In-silico studies in Chinese herbal medicines' research: evaluation of in-silico methodologies and phytochemical data sources, and a review of research to date.

The available databases that catalogue information on traditional Chinese medicines are reviewed in terms of their content and utility for in-silico research on Chinese herbal medicines, as too are the various protein database resources, and the software available for use in such studies. The software available for bioinformatics and 'omics studies of Chinese herbal medicines are summarised, and a critical evaluation given of the various in-silico methods applied in screening Chinese herbal medicines, including classification trees, neural networks, support vector machines, docking and inverse docking algorithms. Recommendations are made regarding any future in-silico studies of Chinese herbal medicines.

In vitro study of aortic valves treated with neo-chordae grafts: hydrodynamics and tensile force measurements.

Reparative surgery of the aortic root functional unit (ARFU) allows for a better preservation of the functionality of the native structure compared to prosthesis implantation. Post-operative results are satisfactory, whereas mid- and long-term results are challenging, for example in terms of cusps prolapse recurrence. At the Cardiothoracic Surgery Unit of the Sacco Hospital, a new surgical technique aimed at the stabilization in time of the results of standard ARFU repair operations has been applied. This technique, inspired by the mitral neo-chordae (NC) implantation, consists of implanting an e-PTFE suture thread between the prolapsed cusp and the sinotubular junction. Aim of this study was to assess the influence of NC implantation on the ARFU functioning by evaluating with an owned pulsatile in vitro apparatus the force magnitude acting on the NC and the dynamic behavior of porcine ARFUs treated according to the operating-room procedures. The maximum recorded values of the mean and peak diastolic forces were 0.064 and 0.186 N, respectively, and were linearly dependent upon the mean diastolic pressure across the valve. In addition, the measurements of the opening-closing times and valve leakage volumes, performed at pre- and post-surgeries, yielded that the valve functionality was not significantly influenced by NC implantation.

Old and new targets for innovative antimalarial compounds: the different strategies of the Italian Malaria Network.

Clinical treatment-failures to affordable drugs encouraged new investigation for discovery and development of new prophylactic and therapeutic interventions against malaria. The Drug Discovery Cluster (DDcl) of the Italian Malaria Network gathers several highly integrated and complementary laboratories from different Italian Institutions to identify, synthesise, screen in vitro and in vivo new antimalarial molecules directed against the intraerythrocytic stage of P. falciparum parasites and/or with transmission blocking activity to select lead compounds for further development. Complementary research activities, both in vitro and in the clinics, aim at investigating the pathogenetic mechanisms of severe malaria anaemia and the different manifestations of the disease in malaria-HIV co-infected patients to identify new therapies and improve survival.

A structure-activity study for the inhibition of metalloproteinase-9 activity and gene expression by analogues of gallocatechin-3-gallate.

Catechins are able to modulate the gelatinolytic activity of matrix metalloproteinase-9 (MMP-9) by reducing its release from macrophages. Gallocatechins decrease MMP-9 secretion by lowering MMP-9 promoter activity and mRNA levels. The effect appears to be dependent on some structural and stereochemical requirements. In this study, the relationship between chemical structure and activity was studied by testing the effect of analogues of (+/-)-gallocatechin-3-gallate (+/-)-GCG, selectively deprived of hydroxyl groups, on MMP-9 activity, transcription, and secretion. Our results indicate that (+/-)-GCG and (+/-)-catechin-3-gallate are characterized by a substitution pattern compatible with direct inhibition of MMP-9 activity. Conversely, when transcription was the target, (+/-)-trans-3-flavanol-3-benzoate, lacking all the hydroxyl groups, was the most effective both in lowering MMP-9 promoter activity and consequently protein secretion, and in inhibiting nuclear-factor-kappaB-driven transcription. Our results suggest that the structural requirements for enzyme inhibition are different from those necessary for targeting gene expression.

Analysis by high-performance liquid chromatography of teucrin A in beverages flavoured with an extract of Teucrium chamaedrys L.

