CYP3A4 and CYP2D6 inhibitory activities of Indonesian medicinal plants

Thirty samples of Indonesian medicinal plants were analyzed for their capacity to inhibit in vitro metabolism by human cytochrome P450 3A4 (CYP3A4) and CYP2D6 with a radiometric assay. The MeOH-soluble fractions of 25 samples, prepared from water extracts, demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, and 21 samples on the metabolism mediated by CYP2D6. Among the MeOH-soluble fractions, Piper nigrum leaf showed the highest inhibitory activity against CYP3A4 (91.7%), and Punica granatum against CYP2D6 (98.1%). The water extracts of which MeOH-soluble fraction showed inhibitory activity more than 70% were fractionated with EtOAc. From the EtOAc-soluble fractions, Curcuma heyneana (67.0%), Pi. cubeba (75.0%), Pi. nigrum fruit (84.0%), Pi. nigrum leaf (85.8%), and Zingiber aromaticum (75.3%) demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, but only Pi. nigrum fruit (72.8%) and Pi. nigrum leaf (69.1%) showed strong inhibitory activity against CYP2D6. For samples that showed more than 70% inhibition, their IC(50) values were determined. The most potent inhibitory activity against CYP3A4 (IC(50) value of 25 microg/ml) was found for the extract of Pi. nigrum leaf, while that of Catharanthus roseus showed the most potent inhibitory effect against CYP2D6 (IC(50) value of 11 microg/ml). These results should indicate once more the possibility of potential medicinal plant-drug interactions.     

Evaluation of phytochemical and antimicrobial properties of leaf extract of Tapinanthus sessilifolius (P. Beauv) van Tiegh

Leaf extracts of T. sessilifolius growing on five different host plants (Psidium guajava, Citrus lemon, Vernonia amygdalina, Persea americana and Jatropa curcas) were evaluated for antimicrobial activity of the plant. Powdered leaves of T. sessilifolius collected from each host plant was divided into two portions. One portion was used for aqueous infusion and the other portion was successively extracted with hexane, ethylacetate and methanol. Infusion of aqueous extract of powdered leaves did not show antimicrobial effect even at the concentration of 1000 and 2000 microg/ml on test microorganisms (Staph. aureus, E. coli, Bacillus subtilis, Pseudomonas aeruginosa and Candida albicans). However in broth culture, methanolic and hexane extract had MIC range of 62.5-500 microg/ml and ethylacetate extract had 250-500 microg/ml. Phytochemical screening of leaf samples of T. sessilifolius collected from different host plants showed positive test for hydrolysable tannins, saponins, flavonoids, terpenes, cardiac glycoside, reducing sugars and proteins. LD50 concentration was found to be > 1.500 mg/kg for samples from P. guajava; 489.89 mg/kg for J. curcas and C. lemon; and 692 mg/kg for V. amydalina in mice.     

The in vitro biological activity of Lepidium meyenii extracts

The biological activity of methanolic and aqueous extracts from dehydrated hypocotyls of Lepidium meyenii (Brassicaceae, vernacular name “maca”), was studied on rat hepatocytes and human breast cancer MCF-7 cells. The extracts did not exhibit cytotoxicity in hepatocyte primary cultures up to 10 mg/ml as measured by the MTT viability test, and lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) leakage. Moreover, after 72 h, extracts inhibited LDH and AST leakage from the hepatocytes. When hepatocytes were intoxicated by t-butyl hydroperoxide, neither extract prevented oxidative damage. Both extracts showed weak antioxidant activity in the DPPH radical scavenging test with IC₅₀ values of 3.46 ± 0.16 and 0.71 ± 0.10 mg/ml, for aqueous and methanolic extracts, respectively. Thus, the observed effect on spontaneous enzyme leakage is probably mediated through mechanisms other than antioxidant activity. Both methanolic and aqueous extracts have shown estrogenic activity comparable with that of silymarin in MCF-7 cell line. Maca estrogenicity was exhibited in the range from 100 to 200 μg of extract per ml. The findings in the present study show that maca does not display in vitro hepatotoxicity. In contrast, a slight cytoprotective effect, probably not mediated by antioxidant capacity, was noted. Maca extracts exhibited estrogenic activity comparably to the effect of silymarin in MCF-7 cells.     

