Inflammatory and Apoptotic Regulatory Activity of Tanshinone IIA in Helicobacter pylori-Infected Cells.

Helicobacter pylori infections induce host cell inflammation and apoptosis, however, they are conflicting. Tanshinone IIA is an active compound of Salvia miltiorrhiza Bge. In this study, we investigated the regulatory effects of tanshinone IIA on H. pylori-induced inflammation and apoptosis in vitro. Tanshinone IIA treatments (13.6-54.4[Formula: see text][Formula: see text]M) significantly decreased nuclear factor kappa B (NF-kB) and mitogen-activated protein kinase (MAPK) [p-38 and C-terminal Jun-kinase 1/2 (JNK1/2)] protein expressions and inflammatory substance [cyclooxygenase-2 (COX-2), 5-lipooxygenase (5-LOX), intercellular adhesion molecule-1 (ICAM-1), reactive oxygen species (ROS), nitric oxide (NO), inducible nitric oxide synthase (iNOS), interleukin-1[Formula: see text] (IL-1[Formula: see text], IL-6, and IL-8] production in the H. pylori-infected cells. In contrast, tanshinone IIA treatments significantly increased apoptotic relevant protein [Bcl-2-associated X protein (Bax) and caspase 9] expressions and increased mitochondrial transmembrane potential ([Formula: see text] disruption, mitochondrial cytochrome [Formula: see text] (cyt [Formula: see text] release, and caspase cascades. Tanshinone IIA treatments effectively decreased H. pylori-induced inflammation and significantly promoted H. pylori-induced intrinsic apoptosis through NF-kB and MAPK (p-38 and JNK) pathways. Tanshinone IIA has great potential as a candidate to protect host cells from H. pylori-induced severe inflammation and gastric cancer.

Medicinal plant activity on Helicobacter pylori related diseases.

More than 50% of the world population is infected with Helicobacter pylori (H. pylori). The bacterium highly links to peptic ulcer diseases and duodenal ulcer, which was classified as a group I carcinogen in 1994 by the WHO. The pathogenesis of H. pylori is contributed by its virulence factors including urease, flagella, vacuolating cytotoxin A (VacA), cytotoxin-associated gene antigen (Cag A), and others. Of those virulence factors, VacA and CagA play the key roles. Infection with H. pylori vacA-positive strains can lead to vacuolation and apoptosis, whereas infection with cagA-positive strains might result in severe gastric inflammation and gastric cancer. Numerous medicinal plants have been reported for their anti-H. pylori activity, and the relevant active compounds including polyphenols, flavonoids, quinones, coumarins, terpenoids, and alkaloids have been studied. The anti-H. pylori action mechanisms, including inhibition of enzymatic (urease, DNA gyrase, dihydrofolate reductase, N-acetyltransferase, and myeloperoxidase) and adhesive activities, high redox potential, and hydrophilic/hydrophobic natures of compounds, have also been discussed in detail. H. pylori-induced gastric inflammation may progress to superficial gastritis, atrophic gastritis, and finally gastric cancer. Many natural products have anti-H. pylori-induced inflammation activity and the relevant mechanisms include suppression of nuclear factor-κB and mitogen-activated protein kinase pathway activation and inhibition of oxidative stress. Anti-H. pylori induced gastric inflammatory effects of plant products, including quercetin, apigenin, carotenoids-rich algae, tea product, garlic extract, apple peel polyphenol, and finger-root extract, have been documented. In conclusion, many medicinal plant products possess anti-H. pylori activity as well as an anti-H. pylori-induced gastric inflammatory effect. Those plant products have showed great potential as pharmaceutical candidates for H. pylori eradication and H. pylori induced related gastric disease prevention.

Apigenin has anti-atrophic gastritis and anti-gastric cancer progression effects in Helicobacter pylori-infected Mongolian gerbils.