Due to its liver toxicity, the medicinal use of germander (Teucrium chamaedrys L.) was banned in some countries. Nevertheless, alcoholic extracts are still permitted as flavour ingredients since they are fundamental in providing a bitter aromatic taste. Teucrin A represents the substance of major concern regarding the potential toxicity of germander. Hence, teucrin A represents the best analytical and toxicological marker of alcoholic extracts of T. chamaedrys. A sensitive high-performance liquid chromatography method to detect teucrin A in beverages is reported. Teucrin A was prepared by isolation from the plant extract using column chromatography and crystallization. The identity and purity (99%) were established by melting point, nuclear magnetic resonance and liquid chromatography-mass spectrometry. The high-performance liquid chromatography procedure was validated and its intra- and interday performance was established (relative standard deviation < or = 13% and error < or = 10%). In-house validation was carried out by analysing samples of beverages not containing T. chamaedrys spiked with a range of concentrations of teucrin A. The limit of detection was 0.1 ppm and the limit of quantification was 0.3 ppm. Teucrin A accounted for about 70% of the neo-clerodane diterpenoids found in the total extract of a specimen of T. chamaedrys. The content (+/- standard deviation) in 18 batches of different geographical origin was 2338 +/- 740 ppm, per cent coefficient of variation = 32, minimum-maximum = 999 - 3445 ppm. The mean level of teucrin A in 10 bottles of the same brand was 6.1 +/- 0.8 ppm, per cent coefficient of variation = 12. In 10 different brands found on the Italian market, the content of teucrin A ranged from not detectable to 10 ppm.

Inhibition of metalloproteinase-9 activity and gene expression by polyphenolic compounds isolated from the bark of Tristaniopsis calobuxus (Myrtaceae).

Excessive breakdown of extracellular matrix by metalloproteinases (MMPs) occurs in many pathological conditions, and thus inhibition of MMP activity might have therapeutic potential. The methanolic extract and the identified compounds from the bark of Tristaniopsis calobuxus Brongniart & Gris (Myrtaceae) were tested on the activity, production, and gene expression of MMP-9. The extract produced a concentration-dependent inhibition (50-95% at 10-50 microg/ml) of MMP-9 activity. The inhibitory activity was retained in the ethyl acetate-soluble fraction (50-95% inhibition at 10-50 microg/ml) which also reduced the release of MMP-9 by mouse peritoneal macrophages up to 80%. In the ethyl acetate-soluble fraction, two active fractions, 5A and 5B were identified. HPLC-MS and NMR analyses of these fractions indicated the presence of gallocatechin, ellagic acid, and its glycoside derivatives. Since the absolute configuration of gallocatechin was not determined, in the next experiments both (+)-gallocatechin (2R,3S) and (-)-gallocatechin (2S,3R) were tested, and (-)-epigallocatechin (2R,3R) was included for comparison. 5A and 5B inhibited MMP-9 secretion, an observation which correlated with the decrease of MMP-9 promoter activity and the downregulation of mRNA levels. All compounds decreased MMP-9 mRNA levels and secretion. Ellagic acid, (+)-gallocatechin and (-)-epigallocatechin, but not (-)gallocatechin inhibited promoter-driven transcription. Thus configuration at C2 (R) of the flavanol seem to be critical for the interaction with the promoter.

In vitro antiplasmodial activity of extracts of Tristaniopsis species and identification of the active constituents: ellagic acid and 3,4,5-trimethoxyphenyl-(6'-O-galloyl)-O-beta-D-glucopyranoside.

Screening of plants from New Caledonia for antiplasmodial activity against Plasmodium falciparum revealed that methanolic extracts of the leaves and bark of Tristaniopsis calobuxus, T. yateensis, and T.glauca inhibited the growth of chloroquine-sensitive and -resistant clones. Ellagic acid and the new compound 3,4,5-trimethoxyphenyl-(6'-O-galloyl)-O-beta-D-glucopyranoside were identified as the active constituents (IC50 0.5 and 3.2 microM, respectively). The growth inhibition of both clones was comparable. The compounds showed negligible or very low cytotoxicity to human skin fibroblasts and Hep G2 cells when tested at concentrations ranging from 0.5 to 100 microM.

Screening of plants from new caledonia and vanuatu for inhibitory activity of xanthine oxidase and elastase.

A series of 38 plants (55 plant extracts) from New Caledonia and 22 plants (40 plant extracts) from Vanuatu (Efate and Erromango islands) were screened for xan-thine oxidase (XOD) and elastase inhibitory activity. Of the crude extracts 82% were found to possess xanthine oxidase inhibitory activity, and 23% were active against elastase, at a concentration of 50 µg/ml. The methanol extracts of Cunonia montana Schlechter (Cunoniaceae) and Amyema scandens Danser (Loranthaceae), bark and leaves, respectively, exhibited the highest activity in both the assays. C. montana bark extract at 50 µg/ml exhibited 85 and 84% inhibition of XOD and elastase, respectively. IC 50 values were 23 ± 0.82 and 41 ± 3 µg/ml, respectively, for XOD and elastase. A. scandens leaf extract, at 50 µg/ml, exhibited 88 and 71% inhibition of XOD and elastase, respectively. IC 50 values were 13 ± 0.48 and 44 ± 2.2 µg/ml respectively, for XOD and elastase.