Hepatoprotective properties of Caesalpinia sappan Linn. heartwood on carbon tetrachloride induced toxicity

Aim of the study was to investigate the methanol and aqueous extracts of heartwood of C. sappan for its hepatoprotective activity against CCl4 induced toxicity in freshly isolated rat hepatocytes and animals. Freshly isolated rat hepatocytes were exposed to CCl4 (1%) along with/without various concentrations of methanolic and aqueous extract of C. sappan (1000-800 microg/ml) and the levels of selected liver enzymes were estimated. Antihepatotoxic effect of methanolic extract was observed in freshly isolated rat hepatocytes at concentrations 1000-800 microg/ml and was found to be similar to that of standard drug silymarin. Wistar strain albino rat model was used for the investigation of in vivo hepatoprotective properties of aqueous and methanolic extract of C. sappan (100 and 200 mg/kg body weight). Liver damage was induced by ip administration of CCl4 (30%) suspended in olive oil (1 ml/kg body weight). Both the tested extracts showed potent hepatoprotective activity at 200 mg/kg body weight test dose which was comparable with that of the standard silymarin used in similar test dose. The methanolic and aqueous extract was able to restore the biochemical levels to normal which were altered due to CCl4 intoxication in freshly isolated rat hepatocytes and also in animals.     

An aqueous extract of Rubus chingii fruits protects primary rat hepatocytes against tert-butyl hydroperoxide induced oxidative stress

Different in vitro free radical generating systems were used to assess the antioxidative activity of aqueous extracts of the five herbal components of Wu-zi-yan-zong-wan, a traditional Chinese medicinal formula with a long history of use for tonic effects. Fructus Rubi [Rubus chingii (Rosaceae) fruits] was found to be the most potent. It was further investigated using the primary rat hepatocyte system. tert-Butyl hydroperoxide (t-BHP) was used to induce oxidative stress. Being a short chain analog of lipid hydroperoxide, t-BHP is metabolized into free radical intermediates by the cytochrome P450 system in hepatocytes, which in turn, initiate lipid peroxidation, glutathione depletion and cell damage. Pre-treatment of hepatocytes with Fructus Rubi extract (50 microg/ml to 200 microg/ml) for 24 h significantly reversed t-BHP-induced cell viability loss, lactate dehydrogenase leakage and the associated glutathione depletion and lipid peroxidation. The amount of reactive oxygen species formed was also decreased as visualized by the fluorescence probe 2',7'-dichlorofluorescin diacetate. These results suggested that Fructus Rubi was useful in protecting against t-BHP-induced oxidative damage and may also be capable of attenuating cytotoxicity of other oxidants.     

Modulation of P450 enzymes by Cuban natural products rich in polyphenolic compounds in rat hepatocytes

This paper reports cytotoxic effects and changes in the P450 system after exposing rat hepatocytes to four polyphenol-rich products widely used in Cuban traditional medicine (Mangifera indica L. (MSBE), Thalassia testudinum (Tt), Erythroxylum minutifolium and confusum extracts). Effects of mangiferin, the main polyphenol in MSBE, were also evaluated. Cytotoxicity was assayed by the MTT test after exposure of cells to the products (50-1000 microg/mL) for 24 or 72 h. The results showed that 500 microg/mL MSBE was moderately cytotoxic after 72 h, while mangiferin was not. Marked reductions in cell viability were produced by Erythroxylum extracts at concentrations > or = 200 microg/mL, whereas only moderate effects were induced by 1000 microg/mL Tt. Seven specific P450 activities were evaluated after 48 h exposure of cells to the products. MSBE reduced phenacetin O-deethylation (POD; CYP1A2) activity in a concentration-dependent manner (IC(50)=190 microg/mL). No decreases were observed in other activities. In contrast, mangiferin produced reductions in five P450 activities: IC(50) values of 132, 194, >200, 151 and 137 microg/ml for POD (CYP1A2), midazolam 1'-hydroxylation (M1OH; CYP3A1), diclofenac 4'-hydroxylation (D4OH; CYP2C6), S-mephenytoin 4'-hydroxylation (SM4OH), and chlorzoxazone 6-hydroxyaltion (C6OH; CYP2E1), respectively. E. minutifolium, E. confusum and Tt extracts produced small reductions in SM4OH and C6OH activities, but no significant changes were noted in the other P450 activities. On the other hand, all the products increased the benzyloxyresorufin O-debenzylation (BROD; CYP2B1) activity, with MSBE, mangiferin or E. minutifolium showing the highest effects (about 2-fold over control). Our results showed in vitro effects of these natural products on P450 systems, possibly leading to potential metabolic-based interactions.     