ETHNOPHARMACOLOGICAL RELEVANCE: Apigenin, one of the most common flavonoids, is abundant in celery, parsley, chamomile, passionflower, and other vegetables and fruits. Celery is recognized as a medicinal vegetable in Oriental countries to traditionally treat inflammation, swelling, blood pressure, serum lipid, and toothache. In this study, we investigated apigenin treatment effects on Helicobacter pylori-induced atrophic gastritis and gastric cancer progression in Mongolian gerbils. MATERIALS AND METHODS: Five to eight-week-old Mongolian gerbils were inoculated with Helicobacter pylori for four weeks without (atrophic gastritis group) or with N'-methyl-N'-nitro-N-nitroso-guanidine (MNNG) (gastric cancer group) in drinking water, and were then rested for two weeks. During the 7th-32th (atrophic gastritis group) or the 7th-52th (gastric cancer group) weeks, they were given various doses (0-60 mg/kgbw/day) of apigenin. At the end of the 32th (atrophic gastritis group) or the 52th (atrophic gastritis group) week, all Mongolian gerbils were sacrificed using the CO2 asphyxia method. The histological changes of Helicobacter pylori colonization, neutrophil and monocyte infiltrations, and atrophic gastritis in both atrophic gastritis and gastric cancer Mongolian gerbils were examined using immunohistochemistry stain and Sydney System scoring. RESULTS: Apigenin treatments (30-60 mg/kgbw/day) effectively decreased atrophic gastritis (atrophic gastritis group) and dysplasia/gastric cancer (gastric cancer group) rates in Mongolian gerbils. Apigenin treatment (60 mg/kgbw/day) significantly decreased Helicobacter pylori colonization and Helicobacter pylori-induced histological changes of neutrophil and monocyte infiltrations and atrophic gastritis in both atrophic gastritis and gastric cancer Mongolian gerbils. CONCLUSIONS: Apigenin has the remarkable ability to inhibit Helicobacter pylori-induced atrophic gastritis and gastric cancer progression as well as possessing potent anti-gastric cancer activity.

Low density supercritical fluids precipitation of 9-cis and all trans-ß-carotenes enriched particulates from Dunaliella salina.

In this study, supercritical anti-solvent (SAS) pulverization coupled with reverse phase elution chromatography was employed to isolate 9-cis and trans-ß-carotenes from Dunaliella salina. Total concentration of 9-cis (134.7mg/g) and trans-ß-carotene (204.2mg/g) was increased from 338.9mg/g of the ultrasonic extract to 859.7mg/g (338.9 for 9-cis and 520.8 for trans) of the elution fraction. The SAS pulverization of the collected fraction further produced submicron-sized particulates containing 932.1mg/g (355.6 for 9-cis and 576.5 for trans) of total ß-carotenes with a recovery of 86.3% (83.9% for cis and 87.8% for trans). Effects of two SAS operational conditions on the purity, recovery of total ß-carotenes, mean size and morphology of the precipitates were obtained from an experimentally designed method. Generation of micronized particulates enriched with 9-cis and trans-ß-carotenes by low-density SAS was proved to be feasible and environmental benign.

In vitro anti-inflammatory effect of apigenin in the Helicobacter pylori-infected gastric adenocarcinoma cells.

Infection with Helicobacter pylori causes extensive gastric epithelial cell inflammation which may progress to atrophic gastritis, intestinal metaplasia, and even gastric adenocarcinoma. Apigenin (4',5,7-trihydroxyflavone) is widely distributed in fruits and vegetables, and is a well-known antiinflammatory supplement with low cytotoxicity. In this study, we investigated the anti-inflammatory effects of apigenin in H. pylori-infected MKN45 cells, for which IκBα, cyclooxygenase-2 (COX-2), intercellular adhesion molecule-1 (ICAM-1), reactive oxygen species (ROS), interleukin-8 (IL-8), IL-6, IL-1ß, and mucin-2 (MUC-2) expressions were examined. Apigenin treatments (9.3-74 µM) significantly increased the IκBα expression, and thus inhibited nuclear factor kappa B (NF-κB) activation, and the inflammatory factor (COX-2, ICAM-1, ROS, IL-6, and IL-8) expressions decreased. The ROS levels decreased partially based on the intrinsic scavenging property of apigenin. In summary, apigenin treatments effectively inhibited NF-κB activation and the related inflammatory factor expressions, as well as increased MUC-2 expression in the H. pylori-infected MKN45 cells. The compound shows great potential as a candidate agent for the inhibition of H. pylori-induced extensive gastric epithelial cell inflammation.

Production of all trans-beta-carotene by using impinging flow of supercritical carbon dioxide anti-solvent pulverization.

This work investigated column elution chromatography coupled with supercritical anti-solvent precipitation to produce carotenoid rich microsized particulates from microalgal Dunaliella salina species. The extract contained carotenoids ranging from 61.3 mg/g(salina) to 72.5 mg/g(salina) using ultrasonic stirred ethyl ether or tetrahydrofuran (THF) extraction. When 10 L of ethyl alcohol was employed to elute the THF extract, purity of trans-ß-carotene is 823.6 mg/g with a recovery of 86.2%. It was found that the supercritical anti-solvent of THF solution at 160 bar and 318 K produced powdered particulates with a purity of carotenoids above 90%. Subsequently, a central composite response surface design method was used to design supercritical anti-solvent precipitation of carotenoid-rich THF solution. This was accomplished by increasing the pressure from 140 bar to 180 bar and the time from 40 min to 60 min at a feed flow rate of 0.2 mL/min. A CO(2) flow rate of 15 L/min and a temperature of 318 K were also used to determine the effects on purity and recovery of trans-ß-carotene. The combined process produced micronized precipitates with a mean particle size ranging from 3.5 µm to 19 µm and the purity of trans-ß-carotene attained was 926.8 mg/g with a recovery of 54%.

