Isolation of caffeic acid from Perilla frutescens and its role in enhancing γ-glutamylcysteine synthetase activity and glutathione level

Perilla frutescens is an annual herbaceous plant native to Asia, where its leaves are used in Asian gourmet food. Our previous study showed that the inhibition of γ-glutamylcysteine synthetase (γ-GCS) activity was remarkably recovered by pretreatment with perilla leaf extract (PLE). The objective was to fractionise PLE, and to identify the active component that is responsible for the enhancement of γ-GCS activity and glutathione (GSH) concentration. Among the five fractions from PLE, PLE-III of the ethyl acetate fraction showed the highest γ-GCS activity in a HepG2 cell experiment, and was further chromatographed. The purified compound, which enhanced γ-GCS activity, was finally identified as caffeic acid. We first report the enhancement of γ-GCS activity and GSH level in HepG2 cells by caffeic acid obtained from PLE. Our results suggest that caffeic acid may be a key factor in the chemopreventive potential of perilla leaf components by increasing de novo synthesis of GSH.     

Protective effects of four Iranian medicinal plants against free radical-mediated protein oxidation

The role of oxidative protein damages in the pathophysiology of human diseases is currently a topic of considerable interest as oxidised proteins has been implicated in a wide spectrum of clinical disorders. In this study, the antioxidant activities of four Iranian medicinal plants, namely Teucrium polium, Cyperus rotundus, Anethum graveolens and Nasturtium officinale against metal–catalysed protein oxidation were evaluated by pro-oxidant model (Fe²⁺/ascorbate) in rat liver homogenates. The addition of Fe²⁺/ascorbate to the liver homogenate significantly increased the extent of protein oxidation, such as protein carbonyl (PCO) formation and loss of protein-bound sulphydryl (P-SH) groups. Furthermore, the rates of reactive oxygen species (ROS) formation and lipid peroxidation (LPO) were also increased. The plant extracts showed inhibitory effects against PCO formation, P-SH oxidation, ROS formation and LPO to varying degrees. Based on this study, the order of antioxidant activity against protein oxidation was found to be: T. polium > C. rotundus > A. graveolens > N. officinale. The protective effects of each plant extract could be due to its polyphenolic content. In that respect, the T. polium extract with highest polyphenolic content has more antioxidant activity against protein oxidation.     

Phycobiliproteins or C-phycocyanin of Arthrospira (Spirulina) maxima protect against HgCl2-caused oxidative stress and renal damage

Our objective was to determine if the phycobiliproteins of Arthrospira (Spirulina) maxima protect renal cells against mercury-caused oxidative stress and cellular damage in the kidney. We used 40 male mice that were assigned into eight groups: (1) a control group that received 100 mM phosphate buffer (PB) ig and 0.9% saline ip, (2) PB + HgCl₂ (5 mg/kg ip), (3) PB plus phycobiliproteins (100 mg/kg ig), (4) PB plus C-phycocyanin (100 mg/kg ig), and four groups receiving HgCl₂ + phycobiliproteins or C-phycocyanin (50, and 100 mg/kg ig). The left kidneys were used to determine lipid peroxidation, quantification of reactive oxygen species, and reduced glutathione and oxidised content. The right kidneys were processed for histology. The HgCl₂ caused oxidative stress and cellular damage. All doses of phycobiliproteins or C-phycocyanin prevented enhancement of oxidative markers and they protected against HgCl₂-caused cellular damage.     

Induction of apoptosis in U937 human leukaemia cells by the hexane fraction of an extract of immature Citrus grandis Osbeck fruits

The antiproliferative effect of an immature Citrus grandis Osbeck fruit extract was investigated using U937 human leukaemia cells. Maximum cytotoxicity was observed using the hexane fraction (HF) of the extract. Cell death was dose-dependent (IC₅₀ = ca. 60 μg/ml) and was characterised by chromatin condensation, apoptotic body formation, and DNA fragmentation. The induction of apoptosis was confirmed by caspase-3 activity assays and by immunoblotting using antibodies against Bcl-2, Bax, poly(ADP-ribose) polymerase (PARP), caspase-9, and caspase-3. The molecular mechanism underlying HF-induced apoptosis in U937 cells may involve a mitochondria-mediated signalling pathway, as demonstrated by an increase in the Bax/Bcl-2 expression ratio. Analyses of the HF by gas chromatography (GC) and GC-mass spectrometry (MS) tentatively identified 19 compounds, including γ-sitosterol (17.5%), 7-methoxy-8-(2-oxo-3-methylbutyl) coumarin (6.8%), stigmasterol (3.8%), and campesterol (3.4%). Together, our results provide the first evidence that the HF of an immature C. grandis Osbeck fruit extract induces apoptosis in U937 cells.     

