Protective effects of citrus and rosemary extracts on UV-induced damage in skin cell model and human volunteers.

Ultraviolet radiation absorbed by the epidermis is the major cause of various cutaneous disorders, including photoaging and skin cancers. Although topical sunscreens may offer proper skin protection, dietary plant compounds may significantly contribute to lifelong protection of skin health, especially when unconsciously sun UV exposed. A combination of rosemary and citrus bioflavonoids extracts was used to inhibit UV harmful effects on human HaCaT keratinocytes and in human volunteers after oral intake. Survival of HaCaT cells after UVB radiation was higher in treatments using the combination of extracts than in those performed with individual extracts, indicating potential synergic effects. The combination of extracts also decreased UVB-induced intracellular radical oxygen species (ROS) and prevented DNA damage in HaCaT cells by comet assay and decreased chromosomal aberrations in X-irradiated human lymphocytes. The oral daily consumption of 250 mg of the combination by human volunteers revealed a significant minimal erythema dose (MED) increase after eight weeks (34%, p<0.05). Stronger protection was achieved after 12 weeks (56%, p<0.01). The combination of citrus flavonoids and rosemary polyphenols and diterpenes may be considered as an ingredient for oral photoprotection. Their mechanism of action may deserve further attention.

Effect of the phenolic compounds apigenin and carnosic acid on oral carcinogenesis in hamster induced by DMBA.

OBJECTIVE: To investigate oral carcinogenesis in hamster induced by the topical application of 7,12-dimethyl benzanthracene (DMBA) to evaluate the different lesions produced and the possible preventive effects of the phenolic compounds apigenin (flavone) and carnosic acid (diterpene). MATERIALS AND METHODS: Thirty-two Syrian hamsters were divided into three groups: I: 0.5% DMBA (n = 12); II: 0.5% DMBA + potassium apigenin (n = 8); III: 0.5% DMBA + carnosic acid (n = 12). All the animals were sacrificed after 11 weeks, and a macroscopic and light microscopic study was made of the lesions. RESULTS: The largest number of neoplasms, showing the most aggressive biological behavior, corresponded to the control group. The group treated with potassium apigenin ranked second in tumor incidence, although the tumors were not very aggressive behavior. In the group treated with carnosic acid, only one malignancy was recorded, showing the smallest volume of all the recorded tumor lesions. CONCLUSIONS: Our findings indicate that both potassium apigenin and carnosic acid have chemoprotective effects against carcinogenesis induced by DMBA in hamster.

Chemical genoprotection: reducing biological damage to as low as reasonably achievable levels.

OBJECTIVES: The aim of this study was to evaluate the antioxidant substances present in the human diet with an antimutagenic protective capacity against genotoxic damage induced by exposure to X-rays in an attempt to reduce biological damage to as low a level as reasonably possible. METHODS: Ten compounds were assessed using the lymphocyte cytokinesis-block micronucleus (MN) cytome test. The compounds studied were added to human blood at 25 µM 5 min before exposure to irradiation by 2 Gy of X-rays. RESULTS: The protective capacity of the antioxidant substances assessed was from highest to lowest according to the frequency of the MN generated by X-ray exposure: rosmarinic acid = carnosic acid = δ-tocopherol = l-acid ascorbic = apigenin = amifostine (P < 0.001) > green tea extract = diosmine = rutin = dimetylsulfoxide (P < 0.05) > irradiated control. The reduction in genotoxic damage with the radiation doses administered reached 58%, which represents a significant reduction in X-ray-induced chromosomal damage (P < 0.001). This degree of protection is greater than that obtained with amifostine, a radioprotective compound used in radiotherapy and which is characterised by its high toxicity. CONCLUSION: Several antioxidant substances, common components of the human diet and lacking toxicity, offer protection from the biological harm induced by ionizing radiation. Administering these protective substances to patients before radiological exploration should be considered, even in the case of small radiation doses and regardless of the biological damage expected.

Liposoluble antioxidants provide an effective radioprotective barrier.