Countercurrent flow of supercritical anti-solvent in the production of pure xanthophylls from Nannochloropsis oculata.

This study examined pilot scaled elution chromatography coupled with supercritical anti-solvent precipitation (using countercurrent flow) in generating zeaxanthin-rich particulates from a micro-algal species. Ultrasonic agitated acetone extract subjected to column fractionation successfully yielded a fraction containing 349.4 mg/g of zeaxanthin with a recovery of 85%. Subsequently, supercritical anti-solvent (SAS) precipitation of the column fraction at 150 bar and 343 K produced submicron-sized particulates with a concentration of 845.5mg/g of zeaxanthin with a recovery of 90%. Experimental results from a two-factor response surface method SAS precipitation indicated that purity, mean size and morphology of the precipitates were significantly affected by the flow type configuration, feed flow rate and injection time.

Anti-gastric adenocarcinoma activity of 2-Methoxy-1,4-naphthoquinone, an anti-Helicobacter pylori compound from Impatiens balsamina L.

2-Methoxy-1,4-naphthoquinone (MeONQ) from Impatiens balsamina L. exhibited strong anti-H. pylori activity in our previous study. In this study, we investigated the cytotoxicity of MeONQ against gastric adenocarcinoma (MKN45 cell line) and propose the relevant mechanisms. MeONQ resulted in serious necrosis via superoxide anion catastrophe when the treatment doses were higher than 50µM, whereas apoptosis occurred at low treatment doses (25-50µM) through the caspase-dependent apoptosis pathway. Necrosis is the dominant mode of cell death. MeONQ exhibited high ability to induce gastric adenocarcinoma necrosis, showing good potential as a candidate agent for H. pylori infection related disease therapy.

Preparation of highly pure zeaxanthin particles from sea water-cultivated microalgae using supercritical anti-solvent recrystallization.

Xanthophylls, including zeaxanthin, are considered dietary supplements with a potentially positive impact on age-related macular degeneration. Using pilot-scale column fractionation coupled with supercritical anti-solvent (SAS) recrystallization, highly pure zeaxanthin particulates were prepared from ultrasonic extracts of the microalgae, Nannochloropsis oculata, grown in sea water. Column partition chromatography increased the concentration of zeaxanthin from 36.2 mg/g of the ultrasonic extracts to 425.6 mg/g of the collected column fractions. A response surface methodology was systematically designed for the SAS process by changing feed concentration, CO(2) flow rate and anti-solvent pressure. Zeaxanthin-rich particles with a purity of 84.2% and a recovery of 85.3% were produced using supercritical anti-solvent recrystallization from the column eluate at a feed concentration of 1.5 mg/mL, CO(2) flow rate of 48.6 g/min and pressure of 135 bar.

In vitro activity of Paris polyphylla smith against enterovirus 71 and coxsackievirus B3 and its immune modulation.

Enterovirus 71 (EV71) and coxsackievirus B3 (CVB3) have resulted in severe pathogenesis caused by the host's immune response, including the cytokine cascade. Paris polyphylla Smith is a folk medicinal plant in Asia traditionally prescribed for the reduction of pain and elimination of poisoning. In this study, we investigated the anti-EV71 and CVB3 activity of P. polyphylla Smith as well as its immune modulation. The IC(50) for the P. polyphylla Smith 95% ethanol extract against EV71 and CVB3 were 12.5-23% and 99-156% of that of ribavirin, a positive control. Prevention of viral infection, viral inactivation, and anti-viral replication effects against both EV71 and CVB3 were demonstrated by the extract, the anti-viral replication effect being dominant. The extract significantly increased IL-6 production in both EV71- and CVB3-infected cells. A high correlation was possibly demonstrated between the high amounts of IL-6 induction in the EV71 and CVB3-infected cells and the anti-viral replication activity of the extract. In conclusion, good anti-EV71 and CVB3 activity was observed in the P. polyphylla Smith 95% ethanol extract. The high amounts of IL-6 induction in the virus-infected cells played a key role in the anti-viral activity of the extract.