Antioxidant and protective properties of six Tanacetum species against hydrogen peroxide-induced oxidative stress in K562 cell line: A comparative study

The antioxidant activities of ethanolic extract of six species of Tanacetum (T. budjnurdense, T. hololeucum, T. chiliophyllum, T. sonboli, T. tabrisianum, T. kotschyi) from Iran were examined by employing various established in vitro assay systems. The results showed that all Tanacetum extracts displayed antioxidant activities, with IC₅₀ values ranging from 59.55 to 157.24 μg/ml, using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The extract of T. hololeucum, with an IC₅₀ value of 59.55 μg/ml was nearly two or three times more active than the other used Tanacetum extracts. The amounts of total phenolics and total flavonoids were also determined by spectrophotometer. A similar order of the amounts of phenolics and flavonoids in all plant extracts were found. The results showed that the extent of antioxidant activities is in accordance with the amounts of phenolics and flavonoids existing in all plant extracts. Regarding high performance liquid chromatography (HPLC) data, caffeic acid, ferulic acid, luteolin, apigenin and rutin were detected as major phenolic compounds in all the species investigated. Pretreatment of K562 cells with various concentrations of Tanacetum extracts (10–100 μg/ml) for 16 h prevent cell damage and enhanced activity of antioxidant enzymes induced by a treatment with H₂O₂. Moreover, lower level of glutathione caused by hydrogen peroxide in K562 cells were partly recovered by a pretreatment of cells with various Tanacetum species extract. These results may show the significant protection effect of Tanacetum sp. against oxidation of the cells.     

Major flavonoids with antioxidant activity from Teucrium polium L.

Teucrium polium L. (Lamiaceae) aerial parts are used widely in the daily diet and for medicinal purposes. This plant is used also as a spice and refreshing beverage. Phytochemical and bioactivity studies of this plant have been carried out. Aerial parts of the plant were extracted with petroleum ether, chloroform, methanol and water successively. Fractionation of the methanol extract yielded four major flavonoids. The crude extracts and isolated compounds were screened for their antioxidant and free radical scavenging activities using DPPH radical-scavenging, beta-carotene/linoleic acid and ammonium thiocyanate methods. Methanol extract, rutin and apigenin were found to be the most active fractions as radical-scavengers with IC₅₀ values of 20.1 ± 1.7, 23.7 ± 1.9 and 30.3 ± 2.1 μg/ml, respectively. The samples with the highest inhibition of oxidation of beta-carotene and lipid peroxidation in ammonium thiocyanate methods were also found to be methanol extract, rutin and apigenin. Methoxylated flavonoids exhibited a lesser antioxidant activity.     

Phytochemical composition,anti-inflammatory and antitumour activities of four Teucrium essential oils from Greece

The essential oils of four Teucrium species were studied and 150 components, in all, were identified. All oils were rich in sesquiterpenes (50.1–55.8%). Spathulenol and δ-cadinene were the main compounds of Teucrium brevifolium oil; caryophyllene and 4-vinyl guaiacol predominated in Teucrium flavum. Carvacrol and caryophyllene oxide predominated in Teucrium montbretii ssp. heliotropiifolium, while carvacrol and caryophyllene were the most abundant components in Teucrium polium ssp. capitatum. The oil which most effectively inhibited LPS-induced NO production in macrophage cell line RAW 264.7 was that from T. brevifolium (IC₅₀ = 7.1 μg/ml), followed by T. montbretii ssp. heliotropiifolium and T. polium ssp. capitatum (IC₅₀ = 16.5 and 29.4 μg/ml, respectively). The in vitro cytotoxic assay on three human cancer cell lines showed that the most antiproliferative oils were those from T. polium ssp. capitatum and T. montbretii ssp. heliotropiifolium on CACO-2 cell lines (IC₅₀ = 52.7 and 92.2 μg/ml, respectively). The T. brevifolium oil showed a selective cytotoxicity on COR-L23 while significant activity was exerted by T. polium oil on C32.     

