extracts. Apothecia extract showed stronger antioxidant activity as compared to thallus extract. Tested extracts at concentration of 10 mg mL-1 exhibited inhibitory effect (16.5% for thallus and 12.8% for apothecia) on pooled human serum cholinesterase. P. horizontalis acetone extracts had no activity against the tested five bacteria strains.

Hypogymnia tubulosa extracts: chemical profile and biological activities.

This study reports for the first time in the chemical composition of acetone, ether, ethyl acetate and dichloromethane extracts of Hypogymnia tubulosa determined by HPLC-UV, GC-FID and GC-MS as well as effect of H. tubulosa acetone extract on micronucleus distribution on human lymphocytes and on cholinesterase activity. Additionally, antioxidant (estimated via DPPH, ABTS, TRP, CUPRAC and TPC assays) and antibacterial activity against two Gram-positive and three Gram-negative bacteria were also determined. The HPLC-UV analysis revealed the presence of depsidones, 3-hydroxyphysodic, 4-O-methyl physodic acid, physodic and physodalic acid together with two depsides, atranorin and chloroatranorin. GC-FID and GC-MS analyses enabled the identification of atranol, chloroatranol, atraric acid, olivetol, olivetonide and 3-hydroxyolivetonide as the main components. The results of present study show that H. tubulosa acetone extract is a promising candidate for in vivo experiments considering antioxidant activity.

Ramalina capitata (Ach.) Nyl. acetone extract: HPLC analysis, genotoxicity, cholinesterase, antioxidant and antibacterial activity.

In the present investigation, effects of Ramalina capitata acetone extract on micronucleus distribution on human lymphocytes, on cholinesterase activity and antioxidant activity (by the CUPRAC method) were examined, for the first time as well as its HPLC profile. Additionally, total phenolic compounds (TPC), antioxidant properties (estimated via DPPH, ABTS and TRP assays) and antibacterial activity were determined. The predominant phenolic compounds in this extract were evernic, everninic and obtusatic acids. Acetone extract of R. capitata at concentration of 2 µg mL-1 decreased a frequency of micronuclei (MN) for 14.8 %. The extract reduces the concentration of DPPH and ABTS radicals for 21.2 and 36.1 % (respectively). Values for total reducing power (TRP) and cupric reducing capacity (CUPRAC) were 0.4624 ± 0.1064 µg ascorbic acid equivalents (AAE) per mg of dry extract, and 6.1176 ± 0.2964 µg Trolox equivalents (TE) per mg of dry extract, respectively. The total phenol content was 670.6376 ± 66.554 µg galic acid equivalents (GAE) per mg of dry extract. Tested extract at concentration of 2 mg mL-1 exhibited inhibition effect (5.2 %) on pooled human serum cholinesterase. The antimicrobial assay showed that acetone extract had inhibition effect towards Gram-positive strains. The results of manifested antioxidant activity, reducing the number of micronuclei in human lymphocytes, and antibacterial activity recommends R. capitata extract for further in vivo studies.

Phenolic Compounds from Atriplex littoralis and Their Radiation-Mitigating Activity.

From the aerial parts of Atriplex littoralis, three new flavonoid glycosides named atriplexins I-III (1-3), a known flavonoid glycoside, spinacetin 3-O-ß-d-glucopyranoside (4), arbutin (5), and 4-hydroxybenzyl-ß-d-glucopyranoside (6) were isolated. Their structures were elucidated on the basis of detailed spectroscopic analysis, including 1D and 2D NMR (COSY, NOESY, TOCSY, HSQC, HMBC) and HRESITOF MS data. The compounds were tested for in vitro protective effects on chromosome aberrations in peripheral human lymphocytes using a cytochalasin-B-blocked micronucleus (MN) assay in a concentration range of 0.8-7.4 µM of final culture solution. Chromosomal damage was induced by 2 Gy of γ-radiation on binucleated human lymphocytes, and the effects of the compounds were tested 2 to 19 h after irradiation. The frequency of micronuclei (MNi) was scored in binucleated cells, and the nuclear proliferation index was calculated. The highest prevention of in vitro biochemical and cytogenetic damage of human lymphocytes induced by γ-radiation was exhibited by 3 (reduction of MN frequency by 31%), followed by 4 and 6.

Synthesis of novel tetrahydrobenzazepine derivatives and their cytoprotective effect on human lymphocytes.

