Influence of the composition of essential oils on their antioxidant and antiradical properties

The antiradical and antioxidant properties of essential oil mixtures (EOMs) with various compositions in model systems of hexanal autooxidation, thermal oxidation of methyl linoleate and β-carotene, and in a reaction with the stable diphenylpicrylhydrazyl radical were studied and compared. It was found that all studied EOMs exhibited antiradical activity. The highest antiradical activity was observed for an EOM containing monoterpene hydrocarbons as the main components (the phenol content was low). The antioxidant activity of all EOMs was from 60 to 98% and depended on the model system composition and the method of assay. The mixture with the maximum phenol content exhibits the highest antioxidant activity level in the hexanal autooxidation system. EOMs with a high content of phenols and α- and γ-terpinenes were the most efficient antioxidants in the β-carotene model system. The study confirmed the possibility to vary the antioxidant and antiradical properties of essential oils by preparing their mixtures with a specific composition.     

Effect of traumatic acid on antioxidant activity in <Emphasis Type="Italic">Chlorella vulgaris</Emphasis> (Chlorophyceae)

The present study was undertaken to test the influence of exogenously applied traumatic acid (TA) upon the activity of several antioxidant enzymes as well as lipid and protein peroxidation in green algae Chlorella vulgaris. Treatment with TA in concentration range of 10⁻⁶–10⁻⁵ M resulted in an increase of antioxidant enzyme (sodium dismutase, catalase, ascorbate peroxidase, NADH peroxidase, glutathione reductase) activity. Moreover, TA suppressed lipid peroxidation and oxidative destruction of proteins belonging to the SH groups. This data suggest that TA plays an important role in the metabolism of C. vulgaris and probably in its high ability to adapt to various environmental stress factors.     

Volatile constituents of glutathione--ribose model system and its antioxidant activity

Summary.  Reaction between glutathione and ribose was carried out to study the volatiles formed via Maillard reaction and their antioxidant activity as well as their role in inhibition of LDL oxidation. The simultaneous distillation – extraction technique was used for trapping the volatile components followed by GC – MS analysis. Thirty six compounds were identified with the predominance of carbonyls and sulfur – containing compounds in the volatiles of this model system. Sensory evaluation was performed for the model system product according to the International Standard Methods (ISO). The results showed a high decrease in roasted and burnt attributes and remarkable increase in the like – boiled and roasted meat attributes. The sensory results of the model system product were confirmed by the presence of high concentrations of some volatile compounds having meat – like aroma such as 2-methyl-3-furanthiol and 2-furylmethanethiol. The radical scavenging activity of glutathione – ribose model system was quantified spectrophotometrically, using DPPH radical. The activity of the model system product was found to be slightly lower than that of gallic acid and BHA, but it was much higher than that of cinnamic acid (200 ppm. for each). A highly antioxidative activity was recorded by the model system product during the inhibition of LDL – oxidation in comparison with L-ascorbic acid as well as reduced glutathione (as a concentration of 0.5 μmol/L, for each) which may be due to the presence of some compounds such as 2-furylmethanethiol, 2-acetyl thiazole, 4-hydroxy-5-methyl-3(2H)-furanone. Received October 15, 2001 Accepted April 3, 2002 Published online September 4, 2002 Authors' address:  Khaled Farouk El-massry, Flavour and Aromatic Chemistry Department, National Research Center, Tahrir st., Dokki, Cairo, Egypt, Fax: 002 02 337 0 931, E-mail: kfarouk@yahoo.com     

Glycation-induced inactivation of aspartate aminotransferase, effect of uric acid

Glycation is common posttranslational modification of proteins impairing their function, which occurs during diabetes mellitus and aging. Beside extracellular glycation of long-lived proteins, intracellular modifications of short-lived proteins by more reactive sugars like fructose are possible. The process includes free oxygen radicals (glycoxidation). In an attempt to reduce glycoxidation and formation of advanced glycation products (AGE), influence of 0.2–1.2 mM uric acid as endogenous antioxidant on glycoxidation of purified pig heart aspartate aminotransferase (AST) by 50 mM and 500 mM D-fructose in vitro was studied. Uric acid at 1.2 mM concentration reduced AST activity decrease and formation of total AGE products caused by incubation in vitro of the enzyme with sugar up to 25 days at 37 °C. The results thus support the hypothesis that uric acid has beneficial effects in controlling protein glycoxidation. The in vitro system AST-fructose proved to be a useful tool for investigation of glycation process. (Mol Cell Biochem 278: 85–92, 2005)     

