Dandelion (Taraxacum officinale) flower extract suppresses both reactive oxygen species and nitric oxide and prevents lipid oxidation in vitro

Flavonoids and coumaric acid derivatives were identified from dandelion flower (Taraxacum officinale). Characteristics of chain-breaking antioxidants, such as extended lag phase and reduced propagation rate, were observed in oxidation of linoleic acid emulsion with the addition of dandelion flower extract (DFE). DFE suppressed both superoxide and hydroxyl radical, while the latter was further distinguished by both site-specific and non-specific hydroxyl radical inhibition. DPPH-radical-scavenging activity and a synergistic effect with alpha-tocopherol were attributed to the reducing activity derived from phenolic content of DFE. A significant (p < 0.05) and concentration-dependent, reduced nitric oxide production from acterial-lipopolysaccharide-stimulated mouse macrophage RAW264.7 cells was observed with the addition of DFE. Moreover, peroxyl-radical-induced intracellular oxidation of RAW264.7 cells was inhibited significantly (p < 0.05) by the addition of DFE over a range of concentrations. These results showed that the DFE possessed marked antioxidant activity in both biological and chemical models. Furthermore, the efficacy of DFE in inhibiting both reactive oxygen species and nitric oxide were attributed to its phenolic content.     

崇左金花茶花朵和叶片类黄酮UPLC-Q-TOF-MS分析

以崇左金花茶（Camellia chuangtsoensis）为材料,利用超高效液相色谱-四极杆-飞行时间质谱（UPLC-Q-TOF-MS）联用技术定性定量分析其花朵（花瓣、雄蕊）和叶片（老叶、新叶）中类黄酮成分与含量。结果表明,崇左金花茶中共检测到14种类黄酮成分,木犀草素、木犀草素-7-O-芸香糖苷、槲皮素-3,7-O-二葡萄糖苷、芸香柚皮苷、圣草素和染料木苷为山茶属金花茶组植物中首次发现,其中槲皮素-3,7-O-二葡萄糖苷、芸香柚皮苷、圣草素和染料木苷主要存在于花朵中,木犀草素和木犀草素-7-O-芸香糖苷在花朵中含量高于叶片,雄蕊中高于花瓣;槲皮素-3-O-葡萄糖苷、槲皮素-7-O-葡萄糖苷、槲皮素-3-O-芸香糖苷和山柰酚-3-O-葡萄糖苷为金花茶组植物叶片中首次发现,其叶片中含量远低于花朵,老叶中远低于新叶,雄蕊中远低于花瓣;儿茶素和表儿茶素在花朵中含量高于叶片,雄蕊中高于花瓣;槲皮素和山萘酚在花朵和叶片中含量均较低。崇左金花茶花瓣和雄蕊中含量较高的类黄酮为儿茶素类、木犀草素类和槲皮素类,主要是表儿茶素、木犀草素和槲皮素-3-O-葡萄糖苷;叶片中为儿茶素类和木犀草素类,主要是表儿茶素、木犀草素和木犀草素-7-O-芸香糖苷。崇左金花茶花瓣和雄蕊中儿茶素类、木犀草素类及类黄酮总量均高于叶片,且雄蕊高于花瓣;花瓣和雄蕊中槲皮素类远高于叶片,且花瓣中远高于雄蕊。     

A new hepatoprotective flavone glycoside from the flowers of Onopordum alexandrinum growing in Egypt

A bioactivity-guided fractionation of the ethyl acetate fraction of the flowers of Onopordum alexandrinum L. (Asteraceae) yielded a new flavonoidal glycoside designated as acacetin-7-O-galacturonide (9), alongside with nine known flavonoids; 6-methoxy-apigenin (hispidulin) (1), acacetin (2), apigenin (3), luteolin (4), kaempferol (5), eriodictyol (6), apigenin-7-O-glucoside (7), luteolin-7-O-glucoside (8), and kaempferol-3-O-rutinoside (10). The compounds were assayed for their hepatoprotective activity against CCl4-induced hepatic cell damage in rats and free radical scavenging activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH). Compounds 4, 6, 9, and 10 have not been previously reported from flowers of O. alexandrinum L., and this is the first report of acacetin-7-O-galacturonide (9) in nature which has also shown significant hepatoprotective and free radical scavenging effects. The isolated compounds were identified using different spectroscopic methods (UV, 1H NMR, 13C NMR, HMQC, HMBC, and COSY).     

