Improvement in the Biosynthesis of Antioxidant-Active Metabolites in In Vitro Cultures of Isatis tinctoria (Brassicaceae) by Biotechnological Methods/Elicitation and Precursor Feeding.

This study aimed to establish the in vitro shoot culture of Isatis tinctoria L. and its ability to produce antioxidant bioactive compounds. The Murashige and Skoog (MS) medium variants, containing different concentrations (0.1-2.0 mg/L) of benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) were tested. Their influence on the growth of biomass, accumulation of phenolic compounds, and antioxidant potential was evaluated. To improve the phenolic content, agitated cultures (MS 1.0/1.0 mg/L BAP/NAA) were treated with different elicitors, including the following: Methyl Jasmonate, CaCl2, AgNO3, and yeast, as well as with L-Phenylalanine and L-Tyrosine-precursors of phenolic metabolites. The total phenolic content (TPC) of hydroalcoholic extracts (MeOH 70%) obtained from the biomass grown in vitro was determined spectrophotometrically; phenolic acids and flavonoids were quantified by RP-HPLC. Moreover, the antioxidant potential of extracts was examined through the DPPH test, the reducing power, and the Fe2+ chelating assays. The biomass extracts obtained after 72 h of supplementation with Tyr (2 g/L), as well as after 120 and 168 h with Tyr (1 g/L), were found to be the richest in TPC (49.37 ± 0.93, 58.65 ± 0.91, and 60.36 ± 4.97 mg GAE/g extract, respectively). Whereas among the elicitors, the highest TPC achieved was with CaCl2 (20 and 50 mM 24 h), followed by MeJa (50 and 100 µM, 120 h). The HPLC of the extracts led to the identification of six flavonoids and nine phenolic acids, with vicenin-2, isovitexin, syringic, and caffeic acids being the most abundant compounds. Notably, the amount of all flavonoids and phenolic acids detected in the elicited/precursor feeding biomass was higher than that of the leaves of the parental plant. The best chelating activity was found with the extract of biomass fed with Tyrosine 2 g/L, 72 h (IC50 0.27 ± 0.01 mg/mL), the strongest radical scavenging (DPPH test) for the extract obtained from biomass elicited with CaCl2 50 mM, after 24 h of incubation (25.14 ± 0.35 mg Trolox equivalents (TE)/g extract). In conclusion, the in vitro shoot culture of I. tinctoria supplemented with Tyrosine, as well as MeJa and/or CaCl2, could represent a biotechnological source of compounds with antioxidant properties.

Different Types of Hypericum perforatum cvs. (Elixir, Helos, Topas) In Vitro Cultures: A Rich Source of Bioactive Metabolites and Biological Activities of Biomass Extracts.

Microshoot agitated and bioreactor cultures (PlantForm bioreactors) of three Hypericum perforatum cultivars (Elixir, Helos, Topas) were maintained in four variants of Murashige and Skoog medium (MS) supplemented with 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) (in the range of 0.1-3.0 mg/L). In both types of in vitro cultures, the accumulation dynamics of phenolic acids, flavonoids, and catechins were investigated during 5- and 4-week growth cycles, respectively. The contents of metabolites in methanolic extracts from biomasses collected in 1-week intervals were estimated by HPLC. The highest total contents of phenolic acids, flavonoids, and catechins were 505, 2386, and 712 mg/100 g DW, respectively (agitated cultures of cv. Helos). The extracts from biomass grown under the best in vitro culture conditions were examined for antioxidant and antimicrobial activities. The extracts showed high or moderate antioxidant activity (DPPH, reducing power, and chelating activity assays), high activity against Gram-positive bacteria, and strong antifungal activity. Additionally, experiments with phenylalanine feeding (1 g/L) in agitated cultures were performed reaching the highest enhancement of the total contents of flavonoids, phenolic acids, and catechins on day 7 after the addition of the biogenetic precursor (2.33-, 1.73- and 1.33-fold, respectively). After feeding, the highest accumulation of polyphenols was detected in the agitated culture of cv. Elixir (4.48 g/100 g DW). The high contents of metabolites and the promising biological properties of the biomass extracts are interesting from a practical point of view.