Free-radical scavenging by Ouratea parviflora in experimentally-induced liver injuries

The antioxidant potential of crude extracts and fractions from leaves of Ouratea parviflora, a Brazilian medicinal plant used for the treatment of inflammatory diseases, was investigated in vitro through the scavenging of radicals 2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH), hydroxyl radical (HO*), superoxide anion (O2*-), and lipid peroxidation in rat liver homogenate. The crude extract (CEOP) and hydro-alcoholic fraction (OP4) showed strong inhibitory activity toward lipid peroxidation induced by tert-butyl peroxide (IC50 = 2.3 +/- 0.2 and 1.9 +/- 0.1 microg/ml, respectively). The same products exhibited a strong concentration-dependent inhibition of deoxyribose oxidation (14.9 +/- 0.2 and 0.2 +/- 0.1 microg/ml, respectively), and also showed a considerable antioxidant activity against O2*- (87.3 +/- 0.1 and 73.1 +/- 0.4 microg/ml, respectively) and DPPH radicals (55.4 +/- 0.3 and 38.3 +/- 0.4 microg/ml, respectively). The protective effects of CEOP and OP4 were also studied in mouse liver. CCl4 significantly increased (by 90%) levels of lipid hydroperoxides, carbonyl protein content (64%), DNA damage index (133%), aspartate aminotransferase (261%), alanine aminotransferase (212%), catalase activity (23%), and also caused a decrease of 60% in GSH content. The results showed that CEOP and OP4 exerted cytoprotective effects against oxidative injury caused by CCl4 in rat liver, probably related to the antioxidant activity showed by the in vitro free radical scavenging property.     

Immunomodulating properties of Argentine plants with ethnomedicinal use

Five Argentine medicinal plants selected according to folk traditional or ethnomedical use, references and primary pharmacological screening; were chosen to elucidate their immunomodulating properties. Dichloromethane, methanolic and aqueous extracts of the aerial parts of Achyrocline flaccida (A. flaccida), Eupatorium arnottianum (E. arnottianum) and Eupatorioum buniifolium (E. buniifolium), leaves of Lithraea molleoides (L. molleoides) and leaves and stems of Phyllanthus sellowianus (P. sellowianus) were analyzed to disclose their effects on murine normal and tumor cell growth as well as on complement hemolytic activity. Modulation of cell growth was evaluated by tritiated thymidine incorporation while inhibition of complement activity was measured on both classical and alternative complement pathways (CP and AP respectively). The results obtained show that most of the extracts exerted inhibitory effect on tumor as well as on mitogen activated normal spleen cell growth. On tumor cells, IC50 ranged between 1-75 microg/ml for most of the extracts with the exception of dichloromethane of L. molleoides and P. sellowianus which required concentrations higher than 100 microg/ml to produce the effect. On mitogenic activated splenocytes, IC50 ranged between < 1 to 85 microg/ml with the exception of methanolic extract of E. buniifolium or P. sellowianus which were not effective on ConA or LPS stimulated splenocytes respectively. Only E. buniifolium was active on murine normal splenocytes proliferation (IC50 0.5-1.5 microg/ml). Finally, one (7%) of 15 extracts showed inhibition of complement activity on CP and 6 extracts (40%) presented moderate activity on CP. The dichloromethane extract of E. arnottianum was the most active (IC50 5 microg/ml), although remarkable effect was also obtained with dichloromethane and methanolic extracts of P. sellowianus (IC50 11.2 and 17.3 microg/ml respectively). Besides, 2 extracts (13%), dichloromethane extract of E. arnottianum and aqueous extract of P. sellowianus, showed moderate inhibition on AP.     