Inhibition of cAMP-phosphodiesterase by biflavones of Ginkgo biloba in rat adipose tissue.

This work compares the inhibition of cAMP-phosphodiesterase in rat adipose tissue by a mixture of Ginkgo biloba biflavones with the effect of individual dimeric flavonoids. The degree of enzyme inhibition by G. biloba biflavones was amentoflavone > bilobetin > sequoiaflavone > ginkgetin = isoginkgetin. Sciadopitysin was almost inactive.

LC-UV-electrospray-MS-MS mass spectrometry analysis of plant constituents inhibiting xanthine oxidase.

PURPOSE: A previous screening showed that Amyema scandensi [corrected] Danser (Loranthaceae) efficiently inhibited XOD. The aim of this study was to identify the compounds with anti-XOD properties. For this purpose, Electrospray Tandem Mass Spectrometry (ESI-MS-MS) coupled with UV and Diode Array LC techniques were employed. METHODS: Leaves were delipidized with petroleum ether and extracted with acetone:water 70:30, v:v. The extract was fractionated into the ethyl acetate and water soluble phases. Chemical investigation was performed following the bioactivity guided fractionation. Two fractions with anti-XOD activity were isolated by silica gel column chromatography of the ethyl acetate phase and analyzed by LC-UV-ESI-MS-MS. RESULTS: The compounds identified with authentic standards were: catechin, epicatechin, epicatechin-3-gallate, quercetin-3-O-glucoside, quercetin-3-O-rhamnoside, and isorhamnetin-3-O-glucoside. Other constituents, only partially characterized, were a procyanidin dimer, a procyanidin trimer, three dimers epi/catechine-epi/catechine gallate and isorhamnetin-O-deoxyhexose. The anti-XOD activity was mainly due to galloyl-containing oligomeric proanthocyanidins. CONCLUSIONS: The coupling of UV Diode Array-HPLC with ESI-MS-MS represents a versatile tool for the rapid characterization of compounds in complex mixtures, avoiding time-consuming previous isolation.

Extracts of Ginkgo biloba L. leaves and Vaccinium myrtillus L. fruits prevent photo induced oxidation of low density lipoprotein cholesterol.

Oxidation of low density lipoproteins (LDL) favours cholesterol loading in macrophages and formation of "foam cells", typical of the early atheroma lesions. LDL cholesterol oxidation generates oxysterols, extremely cytotoxic molecular species with diverse biological activities. Vegetable polyphenols are dietary components of pharmacological interest for their anti-oxidant properties. Ginkgo biloba L. (Gingkoaceae) leaves and Vaccinium myrtillus L. (Ericaceae) fruits are known for their beneficial effects in the treatment of various diseases involving free radicals and oxidative damage to biological lipids. In this study we investigated the effect of Ginkgo biloba L. and Vaccinium myrtillus L. extracts on the formation of cholesterol oxides during the photo induced oxidation of human LDL. The results demonstrate a concentration dependent inhibition of oxysterol formation in the presence of both extracts. Protection against oxidation was confirmed by the partial restoration of the normal electrophoretic mobility of LDL, which has been influenced by the UV irradiation. These effects extend knowledge of the therapeutic action of Ginkgo biloba L. and Vaccinium myrtillus L. as agents in anti-atherosclerotic regimens.

Guiera senegalensis J. F. Gmelin (Combretaceae): Biological activities and chemical investigation.

Guiera senegalensis J. F. Gmelin (Combretaceae) leaves are used in African traditional medicine for gastrointestinal disorders, cough and topically for wound healing. This paper regards the evaluation of antiradical, antielastase, antimicrobial, genotoxic and antimutagenic activities of the leaf extracts and the determination of chemical structure of the elastase inhibitors. Antimicrobial activity was tested against Gram positive and negative bacteria, moulds and yeasts. Genotoxic potential was assayed with Bacillus subtilis rec -assay and Salmonella-microsome test. The latter was used also for determining antimutagenic activity. Antiradical properties were evaluated as inhibition of ADP-Fe(2+) induced lipoperoxidation in rat liver microsomes. Porcine pancreatic elastase was used to test enzyme inhibition. The methanolic extract was fractionated with dichloromethane, w-butanol and water and these fractions were tested for the above mentioned activities. The crude extract possessed a mild antimicrobial effect only on Gram positive bacteria (MIC 0.8-1.5 mg/ml) and the effect was associated to dichloromethane and n-butanol fractions. The crude extract and the dichloromethane and n-butanol fractions were weakly genotoxic but showed also a significant antimutagenicity. Inhibition of lipoperoxidation was assignable mainly to the n-butanol fraction. Elastase was inhibited (IC(50) 181 µg/ml) and the inhibition was retained in the water soluble fraction (IC(50) 37µg/ml). The compounds responsible for the enzyme inhibition were a mixture of proanthocyanidins constituted predominantly by (-)-epicatechin and (-)-epigallocatechin units. The mean degree of polymerization was 2-6.