Dioscin: A synergistic tyrosinase inhibitor from the roots of Smilax china

In our study, we investigated the inhibition of mushroom tyrosinase in Smilax china. A methanol (MeOH) extract of S. china was partitioned into hexane, ethyl acetate (EtOAc) and water. Of the three fractions, EtOAc extract showed the strongest inhibition of tyrosinase activity with l-tyrosine or l-DOPA as a substrate. Two compounds were isolated from a final active fraction by activity-guided column chromatography. These compounds were identified as dioscin and oxyresveratrol by comparing their mass, ¹H- and ¹³C-NMR spectral data with those reported in the literature. Dioscin showed little inhibition activity of tyrosinase, whereas oxyresveratrol, a known tyrosinase inhibitor, showed a strong tyrosinase inhibitory activity. We discovered that a mixture of oxyresveratrol and dioscin (IC₅₀ = 5.1 and 5.7 μg/ml) highly increased the inhibition of tyrosinase activity with l-tyrosine or l-DOPA as the substrate as compared to either oxyresveratrol (IC₅₀ = 7.8 and 10.9 μg/ml) or dioscin (IC₅₀ > 100 and 100 μg/ml) alone.     

Antifungal activity of the honey flavonoid extract against Candida albicans

Candida albicans is the most prevalent dimorphic pathogen in humans. We investigated the potential protective effect of honey flavonoid extract (HFE) on Candida dimorphism induced by RPMI 1640 medium. Yeast to hyphal transition was dose dependently prevented when Candida cultures were treated with HFE for 6 h (germ-tube formation) and 18 h (hypha elongation). Since during hyphal growth remarkable amounts of reactive oxygen species (ROS) are generated, we investigated whether HFE affects the generation of ROS, the glutathione level and the activity of glutathione-dependent enzymes. Treatment of Candida cells with 48 μg/ml (MIC₅₀) of HFE for 6 or 18 h inhibited the dimorphic conversion by supporting of the intracellular glutathione level. HFE exerts a dual protective effect inhibiting both ROS generation during germ-tube formation and γ-glutamyltranspeptidase activity, responsible for GSH degradation, during hypha elongation. These results show that HFE confers a significant protection against yeast to hyphal transition of C. albicans.     

Inhibition of cell growth by Stellera chamaejasme extract is associated with induction of autophagy and differentiation in chronic leukemia K562 cells

Stellera chamaejasme has been used as a therapeutic plant for treatment of various inflammatory diseases and solid tumors. Our study was particularly interested in the differentiation inducing activity of the aerial parts of S.chamaejasme using human chronic leukemia cell line K562. The ethanol extract has been shown to be highly cytotoxic against K562 cells at > 0.008% (v/v) doses. Cell cycle arrest at G₀/G₁ phase, not detectable DNA fragmentation as well as positive staining with acridine orange and expression of beclin-1 protein in the treated cells led as to detect autophagic cell death during the plant extract treatment of K562 cells. Moreover, differentiation marker CD11b was also expressed in the treated cells.     

In vitro activity of the essential oils of Origanum vulgare, Satureja montana and their main constituents in peroxynitrite-induced oxidative processes