Antibacterial activity of extracts and neolignans from Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck

The evaluation of the activity of the aqueous and ethyl acetate extracts of the leaves of Piper regnellii was tested against gram-positive and gram-negative bacteria. The aqueous extract displayed a weak activity against Staphylococcus aureus and Bacillus subtilis with minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of 1000 micrograms/ml. The ethyl acetate extract presented a good activity against S. aureus and B. subtilis with MIC and MBC at 15.62 micrograms/ml. In contrast to the relative low MICs for gram-positive bacteria, gram-negative bacteria were not inhibited by the extracts at concentrations < or = 1000 mg/ml. The ethyl acetate extract was fractionated on silica gel into nine fractions. The hexane and chloroform fractions were active against S. aureus (MIC at 3.9 micrograms/ml) and B. subtilis (MIC at 3.9 and 7.8 micrograms/ml, respectively). Using bioactivity-directed fractionation, the hexane fraction was rechromatographed to yield the antimicrobial compounds 1, 2, 5, and 6 identified as eupomatenoid-6, eupomatenoid-5, eupomatenoid-3, and conocarpan, respectively. The pure compounds 1 and 2 showed a good activity against S. aureus with MIC of 1.56 micrograms/ml and 3.12 micrograms/ml, respectively. Both compounds presented MIC of 3.12 micrograms/ml against B. subtilis. The pure compound 6 named as conocarpan was quite active against S. aureus and B. subtilis with MIC of 6.25 micrograms/ml. The antibacterial properties of P. regnellii justify its use in traditional medicine for the treatment of wounds, contaminated through bacteria infections.     

Quantitative determination of enhydrin in leaf rinse extracts and in glandular trichomes of Smallanthus sonchifolius (Asteraceae) by reversed-phase high-performance liquid chromatography

A simple, reliable and rapid reversed-phase HPLC-PAD procedure for the characterisation and quantitative determination of the anti-diabetic sesquiterpene lactone enhydrin (1) from Smallanthus sonchifolius (yacón) has been evaluated and validated. The approach focused on the analysis of various leaf rinse extracts, as well as the glandular trichomes of intact leaves, in which 1 was the major compound detected. The best sample preparation of a rinse extract yielded 0.67 mg/mL of 1, whilst a rapid rinse of a small piece of one dried leaf gave 0.09 mg/mL of 1; the highest concentration obtained from a glandular extract was 0.07 mg/mL. The dried leaves of S. sonchifolius were found to contain a total of 0.97% of 1.     

Simple 1,4-benzoquinones with antibacterial activity from stems and leaves of Gunnera perpensa

From the dichloromethane extract of the leaves and stems of Gunnera perpensa two new, simple 1,4-benzoquinones and a known benzopyran-6-ol were isolated. From the methanol extract phytol was obtained. The two benzoquinones, 2-methyl-6-(-3-methyl-2-butenyl)benzo-1,4-quinone (1) and 3-hydroxy-2-methyl-5-(3-methyl-2-butenyl)benzo-1,4-quinone (2) and the benzopyran, 6-hydroxy-8-methyl-2,2-dimethyl-2H-benzopyran (3) were examined for antimicrobial properties together with the crude stem, leaf and root extracts. Minimum inhibitory concentration (MIC) assays were used to quantify antimicrobial activity and the MIC values for the crude extracts of stems, roots and leaves ranged between 100 microg and >16 mg/ml against the eight microorganisms investigated. Compound 1 showed significant antimicrobial activity with the most sensitive organism being Staphylococcus epidermidis with an MIC of 9.8 microg/ml. For compound 2, no activity was noted. Compound 3 exhibited good activity against the yeasts Cryptococcus neoformans (75 microg/ml) and Candida albicans (37.5 microg/ml).     

