Clovamide and Flavonoids from Leaves of Trifolium pratense and T. pratense subsp. nivale Grown in Italy.

The phenolic content and composition in leaves of Trifolium pratense (red clover) and T. pratense subsp. nivale (snow clover) grown in Italy were evaluated by means of ultraperformance liquid chromatography (UPLC) coupled with photodiode array and mass spectrometry detectors. Compound identification was based on UV and MS data comparing results with those of reference compounds. Quantitative evaluation of all detected compounds was based on calibration curves obtained with available standards. Several phenolics were identified in both extracts, including clovamide, flavonols and isoflavones as their glycosilated and malonated derivatives. The total phenolic content was higher in red clover (53.7 ± 2.2 mg/g dry weight) than in snow clover (44.4 ± 4.9 mg/g dry weight). Red clover contained higher amounts of clovamide and isoflavones (15.6 ± 0.6 and 24.6 ± 1.6 mg/g dry weight, respectively) than snow clover (8.2 ± 0.1 mg/g and 16.9 ± 0.4 mg/g dry weight, respectively), while flavonols were quantified almost in the same amount in both extracts (13.2 ± 0.6 mg/g and 15.8 ± 0.6 mg/g dry weight in red clover and snow clover, respectively). Red clover was characterized by the presence of quercetin, formononetin and biochanin A derivatives as the most abundant flavonoids, whereas snow clover was characterized by higher amounts of quercetin and prunetin derivatives. This investigation, conducted for the first time on phenolics from T. pratense subsp. nivale, revealed the presence in this plant of several flavonoid derivatives the same as in T. pratense. The higher amount of prunetin in snow clover suggest a possible role of this isoflavone as a chemotaxonomic marker for this subspecies. Moreover, snow clover may represent an interesting new source of natural isoflavones with a different concentration pattern than in red clover.

In vivo anti-inflammatory activity of some naturally occurring O- and N-prenyl secondary metabolites.

A series of O- and N-prenyl secondary metabolites of insect, fungal, and plant origin have been evaluated for their topical anti-inflammatory activity using the Croton oil ear test in mice as a model of acute inflammation. Some of the tested compounds revealed an effect (ID50 = 0.31 divided by 0.56 micromol/cm2) comparable with that of the reference non-steroidal anti-inflammatory drug indomethacin (ID50 = 0.23 micromol/cm2).

Plant polyphenols attenuate hepatic injury after hemorrhage/resuscitation by inhibition of apoptosis, oxidative stress, and inflammation via NF-kappaB in rats.

PURPOSE: Oxidative stress and inflammation contribute to hepatic injury after hemorrhage/resuscitation (H/R). Natural plant polyphenols, i.e., green tea extract (GTE) possess high anti-oxidant and anti-inflammatory activities in various models of acute inflammation. However, possible protective effects and feasible mechanisms by which plant polyphenols modulate pro-inflammatory, apoptotic, and oxidant signaling after H/R in the liver remain unknown. Therefore, we investigated the effects of GTE and its impact on the activation of NF-kappaB in the pathogenesis of hepatic injury induced by H/R. METHODS: Twenty-four female LEWIS rats (180-250 g) were fed a standard chow (ctrl) or a diet containing 0.1% polyphenolic extracts (GTE) from Camellia sinensis starting 5 days before H/R. Rats were hemorrhaged to a mean arterial pressure of 30 ± 2 mmHg for 60 min and resuscitated (H/R and GTE H/R groups). Control groups (sham, ctrl, and GTE) underwent surgical procedures without H/R. Two hours after resuscitation, tissues were harvested. RESULTS: Plasma alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) increased 3.5-fold and fourfold, respectively, in vehicle-treated rats as compared to GTE-fed rats. Histopathological analysis revealed significantly decreased hepatic necrosis and apoptosis in GTE-fed rats after H/R. Real-time PCR showed that GTE diminished gene expression of pro-apoptotic caspase-8 and Bax, while anti-apoptotic Bcl-2 was increased after H/R. Hepatic oxidative (4-hydroxynonenal) and nitrosative (3-nitrotyrosine) stress as well as systemic IL-6 level and hepatic IL-6 mRNA were markedly reduced in GTE-fed rats compared with controls after H/R. Plant polyphenols also decreased the activation of both JNK and NFκB. CONCLUSIONS: Taken together, GTE application blunts hepatic damage, apoptotic, oxidative, and pro-inflammatory changes after H/R. These results underline the important roles of JNK and NF-kappaB in inflammatory processes after H/R and the beneficial impact of plant polyphenols in preventing their activation.