Ionising radiation causes the massive generation of reactive oxygen species and induces cellular DNA damage. The antioxidant, protective effects of several compounds against gamma-ray-induced chromosomal damage were determined by the micronucleus test, evaluating the reduction in the frequency of micronuclei in cytokinesis-blocked human lymphocytes. The compounds studied were added to human blood at 25 microM, 5 min before or after irradiation with 2 Gy of caesium-137. The results suggest that different protective mechanisms are operating in each case. When the phenolic compounds are added before gamma-irradiation, their protective antimutagenic activity is based on their scavenging capacity against superoxide anion (O(2)(.-)) and, especially, hydroxyl radical ((.)OH), regardless of whether they are oil- or water-soluble compounds. When the phenolic compounds are added after gamma-irradiation treatment, the protective effect relies on activity against reactive oxygen species present in cells, i.e. lipoperoxy radicals (R(-)OO(.)), which are mainly responsible for continuous chromosomal oxidative damage. In addition, ionising radiation enhances lysosomal enzyme secretion and arachidonate release from membranes through lipo-oxygenase, cyclo-oxygenase and phospholipase activities, thus increasing the inflammatory cell response. Only oil-soluble compounds, such as carnosic acid, carnosol and delta-tocopherol, provide a significant protective antimutagenic activity. The most powerful water-soluble antioxidants lack the capacity to protect against gamma-ray-induced damage. The difference between anti-radical and anti-lipoperoxidant activities could explain the different behaviour of the compounds tested in terms of protecting against the lipid peroxidative processes. This anti-lipoperoxidant activity depends on several factors, but it is clear that only the lipo-antioxidants are effective in protecting human cells against oxidative damage, even when administered after exposure to ionising radiation.

Rosmarinic acid, a photo-protective agent against UV and other ionizing radiations.

Solar UV and other ionizing radiations cause a generation of reactive oxygen species, induce cellular DNA damage and alter skin homeostasis. The use of exogenous antioxidants is increasingly frequents, we attempt to demonstrate that a rosmarinic acid extract acts as photo-protector; both free radical scavenger as an inducer of the body's own endogenous defence mechanisms by regulating tyrosinase activity and stimulating melanin production. Malonyldialdehyde formation (TBARS) was delayed when RA was used. The protection factor was 3.24 times vs AA. TEAC value for RA was 1.6 times vs AA. The radioprotective-antimutagenic effects of RA were measure using the micronucleus test. The level of micronucleous for treatments before irradiation was: RA [14]

Update on uses and properties of citrus flavonoids: new findings in anticancer, cardiovascular, and anti-inflammatory activity.

Significantly, much of the activity of Citrus flavonoids appears to impact blood and microvascular endothelial cells, and it is not surprising that the two main areas of research on the biological actions of Citrus flavonoids have been inflammation and cancer. Epidemiological and animal studies point to a possible protective effect of flavonoids against cardiovascular diseases and some types of cancer. Although flavonoids have been studied for about 50 years, the cellular mechanisms involved in their biological action are still not completely known. Many of the pharmacological properties of Citrus flavonoids can be linked to the abilities of these compounds to inhibit enzymes involved in cell activation. Attempts to control cancer involve a variety of means, including the use of suppressing, blocking, and transforming agents. Suppressing agents prevent the formation of new cancers from procarcinogens, and blocking agents prevent carcinogenic compounds from reaching critical initiation sites, while transformation agents act to facilitate the metabolism of carcinogenic components into less toxic materials or prevent their biological actions. Flavonoids can act as all three types of agent. Many epidemiological studies have shown that regular flavonoid intake is associated with a reduced risk of cardiovascular diseases. In coronary heart disease, the protective effects of flavonoids include mainly antithrombotic, anti-ischemic, anti-oxidant, and vasorelaxant. It is suggested that flavonoids decrease the risk of coronary heart disease by three major actions: improving coronary vasodilatation, decreasing the ability of platelets in the blood to clot, and preventing low-density lipoproteins (LDLs) from oxidizing. The anti-inflammatory properties of the Citrus flavonoids have also been studied. Several key studies have shown that the anti-inflammatory properties of Citrus flavonoids are due to its inhibition of the synthesis and biological activities of different pro-inflammatory mediators, mainly the arachidonic acid derivatives, prostaglandins E 2, F 2, and thromboxane A 2. The anti-oxidant and anti-inflammatory properties of Citrus flavonoids can play a key role in their activity against several degenerative diseases and particularly brain diseases. The most abundant Citrus flavonoids are flavanones, such as hesperidin, naringin, or neohesperidin. However, generally, the flavones, such as diosmin, apigenin, or luteolin, exhibit higher biological activity, even though they occur in much lower concentrations. Diosmin and rutin have a demonstrated activity as a venotonic agent and are present in several pharmaceutical products. Apigenin and their glucosides have been shown a good anti-inflammatory activity without the side effects of other anti-inflammatory products. In this paper, we discuss the relation between each structural factor of Citrus flavonoids and the anticancer, anti-inflammatory, and cardiovascular protection activity of Citrus flavonoids and their role in degenerative diseases.