Biochemical responses of Hyacinth bean (<Emphasis Type="Italic">Lablab purpureus)</Emphasis> to salinity stress

Effect of salinity on Hyacinth bean, Lablab purpureus (HA-4 cultivar) was evaluated in 10-day old seedlings with 100–500 mM NaCl over 72 h of exposure. The stress reduced dry and fresh weight, leaf surface area, root and shoot length, total chlorophyll, and RWC. Oxidative stress markers, H₂O₂, glutathione, TBARS, proline, ascorbic acid, total phenols, and total soluble sugar contents were significantly elevated. Salinity enhanced antioxidant enzymes, POX, and GR activities and reduced that of CAT in concentration and time dependent manner in leaves. Antioxidant enzymes in roots showed inverse relationship with concentration and time of exposure. Metabolic enzyme β-amylase activity increased in both leaves and roots. Acid phosphatase decreased in leaves and elevated in roots. Intensity of constitutive isozymes correlated with in vitro levels under stress, but the protein band patterns differed from controls. Lablab showed reasonable tolerance up to 300 mM NaCl, but leaves and roots differed in their response.     

Effect of 28-homobrassinolide on the drought stress-induced changes in photosynthesis and antioxidant system of <Emphasis Type="Italic">Brassica juncea</Emphasis> L.

To investigate the effect of exogenously applied 28-homobrassinolide (HBL) on drought-stressed plants, photosynthesis and antioxidant systems were examined in Indian mustard (Brassica juncea L.). Seedlings of Indian mustard were subjected to drought stress for 7 days at the 8–14 (DS1)/15–21 (DS2) days’ stage of growth and then returned to normal conditions of growth. These seedlings were sprayed with HBL (0.01 μM) at the 30-day stage and were sampled at 60 days to assess the changes in growth, photosynthesis and antioxidant enzymes. Plants exposed to stress at either of the stages of growth exhibited a significant decrease in growth and photosynthesis. The exposure of plants to stress at an earlier stage (DS1) was more inhibitory than that at a later stage (DS2). However, the follow-up treatment with HBL significantly improved the values of these parameters and also overcame the inhibitory effect of water stress. The activity of antioxidant enzymes [catalase (E.C. 1.11.1.6), peroxidase (E.C. 1.11.1.7) and superoxide dismutase (E.C. 1.15.1.1)] and proline content in leaves exhibited an increase in response to both the treatment factors, where their interaction had an additive effect. It was, therefore, concluded that the elevated antioxidant system, at least in part, was responsible for amelioration of the drought stress.     

The involvement of low-molecular antioxidants in cross-adaptation of medicine plants to successive action of UV-B radiation and salinity

Artemisia (Artemisia lercheana Web.), common basil (Ocimum basilicum L.), and black cumin (Nigella sativa L.) plants grown in water culture until the stage of 4–5 true leaves were subjected to 10-min UV-B irradiation, treated with 100 mM NaCl, or subjected to the successive action of both stressors. The contents of proline, anthocyanins, flavonoids, soluble phenols, and carotenois were measured. Superoxide dismutase activity was also assayed. Experimental plants could tolerate UV-B irradiation due to the accumulation of phenolic compounds (anthocyanins, soluble phenols, and flavonoids). Anthocyanins contributed mostly in the defnse effect; their content in black cumin and common basil increased 3–5-fold after irradiation. Dynamics of the anthocyanin content in tested plants of all treatments indicates the activation of their biosynthesis by UV-B irradiation and suppression by salinity. Successive action of stressors resulted in synergism of their effects on accumulation of low-molecular compounds in artemisia and common cumin plants. When these plants were irradiated with UV-B and then treated with NaCl, they accumulated more low-molecular compounds than after separate treatment with these stressors, especially in Artemisia. Plant pretreatment with UV-B reduced the adverse action of salinity; this was manifested in the turgor maintenance in salt-treated plants after preliminary irradiation. For basil, salinity was the stronger stressor than UV-B, which was manifested in a decrease in the content of low-molecular compounds.     

