Evaluation of antioxidant activity of extracts from the roots and shoots of Scutellaria alpina L. and S. altissima L. in selected blood cells.

BACKGROUND: It is widely known that reactive oxygen species (ROS) can cause oxidative damage in cells and have been linked to the pathogenesis of oxidative diseases, such as atherosclerosis, ischemia, neurodegenerative disease, diabetes, or cancer. Recently, much attention has been focused on preventive strategies for oxidative stress and related diseases. Plants represent a source of bioactive compounds whose antioxidant activity may be useful in protecting against pro-oxidative reactions. OBJECTIVES: The study determines the in vitro biological activity of the ethanolic extracts from the shoots and roots of Scutellaria species (S. altissima and S. alpina) in selected blood cells (blood platelets and lymphocytes). MATERIAL AND METHODS: Platelet activity, both resting and after thrombin stimulation, was used to indicate the ability of the plant extracts to inhibit the production of superoxide anion radicals (O2 •-) and platelet lipid peroxidation. The generation of superoxide anion radicals was measured by cytochrome c reduction. Lipid peroxidation in blood platelets was measured by the level of thiobarbituric acid reactive substances (TBARS). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay was used to determine the protective effect of Scutellaria extracts on lymphocyte cells against oxidative damage induced by hydroxyl radicals. RESULTS: Extracts (5-50 µg/mL) containing phenolic compounds from both Scutellaria species distinctly reduced nonenzymatic lipid peroxidation and arachidonic acid metabolism by blood platelets in vitro. When given at the tested concentration, the extracts reduced the generation of O2 •- in resting blood platelets and platelets activated by thrombin in vitro. All Scutellaria extracts (10 µg/mL) containing phenolic compounds also protected human lymphocytes against oxidative stress induced by hydrogen peroxide (H2O2). CONCLUSIONS: The present study suggests that the natural extracts from S. altissima and S. alpina have antioxidant properties and, therefore, may be beneficial in the prevention of diseases in which blood platelets and lymphocytes are involved, i.e., cancer or inflammatory and infective diseases.

Induction of apoptosis in human glioma cell lines of various grades through the ROS-mediated mitochondrial pathway and caspase activation by Rhaponticum carthamoides transformed root extract.

The present study is the first investigation of the inhibitory effect of Rhaponticum carthamoides transformed roots (TR) extract on the proliferation of grade II and III human glioma cells. TR extract showed the cytotoxic effect and inhibited the colony formation of both glioma cell lines in dose-dependent manner. The root extract induced apoptosis by increasing of the reactive oxygen species (about threefold compared to the control cells) leading to a disruption of mitochondrial membrane potential. Additionally, the mRNA levels of the apoptotic factors such as Bax, Tp53, caspase-3, and caspase-9 were observed to increase. These results indicate that the TR extract possesses anticancer activity by inhibiting glioma cell proliferation and inducing apoptotic cell death, and may be used as a promising anticancer agent.

Antibacterial, Anti-Inflammatory, Antioxidant, and Antiproliferative Properties of Essential Oils from Hairy and Normal Roots of Leonurus sibiricus L. and Their Chemical Composition.

Essential oils obtained from the NR (normal roots) and HR (hairy roots) of the medicinal plant Leonurus sibiricus root were used in this study. The essential oil compositions were detected by GC-MS. Eighty-five components were identified in total. Seventy components were identified for NR essential oil. The major constituents in NR essential oil were ß-selinene (9.9%), selina-4,7-diene (9.7%), (E)-ß-caryophyllene (7.3%),myli-4(15)-ene (6.4%), and guaia-1(10),11-diene (5.9%). Sixty-seven components were identified in HR essential oil, the main constituents being (E)-ß-caryophyllene (22.6%), and germacrene D (19.8%). The essential oils were tested for cytotoxic effect, antimicrobial, anti-inflammatory, and antioxidant activities. Both essential oils showed activity against grade IV glioma cell lines (IC50 = 400 µg/mL), antimicrobial (MIC and MFC values of 2500 to 125 µg/mL), and anti-inflammatory (decreased level of IL-1ß, IL-6, TNF-α, and IFN-γ in LPS-stimulated cells).The essential oils exhibited moderate antioxidant activity in ABTS (EC50 = 98 and 88 µg/mL) assay. This is the first study to examine composition of the essential oils and their antimicrobial, antioxidant, antiproliferative, and anti-inflammatory activities. The results indicate that essential oils form L. sibiricus root may be used in future as an alternative to synthetic antimicrobial agents with potential application in the food and pharmaceutical industries.