In Vitro Cultures of Scutellaria brevibracteata subsp. subvelutina as a Source of Bioactive Phenolic Metabolites.

Some of the more than 350 Scutellaria species, such as S. baicalensis and S. lateriflora, have been used in traditional medicine and today play an important role in official phytotherapy. Other species have been less investigated, and their therapeutic potential is unknown. This is one of the few studies on Scutellaria brevibracteata subsp. subvelutina, and the first research of this species' in vitro cultures. The aim of this study was to establish an in vitro culture and analyse its phytochemical profile and biological activity. In the methanolic extracts from biomass cultured on six solid Murashige and Skoog (MS) medium variants supplemented with different combinations of 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) in the range 0.5-3 mg/L analysed by HPLC, the presence of specific flavonoids (baicalein, baicalin, wogonin, wogonoside, scutellarin, chrysin), phenylpropanoid glycosides (verbascoside, isoverbascoside), and phenolic acids (p-hydroxybenzoic, caffeic, ferulic, m-coumaric acids) was confirmed. The dominant metabolites were wogonoside and verbascoside with the highest content of 346 and 457 mg/100 g DW, respectively. Thus, the extract with the highest content of bioactive metabolites was selected for further research and subjected to evaluation of antioxidant and antimicrobial potential. The extract exhibited good free radical scavenging activity (IC50 = 0.92 ± 0.01 mg/mL) and moderate reducing power and chelating activity. The brine shrimp lethality bioassay proved its lack of biotoxicity. Antimicrobial activity was tested against sixteen strains of Gram-positive and Gram-negative bacteria and fungi. The strongest growth inhibitory activity was observed against Trichophyton tonsurans.

Hydroalcoholic Leaf Extract of Isatis tinctoria L. via Antioxidative and Anti-Inflammatory Effects Reduces Stress-Induced Behavioral and Cellular Disorders in Mice.

Stress that can occur at different levels of a person's life can cause and exacerbate various diseases. Oxidative stress and inflammation underlie this process at the cellular level. There is an urgent need to identify new and more effective therapeutic targets for the treatment of stress-induced behavioral disorders and specific drugs that affect these targets. Isatis tinctoria L. is a herbaceous species in the Brassicaceae family. Due to its potential antioxidant, nitric oxide- (NO-) inhibiting, anti-inflammatory, and neuroprotective properties, I. tinctoria could be used to treat depression, anxiety, and stress resistance. Hence, the present study is aimed at delineating whether administration of I. tinctoria leaf extract may improve stress-induced disorders in mice. A set of four behavioral tests was selected that together are suitable for phenotyping acute restraint stress-associated behaviors in mice, namely locomotor activity, social integration, dark/light box, and splash tests. The plasma and brains were collected. A brain-derived neurotrophic factor, tumor necrosis factor-alpha, C-reactive protein, corticosterone, NO, reactive oxygen species levels, superoxide dismutase and catalase activity, and ferric-reducing antioxidant power were measured. In mice stressed by immobilization, decreased locomotor activity, anxiety-like behavior, and contact with other individuals were observed, as well as increased oxidative stress and increased levels of nitric oxide in the brain and plasma C-reactive protein. A single administration of I. tinctoria leaf extract was able to reverse the behavioral response to restraint by a mechanism partially dependent on the modulation of oxidative stress, neuroinflammation, and NO reduction. In conclusion, Isatis tinctoria hydroalcoholic leaf extract can reduce stress-induced behavioral disturbances by regulating neurooxidative, neuronitrosative, and neuroimmune pathways. Therefore, it could be recommended for further research on clinical efficacy in depression and anxiety disorder treatment.

Antioxidant Potential and Enhancement of Bioactive Metabolite Production in In Vitro Cultures of Scutellaria lateriflora L. by Biotechnological Methods.