A substance inducing teliospore production in wheat leaf rust,Puccinia recondita f. sp.tritici

A substance inducing teliospore production inPuccinia racondita f. sp.tritici was found in water and methanol extracts of wheat leaves with telia of the wheat leaf rust just before harvest time. Methanol (MeOH) and water extracts from uninfected wheat leaves also showed telia-inducing activity. However, the MeOH and water extracts from wheat leaves covered with telia showed much stronger activity than those from uninfected wheat leaves. We obtained a fraction (0.2 mg) showing activity at 2 ng/ml by purification of the water extract.     

Free radical scavenging profile and myeloperoxidase inhibition of extracts from antidiabetic plants: Bauhinia forficata and Cissus sicyoides

There is abundant evidence that reactive oxygen species are implicated in several physiological and pathological processes. To protect biological targets from oxidative damage, antioxidants must react with radicals and other reactive species faster than biological substrates do. The aim of the present study was to determine the in vitro antioxidant activity of aqueous extracts from leaves of Bauhinia forficata Link (Fabaceae-Caesalpinioideae) and Cissus sicyoides L. (Vitaceae) (two medicinal plants used popularly in the control of diabetes mellitus), using several different assay systems, namely, 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) decolorization, superoxide anion radical (O2(.-)) scavenging and myeloperoxidase (MPO) activity. In the ABTS assay for total antioxidant activity, B. forficata showed IC50 = 8.00+/-0.07 microg/mL, while C. sicyoides showed IC50 = 13.0+/-0.2 microg/mL. However, the extract of C. sicyoides had a stronger effect on O2(.-) (IC50 = 60.0+/-2.3 microg/mL) than the extract of B. forficata (IC50 = 90.0+/-4.4 microg/mL). B. forficata also had a stronger inhibitory effect on MPO activity, as measured by guaiacol oxidation, than C. sicyoides. These results indicate that aqueous extracts of leaves of B. forficata and C. sicyoides are a potential source of natural antioxidants and may be helpful in the prevention of diabetic complications associated with oxidative stress.     

Smallanthus sonchifolius and Lepidium meyenii - prospective Andean crops for the prevention of chronic diseases

Smallanthus sonchifolius (yacon) and Lepidium meyenii (maca) were the traditional crops of the original population of Peru where they are also still used in folk medicine. These plants are little known in Europe and Northern America although at least yacon can be cultivated in the climatic conditions of these regions. This article deals with the botany and the composition, the structure of main constituents, biological activity of these plants and the cultivation of yacon in the Czech Republic. The potential of yacon tubers to treat hyperglycemia, kidney problems and for skin rejuvenation and the antihyperglycemic and cytoprotective activity of its leaves seems to be related mostly to its oligofructan and phenolic content, respectively. Maca alkaloids, steroids, glucosinolates, isothicyanates and macamides are probably responsible for its aptitude to act as a fertility enhancer, aphrodisiac, adaptogen, immunostimulant, anabolic and to influence hormonal balance. Yacon and maca are already on the European market as prospective functional foods and dietary supplements, mainly for use in certain risk groups of the population, e.g. seniors, diabetics, postmenopausal women etc.     

Hepatocellular toxicity of kava leaf and root extracts.

Kava extracts are used widely for different purposes and were thought to be safe. Recently, several cases of hepatotoxicity have been published. To explore possible mechanisms of kava hepatotoxicity, we prepared and analyzed three different kava extracts (a methanolic and an acetonic root and a methanolic leaf extract), and investigated their toxicity on HepG2 cells and isolated rat liver mitochondria. All three extracts showed cytotoxicity starting at a concentration of 50 microg/ml (lactate dehydrogenase leakage) or 1 microg/ml (MTT test). The mitochondrial membrane potential was decreased (root extracts starting at 50 microg/ml) and the respiratory chain inhibited and uncoupled (root extracts) or only uncoupled (leaf extract) at 150 microg/ml, and mitochondrial beta-oxidation was inhibited by all extracts starting at 100 microg/ml. The ratio oxidized to reduced glutathione was increased in HepG2 cells, whereas the cellular ATP content was maintained. Induction of apoptosis was demonstrated by all extracts at a concentration of 150 microg/ml. These results indicate that the kava extracts are toxic to mitochondria, leading to inhibition of the respiratory chain, increased ROS production, a decrease in the mitochondrial membrane potential and eventually to apoptosis of exposed cells. In predisposed patients, mitochondrial toxicity of kava extract may explain hepatic adverse reactions of this drug.     