Clovamide-rich extract from Trifolium pallidum reduces oxidative stress-induced damage to blood platelets and plasma.

Numerous plants (including clovers) have been widely used in folk medicine for the treatment of different disorders. This in vitro study was designed to examine the antioxidative effects of the clovamide-rich fraction, obtained from aerial parts of Trifolium pallidum, in the protection of blood platelets and plasma against the nitrative and oxidative damage, caused by peroxynitrite (ONOO(-)). Carbonyl groups and 3-nitrotyrosine in blood platelet and plasma proteins were determined by ELISA tests. Thiol groups level was estimated by using 5,5'-dithio-bis(2-nitro-benzoic acid, DTNB). Plasma lipid peroxidation was measured spectrophotometrically as the production of thiobarbituric acid reactive substances. The results from our work indicate that clovamide-rich T. pallidum extract may reveal the protective properties in the prevention against oxidative stress. The presence of clovamide-rich T. pallidum extract (12.5-100 µg/ml) partly inhibited ONOO(-)-mediated protein carbonylation and nitration. All the used concentrations of T. pallidum extract reduced lipid peroxidation in plasma. The antioxidative action of the tested extract in the protection of blood platelet lipids was less effective; the extract at the lowest final concentration (12.5 µg/ml) had no protective effect against lipid peroxidation. The present results indicate that the extract from T. pallidum is likely to be a source of compounds with the antioxidative properties, useful in the prevention against the oxidative stress-related diseases.

Two new compounds from an endophytic fungus Aspergillus sp. HS-05.

Two new compounds, N-[4'-hydroxy-(E)-cinnamoyl]-l-tyrosine methyl ester (1) and methyl 4-methoxy-3-(3'-hydroxy-2'-methyl)propionyloxy-benzoate (2), were isolated from EtOAc extract of the fermentation broth of the endophytic fungus Aspergillus sp. HS-05. Their structures were elucidated by NMR, IR, UV, MS, and CD methods. Compounds 1 and 2 showed no anticancer activities against HL-60 cell lines (both of IC(50)>100 µM) in vitro.

Dibromotyrosine and histamine derivatives from the tropical marine sponge Aplysina sp.

Two new compounds, 3-amino-7,8-dihydroimidazo-[1,5-c]-pyrimidin-5(6H)-one (1) and ethyl 3-(2-amino-1H-imidazol-4-yl)propylcarbamate (2), along with the previously known 7,8-dihydroimidazo-[1,5-c]-pyrimidin-5(6H)-one (3), aeroplysinin-1 (4), dibromoverongiaquinol (5), bisoxazolidinone derivative (6), aerophobins-1 (7) and -2 (8), purealidins J (9) and L, have been isolated from Aplysina sp. from the South China Sea. The structures were elucidated on the basis of 1H, 13C NMR, MS and IR analyses. The histamine-derived alkaloids 1-3 may be unknown bioconversion products of purealidin J (9), aerophobin-2 (8) and aerophobin-1 (7), respectively, when 7-9 are cleaved at C-8-C-9 in reactions of activated chemical defense in Aplysina sponge.

N-(jasmonoyl)tyrosine-derived compounds from flowers of broad beans (Vicia faba).

