The Impact of Nigella sativa Essential Oil on T Cells in Women with Hashimoto's Thyroiditis.

BACKGROUND: Hashimoto's thyroiditis (HT) is an autoimmune disease mediated by T cells. It is characterized by the presence of thyroid autoantibodies in the serum, such as anti-thyroid peroxidase antibodies (TPO-Ab) and anti-thyroglobulin antibodies (TG-Ab). The essential oil extracted from Nigella sativa seeds is rich in bioactive substances, such as thymoquinone and cymene. METHODS: Therefore, we examined the effect of essential oil from Nigella sativa (NSEO) on T cells from HT patients, especially their proliferation capacity, ability to produce cytokines, and susceptibility to apoptosis. RESULTS: The lowest ethanol (EtOH) dilution (1:10) of NSEO significantly inhibited the proliferation of CD4+ and CD8+ T cells from HT patients and healthy women by affecting the percentage of dividing cells and the number of cell divisions. In addition, 1:10 and 1:50 NSEO dilutions induced cell death. Different dilutions of NSEO also reduced the concentration of IL-17A and IL-10. In healthy women, the level of IL-4 and IL-2 significantly increased in the presence of 1:10 and 1:50 NSEO dilutions. NSEO did not influence the concentration of IL-6 and IFN-γ. CONCLUSIONS: Our study demonstrates that NSEO has a strong immunomodulatory effect on the lymphocytes of HT patients.

Evaluation of the yield, chemical composition and biological properties of essential oil from bioreactor-grown cultures of Salvia apiana microshoots.

Microshoot cultures of the North American endemic Salvia apiana were established for the first time and evaluated for essential oil production. Stationary cultures, grown on Schenk-Hildebrandt (SH) medium, supplemented with 0.22 mg/L thidiazuron (TDZ), 2.0 mg/L 6-benzylaminopurine and 3.0% (w/v) sucrose, accumulated 1.27% (v/m dry weight) essential oil, consisting mostly of 1,8-cineole, ß-pinene, α-pinene, ß-myrcene and camphor. The microshoots were adapted to agitated culture, showing biomass yields up to ca. 19 g/L. Scale-up studies demonstrated that S. spiana microshoots grow well in temporary immersion systems (TIS). In the RITA bioreactor, up to 19.27 g/L dry biomass was obtained, containing 1.1% oil with up to ca. 42% cineole content. The other systems employed, i.e. Plantform (TIS) and a custom made spray bioreactor (SGB), yielded ca. 18 and 19 g/L dry weight, respectively. The essential oil content of Plantform and SGB-grown microshoots was comparable to RITA bioreactor, however, the content of cineole was substantially higher (ca. 55%). Oil samples isolated from in vitro material proved to be active in acetylcholinesterase (up to 60.0% inhibition recorded for Plantform-grown microshoots), as well as hyaluronidase and tyrosinase-inhibitory assays (up to 45.8 and 64.5% inhibition observed in the case of the SGB culture).

The influence of Nigella sativa essential oil on proliferation, activation, and apoptosis of human T lymphocytes in vitro.

In previous work, we tested the immunomodulatory effect of Nigella sativa (NS) fatty oil. Our results demonstrated that unrefined, obtained by cold pressing black cumin seed oil inhibited lymphocytes' proliferation and induced their apoptosis in a dose-dependent manner. In this study, we examined the immunomodulatory properties of essential oil (EO) obtained from the NS seeds by hydrodistillation and its two main constituents: thymoquinone (TQ) and p-cymene. We analyzed the proliferation, activation phenotype, and apoptosis rates of human T lymphocytes stimulated with an immobilized monoclonal anti-CD3 antibody in the presence of serial ethanol dilutions of tested oil or serial distilled water dilutions of tested compounds with flow cytometry. Our results showed that NSEO significantly inhibited the proliferation of CD4+ and CD8+ T lymphocytes, induced cell death in a dose-dependent manner, and reduced the expression of CD28 and CD25 antigens essential for lymphocyte activation. TQ inhibited the proliferation of T lymphocytes and induced cell death, particularly in high concentrations. Meanwhile, p-cymene did not influence lymphocyte proliferation. However, its high concentration induced cell necrosis. These results show that the essential oil from Nigella sativa has powerful immunomodulatory properties, which, at least partially, are related to the TQ component.

Iris pseudacorus as an easily accessible source of antibacterial and cytotoxic compounds.