Giardia lamblia: the effects of extracts and fractions from Mentha x piperita Lin. (Lamiaceae) on trophozoites

Giardia lamblia is a parasite that causes giardiasis in humans and other mammals. The common treatment includes different classes of drugs, which were described to produce unpleasant side effects. Mentha x piperita, popularly known as peppermint, is a plant that is frequently used in the popular medicine to treat gastrointestinal symptoms. We examined the effects of crude extracts and fractions from peppermint against G. lamblia (ATCC 30888) on the basis of trophozoite growth, morphology and adherence studies. The methanolic, dichloromethane and hexanic extracts presented IC(50) values of 0.8, 2.5 and 9.0microg/ml after 48h of incubation, respectively. The aqueous extract showed no effect against the trophozoites with an IC(50)>100microg/ml. The aqueous fraction presented a moderate activity with an IC(50) of 45.5microg/ml. The dichloromethane fraction showed the best antigiardial activity, with an IC(50) of 0.75microg/ml after 48h of incubation. The morphological and adhesion assays showed that this fraction caused several alterations on plasma membrane surface of the parasite and inhibited the adhesion of G. lamblia trophozoites. Cytotoxic assays showed that Mentha x piperita presented no toxic effects on the intestinal cell line IEC-6. Our results demonstrated antigiardial activity of Mentha x piperita, indicating its potential value as therapeutic agent against G. lamblia infections.     

Antioxidant activities of cultured Armillariella mellea

This study aimed to evaluate the antioxidant activities of a cultured medicinal fungus--Armillariella mellea (Vahl. ex Fr.) Karst. (AM). Three antioxidant assay systems, namely cytochrome c, xanthine oxidase inhibition and FeCl2-ascorbic acid stimulated lipid peroxidation in rat tissue homogenate tests, were used. Total flavonoid and phenol contents of AM extracts were also analyzed. Results showed that both aqueous (AM-H2O) and ethanolic (AM-EtOH) extracts of solid state cultured AM showed antioxidant activities in a concentration-dependent manner. At concentrations 1-100 microg/ml, the free radical scavenging activity was 73.7-92.1% for AM-H2O, and 60.0-90.8% for AM-EtOH. These extracts also showed an inhibitory effect on xanthine oxidase activity, but with a lesser potency (IC50 - 9.17 microg/ml for AM-H2O and 7.48 microg/ml for AM-EtOH). In general, AM-H2O showed a stronger anti-lipid peroxidation activity on different rat's tissues than AM-EtOH. However, both AM extracts displayed a weak inhibitory effect on lipid peroxidation in plasma. Interestingly, the anti-lipid peroxidation activity of AM-H2O (IC50 - 6.66 microg/ml) in brain homogenate was as good as alpha-tocopherol (IC50 - 5.42 microg/ml). AM-H2O (80.0 mg/g) possessed a significant higher concentration of total flavonoids than AM-EtOH (30.0 mg/g), whereas no difference was noted in the total phenol content between these two extracts. These results conclude that AM extracts possess potent free radical scavenging and anti-lipid peroxidation activities, especially the AM-H20 in the brain homogenate.     