In Vitro Activity of 2-methoxy-1,4-naphthoquinone and Stigmasta-7,22-diene-3ß-ol from Impatiens balsamina L. against Multiple Antibiotic-Resistant Helicobacter pylori.

Infection with Helicobacter pylori is strongly associated with gastric cancer and gastric adenocarcinoma. WHO classified H. pylori as a group 1 carcinogen in 1994. Impatiens balsamina L. has been used as indigenous medicine in Asia for the treatment of rheumatism, fractures and fingernail inflammation. In this study, we isolated anti-H. pylori compounds from this plant and investigated their anti- and bactericidal activity. Compounds of 2-methoxy-1,4-naphthoquinone (MeONQ) and stigmasta-7,22-diene-3ß-ol (spinasterol) were isolated from the pods and roots/stems/leaves of I. balsamina L., respectively. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) for MeONQ were in the ranges of 0.156-0.625 and 0.313-0.625 µg mL(-1), respectively, and in the ranges of 20-80 µg mL(-1) both of MICs and MBCs for spinasterol against antibiotic (clarithromycin, metronidazole and levofloxacin) resistant H. pylori. Notably, the activity of MeONQ was equivalent to that of amoxicillin (AMX). The bactericidal H. pylori action of MeONQ was dose-dependent. Furthermore, the activity of MeONQ was not influenced by the environmental pH values (4-8) and demonstrated good thermal (121°C for 15 min) stability. MeONQ abounds in the I. balsamina L. pod at the level of 4.39% (w/w db). In conclusion, MeONQ exhibits strong potential to be developed as a candidate agent for the eradication of H. pylori infection.

In vitro activity of Impatiens balsamina L. against multiple antibiotic-resistant Helicobacter pylori.

Impatiens balsamina L. has been used as indigenous medicine in Asia for the treatment of rheumatism, fractures, and fingernail inflammation. In this study, anti-H. pylori activity of I. balsamina L. was investigated. The MICs, MBCs, time-kill assay, and effect of environmental pH for the plant extracts were determined. The test H. pylori strains have resistance to clarithromycin (CLR), metronidazole (MTZ), and levofloxacin (LVX). From our results, all part (root/stem/leaf, seed, and pod) extracts of I. balsamina L. exhibited bactericidal H. pylori activity. Specifically, the pod extract had significantly lower MICs and MBCs (1.25-2.5 and 1.25-5.0 microg/ml, respectively). Of the five pod-extraction solvents, both ethyl acetate and acetone were the most efficient for the anti-H. pylori compounds of the pod extraction. The dose-dependency of the pod extract's bactericidal activity was H. pylori strain-dependent. Bactericidal H. pylori activity of the pod extract was not affected by the environmental pH (2-8). In summary, the acetone and ethyl acetate pod extracts of I. balsamina L. exhibited very strong anti-H. pylori activity. This activity exceeded that of MTZ and approximated to that of AMX.

Simultaneous quantification of flavonoids and triterpenoids in licorice using HPLC.

Numerous bioactive compounds are present in licorice (Glycyrrhizae Radix), including flavonoids and triterpenoids. In this study, a reversed-phase high-performance liquid chromatography (HPLC) method for simultaneous quantification of three flavonoids (liquiritin, liquiritigenin and isoliquiritigenin) and four triterpenoids (glycyrrhizin, 18alpha-glycyrrhetinic acid, 18beta-glycyrrhetinic acid and 18beta-glycyrrhetinic acid methyl ester) from licorice was developed, and further, to quantify these 7 compounds from 20 different licorice samples. Specifically, the reverse-phase HPLC was performed with a gradient mobile phase composed of 25 mM phosphate buffer (pH 2.5)-acetonitrile featuring gradient elution steps as follows: 0 min, 100:0; 10 min, 80:20; 50 min, 70:30; 73 min, 50:50; 110 min, 50:50; 125 min, 20:80; 140 min, 20:80, and peaks were detected at 254 nm. By using our technique, a rather good specificity was obtained regarding to the separation of these seven compounds. The regression coefficient for the linear equations for the seven compounds lay between 0.9978 and 0.9992. The limits of detection and quantification lay in the range of 0.044-0.084 and 0.13-0.25 microg/ml, respectively. The relative recovery rates for the seven compounds lay between 96.63+/-2.43 and 103.55+/-2.77%. Coefficient variation for intra-day and inter-day precisions lay in the range of 0.20-1.84 and 0.28-1.86%, respectively. Based upon our validation results, this analytical technique is a convenient method to simultaneous quantify numerous bioactive compounds derived from licorice, featuring good quantification parameters, accuracy and precision.