Iris pseudacorus is one of the most widespread iris species and possesses complex secondary metabolites. Our study showed that its rhizomes are abundant with phenolic compounds of which 80 % belong to the tannin group. Methanolic extracts from garden cultured iris rhizomes possessed antibacterial activity against human Gram positive Staphylococcus aureus and Enterococcus faecalis and Gram negative Pseudomonas aeruginosa and Klebsiella pneumoniae pathogens including clinical isolates resistant to commercially available antibiotics. Moreover the extract from rhizome, in concentration 3.125 mg dry weight/mL, containing gallocatechin (1), effectively combats S. aureus biofilm. The same rhizome extract acts against human cancer cell lines, especially against estrogen positive MCF-7 breast cancer cell line (IC50 = 11.75 µg/mL). In vitro culture of excised, anatomical roots of I. pseudacorus excreted three antistaphylococcal compounds into the plant medium, detected by using TLC-overlayer bioautography. By the use of HPLC-DAD-ESIMS system 2 active compounds were identified as 5,7,4'-trihydroxy-6,3'-dimethoxy-isoflavone (7) and unknown dimethoxy-dihydroxy-isoflavone (9). I. pseudacorus as a non-edible plant might be considered to be new, easy accessible, non-wood source of biologically active polyphenolics.

Production of Verbascoside, Isoverbascoside and Phenolic Acids in Callus, Suspension, and Bioreactor Cultures of Verbena officinalis and Biological Properties of Biomass Extracts.

Callus, suspension and bioreactor cultures of Verbena officinalis were established, and optimized for biomass growth and production of phenylpropanoid glycosides, phenolic acids, flavonoids and iridoids. All types of cultures were maintained on/in the Murashige and Skoog (MS) media with 1 mg/L BAP and 1 mg/L NAA. The inoculum sizes were optimized in callus and suspension cultures. Moreover, the growth of the culture in two different types of bioreactors-a balloon bioreactor (BB) and a stirred-tank bioreactor (STB) was tested. In methanolic extracts from biomass of all types of in vitro cultures the presence of the same metabolites-verbascoside, isoverbascoside, and six phenolic acids: protocatechuic, chlorogenic, vanillic, caffeic, ferulic and rosmarinic acids was confirmed and quantified by the HPLC-DAD method. In the extracts from lyophilized culture media, no metabolites were found. The main metabolites in biomass extracts were verbascoside and isoverbascoside. Their maximum amounts in g/100 g DW (dry weight) in the tested types of cultures were as follow: 7.25 and 0.61 (callus), 7.06 and 0.48 (suspension), 7.69 and 0.31 (BB), 9.18 and 0.34 (STB). The amounts of phenolic acids were many times lower, max. total content reached of 26.90, 50.72, 19.88, and 36.78 mg/100 g DW, respectively. The highest content of verbascoside and also a high content of isoverbascoside obtained in STB (stirred-tank bioreactor) were 5.3 and 7.8 times higher than in extracts from overground parts of the parent plant. In the extracts from parent plant two iridoids-verbenalin and hastatoside, were also abundant. All investigated biomass extracts and the extracts from parent plant showed the antiproliferative, antioxidant and antibacterial activities. The strongest activities were documented for the cultures maintained in STB. We propose extracts from in vitro cultured biomass of vervain, especially from STB, as a rich source of bioactive metabolites with antiproliferative, antioxidant and antibacterial properties.

Naturally occurring xanthone and benzophenone derivatives exert significant anti-proliferative and proapoptotic effects in vitro on synovial fibroblasts and macrophages from rheumatoid arthritis patients.

There is a need for novel, safer and cheaper drugs for the therapy of rheumatoid arthritis (RA), better targeted against the cellular processes involved in the disease pathogenesis. Using advanced analysis of microscopic images and flow cytometry, we demonstrate that naturally occurring xanthone and benzophenone derivatives exert strong, dose- and O2 concentration-dependent anti-proliferative and pro-apoptotic effects on RA patients' fibroblast-like synoviocytes (FLS) and macrophages. Suspensions containing fibroblasts, macrophages and other infiltrating cells were obtained from inflamed synovial tissue collected from female RA patients. Cells were grown in the presence of xanthone (mangiferin, isomangiferin, neomangiferin, norathyriol) or benzophenone (iriflophenone 3-C-glucoside, maclurin) derivatives for 48h or 7days, at 5% or 21% O2. Proportions of macrophages, FLS and infiltrating T cells undergoing apoptosis (annexin- or annexin and 7-AAD-positive) were determined by flow cytometry. The extent of late apoptosis (DNA degradation) was assessed by fluorescent microscopy and image analysis in cultures where DNA was stained with Hoechst 33342. Majority of tested compounds exert anti-proliferative and pro-apoptotic, O2-dependent effects on T cells, FLS and macrophages. The results indicate that xanthone- and benzophenone-rich plant products provide a basis for the development of dietary strategy for rheumatoid arthritis management.

Schisandra lignans production regulated by different bioreactor type.