Antioxidant activity of oxygen-containing aromatic compounds

Inhibition efficiency (antioxidant activity) of 26 oxygen-containing aromatic compounds was studied in methemalbumin-H₂O₂-o-phenylenediamine (PDA) or tetramethylbenzidine (TMB) pseudoperoxidase system at 20°C in buffered physiological solution (pH 7.4) containing 6% DMF and 0.25% DMSO. The inhibitor’s efficiency was quantitatively characterized by the inhibition constants (K _i, μM) or the inhibition degree (%). K _i values varied in the range of 4 to 500 μM and were influenced by a substrate, the structure of an inhibitor, hydroxyl groups, electron-donating substituents in aromatic ring, and steric hindrances. The type of inhibition at cooxidation of eight pairs was noncompetitive, and that of five pairs was mixed and determined by the substrate nature and the inhibitor structure. Lignin phenolic compounds of guaiacyl and syringal series exhibited high antioxidant activity (K _i in the range of 10–300 μM), and their efficiency decreased in the following order: caffeic acid > synapaldehyde > syringic acid > coniferyl aldehyde > para-hydroxycoumaric acid.     

UV-A Mediated Modulation of Photosynthetic Efficiency,Xanthophyll Cycle and Fatty Acid Production of <Emphasis Type="Italic">Nannochloropsis</Emphasis>

Nannochloropsis, a green microalga, is a source for commercially valuable compounds as extensively described and, in particular, is recognised as a good potential source of eicosapentaenoic acid (20:5ϖ3), an important polyunsaturated fatty acid for human consumption for prevention of several diseases. Climate change might include variation in the ultraviolet (UV) levels as one of the consequences derived from the anthropogenic activity. This paper shows the response of Nannochloropsis cultures exposed for 7 days to UV-A (320–400 nm) added to photosynthetically active radiation (PAR; 400–700 nm). Growth rates and photosynthetic activity were assessed to determine the impact of UV-A increased levels on the cell growth and basic metabolism activity. Xanthophyll pigments (zeaxanthin and violaxanthin), carotenoids (canthaxanthin and β-carotene) and polyunsaturated fatty acids (myristic, palmitic, palmitoleic, arachidonic and eicosapentaenoic acids) were measured for assessing the antioxidant response of the microalgae to added UV-A radiation to PAR. The results show that the modulated use of UV-A radiations can lead to increased growth rates, which are sustained in time by an increased light transduction activity. The expected antioxidant response to the incident UV-A radiation consisted of increases in zeaxanthin and β-carotene contents—synthesis of antioxidant carotenoids—and increases in the saturated fatty acids to polyunsaturated fatty acids ratio. The results suggest that modulated UV-A radiation can be used as a tool to stimulate value molecules accumulation in microalgae through an enhanced both light transduction process and antioxidant response, while sustaining cell growth.     

Effects of aluminium on ultrastructure and antioxidant activity in leaves of tea plant

Seedlings of Camellia sinensis (L.) were grown hydroponically to study the effect of aluminium (Al) on leaf antioxidant defence system and cell ultrastructure. We found that malondialdehyde (MDA) content decreased at 0–0.32 mM Al, but increased significantly at 0.53 mM Al. Like MDA, hydrogen peroxide (H₂O₂) content increased at 0.53 mM Al; however, no differences were observed at 0–0.32 mM Al. Superoxide dismutase (SOD, EC1.15.1.1) activity remained practically constant at 0–0.32 mM Al, but increased sharply at 0.53 mM Al; catalase (CAT, EC1.11.1.6) and guaiacol peroxidase (GPX, EC1.11.1.7) activities decreased following an initial increase, reaching their peaks at 0.32 mM Al. Ascorbate peroxidase (APX, EC 1.11.1.11) activity increased and glutathione (GR, EC 1.6.4.2) level fluctuated with increasing Al concentrations. Transmission electron microscope analysis of Al-treated leaves showed that although cell ultrastructural integrity was maintained at 0–0.32 mM, significant membrane damage was observed at 0.53 mM. Our results suggest that at low Al concentrations, the leaf antioxidant defence system can scavenge reactive oxygen species and sufficiently protect cells from free radical injury. However, at higher Al concentrations (0.53 mM), the balance between formation and detoxification of ROS is lost, resulting in the destruction of cell ultrastructure.     