Apigenin inhibits platelet adhesion and thrombus formation and synergizes with aspirin in the suppression of the arachidonic acid pathway.

Previous studies using washed platelets demonstrated that certain flavonoids inhibit platelet function through several mechanisms including blockade of TxA(2) receptors (TPs). We aimed to analyze the binding capacity of flavonoids to TPs in platelet rich plasma (PRP), investigated their effect in flowing blood, and evaluated the ability of apigenin to improve the efficacy of aspirin in the inhibition of platelet aggregation. The binding of flavonoids to TPs in PRP was explored using binding assays and the TP antagonist [ (3)H]SQ29548. Effects of flavonoids on platelet adhesion were assessed using arterial subendothelium with annular plate perfusion chambers, and global evaluation of apigenin on high-shear-dependent platelet function was determined by the PFA-100. To evaluate the ability of apigenin to potentiate the effect of aspirin, arachidonic acid-induced platelet aggregation was measured prior to and after consumption of subaggregatory doses of aspirin in the presence or absence of apigenin. Binding assays revealed that apigenin was an efficient competitor of [ (3)H]SQ29548 binding to PRP ( K i = 155.3 +/- 65.4 microM), and perfusion studies showed that apigenin, genistein, and catechin significantly diminished thrombus formation when compared to control (26.2 +/- 3.8, 33.1 +/- 5.2, and 26.2 +/- 5.2 vs 76.6 +/- 2.6%, respectively; p < 0.05). Apigenin, similarly to the TP antagonist SQ29548, significantly prolonged collagen epinephrine-induced PFA-100 closure time in comparison to the control and, when added to platelets that had been exposed in vivo to aspirin, potentiated its inhibitory effect on platelet aggregation. The inhibitory effect of some flavonoids in the presence of plasma, particularly apigenin, might in part rely on TxA(2) receptor antagonism. There is a clear increase in the ex vivo antiplatelet effect of aspirin in the presence of apigenin, which encourages the idea of the combined use of aspirin and certain flavonoids in patients in which aspirin fails to properly suppress the TxA(2) pathway.

Beneficial action of Citrus flavonoids on multiple cancer-related biological pathways.

Attempts to control cancer involve a variety of means, including the use of suppressing, blocking and transforming agents. Suppressing agents prevent the formation of new cancers from pro-carcinogens, blocking agents prevent carcinogenic compounds from reaching critical initiation sites, while transformation agents act to facilitate the metabolism of carcinogenic components into less toxic materials or to prevent their biological actions. Flavonoids can act as all three types of agent. Epidemiological and animal studies suggest that flavonoids have a protective effect against cardiovascular diseases and some types of cancer. Although flavonoids have been studied for about 50 years, the cellular mechanisms involved in their biological action are still not completely understood. In recent years, experimental studies have provided growing evidence supporting the beneficial action of flavonoids on multiple cancer-related biological pathways (carcinogen bio-activation, cell-signaling, cell cycle regulation, angiogenesis and inflammation). Within the last decade, reports on flavonoid activity have largely associated with enzyme inhibition and anti-proliferative activity. Many of these studies have pointed to a structural-functional relationship, in that the antioxidant, enzyme-inhibition or antiproliferative activities of flavonoids are dependent on particular structural motifs. Our own studies have shown that structural factors would explain the antioxidant, antiproliferative and antimetastatic properties of some citrus flavonoids. In this paper we discuss the relation between each structural factor and the anticancer activity of Citrus flavonoids.

Radioprotective-antimutagenic effects of rosemary phenolics against chromosomal damage induced in human lymphocytes by gamma-rays.