Studies carried out using three different in vitro assays and a biological setting (Escherichia coil) demonstrated the antioxidant activity of Scutellaria lateriflora microshoot extract. Moreover, the extract exhibited no toxicity in a brine shrimp lethality bioassay. These results indicated that microshoots are a rich, safe source of antioxidants, which encouraged us to enhance their production in vitro. In agar and agitated cultures, two biotechnological strategies were applied: feeding the cultures with the biogenetic precursors of the phenolics-phenylalanine and tyrosine, and eliciting them with methyl jasmonate. Specific Scutellaria flavonoids and verbascoside were analysed by HPLC. Feeding with precursors (1 g/L) in agar cultures decreased the production of the metabolites. In agitated cultures, different concentrations of precursors (1.0-2.5 g/L) and the elicitor (10; 50; 100 µM) were tested. Additionally, parallel feeding with the precursor and elicitor in a concentration of 50 µM were applied. The best strategy for total flavonoid and verbascoside production was phenylalanine feeding (1.5 g/L), max. 3765 and 475 mg/100 g DW, respectively, after 7 days. This is the first report documenting the high antioxidant production in S. lateriflora microshoots after feeding with phenylalanine. Moreover, for the first time, bioreactor cultures were successfully maintained, obtaining attractive results (max. total flavonoid content 2348 and verbascoside 485 mg/100 g DW).

Phenylalanine Increases the Production of Antioxidant Phenolic Acids in Ginkgo biloba Cell Cultures.

The aims of this study were to evaluate the antioxidant properties, to investigate the content of major secondary metabolites in Ginkgo biloba cell cultures, and to determine the change in the production of phenolic acids by adding phenylalanine to the culture medium. Three in vitro methods, which depend on different mechanisms, were used for assessing the antioxidant activity of the extract: 1,1-diphenyl-2-picrylhydrazil (DPPH), reducing power and Fe2+ chelating activity assays. The extract showed moderate activity both in the DPPH and in the reducing power assays (IC50 = 1.966 ± 0.058 mg/mL; ASE/mL = 16.31 ± 1.20); instead, it was found to possess good chelating properties reaching approximately 70% activity at the highest tested dose. The total phenolic, total flavonoid, and condensed tannin content of G. biloba cell culture extract was spectrophotometrically determined. The phenolic acid content was investigated by RP-HPLC, and the major metabolites-protocatechuic and p-hydroxybenzoic acids-were isolated and investigated by 1H NMR. The results showed that phenylalanine added to G. biloba cell cultures at concentrations of 100, 150, and 200 mg/150 mL increased the production of phenolic acids. Cultures that were grown for 3 weeks and collected after 4 days of phenylalanine supplementation at high concentration showed maximal content of phenolic acids (73.76 mg/100 g DW).

In Vitro Cultures of Some Medicinal Plant Species (Cistus × incanus, Verbena officinalis, Scutellaria lateriflora, and Scutellaria baicalensis) as a Rich Potential Source of Antioxidants-Evaluation by CUPRAC and QUENCHER-CUPRAC Assays.

Comparative estimations of the antioxidant activity of methanolic extracts from biomasses of different types of in vitro cultures of Cistus × incanus, Verbena officinalis, Scutellaria lateriflora, and S. baicalensis and also from plant raw materials were performed. The antioxidant measurements were based on the modern assays-cupric ion reducing antioxidant capacity (CUPRAC) and quick, easy, new, cheap, and reproducible CUPRAC (QUENCHER-CUPRAC). The total extractable antioxidants (CUPRAC assay) ranged from 10.4 to 49.7 mmol (100 g)-1 of dry weight (DW) expressed as Trolox equivalent antioxidant capacity (TEAC), and the global antioxidant response (QUENCHER-CUPRAC assay) ranged from 16.0 to 79.1 mmol (100 g)-1 DW for in vitro cultures, whereas for plant raw materials the total extractable antioxidants ranged from 20.9 to 69.5 mmol (100 g)-1 DW, and the global antioxidant response ranged from 67.2 to 97.8 mmol (100 g)-1 DW. Finally, the in vitro cultures could be regarded as an antioxidant-rich alternative resource for the pharmaceutical, health food and cosmetics industries.