High-performance liquid chromatography for quantification of plumbagin, an anti-Helicobacter pylori compound of Plumbago zeylanica L.

The plant Plumbago zeylanica L. is a semi-climbing shrub that grows throughout Asia and Africa. In our previous study, P. zeylanica L. exhibited high anti-Helicobacter pylori and good bactericidal activities over a wide pH range (pH 2-7). Plumbagin - the major ingredient derived from the roots of P. zeylanica L. - is a naphthoquinone compound. In this study, we investigated plumbagin's anti-H. pylori activity and developed a reversed-phase high-performance liquid chromatography (HPLC) method for quantification of plumbagin from P. zeylanica L. We also observed that plumbagin has strong anti-H. pylori activity, with 0.02-0.16 mg/ml as minimum inhibitory concentrations and 0.16-1.28 mg/ml as minimum bactericidal concentrations. Reversed-phase HPLC was performed with a gradient mobile phase composed of water and methanol, and peaks were detected at 254 nm. Standard curves were linearized in the range of from 10 to 200 microg/ml (regression coefficient r2 = 0.99995). After spikes of 50, 100, and 150 microg/ml of plumbagin standard solution, recovery rates were between 97.45 and 99.24%. Both intra- and inter-day precisions had coefficient variation of less than 1% at concentrations of 50, 100, and 150 microg/ml. The limits of detection and quantitation were 0.02 and 0.06 microg/ml, respectively. Based on validation results, this analytical method is a precise, accurate and stable method to quantify plumbagin derived from P. zeylanica L.

Anti-Helicobacter pylori activity of Plumbago zeylanica L.

It has been shown that the presence of infection by Helicobacter pylori is strongly associated with gastric cancer and peptic ulceration. In western medicine, a 3-fold therapeutic regimen, emphasizing the use of antibiotics, is typically used to suppress H. pylori activity. However, antibiotic drug resistance frequently develops as a consequence of such treatment. In our previous study, 50 Taiwanese folk medicinal plants were screened for their anti-H. pylori activities. The results revealed that Plumbago zeylanica L. had the highest inhibitory effects against H. pylori. In this study, therefore, we have focused on establishing the anti-H. pylori activities of P. zeylanica L. Water and the organic solvents ethanol, ethyl acetate and acetone were used for P. zeylanica L. extraction, obtaining yields of 1.66-6.84% (w/w). Excluding the water extract, higher anti-H. pylori activity was demonstrated for all the extracts, both using the agar diffusion and dilution methods. The ethyl acetate extract exhibited the lowest minimum inhibitory concentrations against five H. pylori strains, of which ranged from 0.32 to 1.28 mg ml-1, followed, in ascending order, by the acetone, ethanol and water analogs. Bactericidal activity was determined for P. zeylanica L. extracts, with the lowest minimum bactericidal concentrations (5.12-20.48 mg ml-1) demonstrated for the ethyl acetate, followed, in ascending order, by the acetone and ethanol analogs. Bactericidal activity appeared to be in a dose-dependent manner. Through a broad pH range (2-7), bactericidal activity was not affected when extract concentrations were greater than or equal to the minimum bactericidal concentration. High stability was demonstrated for the ethyl acetate P. zeylanica L. extract within pH range of 1-7, exhibiting all pH treatments bactericidal activity.

Screening of anti-Helicobacter pylori herbs deriving from Taiwanese folk medicinal plants.

In this study, extracts from 50 Taiwanese folk medicinal plants were examined and screened for anti-Helicobacter pylori activity. Ninety-five percent ethanol was used for herbal extraction. Paederia scandens (Lour.) Merr. (PSM), Plumbago zeylanica L. (PZL), Anisomeles indica (L.) O. Kuntze (AIOK), Bombax malabaricum DC. (BMDC) and Alpinia speciosa (J. C. Wendl.) K. Schum. (ASKS) and Bombax malabaricum DC. (BMDC) all demonstrated strong anti-H. pylori activities. The minimum inhibitory concentration values of the anti-H. pylori activity given by the five ethanol herb extracts ranged from 0.64 to 10.24 mg ml(-1). Twenty-six herbs, including Artemisia argvi Levl. et Vant (AALEV), Phyla nodiflora (Linn.) Greene (PNG) and others, showed moderate anti-H. pylori activity. The additional 19 herbs, including Areca catechu Linn. (ACL), Euphorbia hirta Linn. (EHL) and Gnaphalium adnatum Wall. ex DC. (GAWEDC), possessed lower anti-H. pylori effects. About half of the Taiwanese folk medicinal plants tested, demonstrated to possess higher anti-H. pylori activity.