Antioxidant activity of extracts from Thalictrum minus suspension culture

Low meadow-rue (Thalictrum minus L.) antioxidant complex was studied in cell extracts and culture medium. Its activity was expressed as total polyphenol content in ferulic acid equivalents. In these model systems (cell extracts and culture medium) the inhibition of lipid oxidation and diphenylpicrylhydrazine reduction (EC₅₀ = 12–15 μg/ml) were observed. At the phenolic compound concentration of 8–15 μg/ml, the reducing capacity of cell extracts was equivalent to 1.5 mM ascorbic acid. At the same time, berberine, a major alkaloid synthesized by the culture, manifested a low antioxidant activity. The analysis of phenolic acid composition in low meadow-rue showed that one of the main components of its antioxidant system were caffeic, gallic, chlorogenic, and ferulic acids.     

Evaluation of 28 marine algae from the Qingdao coast for antioxidative capacity and determination of antioxidant efficiency and total phenolic content of fractions and subfractions derived from Symphyocladia latiuscula (Rhodomelaceae)

The extracts obtained from 28 species of marine algae were evaluated for their antioxidant activity (AA) versus the positive controls butylated hydroxytoluene (BHT), gallic acid (GA), and ascorbic acid (AscA). Most of the tested samples displayed antioxidant activity to various degrees. Among them, the extract of Symphyocladia latiuscula exhibited the strongest AA, which was comparable to BHT, GA, and AscA in radical scavenging activity, as shown in the DPPH (α,α-diphenyl-β-picrylhydrazyl) assay, and higher than those of the positive controls in β-carotene-linoleate assay system. In addition, the ethyl acetate-soluble fraction isolated from the crude extract of S. latiuscula exhibited the highest antioxidant activity in both assay systems. This fraction was further fractionated into seven subfractions (F1-F7) by vacuum liquid chromatography (VLC). F1 and F4 were found to be the most effective subfractions in scavenging DPPH radical assay and in the β-carotene-linoleate assay, respectively. The total phenolic content (TPC) and reducing power (RP) for all of the extracts, fractions, and subfractions (F1–F7) were also determined. The TPC of the 28 extracts ranged from 0.10 to 8.00 gallic acid equivalents (mg/g seaweed dry weight) while the RP ranged from 0.07 to 11.60 ascorbic acid equivalents (mg·g⁻¹ seaweed dry weight). Highly positive relationships between AA and TPC as well as between AA and RP were found for the extracts and fractions, while for the subfractions F1–F7 only weak or no such relations were found. The results obtained from this study indicate that further analysis is needed of those marine algal species that contain the most antioxidant activity in order to identify the active principles.     

Antioxidant Properties of Selected <Emphasis Type="Italic">Boletus</Emphasis> Mushrooms

Considering the growing interest for mushrooms and the demand search of natural antioxidants sources, the aim of this study was to investigate the antioxidant properties of two edible widely used Boletus species, Boletus edulis, and Boletus auranticus, collected from Istra region in Croatia in late summer 2007. To evaluate the antioxidant properties and content of antioxidant compounds, scavenging capacity on DPPH˙, OH˙, and O₂˙⁻ radicals, reducing power and capacity to inhibit lipid peroxidation has been investigated. It is determined that content of total phenols (41.82 ± 0.08 mg gallic acid equivalent per gram of dry extract) was higher for B. edulis. Using high performance liquid chromatography/diode array detector analysis, the main antioxidant compound, variegatic acid, has been detected and quantified. 1,1-Diphenyl-2-picryl-hydrazyl-hydrate assay was used as a preliminary free radical–scavenging evaluation. By this assay, it has been found that B. edulis dry mushroom extract exhibits 50% of inhibition value at the extract concentration of 0.016 ± 0.0003 mg/ml. The extracts were capable of reducing iron(III) and, thus, are capable of donating electrons. Using electron paramagnetic resonance spin-trapping and spin-probing techniques, activity against relevant reactive species, ˙OH and O₂˙⁻ radical, was analyzed for both mushroom extracts. Both investigated extracts are determined as good inhibitors for ˙OH radical reduction, and both exhibited significant capacity for scavenging O₂˙⁻ radical and for that could help to prevent or meliorate oxidative damage. Only B. edulis extract prevents lipid peroxidation. Investigated mushroom extracts could represent easily accessible natural antioxidant resource.     