Fermented Vinegars from Apple Peels, Raspberries, Rosehips, Lavender, Mint, and Rose Petals: The Composition, Antioxidant Power, and Genoprotective Abilities in Comparison to Acetic Macerates, Decoctions, and Tinctures.

Acetic fermentation is a method for processing plant material which has been known since antiquity. Balsamic and apple cider vinegars are investigated as antibacterial, anti-obesity, and anti-diabetic remedies. However, there is little information about vinegars fermented from aromatic herbs and edible plants. The aim of this study was to compare extracts used for culinary and medicinal purposes according to their composition, antioxidant power, and genoprotective properties. Fermented vinegars, acetic macerates, decoctions, and tinctures in 70% ethanol from raspberries, apple peels, rosehips, lavender, mint, and rose petals were prepared. Polyphenols, ascorbate, carotenoid concentrations, and antioxidant power were analyzed. The polyphenols were identified using HPLC (high-performance liquid chromatography). The genoprotective properties were measured using a comet assay on lymphocytes. Fermented vinegars were poorest in phytochemicals in comparison to tinctures, decoctions, or acetic macerates, although they contained the highest concentration of metal ions. The antioxidant abilities were correlated to the phenolic content of extract. None of the extracts induced DNA damages into lymphocytes. The rosehip and rose petal extracts revealed the highest genoprotective abilities, while mint and apple fermented vinegars and decoctions had the lowest. Fermented vinegars are not a rich source of phytochemicals and they show weak genoprotective abilities, but, in increasing demand for antioxidants, any form of phytochemical sources is an added-value in diet.

Isatis tinctoria L. (Woad): A Review of its Botany, Ethnobotanical Uses, Phytochemistry, Biological Activities, and Biotechnological Studies.

Isatis tinctoria L. (Brassicaceae), which is commonly known as woad, is a species with an ancient and well-documented history as an indigo dye and medicinal plant. Currently, I. tinctoria is utilized more often as medicinal remedy and also as a cosmetic ingredient. In 2011, I. tinctoria root was accepted in the official European phytotherapy by introducing its monograph in the European Pharmacopoeia. The biological properties of raw material have been known from Traditional Chinese Medicine (TCM). Over recent decades, I. tinctoria has been investigated both from a phytochemical and a biological point of view. The modern in vitro and in vivo scientific studies proved anti-inflammatory, anti-tumour, antimicrobial, antiviral, analgesic, and antioxidant activities. The phytochemical composition of I. tinctoria has been thoroughly investigated and the plant was proven to contain many valuable biologically active compounds, including several alkaloids, among which tryptanthrin, indirubin, indolinone, phenolic compounds, and polysaccharides as well as glucosinolates, carotenoids, volatile constituents, and fatty acids. This article provides a general botanical and ethnobotanical overview that summarizes the up-to-date knowledge on the phytochemistry and biological properties of this valuable plant in order to support its therapeutic potential. Moreover, the biotechnological studies on I. tinctoria, which mainly focused on hairy root cultures for the enhanced production of flavonoids and alkaloids as well as on the establishment of shoot cultures and micropropagation protocols, were reviewed. They provide input for future research prospects.

The impact of media composition on production of flavonoids in agitated shoot cultures of the three Hypericum perforatum L. cultivars 'Elixir,' 'Helos,' and 'Topas'.