Anti-inflammatory and antinociceptive potential of major phenolics from Verbascum salviifolium Boiss

The potential effects of flavonoids, phenylethanoid and neolignan glycosides from the aerial parts of Verbascum salviifolium Boiss. were studied in the p-benzoquinone-induced writhing reflex, for the assessment of the antinociceptive activity, and in carrageenan- and PGE1-induced hind paw edema and 12-O-tetradecanoyl-13-acetate (TPA)-induced ear edema models in mice, for the assessment of the anti-inflammatory activity. Through bioassay-guided fractionation and isolation procedures ten compounds from the aqueous extract of the plant, luteolin 7-O-glucoside (1), luteolin 3'-O-glucoside (2), apigenin 7-O-glucoside (3), chrysoeriol 7-O-glucoside (4), beta-hydroxyacteoside (5), martynoside (6), forsythoside B (7), angoroside A (8), dehydrodiconiferyl alcohol-9'-O-beta-D-glucopyranoside (9) and dehydrodiconiferyl alcohol-9-O-beta-D-glucopyranoside (10), were isolated and their structures were elucidated by spectral techniques. Results have shown that 1, 2, 3 and 5 significantly inhibited carrageenan-induced paw edema at a 200 mg/kg dose, while 1, 2 and 5 also displayed anti-inflammatory activity against the PGE1-induced hind paw edema model. However, all the compounds showed no effect in the TPA-induced ear edema model. The compounds 1 and 2 also exhibited significant antinociceptive activity.     

Bioavailability and pharmacokinetics of caffeoylquinic acids and flavonoids after oral administration of Artichoke leaf extracts in humans

Extracts from artichoke leaves are traditionally used in the treatment of dyspeptic and hepatic disorders. Various potential pharmacodynamic effects have been observed in vitro for mono- and dicaffeoylquinic acids (e.g. chlorogenic acid, cynarin), caffeic acid and flavonoids (e.g. luteolin-7-O-glucoside) which are the main phenolic constituents of artichoke leaf extract (ALE). However, in vivo not only the genuine extract constituents but also their metabolites may contribute to efficacy. Therefore, the evaluation of systemic availability of potential bioactive plant constituents is a major prerequisite for the interpretation of in vitro pharmacological testing. In order to get more detailed information about absorption, metabolism and disposition of ALE, two different extracts were administered to 14 healthy volunteers in a crossover study. Each subject received doses of both extracts. Extract A administered dose: caffeoylquinic acids equivalent to 107.0 mg caffeic acid and luteolin glycosides equivalent to 14.4 mg luteolin. Extract B administered dose: caffeoylquinic acids equivalent to 153.8 mg caffeic acid and luteolin glycosides equivalent to 35.2 mg luteolin. Urine and plasma analysis were performed by a validated HPLC method using 12-channel coulometric array detection. In human plasma or urine none of the genuine target extract constituents could be detected. However, caffeic acid (CA), its methylated derivates ferulic acid (FA) and isoferulic acid (IFA) and the hydrogenation products dihydrocaffeic acid (DHCA) and dihydroferulic acid (DHFA) were identified as metabolites derived from caffeoylquinic acids. Except of DHFA all of these compounds were present as sulfates or glucuronides. Peak plasma concentrations of total CA, FA and IFA were reached within 1 h and declined over 24 h showing almost biphasic profiles. In contrast maximum concentrations for total DHCA and DHFA were observed only after 6-7 h, indicating two different metabolic pathways for caffeoylquinic acids. Luteolin administered as glucoside was recovered from plasma and urine only as sulfate or glucuronide but neither in form of genuine glucosides nor as free luteolin. Peak plasma concentrations were reached rapidly within 0.5 h. The elimination showed a biphasic profile.     

In vitro estrogenic activity of Achillea millefolium L.