All-trans retinoic acid induces free radical generation and modulate antioxidant enzyme activities in rat sertoli cells

In this work we investigated the effects of retinoic acid (RA) in Sertoli cells. Sertoli cells isolated from 15-day-old Wistar rats were previously cultured for 48 h and then treated with RA for 24 h. RA at high doses (1–10 μM) increased TBARS levels and induced a decrease in cell viability. At low doses (0.1–100 nM) RA did not increase TBARS level. RA also did not increase cell death at these doses. In order to investigate changes in antioxidant defenses we measured the CAT, SOD and GPx activities in Sertoli cells treated with RA. Compared to control, RA increased around 200% SOD activity in all doses tested (0.1–100 nM); GPx activity was increased 407.49, 208.98 and 243.88% (0.1, 1 and 10 nM, respectively); CAT activity was increased 127% with RA 1 nM. To clarify if RA induces ROS production per se, we performed experiments in vitro using 2-deoxyribose as specific substrate of oxidative degradation by ^•OH radical as well as TRAP assay. RA at 10 μM increased 2-deoxyribose degradation, suggesting that some of the RA-induced effects are mediated via ^•OH formation. Furthermore, the total reactive antioxidant potential (TRAP) of the RA was determined. At low concentrations RA has induced no redox activity. Conversely, higher concentration of RA (1–10 μM) increased chemiluminescence. The chemiluminescence produced was directly proportional to radical generation. We provide, for the first time, evidence for a free radical generation by RA. Our results demonstrated that RA plays an important role in Sertoli cells and these effects appear to be mediated by ROS.     

Effect of molecular weight on the antioxidant property of low molecular weight alginate from Laminaria japonica

In this study, three alginate fractions with different molecular weights and ratios of mannuronic acid (M) to guluronic acid (G) were prepared by enzymatic hydrolysis and ultrafiltration to assess the antioxidant property of alginates from Laminaria japonica with molecular weight below 10 kDa. The antioxidant properties of different molecular weight alginates were evaluated by determining the scavenging abilities on superoxide, hydroxyl, and hypochlorous acid and inhibitory effect on Fe²⁺-induced lipid peroxidation in yolk homogenate. The results showed that low molecular weight alginates exhibited high scavenging capacities on superoxide, hydroxyl, and hypochlorous acid radicals and good inhibition of Fe²⁺-induced lipid peroxidation in yolk. By comparison, alginate A1 with molecular weight below 1 kDa and M/G of 1.84 had better scavenging activity on superoxide, hydroxyl, and hypochlorous acid radicals in vitro than A2 (1–6 kDa), A3 (6–10 kDa), ascorbic acid, and carnosine. With similar M/G ratio, A2 exhibited better antioxidant activity on superoxide and hypochlorous acid radicals than A3. However, fraction A3 with molecular weight of 6–10 kDa exhibited higher inhibitory ability on lipid peroxidation in yolk in vitro than A1 and A2. The results indicated that molecular weight played a more important role than M/G ratio on alginate to determine the antioxidant ability. By comparison, low molecular weight alginates composed of guluronic acid and mannuronic acid exhibited better antioxidant ability on oxygen free radicals than sulfated polysaccharides from L. japonica in our previous study and represent a good source of marine polysaccharide with potential application as natural antioxidant.     

Effect of glutathione biosynthesis-related modulators on the thiol redox state enzymes and on sclerotial differentiation of filamentous phytopathogenic fungi

In this study, sclerotial differentiation in filamentous phytopathogenic fungi, representing the four main types of sclerotia, was studied in relation to thiol redox state (TRS)-related enzymes and their substrates/products. TRS was altered by the general TRS modulator Ν-acetylcysteine (AcCSH) and by the glutathione (GSH) biosynthesis modulators l-oxo-thiazolidine-4-carboxylate (OTC), and l-buthionine-S,R-sulfoximine (BSO). This study showed that the four studied types of sclerotial differentiation are directly related with the antioxidant –SH groups of GSH and/or CSH, since the decrease of sclerotial differentiation concurred with an increase of these thiols by the GSH biosynthesis modulators AcCSH, OTC, and BSO. Supportive to that conclusion is the fact that, in general, the activities of the TRS-related enzymes GR/GPDH and Ttase decrease in the end of the undifferentiated stage due to the substitution of their antioxidant function by the antioxidant potential of the –SH group providers AcCSH and OTC. Moreover, it was found that BSO expectedly suppressed GSH biosynthesis in the tested fungi, and unexpectedly decreased their sclerotial differentiation by a dose-dependent manner typical for antioxidants. The possible antioxidant role of BSO was supported by the decrease it caused in the antioxidant enzymes GR/GPDH and Ttase. The results of this study are in accordance with our hypothesis that sclerotial differentiation in phytopathogenic fungi is induced by oxidative stress.     