Cytoprotective compounds such as amifostine play an important role in chemo- and radiotherapy due to their ability to reduce the side effects of these treatments. Our work was initiated with the intention to design, synthesise and test a new class of heterocyclic compounds that would have an antioxidative profile with the potential to be further developed as cytoprotective agents. The design was based on the privileged tetrahydrobenzazepine scaffold found in many natural products with a wide range of biological properties. This structure was further functionalised with moieties known to possess antioxidative features such as tertiary amine and styrene double bond. A series of eight tetrahydrobenzazepine derivatives of isoquinoline, 3,4-dihydro-ß-carboline and pyridine were synthesised employing the Heck reaction as a key transformation. Some of the prepared compounds were tested for their in vitro effects on chromosome aberrations in peripheral human lymphocytes using the cytochalasin-B blocked micronucleus (MN) assay. Three tetrahydrobenzoazepine derivatives showed significant cytoprotective properties, comparable or even better to those of the radioprotective agent amifostine.

Venlafaxine versus applied relaxation for generalized anxiety disorder: a randomized controlled study on clinical and electrophysiological outcomes.

Some components of generalized anxiety disorder, such as physical symptoms, are thought to reflect autonomic nervous system arousal. This study primarily assessed the relationships between psychophysiological and clinical measures using venlafaxine extended release or applied relaxation, and secondarily, the impact of combination treatment in patients not remitting after 8 weeks. Fifty-eight patients were randomly assigned to 8 weeks of treatment with either venlafaxine or applied relaxation (Phase I). Non-remitted patients received combination treatment for an additional 8 weeks (Phase II). Assessments included the Hamilton Anxiety Scale (HAM-A), Beck Depression Inventory, Penn State Worry Questionnaire and the Stroop Color-Word Task coupled with electrophysiological measures (skin conductance and frontalis electromyography (EMG)). In Phase 1, a time effect was found for the clinical and skin conductance measures. Thirteen patients from each group were in remission. In Phase 2, seven additional patients remitted. Baseline psychophysiological measures were not associated with baseline clinical variables or with clinical outcomes. Independently of treatment allocation, a reduction in frontal EMG values at week 4 was significantly associated with a decrease in HAM-A scores at week 8. At week 4, responders from the applied relaxation group had lower electrophysiological activity than the venlafaxine group. Baseline psychophysiological measures were not linked with clinical measures at study inclusion or with treatment response. Frontal EMG response at week 4 is a possible predictor of treatment response. Treatment combination enhances treatment response after initial failure.

Diarylheptanoids from green alder bark and their potential for DNA protection.

Nine diarylheptanoids, 1-9, catechin (11), and a phenolic glucoside, 10, were isolated from the bark of green alder (Alnus viridis). Four of the isolated compounds, i.e., 2, 5, 8, 10, are new. The structures of 1-11 were determined on the basis of spectroscopic data. All isolated compounds were evaluated for their in vitro protective effects on chromosome aberrations in peripheral human lymphocytes using cytokinesis-block micronucleus (CBMN) assay. Almost all of them exerted a pronounced effect of decreasing DNA damage of human lymphocytes, acting stronger than the known synthetic protector amifostine.

Clastogenic effect of atranorin, evernic acid, and usnic acid on human lymphocytes.

Three lichen secondary metabolites atranorin (1), evernic acid (2), and usnic acid (3), were evaluated for their in vitro clastogenic and antiproliferative effects on human lymphocytes using the cytochalasin-B blocked micronucleus (CBMN) assay at concentrations of 2 microg/mL, 4 microg/mL and 6 microg/mL of final culture solution. The frequency of micronucleus (MN) was scored in binucleated cells, and cytokinesis-block proliferation index (CBPI) was calculated. Among the tested compounds, 3 exhibited the most prominent effect decreasing the frequency of MN in the range of 42.5% - 48.9%, that is about double of the positive control amifostin WR-2721 that reduces MN frequency for 22.0%. The effect of evernic acid was approximately equal to action of amifostin (23.2% -32.9%). Atranorin at concentrations of 2 microg/mL and 4 microg/mL decreasing the frequency of MN only for 11.1% and 1.8%, while in concentration of 6 microg/mL increases the frequency of MN for 9.6 %. The comparable CBPI values of the investigated compounds and control suggested that they did not show a statistically significant inhibitory effect on lymphocyte cell proliferation at applied concentrations.

Radioprotectors - the evergreen topic.