Transformed Root Extract of Leonurus sibiricus Induces Apoptosis through Intrinsic and Extrinsic Pathways in Various Grades of Human Glioma Cells.

This study determines the influence of transformed root (TR) extract of Leonurus sibiricus L. on various grades (I-III) of human glioma cells derived from patients. This plant occurs in southern Asia and Siberia and is widely used as a medicinal plant with various biological activities. Chromatographic profile of TR extract have revealed the presence of various polyphenolic compounds (4-hydroxybenzoic acid, gentisic acid, vanilic acid, 1,3-dicaffeoylquinic acid, α-resorcylic acid). We found TR root extract to have antiproliferative activity on glioma cells after 24 h of treatment. TR root extract induces apoptosis on various grades (I-III) of human glioma cells by the generation of reactive oxygen species (ROS) along with concurrent loss of mitochondrial membrane potential, enhanced S and G2/M phases of the cell cycle, and altered mRNA levels of Bax, Bcl-2, p53, Cas-3, Cas-8 and Cas-9 factors involved in apoptosis. This work for the first time demonstrate that TR extract from L. sibiricus root has the potential to activate apoptosis in grade I-III human glioma cells through the intrinsic and extrinsic pathways.

Inhibition of human glioma cell proliferation by altered Bax/Bcl-2-p53 expression and apoptosis induction by Rhaponticum carthamoides extracts from transformed and normal roots.

OBJECTIVE: The objective of this study was to determine the cytotoxic effect and apoptotic activity of Rhaponticum carthamoides transformed root (TR) and root of soil-grown plant (NR) extracts in a human glioma primary cells. The effect of these root extracts on cell cycle arrest, mitochondrial membrane potential (ΔΨm) and expression levels of apoptosis-related genes (Bcl-2, Bax and p53) were also examined. METHODS: Cytotoxic activity of root extracts was evaluated by MTT assay. Apoptosis and cell cycle were determined by flow cytometry. Expression levels of apoptosis-related gene were analysed by RT-PCR and Western blotting. ΔΨm was examined by the use of JC-1 reagent. KEY FINDINGS: Rhaponticum carthamoides root extracts inhibit cell growth and induce apoptosis in a dose-dependent manner in human glioma cells. The root extracts were found to up-regulate the pro-apoptotic Bax protein and down-regulate the anti-apoptotic Bcl-2 protein, consequently increasing the ratios of Bax/Bcl-2 protein levels. Moreover, an increase of the p53 protein level and reduction of ΔΨm in glioma cells were observed after treatment with NR and TR extracts. CONCLUSION: The results of this study may offer a new insight into the potential anticancer activity of R. carthamoides root extracts.

Inhibition of Advanced Glycation End-Product Formation and Antioxidant Activity by Extracts and Polyphenols from Scutellaria alpina L. and S. altissima L.

Methanolic extracts from the aerial parts and roots of two Scutellaria species, S. alpina and S. altissima, and five polyphenols from these plants demonstrated a significant ability to inhibit the formation of advanced glycation end-products (AGE) in vitro. S. alpina, which is richer in polyphenolic compounds, had strong antiglycation properties. These extracts demonstrated also high activity in the FRAP (ferric-reducing antioxidant power), antiradical (DPPH) and lipid peroxidation inhibition assays. Among the pure compounds, baicalin was the strongest glycation inhibitor (90.4% inhibition at 100 µg/mL), followed by luteolin (85.4%). Two other flavone glycosides had about half of this activity. Verbascoside was similar to the reference drug aminoguanidine (71.2% and 75.9%, respectively). The strong correlation observed between AGE inhibition and total flavonoid content indicated that flavonoids contribute significantly to antiglycation properties. A positive correlation was also observed between antiglycative and antioxidant activities. The studied skullcap species can be considered as a potential source of therapeutic agents for hyperglycemia-related disorders.