Isolation and biological characterization of pure compounds was used to identify and characterize estrogenic activity and estrogen receptors (ER) preference in chemical components of Achillea millefolium. This medicinal plant is used in folk medicine as an emmenagogue. In vitro assay, based on recombinant MCF-7 cells, showed estrogenic activity in a crude extract of the aerial parts of A. millefolium. After fractionation of the crude extract with increasing polar solvents, estrogenic activity was found in the methanol/water fraction. Nine compounds were isolated and characterized by HR-MS spectra and 1D- and 2D-NMR techniques. In particular, dihydrodehydrodiconiferyl alcohol 9-O-beta-D-glucopyranoside - a glycosyl-neolignan - was isolated for the first time from the genus Achillea in addition to six flavone derivatives, apigenin, apigenin-7-O-beta-D-glucopyranoside, luteolin, luteolin-7-O-beta-D-glucopyranoside, luteolin-4'-O-beta-D-glucopyranoside, rutin, and two caffeic acid derivatives, 3,5-dicaffeoylquinic acid and chlorogenic acid. Apigenin and luteolin, the most important estrogenic compounds among those tested, were studied for their ability to activate alpha or beta estrogen receptors (ERalpha, ERbeta) using transiently transfected cells. Our results suggest that isolation and biological characterization of estrogenic compounds in traditionally used medicinal plants could be a first step in better assessing further (e.g. in vivo) tests of nutraceutical and pharmacological strategies based on phytoestrogens.     

三种山茶属金花茶组植物花朵类黄酮成分研究

该研究以山茶属金花茶组的金花茶、凹脉金花茶和崇左金花茶为材料,利用超高效液相色谱-四极杆-飞行时间质谱联用技术定性定量分析其花朵中类黄酮成分与含量。结果表明:三种植物中检测到15种类黄酮,其中天竺葵素-3-O-葡萄糖苷、木犀草素、木犀草素-7-O-芸香糖苷、槲皮素-3,7-O-二葡萄糖苷、芸香柚皮苷、圣草素和染料木苷为金花茶组首次发现;槲皮素-3-O-葡萄糖苷、槲皮素-7-O-葡萄糖苷、槲皮素-3-O-芸香糖苷和山萘酚-3-O-葡萄糖苷为凹脉金花茶和崇左金花茶中首次发现。儿茶素、表儿茶素、槲皮素-3-O-葡萄糖苷、槲皮素-7-O-葡萄糖苷、槲皮素-3-O-芸香糖苷和山萘酚-3-O-葡萄糖苷为三个物种主体成分;天竺葵素-3-O-葡萄糖苷为金花茶特有,槲皮素-3,7-O-二葡萄糖苷为崇左金花茶特有;木犀草素-7-O-芸香糖苷主要存在于金花茶和崇左金花茶;木犀草素主要存在于凹脉金花茶和崇左金花茶。类黄酮类型主要为儿茶素类、槲皮素类、木犀草素类和山萘酚类;崇左金花茶中槲皮素类、木犀草素类及类黄酮总量远高于金花茶和凹脉金花茶,凹脉金花茶和崇左金花茶儿茶素类高于金花茶,金花茶和崇左金花茶山萘酚类高于凹脉金花茶。     

In vitro anti-inflammatory effect of Carthamus lanatus L

The anti-inflammatory activity of four total extracts, their fractions and two main constituents (alpha-bisabolol beta-D-fucopyranoside and luteolin 7-O-glucoside) of Carthamus lanatus L. aerial parts, were assessed in vitro by determining the inhibitory effects on induced human neutrophils. The dichloromethane extract and its water-alcoholic part exhibited the most significant inhibitory effects.     

Protective Effects of Flavonoids Against Oxidative Stress Induced by Simulated Microgravity in SH-SY5Y Cells

Many lines of evidence suggest that microgravity results in increased oxidative stress in the nervous system. In order to protect neuronal cells from oxidative damage induced by microgravity, we selected some flavonoids that might prevent oxidative stress because of their antioxidant activities. Among the 20 flavonoids we examined, we found that isorhamnetin and luteolin had the best protective effects against H₂O₂ or SIN-1-induced cytotoxicity in SH-SY5Y cells. Using a clinostat to simulate microgravity, we found that isorhamnetin and luteolin treatment protected SH-SY5Y cells by preventing microgravity-induced increases in reactive oxygen species (ROS), nitric oxide (NO) and 3-nitrotyrosine (3-NT) levels, and a decrease in antioxidant power (AP). Moreover, isorhamnetin and luteolin treatment downregulated the expression of inducible nitric oxide synthase (iNOS), and oxidative stress was significantly inhibited by an iNOS inhibitor in SH-SY5Y cells exposed to simulated microgravity (SMG). These results indicate that isorhamnetin and luteolin could protect against microgravity-induced oxidative stress in neuroblastoma SH-SY5Y cells by inhibiting the ROS-NO pathway. These two flavonoids may have potential for preventing oxidative stress induced by space flight or microgravity.     