The aims of the study were to evaluate the effect of media composition on the growth potential and morphology of the in vitro cultured biomass of three cultivars of Hypericum perforatum, and on the production of flavonoids. Agitated shoot cultures were maintained in parallel on Linsmaier and Skoog (LS) and Murashige and Skoog (MS) media supplemented with 0.1-3.0 mg L-1 of α-naphthaleneacetic acid and 6-benzylaminopurine. Methanolic extracts from the biomass collected after 3-wk growth cycles were analyzed quantitatively, for 21 flavonoids using high performance liquid chromatography. Three aglycones (kaempferol, luteolin, and quercetin) and three glycosides of quercetin (hyperoside, quercitrin, and rutoside) were detected in all of the extracts. The total amounts of the estimated compounds increased from 1.18- to 21.66-fold on LS media variants and from 1.52- to 17.34-fold on MS media variants. The main metabolite was quercetin (max. 210.55 mg 100 g-1 dry weight [DW]). The maximum total amounts of all compounds in the biomass of 'Elixir,' 'Helos,' and 'Topas' were 328.53, 255.70, and 166.58 mg 100 g-1 DW, respectively. The shoots of all cultivars cultivated on the LS and MS media containing low levels of plant growth regulators (0.1 mg L-1) accumulated high amounts of flavonoids. The highest amounts were accumulated in shoots of cultivar 'Elixir' grown on MS medium. This is the first comparison of flavonoid production in three cultivars of H. perforatum ('Elixir,' 'Helos,' and 'Topas') cultured in vitro, and the first report of flavonoid production in cultivars 'Elixir' and 'Helos.'

Influence of Culture Medium Composition and Light Conditions on the Accumulation of Bioactive Compounds in Shoot Cultures of Scutellaria lateriflora L. (American Skullcap) Grown In Vitro.

Methanolic extracts from in vitro grown Scutellaria lateriflora shoots cultured on five Murashige and Skoog (MS) medium variants supplemented with different combinations of 6-benzylaminopurine (BAP) and α-naphthaleneacetic acid (NAA) under different light conditions (monochromatic light, white light and no light) were analysed by HPLC for three groups of metabolites: flavonoids (26 compounds), phenolic acids and their precursors (19+2) and phenylethanoid glycosides (2). The analyses revealed the presence of baicalein, baicalin, wogonin, wogonoside, 3,4-dihydroxyphenylacetic acid and verbascoside. There was clear evidence of the influence of plant growth regulators and light conditions on the accumulation of the analysed groups of secondary metabolites. The amounts of the compounds changed within a wide range-for the total flavonoid content, 30.2-fold (max. 1204.3 mg·100 g-1 dry weight (DW)); for 3,4-dihydroxyphenylacetic acid, 5.5-fold (max. 33.56 mg·100 g-1 DW); and for verbascoside, 1.5-fold (169.15 max. mg·100 g-1 DW). The best medium for the production of most of the compounds was the Murashige and Skoog variant with 1 mg l-1 BAP and 1 mg l-1 NAA. For verbascoside, the best 'productive' medium was the MS variant supplemented with 0.5 mg l-1 BAP and 2 mg l-1 NAA. The accumulation of the metabolites was stimulated to the greatest extent by blue light, under which the extracts were found to contain the highest total amount of flavonoids and the highest amounts of flavonoid glucuronides, baicalin and wogonoside, as well as of verbascoside. Their amounts were, respectively, 1.54-, 1.49-, 2.05- and 1.86-fold higher than under the control white light.

S-sulfhydration as a cellular redox regulation.

For many years reactive oxygen and nitrogen species (ROS and RNS) have been recognized as key messengers in the process of thiol-based redox regulation. Relatively recently, literature reports began to mention reactive sulfur species (RSS) and their role in thiol regulation. This review is focused on biogenesis and biological properties of RSS, including: hydropersulfides, polysulfides and hydrogen sulfide (H2S). Based on the most up-to-date literature data, the paper presents biological significance of S-sulfhydration process. In this reaction, sulfane sulfur is transferred to the-SH groups forming hydropersulfides. Protein cysteine residues, called 'redox switches' are susceptible to such reversible modifications. In line with the most recent reports, it was emphasized that sulfane sulfur-containing compounds (mainly hydrogen persulfides and polysulfides) are real and better mediators of S-sulfhydration-based signalling than H2S. We also overviewed proteins participating in the formation and transport of RSS and in mitochondrial H2S oxidation. In addition, we reviewed many reports about proteins unrelated to sulfur metabolism which are modified by S-sulfhydration that influences their catalytic activity. We also addressed the problem of the regulatory function of S-sulfhydration reaction in the activation of KATP channels (vasorelaxant) and transcription factors (e.g. NFκB) as well as in the mechanism of therapeutic action of garlic-derived sulfur compounds. Some aspects of comparison between RNS and RSS are also discussed in this review.