The radioprotective effects of carnosic acid (CA), carnosol (COL), and rosmarinic acid (RO) against chromosomal damage induced by gamma-rays, compared with those of L-ascorbic acid (AA) and the S-containing compound dimethyl sulfoxide (DMSO), were determined by use of the micronucleus test for antimutagenic activity, evaluating the reduction in the frequency of micronuclei (MN) in cytokinesis-blocked cells of human lymphocytes before and after gamma-ray irradiation. With treatment before gamma-irradiation, the most effective compounds were, in order, CA > RO > or = COL > AA > DMSO. The radioprotective effects (antimutagenic) with treatment after gamma-irradiation were lower, and the most effective compounds were CA and COL. RO and AA presented small radioprotective activity, and the sulfur-containing compound DMSO lacked gamma-ray radioprotection capacity. Therefore, CA and COL are the only compounds that showed a significant antimutagenic activity both before and after gamma-irradiation treatments. These results are closely related to those reported by other authors on the antioxidant activity of the same compounds, and the degree of effectiveness depends on their structure. Furthermore, the results for treatments before and after gamma-ray irradiation suggest the existence of different radioprotective mechanisms in each case.

The effect of the flavonoid diosmin, grape seed extract and red wine on the pulmonary metastatic B16F10 melanoma.

OBJECTIVE: To study the effect of different phenolic compounds and red wine on pulmonary metastatic melanoma. METHODS: Swiss mice were inoculated with 500000 melanocytes B16F10 and given oral doses of diosmin, grape seed extract (GSE) and red wine. A macroscopic count was made of the metastatic nodules on the lung surface and a microscopic study by image analysis of five sections, calculating the implantation percentage and tumoral growth and invasion indices. RESULTS: Macroscopically, the group treated with diosmin showed the greatest reduction (52%) in the number of metastatic nodules compared with the control group, which was treated with ethanol, while GSE and red wine caused decreases of 26.07 and 28.81%, respectively. Microscopically, there was a decrease in the implantation percentage after the administration of diosmin (79.4%) and red wine (20.19%), and an increase of 2.12% after the administration of GSE, all relative to the ethanol-treated control. As regards the growth index, diosmin produced a reduction of 67.44% and red wine a reduction of 20.62%, while GSE again produced an increase (25.33%). The reductions in the invasion index were 45.23, 31.65 and 17.57% with diosmin, GSE and red wine, respectively. CONCLUSIONS: Diosmin originated the greatest reduction in pulmonary metastases, both at the macroscopic and microscopic levels.

Flavonoids inhibit platelet function through binding to the thromboxane A2 receptor.

BACKGROUND: Dietary flavonoids are known for their antiplatelet activity resulting in cardiovascular protection, although the specific mechanisms by which this inhibition occurs has not been fully established. OBJECTIVE: The aim of this study was to investigate the interaction of nine flavonoids representative of various chemical classes, with platelet responses dependent on thromboxane A(2) (TxA(2)) generation and on receptor antagonism, and to analyze the structural requirements for such effects. METHODS: The effect of several types of flavonoids on platelet aggregation, serotonin release, and TxA(2) generation was investigated. Competitive radioligand binding assays were used to screen for affinity of these compounds to TxA(2) receptors. RESULTS: Flavones (apigenin and luteolin) and isoflavones (genistein) abrogated arachidonic acid and collagen-induced platelet responses, such as aggregation and secretion, with a less substantial effect on TxA(2) synthesis. These compounds were identified as specific ligands of the TxA(2) receptor in the micromol L(-1) range, this effect accounting for antiplatelet effects related to stimulation with those agonists. Tight binding of flavonoids to the human TxA(2) receptor relies on structural features such as the presence of the double bond in C2-C3, and a keto group in C4. CONCLUSIONS: The inhibition by specific flavonoids of in vitro platelet responses induced by collagen or arachidonic acid seems to be related, to a great extent, to their ability to compete for binding to the TxA(2) receptor. Therefore, antagonism of this TxA(2) receptor may represent an additional mechanism for the inhibitory effect of these compounds in platelet function.

Effects of several polyhydroxylated flavonoids on the growth of B16F10 melanoma and Melan-a melanocyte cell lines: influence of the sequential oxidation state of the flavonoid skeleton.