To protect organisms from ionizing radiation (IR), and to reduce morbidity or mortality, various agents, called radioprotectors, have been utilized. Because radiation-induced cellular damage is attributed primarily to the harmful effects of free radicals, molecules with radical-scavenging properties are particularly promising as radioprotectors. Early development of such agents focused on thiol synthetic compounds, known as WR protectors, but only amifostine (WR-2721) has been used in clinical trials as an officially approved radioprotector. Besides thiol compounds, various compounds with different chemical structure were investigated, but an ideal radioprotector has not been found yet. Plants and natural products have been evaluated as promising sources of radioprotectors because of their low toxicity, although they exhibit an inferior protection level compared to synthetic thiol compounds. Active plant constituents seem to exert the radioprotection through antioxidant and free radical-scavenging activities. Our research established that plants containing polyphenolic compounds (raspberry, blueberry, strawberry, grape, etc.) exhibit antioxidative activities and protect genetic material from IR.

Effect of Hypogymnia physodes extracts and their depsidones on micronucleus distribution in human lymphocytes.

Three lichen depsidones, physodalic acid (1), physodic acid (2), and 3-hydroxy physodic acid (3), were isolated from Hypogymnia physodes diethyl ether extract using column chromatography, and their structures determined by comparing their UV, 1H and 13C NMR spectroscopic and MS data with those given in the literature, as well as with data computed by CHEM draw ultra 11 software. The contents of 1, 2 and 3 were determined in the methanol (ME), acetone (AE), and diethyl ether (EE) extracts using reversed-phase high performance liquid chromatography with photodiode array detection. The extracts, isolated depsidones 1-3 and EE fraction F23 (consisting of 90% 2 and 3, in the ratio 5.5: 1) were evaluated for their in vitro effects on chromosome aberrations in peripheral human lymphocytes using the cytochalasin-B blocked micronucleus (CBMN) assay in doses of 1 microg/mL and 2 microg/mL of final culture solution. The frequency of MN was scored in binucleated cells, and nuclear proliferation index was calculated. It was found that 1, 2, 3, F23, and EE at 1.0 microg/mL exerted a beneficial effect on lymphocyte cells giving a significant decrease of the frequency of MN in comparison with the positive control Amifostin WR-2721.Among the tested depsidones at a concentration of 1 microg/mL, 3 exhibited the most prominent effect decreasing the frequency of MN by 30.3%, followed by 2 (28.2%) and 1 (22.0%). The extracts were less effective than the isolated depsidones.

Diarylheptanoids from Alnus glutinosa bark and their chemoprotective effect on human lymphocytes DNA.

A study of secondary metabolites from the bark of Alnus glutinosa led to the isolation of fourteen diarylheptanoids: oregonin (1), platyphylloside (2), rubranoside A (3), rubranoside B (4), hirsutanonol (5), hirsutenone (6), hirsutanonol-5-O-ß-D-glucopyranoside (7), platyphyllonol-5-O-ß-D-xylopyranoside (8), aceroside VII (9), alnuside A (10), alnuside B (11), 1,7-bis-(3,4-dihydoxyphenyl)-5-hydroxy-heptane-3-O-ß-D-xylopyranoside (12), (5S)-1-(4-hydroxyphenyl)-7-(3,4-dihydroxyphenyl)-5-O-ß-D-glucopyranosyl-heptan-3-one (13), and (5S)-1,7-bis-(3,4-dihydroxyphenyl)-5-O-ß-D-[6-(3,4-dimethoxycinnamoylglucopyranosyl)]-heptan-3-one (14). All of the diarylheptanoids, except 1 and 5, were found in A. glutinosa for the first time, while 13 and 14 were new compounds. The structures were determined by spectroscopic techniques: 1D and 2D NMR, HR-ESI-MS, FTIR, UV, and CD. All isolated compounds were analyzed for an in vitro protective effect on chromosome aberrations in peripheral human lymphocytes using the cytokinesis-block micronucleus assay. The majority of them, including the new compounds 13 and 14, exerted a pronounced effect in decreasing DNA damage in human lymphocytes. Diarylheptanoids 1, 2, 5, 13, and 14 at a concentration of 1 µg/mL decreased the frequency of micronuclei by 52.8 %, 43.8 %, 63.6 %, 44.4 %, and 56.0 %, respectively, exerting a much stronger effect than the synthetic protector amifostine (17.2 %, c = 1 µg/mL).

Antioxidant evaluation of heterocyclic compounds by cytokinesis-block micronucleus assay.

This article summarizes the results of using cytokinesis-block micronucleus (CBMN) assay to evaluate the antioxidant potential of heterocyclic compounds. Most studies were carried out with naturally occurring heterocyclic compounds such as plant polyphenols: flavonoids, xanthones, coumarins, and ellagitannins, or plant derived products (juices, extracts, supplements) rich in bioactive heterocyclic compounds. There are also some studies dealing with synthetic heterocyclic antioxidants. CBMN assay is an in vitro study that has been used to evaluate antioxidant and protective effects of heterocyclic compounds on induced chromosome aberration in human lymphocytes.