Comparative analysis of therapeutically important indole compounds in in vitro cultures of Hypericum perforatum cultivars by HPLC and TLC analysis coupled with densitometric detection.

Five indole compounds (5-hydroxy-L-tryptophan, L-tryptophan, indole-3-acetic acid, melatonin, serotonin) and hypericin were identified and quantified in methanolic extracts of shoot cultures of three Hypericum perforatum cultivars (Helos, Elixir, Topas) growing on two variants of Murashige -Skoog medium differing in concentrations of growth regulators (naphthalene-l-acetic acid and 6-benzylaminopurine). Extracts of the aboveground parts of field-grown plants (Hyperici herba) were also analyzed by HPLC and TLC analysis coupled with densitometric detection. Determination of four compounds was based on our assay described earlier. The methods of determination of 5-hydroxy-L-tryptophan and hypericin were developed and validated in this study. The composition and contents of the metabolites under study differed between the cultivars cultured in vitro and between medium variants containing diverse contents of growth regulators. The contents of individual indole compounds in the biomass from in vitro cultures ranged from 39.6 to 343.2 mg/100 g dry mass. 5-Hydroxy-L- tryptophan was the dominating metabolite (from 78.2 to 343.2 mg/100 g dry mass). Extracts from shoots of the cultivar Helos also contained high contents of serotonin (319.9 and 197.4 mg/100 g dry mass). The contents of indole compounds in Hyperici herba were also diverse (from 7.1 to 55.3 mg/100 g dry mass). 5-Hydroxy-L-tryptophan was the dominating metabolite as well. Hypericin content ofHyperici herba, equaling 12.2 mg/100 g dry mass was from 3.3 to 10 times higher than in extracts from shoots cultured in vitro. The present report is the first analysis of endogenous accumulation of indole compounds in Hyperici herba which involves, apart from melatonin, four other compounds.

In vivo anti-inflammatory activity of lipoic acid derivatives in mice.

BACKGROUND: In mammals lipoic acid (LA) and its reduced form dihydrolipoic acid (DHLA) function as cofactors for multienzymatic complexes catalyzing the decarboxylation of α-ketoacids. Moreover, LA is used as a drug in a variety of diseases including inflammatory diseases. The aim of the study was to examine anti-inflammatory properties of LA metabolites. MATERIAL/METHODS: The present paper reports the chemical synthesis of 2,4-bismethylthio-butanoic acid (BMTBA) and tetranor-dihydrolipoic acid (tetranor-DHLA). BMTBA is one of the biotransformation products of LA, while tetranor-DHLA is an analogue of DHLA. Structural identity of these compounds was confirmed by 1H NMR. These compounds were assessed for their anti-inflammatory activity in mice. For this purpose, the zymosan-induced peritonitis and the carrageenan-induced hind paw edema animal models were applied. RESULTS/CONCLUSIONS: The obtained results indicated that the early vascular permeability measured at 30 min of zymosan-induced peritonitis was significantly inhibited in groups receiving BMTBA (10, 30, 50 mg/kg). The early infiltration of neutrophils measured at 4 hours of zymosan-induced peritonitis was inhibited in the group receiving BMTBA (50 mg/kg) and tetranor-DHLA (50 mg/kg). The results indicated that the increase in paw edema was significantly inhibited in the groups receiving BMTBA (50, 100 mg/kg) and tetranor-DHLA (30, 50 mg/kg). In summary, the present studies clearly demonstrated that both BMTBA and tetranor-DHLA were able to act as anti-inflammatory agents. This is the first study examining in vivo the anti-inflammatory properties of LA metabolites.

Arbutin production via biotransformation of hydroquinone in in vitro cultures of Aronia melanocarpa (Michx.) Elliott.