Regulation of enzymes involved in C4 photosynthesis and the antioxidant metabolism by UV-B radiation in Egeria densa,a submersed aquatic species

Egeria densa, a submersed aquatic species, was exposed to different treatments under UV-B radiation, and the response of phosphoenolpyruvate carboxylase (PEPC) and NADP-malic enzyme (NADP-ME) was determined. Exposure to UV-B radiation for 4 h per day over 7–16 days caused an increase in both enzymes, together with an increase in the activity of some isoforms of several enzymes involved in the antioxidant metabolism, such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD). The content of chlorophylls and carotenoids was considerably decreased, suggesting that degradation or repression of the synthesis of these molecules may be occurring after UV-B exposure. Reactive oxygen species (ROS) were also required for UV-B induction of PEPC and NADP-ME, as the addition of ascorbic acid before UV-B treatment prevented the induction of these enzymes, while salicylic acid was not effective in inducing NADP-ME but increased the expression of the lower molecular mass isoform of PEPC. On the other hand, damage to the photosynthetic machinery may be occurring after exposure to UV-B radiation for 8 per day over 1–2 days, as indicated by a decrease in the levels of Rubisco, PEPC and NADP-ME. Some of the enzymes involved in the antioxidant metabolism, such as CAT and APX, were also sensitive to continuous exposure, evidenced by a decrease in their activity. In this way, in E. densa, several enzymes involved in different metabolic pathways showed a distinct response, depending on the UV-B treatment. This revised version was published online in June 2006 with corrections to the Cover Date.     

Importance of GAD65 peptides and I-Ag7 in the development of insulitis in nonobese diabetic mice

 Insulin-dependent diabetes mellitus (IDDM) develops in nonobese diabetic (NOD) mice through the destruction of the B cells in pancreatic Langerhans islets by islet autoantigen-specific T cells. The islet autoantigen glutamic acid decarboxylase 65 (GAD65) is thought to be a major target autoantigen in IDDM. In the present report, we established GAD65-specific T-cell clones using overlapping peptides that cover the amino acid sequences of mouse GAD65. T-cell epitopes of GAD65 were characterized by proliferation and binding assays using various analogue peptides and wild-type or mutant I-A^g7 transfectants. The efficacy of the peptide vaccine in IDDM was determined by administering T-cell epitope peptides to NOD mice and evaluating the histopathology of their insulitis. We obtained two types of T-cell clone, one specific for peptide p316–335 and another specific for p531–545 of GAD65. The p531–545 site has already been identified, but we report the p316–335 site for the first time. T-cell clones recognized those peptides in the wild-type I-A^g7 but not in the mutant I-A^g7 in which the serine at position 57 of the β-chain was replaced by an aspartic acid. Both the p316–335 and p531–545 peptides bound weakly to I-A^g7. Some peptides with amino acid substitutions had antagonistic activity, and administration of a large amount of wild-type peptide reduced the severity of insulitis in NOD mice. Our results suggest that peptide vaccine therapy may be useful in autoimmune diseases, including IDDM. Received: 19 July 1999 / Revised: 4 January 2000     

Nitrogen nutrition modulates oxidative stress and metabolite production in Hypericum perforatum

Impact of various nitrate concentrations (14.12 mM, 3.53 mM, no nitrate) or ammonium presence (14.12 mM) on physiological and metabolic changes in Hypericum perforatum after 14 days of cultivation was monitored. Nitrate deficiency suppressed growth of shoots but stimulated root growth while ammonium suppressed root growth: concomitant changes of ascorbic acid and glutathione supported these growth changes, e.g., unaltered level in roots under nitrate deficiency but depleted in ammonium treatment. Soluble proteins and water content were more suppressed by nitrate deficiency but total ROS, nitric oxide formation, and antioxidative enzyme activities (APX and SOD) indicate higher sensitivity of plants to ammonium. Though both extreme treatments (NO3− deficiency or ammonium) stimulated accumulation of total soluble phenols and affected PAL activity (in comparison with full or 1/4× nitrate dose), major phenols (chlorogenic acid and three flavonoids) were elevated mainly by NO3– deficiency. At the level of specific metabolites, NO3– deficiency had stimulatory impact on pseudohypericin (but not hypericin) content while hyperforin decreased. Expression of earlier putative gene of hypericin biosynthesis (hyp-1) showed rather partial correlation with pseudohypericin amount. Data indicate that depletion of NO3– is useful to obtain Hypericum plants with higher amount of health-positive secondary metabolites.