Chemical composition of currant seed extracts and their protective effect on human lymphocytes DNA.

UNLABELLED: On the basis of LC/UV/MS analysis, 35 compounds from the extracts of seeds of black, red, and white currants (2 cultivars of each) were identified. Black currants cultivars contained protocatechuic acid and p-hydroxybenzoic acid, and traces of nitril containing phenolic acids. The presence of synapoyl glucoside was characteristic for cv. Malling Jewel. Sesquiterpenoid glycosides and carboxymethylindol glycosides were present mainly in white and red currant cultivars. Blackcurrant seeds contained higher amounts of flavonoids, especially rutin, isoquercetin, and taxifolin. The currant seed extracts were tested for in vitro protective effect on chromosome aberrations in peripheral human lymphocytes using cytochalasin-B blocked micronucleus (CBMN) assay. The frequency of MN was scored in binucleated cells, and the cultivars of black currants showed the best antioxidant potential. At a concentration of only 0.17 mg/mL, extract of the seed cv. Malling Jewel effected a decrease in the frequency of MN of 60% compared with control cell cultures. PRACTICAL APPLICATION: Our results provide evidence of protective effects of currant seed extracts and isolated pure compounds on cytogenetic damages in human lymphocytes. Thus, currant seed extracts could exert beneficial effects in quite a few diseases, for many of the biological actions have been attributed to their antioxidant properties.

Blackberry seed extracts and isolated polyphenolic compounds showing protective effect on human lymphocytes DNA.

The tentative identification of seed extracts from 3 cultivars of blackberry (blackberry seed extracts [BSEs]) constituents was performed by LC/UV/MS technique. The identified compounds belonged to ellagitannins, galic acid derivatives, and ellagic acid derivatives. Two ellagitannins, Lambertianin C and Sanguiniin H-6, and an ellagic acid derivative, 4-α-L-arabinofuranosylellagic acid, were isolated using semipreparative High-performance liquid chromatography. The structure elucidations were based on high resolution-mass spectrometry and nuclear magnetic resonance studies. The BSEs and 3 isolated pure compounds were tested for in vitro protective effect on chromosome aberrations in peripheral human lymphocytes using cytochalasin-B blocked micronucleus (MN) assay. The frequency of MN was scored in binucleated cells, and nuclear proliferation index was calculated. Among the tested extracts, the seeds of cv. Thornfree at concentration of 1 µg/mL exhibit the most prominent effect decreasing the frequency of MN by 62.4%, when compared with the controls cell cultures. Antioxidant potential of pure ellagitannins cannot explain the strong effect of BSEs. The assumption was that better antioxidant effect of BSEs result from synergistic effects of individual compounds contained in the extracts and/or some minor components possessed strong activity. PraCTICAL APPLICATION: Our results provide evidence of protective effects of BSEs and isolated pure compounds on cytogenetic damages in human lymphocytes. Thus, BSEs could exert beneficial effects in quite a few diseases, because many of the biological actions have been attributed to their antioxidant properties.

Protective effect on human lymphocytes of some flavonoids isolated from two Achillea species.

This study was conducted to elucidate the in vitro protective effect of five flavonoids [apigenin (1), apigenin-7-O-glucoside (2), centaureidin (3), jaceidin (4) and quercetin (5)] against chromosomal damage in mitogen-induced human lymphocytes. Using the Cytochalasin-B blocked micronucleus (CBMN) assay, in which the biomarker of chromosome breakage and/or chromosome loss is the elevated frequency of micronucleus (MN) in binucleated (BN) cells, the presence of flavonoid 2 in minimal concentration (3 microg/mL) gave a 35.5% decrease in the frequency of MN when compared with control human lymphocytes. The same concentration of flavonoids 1, 3 and 4, reduced the MN frequency by 24.4%, 28.0% and 28.0%, respectively. Higher concentrations (6 microg/mL and 10 microg/mL) seemed less effective. Flavonoid 5 (3 microg/mL) induced a slight decrease in MN frequency (5%), while higher doses (6 microg/mL and 10 microg/mL) provoked an increase of DNA damage. The comparable values for the cytokinesis-block proliferation index (CBPI) of the tested flavonoids and positive control suggested an inhibitory effect on lymphocyte proliferation. In the DPPH scavenging assay, flavonoids 1-4 demonstrated modest activity, in a dose-dependent manner, compared with the synthetic antioxidants BHT and Trolox, while 5 exhibited comparably high antioxidative activity.