The response of B16F10 melanoma and Melan-a melanocyte cell lines to treatment with five polyhydroxylated flavonoids and gallic acid, after 24 and 72 h of exposure, was determined, and the relationship between any antiproliferative effects observed and the chemical structure is discussed. After 24 h, none of the studied compounds showed significant cytotoxic activity in the B16F10 cell line, whereas compounds with an adjacent trihydroxylated substitution pattern did affect the viability of the Melan-a cell line. After 72 h of exposure, myricetin, baicalein and gallic acid significantly inhibited both B16F10 and Melan-a cell cultures, whereas luteolin and quercetin had only a moderate effect. Eriodictyol only had an effect on Melan-a cell viability, which was reduced slightly. These results suggest that the presence of a C2-C3 double bond and three adjacent hydroxyl groups in the flavonoid A- or B-rings confers greater antiproliferative activity to the flavonoid.

Radioprotective effects in vivo of phenolics extracted from Olea europaea L. leaves against X-ray-induced chromosomal damage: comparative study versus several flavonoids and sulfur-containing compounds.

The radioprotective effects of a polyphenolic extract of Olea europaea L. leaves (OL); the flavonoids diosmin and rutin, which are widely used as pharmaceuticals; and the sulfur-containing compounds dimethylsulfoxide (DMSO) and 6-n-propyl-2-thiouracil (PTU) were determined by using the micronucleus test for anticlastogenic activity, evaluating the reduction of the frequency of micronucleated polychromatic erythrocytes (MnPCEs) in bone marrow of mouse before and after X-ray irradiation. With treatment before X-irradiation, the most effective compounds were, in order, rutin > DMSO > OL > PTU > diosmin. These results showed, for the polyphenols studied, a linear correlation (r(2) = 0.965) between anticlastogenic activity and antioxidant capacity. The magnitude of protection with treatment after X-irradiation were lower, and the most effective compounds were, in order, OL > diosmin > rutin; DMSO and PTU lacked radioprotective activity. Therefore, OL is the only substance that showed a significant anticlastogenic activity both before and after X-ray irradiation treatments. Structurally, the free oxygen radicals and lipoperoxyradicals scavenging capacity and, consequently, the anticlastogenic activity of these polyphenolic compounds are based principally on the presence of specific functional groups, mainly catechol groups (rutin, oleuropein, hydroxytyrosol, verbascoside, luteolin), that also increase the stability of the aroxyl-polyphenol radical generated in the above processes.

Effects of several flavonoids on the growth of B16F10 and SK-MEL-1 melanoma cell lines: relationship between structure and activity.

Although flavonoids seem to be capable of acting at all stages of the carcinogenic process, little information is available on their action in melanoma cell lines. The aim of this study was to assess the response of B16F10 and SK-MEL-1 melanoma cell lines to treatment with six different flavonoids after 24 and 72 h of exposure and to relate the response to their structure. We then compared the findings with those for melphalan treatment. When cultures were treated for 24 h, only slight inhibition at the highest concentrations (25 and 50 microM) of tangeretin and luteolin were observed, whereas melphalan caused a dose-related inhibition of growth at all concentrations. Quercetin, hesperetin, 7,3'-dimethylhesperetin and eriodictyol did not produce any effect at 24 h on B16F10 or SK-MEL-1 cells, results which point to the low toxicity of flavonoids. After 72 h of exposure culture growth was inhibited by 7,3'-dimethylhesperetin at 50 microM, but lower concentrations had no effect. Tangeretin was the most effective of the flavonoids in inhibiting B16F10 and SK-MEL-1 cell growth, showing a clear dose-response curve after 72 h. These results suggest that the absence of the C2-C3 double bond on hydroxylated flavonoids results in a loss of effect on both the cell lines, while the higher activity of tangeretin compared with 7,3'-dimethylhesperetin suggests that the presence of at least three adjacent methoxyl groups confers a more potent antiproliferative effect.

Modulation of the biosynthesis of some phenolic compounds in Olea europaea L. fruits: their influence on olive oil quality.

The phenolic composition of olive fruits (Olea europaea L.) (cv. Picual, Villalonga, Alfafarenca, and Cornicabra) grown in different areas of Spain was studied by high performance liquid chromatography-mass spectrometry. Different levels of tyrosol, catechin, p-coumaric acid, rutin, luteolin, and oleuropein were observed in the different varieties analyzed. Treating the fruit with 0.3% Brotomax 50 days after anthesis had a beneficial effect on fruit size, oil content, levels of polyphenolic compounds, and Trolox-equivalent antioxidant activity (TEAC) in all the varieties analyzed.