Arbutin (hydroquinone ß-D-glucoside) is a compound of plant origin possessing valuable therapeutic (urinary tract disinfection) and cosmetic (skin whitening) properties, which can be obtained from in vitro cultures of plants belonging to different taxa via biotransformation of exogenously supplemented hydroquinone. Agitating cultures of Aronia melanocarpa were maintained on the Murashige and Skoog medium containing growth regulators: the cytokinin - BAP (6-benzylaminopurine), 2 mg/l and the auxin NAA (α-naphthaleneacetic acid), 2 mg/l. The biomass was cultured for 2 weeks and then hydroquinone was supplemented at the following doses: 96, 144, 192, 288 and 384 mg/l either undivided or divided into two or three portions added at 24-hour intervals. The content of the reaction product - arbutin, was determined using an HPLC method in methanolic extracts from biomass and lyophilized medium samples collected 24 hours after the addition of the last precursor dose. The total amounts of arbutin were very diverse, from 2.71 to 8.27 g/100g d.w. The production of arbutin rose with increasing hydroquinone concentration. The maximum content of the product was observed after hydroquinone addition at 384 mg/l divided into two portions. Biotransformation efficiency also varied widely, ranging from 37.04% do 73.80%. The identity of the product - arbutin, after its isolation and purification was confirmed by spectral analysis ((1)H-NMR spectrum). The maximum amount of arbutin obtained was higher than that required by the latest 9(th) Edition of the Polish Pharmacopoeia and by the newest 8th Edithion of European Pharmacopoeia for Uvae ursi folium (7.0 g/100g d.w.), and is interesting from practical point of view.

Acceleration of anaerobic cysteine transformations to sulfane sulfur consequent to γ-glutamyl transpeptidase inhibition.

Toxicity of drugs and radiation in the cells is largely dependent on the level of thiols. In the present studies, an attempt has been made to inhibit γ-glutamyl transpeptidase (γGT) activity in EAT-bearing animals tissue. We have expected that administration of γGT inhibitors: acivicin and 1,2,3,4-tetrahydroisoquinoline (TIQ) may influence GSH/γ-glutamyl transpeptidase (γGT) system in the regulation of cysteine concentration and anaerobic cysteine metabolism in normal and cancer cells. Development of Ehrlich ascites tumor in mice enhances peroxidative processes, diminishes levels of nonprotein thiols (NPSH) and sulfane sulfur, and lowers activities of enzymes involved in its formation and transfer in the liver and kidney. Although γGT inhibitors further decrease NPSH level, they increase cysteine and sulfane sulfur levels. This means that upon γGT inhibition, cysteine can be efficiently acquired by normal liver and kidney cells via another pathway, that is so productive that sulfane sulfur level and intensity of anaerobic cysteine metabolism even rise.

The effects of garlic-derived sulfur compounds on cell proliferation, caspase 3 activity, thiol levels and anaerobic sulfur metabolism in human hepatoblastoma HepG2 cells.

The aim of the present studies was to determine whether the mechanism of biological action of garlic-derived sulfur compounds in human hepatoma (HepG2) cells can be dependent on the presence of labile sulfane sulfur in their molecules. We investigated the effect of allyl sulfides from garlic: monosulfide, disulfide and trisulfide on cell proliferation and viability, caspase 3 activity and hydrogen peroxide (H(2)O(2)) production in HepG2 cells. In parallel, we also examined the influence of the previously mentioned compounds on the levels of thiols, glutathione, cysteine and cysteinyl-glycine, and on the level of sulfane sulfur and the activity of its metabolic enzymes: rhodanese, 3-mercaptopyruvate sulfurtransferase and cystathionase. Among the compounds under study, diallyl trisulfide (DATS), a sulfane sulfur-containing compound, showed the highest biological activity in HepG2 cells. This compound increased the H(2)O(2) formation, lowered the thiol level and produced the strongest inhibition of cell proliferation and the greatest induction of caspase 3 activity in HepG2 cells. DATS did not affect the activity of sulfurtransferases and lowered sulfane sulfur level in HepG2 cells. It appears that sulfane sulfur containing DATS can be bioreduced in cancer cells to hydroperthiol that leads to H(2)O(2) generation, thereby influencing transmission of signals regulating cell proliferation and apoptosis.