Inhibition of nitric oxide synthase expression by a methanolic extract of Crescentia alata and its derived flavonols.

In order to validate the use of Crescentia alata (Bignoniaceae) in the traditional medicine of Guatemala as an antiinflammatory remedy, the methanolic (MeOH) extract has been evaluated in vivo for antiinflammatory activity on carrageenin paw edema in rats and in vitro on Escherichia coli lipopolysaccharide- (LPS)-induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in J774.A1 macrophage cell line. This extract exerted in vivo a significant anti-inflammatory activity at the highest dose tested. The same extract showed in vitro an inhibitory activity on inducible nitric oxide synthase expression and on NO formation in LPS-primed J774.A1 cells. Subsequent fractionation and analysis of the extract has led to the isolation and characterization as major constituents of two flavonol glycosides: quercetin 3-O-alpha-L-rhamnopyranosyl-(1->6)-beta-D-glucopyranoside (rutin) 1, kaempferol 3-O-alpha-L-rhamnopyranosyl-(1->6)-beta-D-glucopyranoside (kaempferol 3-O-rutinoside) 2, and flavonol aglycone, kaempferol 3. Their structures were elucidated by spectral methods. The bioassay-directed analysis of flavonols 1-3 indicated that kaempferol (3) was the most active compound contained in the MeOH extract because it reduced in vitro both NO production and iNOS expression in LPS-primed J774.A1 cells, whereas rutin (1) and kaempferol 3-O-rutinoside (2) showed no significant activity. The MeOH extract and all of flavonoids tested did not show in vitro significant cytotoxic effect in J774.A1 macrophage cell line.     

Deoxypodophyllotoxin content and antioxidant activity of aerial parts of Anthriscus sylvestris Hoffm

Deoxypodophyllotoxin content of the aerial parts of Anthriscus sylvestris Hoffm. growing at different altitudes was evaluated in comparison to the roots. The lignan accumulation in ground parts was at least double compared to aerial ones. In addition antioxidant-guided fractionation of the crude methanol extract of aerial parts was performed with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) test. Active fractions contained mainly luteolin-7-O-glucoside and chlorogenic acid. Antioxidant properties of both crude extract and isolated compounds were also investigated with the Briggs-Rauscher (BR) oscillating reaction. A satisfactory agreement between the results obtained with the two methods was observed.     

Suppressive effects of coumarins from Mammea siamensis on inducible nitric oxide synthase expression in RAW264.7 cells

A methanol extract of the flowers of Mammea siamensis (Calophyllaceae) was found to inhibit nitric oxide (NO) production in lipopolysaccharide-activated RAW264.7 cells. From the extract, two new geranylated coumarins, mammeasins A (1) and B (2), were isolated together with 17 known compounds including 15 coumarins. The structures of 1 and 2 were determined on the basis of their spectroscopic properties as well as of their chemical evidence. Among the isolates, 1 (IC(50)=1.8μM), 2 (6.4μM), surangins B (3, 5.0μM), C (4, 6.8μM), and D (5, 6.2μM), kayeassamins E (7, 6.1μM), F (8, 6.0μM), and G (9, 0.8μM), mammea A/AD (11, 1.3μM), and mammea E/BB (16, 7.9μM) showed NO production inhibitory activity. Compounds 1, 9, and 11 were found to inhibit induction of inducible nitric oxide synthase (iNOS). With regard to mechanism of action of these active constituents (1, 9, and 11), suppression of STAT1 activation is suggested to be mainly involved in their suppression of iNOS induction.     

Antimicrobial evaluation of coumarins and flavonoids from the stems of Daphne gnidium L.