Effects of aging and of chronic obstructive pulmonary disease on RR interval variability.

Clinical and experimental investigations suggest that spectral analysis of RR interval variability might be employed to assess changes in autonomic regulation of the SA node occurring in various chronic, cardiac or respiratory conditions. We studied three groups of subjects: young healthy, older healthy, and old patients with chronic obstructive pulmonary disease (COPD), considering RR interval and breathing, i.e. tidal volume, variability in the frequency domain (AR algorithms), in order to obtain markers of the sympathetic and vagal modulation of the SA node [by way, respectively, of the low (LF) and high frequency (HF) spectral components]. The gain of the relationship between tidal volume and heart period variation (index beta) was also examined. COPD patients demonstrated a reduced resting RR variance with maintained spectral power distribution; upon tilting they did not manifest the usual increase of LF (and attendant decrease of HF) component. Additionally, we observed that the index beta at rest was significantly reduced in older as opposed to younger subjects. This index was also reduced by tilt, in both groups. In patients the index beta was reduced as compared to the group of similar age and younger subjects, and did not diminish further with tilt. This study indicates that in patients with COPD, sympathetic excitatory modulation of the SA node is depressed. Furthermore, the index beta, which provides an assessment of the gain of respiratory modulation of RR interval variability is significantly reduced in patients, as compared to healthy subjects of similar age. These abnormalities of autonomic control, may represent a functional correlation of the hyperinflated state present in this clinical condition which can be assessed non-invasively with spectral analysis of RR interval and respiration.

Pharmacological activities of Chelidonium majus L. (Papaveraceae).

Chelidonium majus L. (Papaveraceae) has a long history as being useful for the treatment of many diseases in European countries. This plant is of great interest for its use also in Chinese herbal medicine. The plant contains, as major secondary metabolites, isoquinoline alkaloids, such as sanguinarine, chelidonine, chelerythrine, berberine and coptisine. Other compounds structurally unrelated to the alkaloids have been isolated from the aerial parts: several flavonoids and phenolic acids. C. majus extracts and its purified compounds exhibit interesting antiviral, antitumour and antimicrobial properties both in vitro and in vivo.

Effect of the flavanolignans of Silybum marianum L. on lipid peroxidation in rat liver microsomes and freshly isolated hepatocytes.

The effect of several flavanolignans (silicristin, silidianin, silybin and isosilybin) present in silymarin, the extract of Silybum marianum fruits, was tested on lipid peroxidation in rat liver microsomes and freshly isolated hepatocytes. In microsomes lipid peroxidation was generated by ADP/Fe2+ and NADPH. All flavanolignans inhibited peroxidation in a concentration dependent manner. In hepatocytes lipid peroxidation was induced by ADP/Fe3+ complex and cell damage was evaluated as LDH activity released in the medium. The inhibition of the peroxidative process by flavanolignans was also evident in this model, even if with a potency order different from that found in microsomes. In contrast, the effect on LDH release was significant only for silybin and isosilybin, the other compounds being inactive on this parameter.

Plasma lipoproteins and cholesterol metabolism in Yoshida rats: an animal model of spontaneous hyperlipemia.

The purpose of this study was to characterize the lipoprotein profile and cholesterol metabolism in Yoshida rats, a strain of inbred genetically hyperlipemic animals. For comparison, Brown Norway rats were used as control animals. Plasma cholesterol and triglycerides were higher in Yoshida as compared to Brown Norway, the elevation of cholesterol being due to a rise in HDL fraction. Triglyceride distribution among lipoproteins showed an increase in VLDL fraction. Hyperlipemia was not related to diabetes, hypothyroidism or nephropathy. Plasma triglycerides production was increased in Yoshida rats, while lipoprotein and hepatic lipases were similar in the two groups. Hypercholesterolemia was associated with a defect of lipoprotein receptor activity and with elevated HMG-CoA reductase and cholesterol 7 alpha - hydroxylase; conversely ACAT activity was lower in Yoshida as compared to Brown Norway rats. Sterol fecal excretion was comparable in the two groups and hypercholesterolemia in Yoshida rats was not associated to an increase of cholesterol saturation of the bile. We suggest that lipoprotein overproduction is the main cause for hyperlipidemia in this strain of rats.