The essential oil obtained from aerial parts of Satureja montana L. and Origanum vulgare L. (Labiatae) along with four of its main components, p-cymene, carvacrol, thymol and γ-terpinene were tested in models of in vitro peroxynitrite-induced formation of both 3-nitrotyrosine and malondialdehyde, two biomarkers of the oxidative stress of recognised pathological and toxicological significance. The essential oils showed a significant activity, thus decreasing 3-nitrotyrosine formation (IC₅₀ values of 43.9 μg/ml for S. montana and 19.2 μg/ml for O. vulgare), and also inhibited the peroxynitrite induced malondialdehyde formation (IC₅₀ values of 27.2 μg/ml and 17.0 μg/ml respectively). Thymol and carvacrol inhibited 3-nitrotyrosine formation (IC₅₀ values of 81.3 μM and 106.3 μM; ascorbic acid IC₅₀ = 400 μM) and reduced malondialdehyde formation (IC₅₀ values of 43.9 μM and 70.1 μM respectively; trolox IC₅₀ = 240 μM). On the contrary, p-cymene and γ-terpinene were completely inactive in both assays under the concentration of 300 μg/ml. These results support, in particular for origanum, the nutraceutical value of these spices and the potential of thymol and carvacrol in preventing the formation of toxic products by the action of reactive nitrogen species.     

Radical scavenging capacities and inhibition of human prostate (LNCaP) cell proliferation by Fortunella margarita

Lyophilised nagami kumquat (Fortunella margarita) powder was extracted with five different solvents. Dried extracts of EtOAc and MeOH-water (4:1, v/v) had the highest and lowest total phenolics, respectively, by Folin-Ciocalteu method. LC-MS analysis confirmed the presence of different levels of rutin, narirutin, poncirin, apigenin 8-C-rutinoside and 3′,5′,di-C-β-glucopyranosyl phloretin in all the extracts except n-hexane. EtOAc and MeOH extracts exhibited the highest and the lowest 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, respectively. The order of antioxidant capacity was found to be MeOH-water (4:1, v/v) > EtOAc > MeOH > n-hexane > acetone by phosphomolybdenum complex and oxygen radical absorbance capacity (ORAC) values. Among five extracts, n-hexane extract exhibited the highest inhibition of human prostate cancer (LNCaP) cells (86.4%) after 96 h at 100 μg/mL, followed by EtOAc (82.8%), MeOH (76.7%) and MeOH-water (4:1, v/v) (68.2%). Fragmentation of DNA suggests the ability of extracts to induce apoptosis in LNCaP cells. The cleavage of caspase-3 was the highest in n-hexane and EtOAc extracts, whereas the ratio of Bax/Bcl2 was the highest in MeOH and MeOH-water (4:1, v/v) extracts. The results of the present study were also supported by fluorescent images of LNCaP cells treated with kumquat extracts. The maximum cell proliferation inhibition activity of n-hexane extract may be due to the presence of one or cumulative effect of β-carotene, β-cubebene and hexadecanoic acid. Remaining four extracts exhibited differential antioxidant activity and cytotoxicity which may be due to the presence of various levels of rutin, narirutin, poncirin, apigenin-8-C-rutinoside and 3′,4′-di-C-β-glucopyranosyl phloretin in each extract.     

Estimation of neuroprotective effects of Laurocerasus officinalis Roem. (cherry laurel) by in vitro methods

Dichloromethane, ethyl acetate, acetone, methanol, and water extracts prepared from the fruits and leaves of Laurocerasus officinalis Roem. (LO) (Rosaceae) were screened for their cholinesterase inhibitory activity against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), the key enzymes in pathogenesis of Alzheimer's disease (AD), using ELISA microplate reader at 50, 100, and 200 ??g mL⁻¹. As AD is associated with oxidative stress, the antioxidant activity of the extracts was also tested by radical-forming methods against 2,2-diphenyl-1-picrylhydrazyl (DPPH), N,N-dimethyl-p-phenylendiamine (DMPD), and superoxide radicals as well as iron-related methods; iron-chelating capacity and ferric-reducing antioxidant power (FRAP) assays. Total phenol and flavonoid quantification was achieved using Folin-Ciocalteau and AlCl₃ reagents, respectively. The highest AChE (44.01 ± 1.75%) and BChE (19.91 ± 0.37%) inhibition was caused by the LO-leaf-methanol extract 200 ??g mL⁻¹, while it showed the best radical-scavenging activity against DPPH at 2000 ??g mL⁻¹. Only, the dichloromethane and water extracts of the fruits and the leaf water extract had an iron-chelating capacity, while the leaf methanol extract displayed the highest FRAP. The leaf methanol extract (113.45 ± 0.71 mg g⁻¹ extract) was found to be the richest in total phenols, while the leaf acetone extract (139.90 ± 4.64 mg g⁻¹ extract) had the most abundant amount of total flavonoids.     