Antioxidant and DNA Repair Stimulating Effect of Extracts from Transformed and Normal Roots of Rhaponticum carthamoides against Induced Oxidative Stress and DNA Damage in CHO Cells.

Rhaponticum carthamoides has a long tradition of use in Siberian folk medicine. The roots and rhizomes of this species are used in various dietary supplements or nutraceutical preparations to increase energy level or eliminate physical weakness. This is the first report to reveal the protective and DNA repair stimulating abilities of R. carthamoides root extracts in Chinese hamster ovary (CHO) cells exposed to an oxidative agent. Both transformed root extract (TR extract) and extract of soil-grown plant roots (NR extract) may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, but CHO cells stimulated with extract from the transformed roots demonstrated significantly stronger properties than cells treated with the soil-grown plant root extract. These differences in biological activity may be attributed to the differences in the content of phenolic compounds in these root extracts. Preincubation of the CHO cells with TR and NR extracts showed an increase in gene expression and protein levels of catalase (CAT) and superoxide dismutase (SOD2). R. carthamoides may possess antioxidant properties that protect CHO cells against oxidative stress.

The Effect of Leonurus sibiricus Plant Extracts on Stimulating Repair and Protective Activity against Oxidative DNA Damage in CHO Cells and Content of Phenolic Compounds.

Leonurus sibiricus L. has been used as a traditional and medicinal herb for many years in Asia and Europe. This species is known to have antibacterial, anti-inflammatory, and antioxidant activity and has demonstrated a reduction of intracellular reactive oxygen species. All tested extracts of L. sibiricus showed protective and DNA repair stimulating effects in Chinese hamster ovary (CHO) cells exposed to H2O2. Preincubation of the CHO cells with 0.5 mg/mL of plant extracts showed increased expression level of antioxidant genes (SOD2, CAT, and GPx). LC-MS/MS and HPLC analyses revealed the presence of nine phenolic compounds in L. sibiricus plant extracts: catechin, verbascoside, two flavonoids (quercetin and rutin), and five phenolic acids (4-hydroxybenzoic acid, chlorogenic acid, caffeic acid, p-coumaric acid, and ferulic acid). The roots and aerial parts of in vitro L. sibiricus plant extracts, which had the strongest antioxidant properties, may be responsible for stimulating CHO cells to repair oxidatively induced DNA damage, as well as protecting DNA via enhanced activation of the antioxidant genes (SOD2, CAT, and GPx) regulating intracellular antioxidant capacity. The content of phenolic compounds in in vitro raised plants was greater than the levels found in plants propagated from seeds.

A preliminary study of apoptosis induction in glioma cells via alteration of the Bax/Bcl-2-p53 axis by transformed and non-transformed root extracts of Leonurus sibiricus L.

Leonurus sibiricus L. is a traditional medicinal plant which occurs in southern Siberia, China, Korea, Japan, and Vietnam. The plant shows several pharmacological effects, but the most interesting is its anti-cancer activity. The aim of our study was to examine the induction of apoptosis in malignant glioma cells, the most aggressive primary brain tumors of the central nervous system, following treatment with transformed root (TR) or non-transformed root (NR) L. sibiricus extracts. Both the NR and TR extracts were found to have cytotoxic activity in the glioma primary cells. The human glioblastoma cell lines obtained from patients were confirmed to be tumorogenic by the following three markers: D10S1709, D10S1172, and D22S283. HPLC and MS analysis revealed the presence of polyphenolic compounds (chlorogenic acid, ferulic acid, caffeic acid, p-coumaric acid, ellagic acid, and verbascoside) in both sets of root extracts. In summary, our findings demonstrate that treatment of the glioma cells with NR and TR extracts resulted (a) in significant cell growth inhibition, (b) S- and G2/M-phase cell cycle arrest, and (c) apoptosis in a dose-dependent fashion by changing Bax/Bcl-2 ratio (about 4-fold increase) and p53 (5-fold increase) activation. These findings indicate that NR and TR extracts exhibit anti-cancer activity through the regulation of genes involved in apoptosis. This is the first report to demonstrate the cytotoxic effect of polyphenolic extracts from L. sibiricus roots against glioma cells, but further studies are required to understand the complete mechanism of its apoptosic activity.