Schisandra chinensis (Chinese magnolia vine) is a rich source of therapeutically relevant dibenzocyclooctadiene lignans with anticancer, immunostimulant and hepatoprotective activities. In this work, shoot cultures of S. chinensis were grown in different types of bioreactors with the aim to select a system suitable for the large scale in vitro production of schisandra lignans. The cultures were maintained in Murashige-Skoog (MS) medium supplemented with 3mg/l 6-benzylaminopurine (BA) and 1mg/l 1-naphthaleneacetic acid (NAA). Five bioreactors differing with respect to cultivation mode were tested: two liquid-phase systems (baloon-type bioreactor and bubble-column bioreactor with biomass immobilization), the gas-phase spray bioreactor and two commercially available temporary immersion systems: RITA® and Plantform. The experiments were run for 30 and 60 days in batch mode. The harvested shoots were evaluated for growth and lignan content determined by LC-DAD and LC-DAD-ESI-MS. Of the tested bioreactors, temporary immersion systems provided the best results with respect to biomass production and lignan accumulation: RITA® bioreactor yielded 17.86g/l (dry weight) during 60 day growth period whereas shoots grown for 30 days in Plantform bioreactor contained the highest amount of lignans (546.98mg/100g dry weight), with schisandrin, deoxyschisandrin and gomisin A as the major constituents (118.59, 77.66 and 67.86mg/100g dry weight, respectively).

Isolation of xanthone and benzophenone derivatives from Cyclopia genistoides (L.) Vent. (honeybush) and their pro-apoptotic activity on synoviocytes from patients with rheumatoid arthritis.

A fast and efficient method for the isolation of the C-glucosidated xanthones mangiferin and isomangiferin from the South-African plant Cyclopia genistoides was developed for the first time. The procedure involved extraction, liquid-liquid partitioning with ethyl acetate and subsequent precipitation of mangiferin and isomangiferin from methanol and acetonitrile-water fractions, respectively. Additionally, two benzophenone derivatives: 3-C-ß-glucosides of maclurin and iriflophenone, were isolated from C. genistoides extracts using semi-preparative HPLC. Apart from the above, the isolation procedure also yielded hesperidin and small amounts of luteolin. The structures of the compounds were determined by 1D and 2D NMR experiments and/or LC-DAD-ESI-MS. The selected Cyclopia constituents were screened for pro-apoptotic activity on TNF-α-stimulated synovial cells isolated from rheumatoid arthritis patients. The strongest effect, measured as percent of apoptotic cells, was recorded for isomangiferin (75%), followed by iriflophenone 3-C-ß-glucoside (71%), hesperidin (67%) and mangiferin (65%). The results are encouraging for further studies on the use of the above compounds in the treatment of rheumatoid arthritis.

Aronia plants: a review of traditional use, biological activities, and perspectives for modern medicine.

The Aronia genus (Rosaceae family, Maloideae subfamily) includes two species of native North American shrubs: Aronia melanocarpa (Michx.) Ell. (black chokeberry) and Aronia arbutifolia (L.) Pers. (red chokeberry). The fruits of A. melanocarpa have been traditionally used by Potawatomi Native Americans to cure colds. In the first half of the 20(th) century, cultivars of black chokeberry were introduced to the Soviet Union and other European countries, providing fruits used by food industry. At present, it is used mainly for juice, jam, and wine production, as well as an ornamental plant. Among other substances, the berries of A. melanocarpa contain anthocyanins and procyanidins, possessing strong antioxidative potential. Numerous health-promoting activities-namely, antioxidative, antimutagenic, anticancer, cardioprotective, hepatoprotective, gastroprotective, antidiabetic, anti-inflammatory, antibacterial, antiviral, radioprotective, and immunomodulatory-have been demonstrated for black chokeberry extracts by both in vitro and in in vivo studies. The presented review summarizes the information concerning botany, cultivation, chemical composition, and pharmacological activities of Aronia plants.

In vitro cultures of Cyclopia plants (honeybush) as a source of bioactive xanthones and flavanones.

In vitro shoot and callus cultures of the endemic South-African shrubs: Cyclopia intermedia E. Mey., Cyclopia subternata Vogel, and Cyclopia genistoides (L.) Vent. (Fabaceae) were established and examined for the presence of polyphenolic compounds. The xanthones mangiferin and isomangiferin, as well as the flavanones hesperidin and eriocitrin were identified by LC-ESI-MS and LC-DAD, and analyzed quantitatively by HPLC. The respective intact plants were analyzed for comparison. From all in vitro cultures, the highest levels of mangiferin (1.55%) and isomangiferin (0.56%) were recorded in C. subternata microshoots, compared to 1.31% and 0.49% found in the intact plant. Callus cultures of all species synthesized only trace amounts of mangiferin and isomangiferin. Hesperidin and eriocitrin contents were significanly lower in all in vitro cultures, in comparison to the respective intact plants. Among the obtained in vitro biomasses, the highest hesperidin content was recorded in C. intermedia (0.9%) and C. subternata (0.87%) microshoots, whereas C. subternata callus was characterized by the best growth parameters and highest hesperidin content (0.69%) from all examined Cyclopia calli.