Bioactivity-guided fractionation of Coronopus didymus: A free radical scavenging perspective

The whole plant aqueous extract of Coronopus didymus Linn. was fractionated on the basis of polarity and resulting fractions were evaluated for free radical scavenging ability. The most non-polar fraction (CDF1) was found to be more active than other fractions in scavenging DPPH, ABTS(-), nitric oxide and hydroxyl radicals in steady-state conditions. Stop-flow spectrometric studies showed 58.13% inhibition of 100 microM DPPH at a concentration of 150 microg/ml of CDF1 in 1000 s and 32.31% scavenging of 960 microM ABTS(-) at a concentration of 300 microg/ml of CDF1 in 100 s. The reaction of CDF1 with hydroxyl radicals produced by pulse radiolysis showed a transient spectrum with absorption peaks at 320, 390 and 400 nm, indicating the presence of flavonoids/related components. Competition kinetics with potassium thiocyanate against scavenging of hydroxyl radicals showed a reactivity of 0.1326 against thiocyanate. CDF1 also protected against Fenton reagent-induced calf thymus DNA damage at a concentration of 400 mg/ml indicating it to be the most potent fraction.     

The effect of extracts from ginger rhizome on inflammatory mediator production

Compounds from rhizomes of Zingiber officinale, commonly called ginger, have been purported to have anti-inflammatory actions. We have used an in vitro test system to test the anti-inflammatory activity of compounds isolated from ginger rhizome. U937 cells were differentiated and exposed to lipopolysaccharide (LPS) from Escherichia coli (1 microg/ml) in the presence or absence of organic extracts or standard compounds found in ginger (6-, 8-, 10-gingerol or 6-shogaol) for 24 h. Supernatants were collected and analyzed for the production of prostaglandin E(2) (PGE(2)) and tumor necrosis factor alpha (TNF-alpha) by standard ELISA assays. Predominant compounds in the organic extracts were identified as 6-, 8- 10-gingerols and 6-, 8-, 10-shogaols. Organic extracts or standards containing gingerols were not cytotoxic, while extracts or standards containing predominantly shogaols were cytotoxic at concentrations above 20 microg/ml. Crude organic extracts of ginger were capable of inhibiting LPS induced PGE(2) (IC(50)<0.1 microg/ml) production. However, extracts were not nearly as effective at inhibiting TNF-alpha (IC(50)>30 microg/ml). Thirty three fractions and subfractions, prepared by column chromatography, were analyzed for bioactivity. Extracts containing either predominantly gingerols or shogaols (identified by HPLC) were both highly active at inhibiting LPS-induced PGE(2) production (IC(50)<0.1 microg/ml), while extracts that contained unknown compounds were less effective (IC(50)<3.2 microg/ml). Extracts or standards containing predominantly gingerols were capable of inhibiting LPS-induced COX-2 expression while shogaol containing extracts had no effect on COX-2 expression. These data demonstrate that compounds found in ginger are capable of inhibiting PGE(2) production and that the compounds may act at several sites.     

Protective properties of artichoke (Cynara scolymus) against oxidative stress induced in cultured endothelial cells and monocytes

It is currently believed that oxidative stress and inflammation play a significant role in atherogenesis. Artichoke extract exhibits hypolipemic properties and contains numerous active substances with antioxidant properties in vitro. We have studied the influence of aqueous and ethanolic extracts from artichoke on intracellular oxidative stress stimulated by inflammatory mediators (TNFalpha and LPS) and ox-LDL in endothelial cells and monocytes. Oxidative stress which reflects the intracellular production of reactive oxygen species (ROS) was followed by measuring the oxidation of 2', 7'-dichlorofluorescin (DCFH) to 2', 7'-dichlorofluorescein (DCF). Agueous and ethanolic extracts from artichoke were found to inhibit basal and stimulated ROS production in endothelial cells and monocytes in dose dependent manner. In endothelial cells, the ethanolic extract (50 microg/ml) reduced ox-LDL-induced intracellular ROS production by 60% (p<0,001) while aqueous extract (50 microg/ml) by 43% (p<0,01). The ethanolic extract (50 microg/ml) reduced ox-LDL-induced intracellular ROS production in monocytes by 76% (p<0,01). Effective concentrations (25-100 microg/ml) were well below the cytotoxic levels of the extracts which started at 1 mg/ml as assessed by LDH leakage and trypan blue exclusion. Penetration of some active substances into the cells was necessary for inhibition to take place as juged from the effect of preincubation time. These results demonstrate that artichoke extracts have marked protective properties against oxidative stress induced by inflammatory mediators and ox-LDL in cultured endothelial cells and monocytes.