The antimicrobial activity of stems methanol extract from Daphne gnidium L. collected from Sardinia (Italy) was evaluated against 6 strains of standard and clinical isolated gram (+/-) bacteria. The antimicrobial effect on two strains of fungi was also tested. The extract in toto exhibited antibacterial activity against Bacillus lentus and Escherichia coli, but was inactive against fungi. Four coumarins (daphnetin, daphnin, acetylumbelliferone, daphnoretin) and seven flavonoids (luteolin, orientin, isoorientin, apigenin-7-O-glucoside, genkwanin, 5-O-beta-D-primeverosyl genkwanine, 2,5,7,4'-tetrahydroxyisoflavanol) present in the plant extract were also investigated against the same strains of bacteria and fungi assayed for the crude extract. The most active compounds were daphnetin, genkwanin, and 2,5,7,4'-tetrahydroxyisoflavanol.     

The Effect of a Spinal Cord Hemisection on Changes in Nitric Oxide Synthase Pools in the Site of Injury and in Regions Located Far Away from the Injured Site

1. The present study was designed to examine the nitric oxide synthase activities (constitutive and inducible) in the site of injury in response to Th10-Th11 spinal cord hemisection and, to determine whether unilateral disconnection of the spinal cord influences the NOS pools on the contra- and ipsilateral sides in segments located far away from the epicentre of injury.2. A radioassay detection was used to determine Ca²⁺-dependent and inducible nitric oxide synthase activities. Somal, axonal and neuropil neuronal nitric oxide synthase was assessed by immunocytochemical study. A quantitative assessment of neuronal nitric oxide synthase immunoreactivity was made by an image analyser. The level of neuronal nitric oxide synthase protein was measured by the Western blot analysis.3. Our data show the increase of inducible nitric oxide synthase activity and a decrease of Ca²⁺-dependent nitric oxide synthase activity in the injured site analysed 1 and 7 days after surgery. In segments remote from the epicentre of injury the inducible nitric oxide synthase activity was increased at both time points. Ca²⁺-dependent nitric oxide synthase activity had decreased in L5-S1 segments in a group of animals surviving for 7 days. A hemisection performed at thoracic level did not cause significant difference in the nitric oxide synthase activities and in the level of neuronal nitric oxide synthase protein between the contra- and ipsilateral sides in C6-Th1 and L5-S1 segments taken as a whole. Significant differences were observed, but only when the spinal cord was analysed segment by segment, and/or was divided into dorsal and ventral parts. The cell counts in the cervicothoracic (C7-Th1) and lumbosacral (L5-S1) enlargements revealed changes in neuronal nitric oxide synthase immunoreactivity on the ipsilateral side of the injury. The densitometric area measurements confirmed the reduction of somal, neuropil and axonal neuronal nitric oxide synthase immunoreactive staining in the ventral part of rostrally oriented segments.4. Our findings provide evidence that the changes in nitric oxide synthase pools are limited not only to impact zone, but spread outside the original lesion. The regional distribution of nitric oxide synthase activity and neuronal nitric oxide synthase immunoreactivity, measured segment by segment shows that nitric oxide may play a significant role in the stepping cycle in the quadrupeds.     

Attenuation of cardiac mitochondrial dysfunction by melatonin in septic mice

The existence of an inducible mitochondrial nitric oxide synthase has been recently related to the nitrosative/oxidative damage and mitochondrial dysfunction that occurs during endotoxemia. Melatonin inhibits both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase activities, a finding related to the antiseptic properties of the indoleamine. Hence, we examined the changes in inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase expression and activity, bioenergetics and oxidative stress in heart mitochondria following cecal ligation and puncture-induced sepsis in wild-type (iNOS(+/+)) and inducible nitric oxide synthase-deficient (iNOS(-/-)) mice. We also evaluated whether melatonin reduces the expression of inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase, and whether this inhibition improves mitochondrial function in this experimental paradigm. The results show that cecal ligation and puncture induced an increase of inducible mitochondrial nitric oxide synthase in iNOS(+/+) mice that was accompanied by oxidative stress, respiratory chain impairment, and reduced ATP production, although the ATPase activity remained unchanged. Real-time PCR analysis showed that induction of inducible nitric oxide synthase during sepsis was related to the increase of inducible mitochondrial nitric oxide synthase activity, as both inducible nitric oxide synthase and inducible mitochondrial nitric oxide synthase were absent in iNOS(-/-) mice. The induction of inducible mitochondrial nitric oxide synthase was associated with mitochondrial dysfunction, because heart mitochondria from iNOS(-/-) mice were unaffected during sepsis. Melatonin treatment blunted sepsis-induced inducible nitric oxide synthase/inducible mitochondrial nitric oxide synthase isoforms, prevented the impairment of mitochondrial homeostasis under sepsis, and restored ATP production. These properties of melatonin should be considered in clinical sepsis.     