Effects of different garlic-derived allyl sulfides on peroxidative processes and anaerobic sulfur metabolism in mouse liver.

Biological activity of garlic has been attributed to organosulfur compounds, most of all to oil-soluble allyl sulfides, such as diallyl sulfide (DAS), diallyl disulfide (DADS) and diallyl trisulfide (DATS). This study evaluated the effectiveness of garlic-derived allyl sulfides in influencing peroxidative processes, levels of thiols and sulfane sulfur and its metabolic enzymes in normal mouse liver cells. Various allyl sulfides (DAS, DADS and DATS) dissolved in corn oil were given intraperitoneally to mice for 10 days. After sacrificing the mice, biochemical assays were performed in liver homogenates and in plasma in order to establish liver function. All allyl sulfides under study had a beneficial effect in the mouse liver since they decreased reactive oxygen species and malondialdehyde levels and increased glutathione S-transferase activity and non-protein sulfhydryl group level. Moreover, DADS and DATS elevated total sulfane sulfur pool and activity of sulfane sulfur biosynthetic enzymes. The increase in sulfane sulfur level entailed augmentation of its antioxidant and regulatory capacities. Garlic-derived allyl sulfides exhibited antioxidant action in the liver and elevated anaerobic cysteine metabolism leading to the formation of sulfane sulfur-containing compounds. Thus, DADS and DATS showed beneficial action in the liver, which can be used for protection of normal liver cells during chemotherapy or for alleviation of liver damage.

Effects of aspirin on the levels of hydrogen sulfide and sulfane sulfur in mouse tissues.

This study was designed to investigate the effect of aspirin (ASA) on anaerobic cysteine metabolism, which yields sulfane sulfur-containing compounds and hydrogen sulfide (H(2)S), in mouse liver and brain. In order to solve this problem, we determined the levels of sulfane sulfur and H(2)S, and the activities of cystathionase, the enzyme directly engaged in H(2)S synthesis, and rhodanese, the enzyme that catalyzes sulfane sulfur transfer to different acceptors. Moreover, we examined the effect of ASA on glial Gomori-positive cells (GGPC) in the brain that contain sulfur-rich glial Gomori-positive material (GGPM). The studies indicated an ASA-induced decrease in H(2)S levels in the brain and an increase in the liver. ASA-treated animals had lower cerebral levels of GGPM-containing GGPCs but the sulfane sulfur level was not affected. Conversely, the sulfane sulfur content in the liver dropped. ASA did not change cystathionase and rhodanese activity in either organ. The obtained results revealed that ASA was able to influence anaerobic cysteine metabolism, leading to the formation of sulfane sulfur and H(2)S in the mouse liver and brain, and to affect the numbers of GGPM-containing GGPCs.

The effect of nitroglycerin tolerance on oxidative stress and anaerobic sulfur metabolism in rat tissues.

The present results indicated that tolerance to nitroglycerin (glycerin trinitrate, GTN) increased the hepatic and renal level of reactive oxygen species, malondialdehyde, and glutathione peroxidase (PO(x)) and decreased superoxide dismutase activity, whereas non-protein thiols remained unchanged in both organs. In the liver (but not in the kidney) glutathione S-transferase (GST), catalase and rhodanese activities decreased and the sulfane sulfur level also dropped. Unlike in the liver, gamma-glutamyl transpeptidase (gammaGT) activity in the kidney declined. On the other hand, hepatic S-nitrosothiols (SNT) rose while renal SNT declined. As no concomitant changes in the nitric oxide (NO) level were observed in either organ, the results suggested that under nitroglycerin tolerance NO was stored in the liver in the form of SNT. In conclusion, the results obtained in the liver and kidney confirm the involvement of oxidative stress in the pathomechanism of GTN tolerance, and reveal the diverse effects of this phenomenon on the gammaGT and GST activity and SNT level in both organs. We observed for the first time that GTN tolerance could be accompanied by the disruption of hepatic anaerobic cysteine metabolism, associated with sulfane sulfur and rhodanese activity.