Taxodione and Extracts from Salvia austriaca Roots as Human Cholinesterase Inhibitors.

Taxodione, an abietane diterpenoid, was isolated from Salvia austriaca transformed roots grown in in vitro conditions. The compound is known to have antibacterial, cytotoxic and anti-tumour properties. This study evaluates the ability of pure taxodione and extracts obtained from the S. austriaca hairy roots and roots from field-grown plants to inhibit human acetylcholinesterase and butyrylcholinesterase. Both extracts were found to have similar actions against acetylcholinesterase. The IC50 for extracts from transformed and untransformed roots were 142.5 and 139.5 µg ml(-1), respectively. The highest activity towards human acetylcholinesterase was demonstrated by taxodione (IC50 = 54.84 µg ml(-1)). With respect to BChE inhibition, the root extracts demonstrated stronger activity (IC50 = 23.6 µg ml(-1): field-grown plants and 41.6 µg ml(-1): transformed roots) than taxodione (IC50 = 195.9 µg ml(-1)). Taxodione showed significant cytotoxicity against A549 cell line (IC50 = 9.1 µg ml(-1)), whereas the activities for the extracts from S. austriaca roots of field-grown plants (IC50 = 75.7 µg ml(-1)) and hairy roots (IC50 = 86.2 µg ml(-1)) were lower. Computer modelling suggests that taxodione should not demonstrate cardiotoxic or genotoxic activity. It also indicates that taxodione should demonstrate very rapid transport from the body with very good blood-brain barrier penetration, but with no cumulative effect on the human body. The obtained results indicate that taxodione is a safe compound and may be used for further investigations in pharmacological activities.

ANTIOXIDANT ACTIVITY OF TISSUE CULTURE-RAISED BALLOTA NIGRA L. PLANTS GROWN EX VITRO.

Antioxidant properties and total phenolic and flavonoid contents were evaluated in methanolic extracts of shoots from Ballota nigra plants initiated in vitro (from nodal explants) and in vivo (from seeds). The plants were grown in greenhouse and in the field, and were analyzed at the vegetative and flowering stages. The shoot extract of wild-grown plants of B. nigra was also investigated. The results indicate that antioxidant potential of the B. nigra extracts seems to be due to their scavenging of free radicals (DPPH assay) and metal reducing (FRAP test), while they were less effective at the prevention of linoleic acid peroxidation (LPO test). The extracts from shoots of in vitro derived plants were found to exhibit the greatest antioxidant properties. The extracts were also characterized by the highest content of phenolic compounds and their level was affected by plant developmental stage. The extracts of shoots collected at the flowering period exhibited higher amounts of phenolics and flavonoids than in the extracts of immature plants. A close correlation between the total phenolic content and flavonoid content and antioxidant activity using the DPPH and FRAP assays was obtained. The results of the present study suggest the use in vitro-derived plants of B. nigra instead of using wild plants for pharmaceutical purposes.

EVALUATION OF ANTIOXIDANT PROPERTIES OF METHANOLIC EXTRACTS FROM LEAVES AND ROOTS OF REHMANNIA GLUTINOSA LIBOSCH. IN HUMAN BLOOD.

Compounds with antioxidant activity may protect different elements of blood from damaging effects of oxidative stress. The present in vitro study was designed to examine the antioxidant activity of methanolic extracts from leaves and roots of R. glutinosa plants against damages induced by oxidative stress. Oxidative stress was stimulated in human plasma and human blood platelet samples by the strong biological oxidant--hydrogen peroxide (H2O2) or H2O2/Fe (the donor of hydroxyl radicals). In experimental trials, the level of biomarker of lipid peroxidation--TBARS was significantly decreased by the action of methanolic extracts from R. glutinosa leaves. Therefore, it seems that the methanolic extract from leaves of the plants may be a new source of bioactive antioxidant natural compounds. It may be also an active pharmacological agent or a food supplement for healthy subjects and for people with different diseases (cardiovascular diseases and cancer) induced by oxidative stress.

Studies on the antioxidant properties of extracts from the roots and shoots of two Scutellaria species in human blood plasma.