Improved water solubility of neohesperidin dihydrochalcone in sweetener blends.

Significant technological advantages in terms of sweetness synergy and hence cost-saving can be obtained if neohesperidin dihydrochalcone (NHDC) is used in sweetener blends with other intense or bulk sweeteners. The combination of NHDC with sodium saccharin or sodium cyclamate is an excellent method to improve the water solubility at room temperature of NHDC. In the case of NHDC-sodium saccharin, two different methods for blend preparation, a simple mixture and a cosolubilized mixture, can be used, with similar results obtained for solubility and solution stability properties. To improve temporally the water solubility of NHDC in combination with sodium cyclamate, it is absolutely necessary to prepare cosolubilized blends.

Radioprotective Effects Against Chromosomal Damage Induced in Human Lymphocytes by gamma-Rays as a Function of Polymerization Grade of Grape Seed Extracts.

The quantitative distribution of flavan-3-ols was determined using high-performance liquid chromatography in several grape seed extracts (GSEs). In all GSEs, polymers of four or more carbon units were the group of procyanidins present in the highest concentration, the real quantity ranging between 60% and 99.5%. In a previous paper we established a relation between antioxidant and anticlastogenic activity of GSEs. A higher grade of polymerization in GSEs allows the existence of a higher number of conjugated structures and higher antioxidant activity. The radioprotective effects of GSEs with various grades of polymerization were determined by use of the micronucleus test for anticlastogenic activity, evaluating the reduction in the frequency of micronuclei in cytokinesis-blocked cells of human lymphocytes exposed to gamma-rays. The radioprotective efficiency of GSEs was according to their grade of polymerization: GSE3 > GSE2 > GSE1 > dimethylsulfoxide. The higher antioxidant capacity and anticlastogenic activity of GSEs can be explained, structurally, by the high number of conjugated structures between the catechol groups in the B-rings and the 3-OH free groups of the polymeric polyphenolic skeleton and, in addition, by the stability of the aroxyl flavonoid radical generated in the processes.

Antioxidant activity and radioprotective effects against chromosomal damage induced in vivo by X-rays of flavan-3-ols (Procyanidins) from grape seeds (Vitis vinifera): comparative study versus other phenolic and organic compounds.

The quantitative distribution of several flavan-3-ols was determined using HPLC in a grape (Vitis vinifera) seed extract (GSE) of four cultivars grown in the region of Murcia. Polymer >/= C(4) units made up the largest group of procyanidins in the GSE (90.92%, expressed as HPLC % area). The antioxidant activity of GSE and other reference compounds was investigated by measuring their ability to scavenge the ABTS(*)(+) radical cation (TEAC). The most effective compounds were, in order: GSE > rutin > (+)-catechin > diosmin >/= ascorbic acid. The radioprotective effects of GSE and other reference compounds were determined by using the micronucleus test for anticlastogenic activity, any reduction of the frequency of micronucleated polychromatic erythrocytes (MnPCEs) being evaluated in the bone marrow of mouse exposed to X-rays. The most effective compounds were, in order: GSE > rutin > dimethyl sulfoxide (DMSO) > ascorbic acid > 6-n-propyl-2-thiouracil-6c (PTU) > diosmin. The higher ABTS(*)(+) scavenging capacity and anticlastogenic activity of GSE can be explained, structurally, by the high number of conjugated structures between the catechol groups in the B-rings and the 3-OH free groups of the polymeric polyphenolic skeleton and, in addition, by the stability of the aroxyl flavonoid radical generated in the above processes.

Distribution of flavone glycoside diosmin in Hyssopus officinalis plants: changes during growth.

A study of the flavonoid composition of Hyssopus officinalis L. (Lamiaceae) plants using high-performance liquid chromatography and NMR spectroscopy revealed the presence of diosmin as the major flavone. The maximum levels of this secondary compound are located in sepals and leaves, which represent 51 and 40.5%, respectively, of the total content of diosmin in whole plant. The presence of isoferulyl D-glucose ester in this plant material was also revealed.