Beneficial effect of Ficus religiosa Linn. on high-fat-diet-induced hypercholesterolemia in rats

Ficus religiosa Linn. (Moraceae) was evaluated as a hypolipidaemic and antioxidant agent. Ethanol and hexane leaves extracts were examined for inhibition of β-hydroxy-β-methylglutaryl coenzyme A reductase and DPPH free radical elevation. The ethanol extract was evaluated for its effect against hypercholesterolaemia in rats, compared with the standard hypolipidaemic lipanthyl. Cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), phospho and total lipids were estimated. Malondialdehyde (MDA), glutathione (GSH) and superoxide dismutase (SOD) were measured. Administration of rats with 30 mg cholesterol five times per week for nine consecutive weeks caused a significant increase in lipid profile, MDA and SOD, while HDL-C and GSH showed significant decreases. Treatment with plant extract (500 mg/kg body weight) or lipanthyl drug (50 mg finofibrate/kg body weight) for the same duration recorded improvement in lipid profile, antioxidant levels, liver function enzymes and hepatic architecture. In conclusion, ethanol extract of F. religiosa exerted hypolipidaemic and antioxidant effects.     

Isolation and synergism of in vitro anti-inflammatory and quinone reductase (QR) inducing agents from the fruits of Morinda citrifolia (noni)

The tropical fruits and fruit products of Morinda citrifolia, commonly known as noni, are consumed as a food or dietary supplement with purported health benefits. The objective of this study was to investigate the potential anti-inflammatory and cancer preventive effects of noni fruit puree extracts. Bioassay-guided fractionation of an ethyl acetate (EtOAc) extract of noni, comprising ~ 2% noni puree solids, led to the isolation of scopoletin (1), rutin (2), and quercetin (3). Quantitative HPLC analysis of the EtOAc extract revealed levels (dry weight basis) of scopoletin at 0.62 ??mol/g, quercetin at 0.26 ??mol/g and rutin at 0.045 ??mol/g. Scopoletin and quercetin inhibited the production of nitric oxide (NO) in a concentration-dependent manner in lipopolysaccharide (LPS)-induced RAW 264.7 macrophage cells and exhibited quinone reductase (QR) induction in cultured Hepa 1c1c7 cells. Increases in QR activity in induced cells were associated with increases in QR protein as confirmed by Western blots. Combinations of scopoletin and quercetin at a low (< 10 ??M) concentration resulted in synergistic suppression in nitric oxide (NO) production and down-regulated inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 expressions in LPS-induced RAW 264.7 macrophage cells. These results suggest that the combinations of noni compounds with different groups of chemical structures might be useful to efficiently suppress inflammatory and carcinogenic processes related to iNOS and COX-2 gene overexpression. These findings may provide some basis for the purported in vitro anti-inflammatory and anti-cancer effects of noni fruits as functional foods and dietary supplements.     

Antioxidative and anti-inflammatory properties of Citrus sulcata extracts

Citrus sulcata was subjected to ultrasound, high-pressure, and Soxhlet extractions. The antioxidant and anti-inflammatory activities of the extracts were evaluated qualitatively and quantitatively. The antioxidant content of the peel extract was twice that of the fruit extract. The quantitative analysis showed that the narirutin and hesperidin contents in the peel extracts were 8.8 and 7.5 mg/100 g, respectively. These extracts had a total phenolic content of 112.22 ± 2.89 gallic acid equivalent (GAE) mg/100 g, a total flavonoid content of 54.09 ± 1.01 rutin equivalent (RE) mg/100 g, DPPH radical scavenging activity of 46%, and antioxidant activity of 213.25 ± 2.82 μM of Trolox equivalents (TEAC). C. sulcata extracts could prevent hydrogen peroxide (H₂O₂)-induced oxidative stress, reduce expression of the inflammatory markers nuclear Factor kappa B (NFκB) and phosphorylated IκBα (p-IκBα) in A549 human lung carcinoma cells, and inhibit Lipopolysaccharide (LPS)-induced THP-1 monocyte differentiation to an extent of 85%.