Two new amide-linked conjugates of jasmonic acid, N-[(3R,7R)-(-)-jasmonoyl]-(S)-dopa (3) and N-[(3R,7R)-(-)-jasmonoyl]-dopamine (5), were isolated in addition to the known compound N-[(3R,7R)-(-)-jasmonoyl]-(S)-tyrosine (2) from the methanolic extract of flowers of broad bean (Vicia faba). Their structures were proposed on the basis of spectroscopic data (LC-MS/MS) and chromatographic properties on reversed and chiral phases and confirmed by partial syntheses. Furthermore, tyrosine conjugates of two cucurbic acid isomers (7, 8) were detected and characterized by LC-MS. Crude enzyme preparations from flowers of V. faba hydroxylated both (+/-)-2 and N-[(3R,7R/3S,7S)-(-)-jasmonoyl]tyramine [(+/-)-4] to (+/-)-3 and (+/-)-5, respectively, suggesting a possible biosynthetic relationship. In addition, a commercial tyrosinase (mushroom) and a tyrosinase-containing extract from hairy roots of red beet exhibited the same catalytic properties, but with different substrate specificities. The conjugates (+/-)-2, (+/-)-3, (+/-)-4, and (+/-)-5 exhibited in a bioassay low activity to elicit alkaloid formation in comparison to free (+/-)-jasmonic acid [(+/-)-1].

[The isolation and identification of a new lignanoside from Selaginella tamariscina (Beauv.) Spring].

AIM: To study the chemical constituents of Selaginella tamariscina (Beauv.) Spring. METHODS: The compounds were isolated and purified by macroporous adsorption resin, Sephadex LH-20 and silica gel column chromatography and identified on the basis of their physicochemical and spectral data. RESULTS: Four compounds were obtained from the n-BuOH fraction of 70% acetone extracts. Their structures were elucidated as (7S, 8R)-7, 8-dihydro-7-(4-hydroxy-3,5-dimethoxyphenyl)-8-hydroxymethyl-[1'-( 7'-hydroxyethyl)-5' methoxyl] benzofuran-4-O-beta-D-glucopyranoside (tamariscinoside C, I), D-mannitol (II), tyrosine (II), shikimic acid (IV). CONCLUSION: Compound I is a new compound, compounds II and III were obtained from the genius for the first time, compound IV was yielded from the plant for the first time.

[A new alkaloid from Opuntia vulgaris].

AIM: To study the chemical constituents of the stems of Opuntia vulgaris Mill(Cactaceae). METHODS: The compounds of Opuntia vulgaris were isolated by chromatography of Amberlite Dowex 50 and silica gel, and identified by means of UV, IR, MS, 1D and 2D NMR. RESULTS: Three compounds were isolated and identified as: opuntin B(I), 4-hydroxyproline(II) and tyrosine(III). CONCLUSION: Compound I is a new alkaloid.

Ortho- and meta-tyrosine formation from phenylalanine in human saliva as a marker of hydroxyl radical generation during betel quid chewing.

The habit of betel quid chewing, common in South-East Asia and the South Pacific islands, is causally associated with an increased risk of oral cancer. Reactive oxygen species formed from polyphenolic betel quid ingredients and lime at alkaline pH have been implicated as the agents responsible for DNA and tissue damage. To determine whether hydroxyl radical (HO.) is generated in the human oral cavity during chewing of betel quid, the formation of o- and m-tyrosine from L-phenylalanine was measured. Both o- and m-tyrosine were formed in vitro in the presence of extracts of areca nut and/or catechu, transition metal ions such as Cu2+ and Fe2+ and lime or sodium carbonate (alkaline pH). Omission of any of these ingredients from the reaction mixture significantly reduced the yield of tyrosines. Hydroxyl radical scavengers such as ethanol, D-mannitol and dimethylsulfoxide inhibited the phenylalanine oxidation in a dose-dependent fashion. Five volunteers chewed betel quid consisting of betel leaf, areca nut, catechu and slaked lime (without tobacco). Their saliva, collected after chewing betel quid, contained high concentrations of p-tyrosine, but no appreciable amounts of o- or m-tyrosine. Saliva samples from the same subjects after chewing betel quid to which 20 mg phenylalanine had been added contained o- and m-tyrosine at concentrations ranging from 1010 to 3000 nM and from 1110 to 3140 nM respectively. These levels were significantly higher (P < 0.005) than those of subjects who kept phenylalanine in the oral cavity without betel quid, which ranged from 14 to 70 nM for o-tyrosine and from 10 to 35 nM for m-tyrosine. These studies clearly demonstrate that the HO. radical is formed in the human oral cavity during betel quid chewing and is probably implicated in the genetic damage that has been observed in oral epithelial cells of chewers.