Galactosamine decreases nitric oxide formation in cultured rat hepatocytes: mechanism of suppression

We have shown that nitric oxide production is dramatically decreased in rat primary hepatocyte cultures exposed to galactosamine. Cotreatment of the cells with uridine, which is known to prevent cytotoxicity, was found to also attenuate NO loss. In the present study, two possible mechanisms for the decreased nitric oxide production were examined. First, we examined the possibility that galactosamine could interfere with the uptake of extracellular arginine by the cultured hepatocytes. Cellular uptake of arginine was determined after addition of 14C-arginine at the time of hepatocyte attachment. Uptake of arginine was rapid in control cultures, and both the rate and level of uptake were unchanged by the addition of a cytotoxic concentration of galactosamine (4 mM). In addition, increased concentrations of arginine in the cell culture medium did not ameliorate the galactosamine-induced decrease in production of nitric oxide. Second, we determined whether the synthesis of inducible nitric oxide synthase in the hepatocyte cultures was inhibited by addition of galactosamine. Hepatocyte levels of inducible nitric oxide synthase were determined immunochemically at various times after the addition of galactosamine (4 mM). In control cultures, inducible nitric oxide synthase was detectable at 7 and 24 hours after attachment. In contrast, no nitric oxide synthase protein was detectable at any time in the galactosamine-treated cultures. Furthermore, addition of galactosamine after inducible nitric oxide synthase had already been synthesized (6.5 h after attachment) did not result in suppression of nitric oxide production in the hepatocyte cultures. The present studies suggest that galactosamine suppresses nitric oxide production in hepatocyte cultures by inhibiting synthesis of inducible nitric oxide synthase, rather than by interference in cellular uptake of arginine.     

Presence and activity of nitric oxide synthase isoforms in ischemia-reperfusion-injured flaps

Nitric oxide is produced from the amino acid L-arginine by nitric oxide synthase, which has three known isoforms: (1) endothelial nitric oxide synthase and (2) brain nitric oxide synthase, both of which are constitutive nitric oxide synthase; and (3) inducible nitric oxide synthase. The authors' hypothesis is that after reperfusion injury, endothelial cell dysfunction leads to disruption of nitric oxide synthase-mediated nitric oxide production and that this may in part explain the deleterious effects of ischemia-reperfusion injury on tissue survival and blood reflow in flaps. An experiment was designed to study the effects of ischemia-reperfusion injury on the bioactivity of all three isoforms of nitric oxide synthase. Buttock skin flaps and latissimus dorsi myocutaneous flaps were elevated in eight pigs. Flaps on one side of the animal were randomized to receive 6 hours of arterial ischemia, whereas flaps on the other side served as controls. At 6 hours of ischemia and at 1, 4, and 18 hours after reflow, tissue biopsy specimens were obtained and were processed for both constitutive nitric oxide synthase and inducible nitric oxide synthase enzyme activity on the basis of the L-citrulline assay. In addition, specimens were processed for Western blot analysis of the three isoforms. The authors' results revealed three key findings: first, there was a statistically significant (p < 0.001) decrease in constitutive nitric oxide synthase activity of ischemia-reperfusion-injured flaps as compared with controls in both skin and muscle for all time intervals measured. Second, Western blot analyses of endothelial nitric oxide synthase and brain nitric oxide synthase showed a significant decrease in the signal intensity in ischemic and reperfused tissue as compared with controls. Third, the inducible nitric oxide synthase isoform's activity and protein remained undetectable in both tissue types for all time points measured. The authors' data demonstrated that following ischemia-reperfusion injury in the pig flap model there was a disruption of constitutive nitric oxide synthase expression and activity, which may lead to decreased nitric oxide production. The significant decrease in nitric oxide synthase activity found in the current study may partly explain the mechanism of tissue damage in flaps subjected to ischemia-reperfusion injury. Knowledge of the kinetics of nitric oxide synthase activity under conditions of ischemia-reperfusion injury has important implications for the choice and timing of delivery of therapeutic agents whose goal is to increase the bioavailability of nitric oxide in reperfused tissue.