Effect of natural and structurally modified bile acids on cholesterol metabolizing enzymes in rat liver microsomes. II.

The effect of ursodeoxycholic acid analogues bearing modifications at the side-chain moiety of the molecule was tested on cholesterol 7 alpha-hydroxylase and HMG-CoA reductase in rat liver microsomes. The compounds included 23 R,S mixture and the single isomers 23R and 23S of 23 methylursodeoxycholic acid (23-methyl UDCA), the isomeric mixture (cis + trans) of 3 alpha,7 beta-dihydroxy-20,22-methylen-5 beta-cholan-23-oic acid (norcypro-UDCA) and the corresponding single isomers. Each steroid was added to liver microsomes as the sodium salt, at concentrations ranging from 25 to 200 microM. Isomers 23R and 23S of 23-methyl-UDCA inhibited cholesterol 7 alpha-hydroxylase in a concentration-dependent manner. The inhibitory capacity was similar for the two isomers. The extent of inhibition of the analogues was greater than that of the parent compound UDCA. Shortening of the side-chain in norcypro-UDCA resulted in a partial loss of the inhibitory effect, as compared to cypro-UDCA (3 alpha,7 beta-dihydroxy-22,23-methylen-5 beta-cholan-24-oic acid). None of these bile acid derivatives affected the activity of the enzyme HMG-CoA reductase.

Phytochemical characterization and radical scavenger activity of flavonoids from Helichrysum italicum G. Don (Compositae).

The glycosidic fraction of the flavonoids extracted from the flowering tops of the Helichrysum italicum G. Don was isolated, purified and characterized. This fraction was constituted by three compounds, which were assigned the structure of 4,2',4',6'-tetrahydroxychalcone-2'-glucoside, kaempferol-3-glucoside and naringenin-glycoside. Radical scavenger properties of the single glycosyl-flavonoids and of the in toto glycosidic fraction were tested with in vitro systems where different reactive oxygen species are generated (superoxide ions, hydroxyl radicals) and on lipid peroxidation induced by ADP/Fe2+ and NADPH or CCl4 in rat liver microsomes. The formation of reactive oxygen species was detected by cytochrome c reduction, salicylic acid hydroxylation and hyaluronic acid depolymerization. The action of the glycosidic fraction on the release of TXB2 and 12-HETE in human platelets, after collagen stimulation, was also evaluated. The glycosidic fraction inhibited in a dose dependent fashion lipid peroxidation in rat liver microsomes treated with ADP/Fe2+ or CCl4. This effect is due to the ability of flavonoids to scavenge free radicals at different stages of the process (superoxide ions, hydroxyl and lipid peroxide radicals). The single glycosyl-flavonoids exhibited a different scavenger activity, depending on the oxygen species and the chemical structure of the compounds. No effect of the fraction was observed on TXB2 and 12-HETE formation at 100 microns concentration.

Effect of natural and structurally modified bile acids on cholesterol metabolizing enzymes in rat liver microsomes.

The effect of chenodeoxycholic (CDCA), ursodeoxycholic (UDCA), tauroursodeoxycholic (TUDCA), cholic (CA), ursocholic (UCA) acids, analogues of CDCA and UDCA with a cyclopropyl ring at C22, C23 (cypro-CDCA and cypro-UDCA) and 23-methylursodeoxycholic acid (MUDCA) on cholesterol 7 alpha-hydroxylase was studied in rat liver microsomes. Cypro-analogues consisted of a mixture of four diasteroisomers, while MUDCA was the racemic mixture of two enantiomers. Each steroid was added to liver microsomes at concentrations ranging from 10 to 200 microM. With the exception of UCA and CA, all the bile acids inhibited cholesterol 7 alpha-hydroxylase activity. The inhibition shown by cypro-CDCA and cypro-UDCA was stronger than that observed with the corresponding natural compounds. 22S,23S cypro-UDCA exhibited an inhibitory effect which was more pronounced than that of the diasteroisomer mixture. The isomer 22R,23S was less effective and decreased cholesterol 7 alpha-hydroxylase activity in a manner comparable to that of UDCA. The effect of CDCA, UDCA and the cyclopropyl analogues was also tested with respect to HMG-CoA reductase and acylCoA cholesterol acyltransferase (ACAT) activities. ACAT was stimulated by the isomer 22S,23S cypro-UDCA but not affected by the other bile acids. No effect was observed as regards HMG-CoA reductase.