We determined the in vitro antioxidant activity of methanolic extracts from the shoots and roots of Scutellaria species (S. altissima and S. alpina) against the action of strong oxidants: hydrogen peroxide (H2O2) and H2O2+Fe(2+) (donor of hydroxyl radicals) on plasma proteins and lipids. Lipid peroxidation in human plasma was measured by the level of thiobarbituric acid reactive species (TBARS). Protein oxidation was measured by quantitation of thiol group. We observed that the extracts (5-50 µg ml(-1)) containing phenolic compounds from both Scutellaria species distinctly reduced oxidation of lipids and proteins in human plasma treated with H2O2. These results also indicated that the extracts have a protective effect against oxidative damage to the human plasma lipids and proteins by induced hydroxyl radical. The main components of the plant materials analysed were flavonoids, present as aglycones (luteolin) or glycosides (cynaroside, baicalin, wogonoside). In all of the extracts, the phenylethanoid verbascoside was also found. The properties of the tested plant extracts were also compared with the action of a well characterised commercial antioxidative polyphenolic extract from the berries of Aronia melanocarpa (Aronox(®)). The comparative studies indicated that the analysed plant extracts were comparable to or even more effective in reducing the oxidation processes than the A. melanocarpa extract. The present study suggests that natural extracts from S. altissima and S. alpina have antioxidant activities and, therefore, may be beneficial in the prevention of diseases related to oxidant stress, such as cancer, cardiovascular, and inflammatory diseases.

Abietane diterpenoids from Salvia sclarea transformed roots as growth inhibitors of pathogenic Acanthamoeba spp.

Amoebae from the genus Acanthamoeba are known agents leading to various diseases such as granulomatous amoebic encephalitis (GAE), a chronic progressive disease of the central nervous system, amoebic keratitis (AK), chronic eye infection, amoebic pneumitis (AP), chronic lung infection, and skin infections. It is known that various synthetic anti-Acanthamoeba substances are ineffective. Therefore, other substances, e.g., natural plant compounds, are the focus of biological investigations regarding anti-parasite activity. In this work, the ability of four abietane diterpenoids (ferruginol, salvipisone, aethiopinone, and 1-oxo-aethiopinone) to inhibit Acanthamoeba growth is reported. All investigated compounds were active against Acanthamoeba growing in vitro. Among them, ferruginol demonstrated the highest activity against Acanthamoeba. This compound inhibited Acanthamoeba growth by about 72% in a 3-day exposure period (IC50 17.45 µM), while aethiopinone and 1-oxo-aethiopinone demonstrated this activity at the level of 55-56%. Salvipisone reduced the growth of Acanthamoeba in vitro culture by 39%. For this compound, the value of IC50 was 701.94 µM after 72 h of exposure.

An unusual taxodione derivative from hairy roots of Salvia austriaca.

From a root culture of Salvia austriaca, transformed with Agrobacterium rhizogenes, a new diterpenoid was isolated and its chemical structure was determined as 7-(2-oxohexyl)-11-hydroxy-6, 12-dioxo-7,9(11),13- abietatriene [= 7-(2-oxohexyl)-taxodione] on the basis of spectroscopic methods, especially 1D and 2D NMR, and by comparison with structurally related compounds. This compound represents a hitherto unknown 2-oxohexyl diterpenoid derivative. Cytotoxic studies revealed that the new compound exhibited high cytotoxic activity against three cancer cell lines with IC(50) values ranging from 0.63 to 0.72µM. Its cytotoxic effectiveness against the cancer cells was ten fold higher than that of taxodione.

Chemical composition and biological activities of essential oil from Salvia sclarea plants regenerated in vitro.

The essential oils obtained by hydrodistillation of dried aerial parts of Salvia sclarea L. plants, regenerated in vitro and reproduced from seeds, were analyzed by GC and GC-MS. The oils from in vitro and in vivo plants were compared in respect to their chemical composition as well as antimicrobial and cytotoxic activities. The chemical profiles of both oils were very similar, although the yield of essential oil from in vitro plants was lower (0.1%, v/w) than the oil yield isolated from in vivo S. sclarea plants (0.2%, v/w). Both oils showed antimicrobial and cytotoxic activity. The oil from in vitro regenerated plants of S. sclarea exhibited stronger cytotoxic action against NALM-6 cell lines in comparison with the essential oil from in vivo plants.

Salvipisone and aethiopinone from Salvia sclarea hairy roots modulate staphylococcal antibiotic resistance and express anti-biofilm activity.

Two diterpenoids, salvipisone (Salv) and aethiopinone (Aeth), isolated from hairy roots of Salvia sclarea, were tested with respect to their activity against methicillin-resistant Staphylococcus aureus (MRSA) and S. epidermidis (MRSE) strains, cultured as planktonic cells or as adherent biofilms. The standard CLSI method, MTT reduction assay or confocal laser scanning microscopy (CLSM) were used for this purpose and also applied for testing the susceptibility to oxacillin, vancomycin, linezolid and their potential synergy with diterpenoids (evaluated as a fractional inhibitory concentration (FIC) index). Salv and Aeth were shown to be bactericidal or bacteriostatic against S. aureus and S. epidermidis planktonic cultures. Both diterpenoids, at the concentrations of 1/2 MIC, showed synergy with antibiotics representing the beta-lactam, glycopeptide and oxazolidinone groups. None of the antibiotics used at a high concentration killed the staphylococcal biofilms. On the contrary, Salv and Aeth decreased the number of live biofilm cells by 45.7 - 77.1% and slightly reduced the biofilm inhibitory concentration of oxacillin. Diterpenoids also changed the parameters of biofilm morphology, as shown by the CLSM image processing package (PHLIP). It was concluded that salvipisone and aethiopinone (relatively highly lipophilic, log P respectively = 3.4; 4.8) synergized the action of beta-lactam antibiotics towards MRSA and MRSE probably by alteration of cell surface hydrophobicity and cell wall/membrane permeability, but not by changing penicillin-binding protein, PBP2a expression and penicillinase production or by direct binding to the cell wall peptidoglycan and teichoic acids.

In vitro propagation and chemical and biological studies of the essential oil of Salvia przewalskii Maxim.

The procedure of Salvia przewalskii shoot multiplication and the ability of regenerated plants to produce essential oil is reported. The essential oil was obtained by hydrodistillation from leaves and flowering stems of field-grown plants, and their chemical composition was examined by GC, GC-MS and 1H NMR. The differences in yield as well as qualitative and quantitative composition between the oils isolated from in vitro and in vivo plants were observed. S. przewalskii essential oil was tested for its antimicrobial and cytotoxic properties. It was found that cytotoxicity against human leukemia HL-60 cells and antimicrobial activity (especially, against Staphylococcus aureus and S. epidermidis strains) of oils isolated from in vitro plants were higher than those for oils from in vivo S. przewalskii plants.

Mist trickling bioreactor for Centaurium erythraea Rafn growth of shoots and production of secoiridoids.

Shoots of Centaurium erythraea Rafn were cultivated in 5 l mist trickling bioreactor for 21 and 28 days increasing their dry weight from 0.54 g to 13.7 g and 18.3 g, respectively. About 6880 shoots from 223 initial shoot-tips in 21-day bioreactor producing cycle were produced. The shoots could be successfully rooted and transferred to soil. Secoiridoid accumulation (expressed as a sum of gentiopicroside, sweroside and swertiamarin) in shoots after 21 days of culture reached about 303 mg l-1.

Sterols and triterpenes in cell culture of Hyssopus officinalis L.

Cell suspension cultures from hypocotyl-derived callus of Hyssopus officinalis were found to produce two sterols i. e. beta-sitosterol (1) and stigmasterol (2), as well as several known pentacyclic triterpenes with an oleanene and ursene skeleton. The triterpenes were identified as oleanolic acid (3), ursolic acid (4), 2alpha,3beta-dihydroxyolean-12-en-28-oic acid (5), 2alpha,3beta-dihydroxyurs-12-en-28-oic acid (6), 2alpha,3beta,24-trihydroxyolean-12-en-28-oic acid (7), and 2alpha,3beta,24-trihydroxyurs-12-en-28-oic acid (8). Compounds 5-8 were isolated as their acetates (6, 8) or bromolactone acetates (5, 7).