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Abstracts

Naringin: 27 Research Abstracts

1. J Biol Chem. 1995 Mar 17;270(11):5830-8. Protection by naringin and some other flavonoids of hepatocytic autophagy and endocytosis against inhibition by okadaic acid. Gordon PB, Holen I, Seglen PO. Department of Tissue Culture, Norwegian Radium Hospital, Montebello, Oslo.

In isolated rat hepatocytes, the protein phosphatase inhibitor okadaic acid exerts a strong inhibitory effect on autophagy, which can be partially overcome by certain protein kinase inhibitors like the isoflavone genistein. To see if other, more specific okadaic acid antagonists could be found among the flavonoids, 55 different flavonoids were tested for their effect on okadaic acid-inhibited autophagy, measured as the sequestration of electroinjected [3H]raffinose. Naringin (naringenin 7-hesperidoside) and several other flavanone and flavone glycosides (prunin, neoeriocitrin, neohesperidin, apiin, rhoifolin, kaempferol 3-rutinoside) offered virtually complete protection against the autophagy-inhibitory effect of okadaic acid. Unlike genistein, these compounds had little or no autophagy-inhibitory effect of their own. Their innocuousness appeared to be related to glycosylation, because the corresponding aglycones (naringenin, eriodictyol, hesperetin, apigenin, kaempferol) were all inhibitory, in particular apigenin (80% inhibition at 100 microM). Naringin, the most potent okadaic acid-antagonistic flavonoid, gave half-maximal protection at 5 microM and maximal effect at 100 microM. Naringin also prevented the okadaic acid-induced inhibition of endogenous, autophagic lysosomal protein degradation and of receptor-mediated asialoglycoprotein uptake and degradation. Naringin and other okadaic acid-antagonistic flavonoids may be useful tools in the study of intracellular protein phosphorylation and could have potential therapeutic value as protectants against pathological hyperphosphorylations, environmental toxins, or side effects of chemotherapeutic drugs.

Alcohol – effects on **

2. Life Sci. 2003 Jul 4;73(7):933-46. Role of naringin supplement in regulation of lipid and ethanol metabolism in rats. Seo HJ, Jeong KS, Lee MK, Park YB, Jung UJ, Kim HJ, Choi MS. Department of Food Science and Nutrition, Kyungpook National University, 1370 Sankyuk Dong Puk-ku, Daegu 702-701, South Korea.

The current study was performed to investigate the effect of naringin supplements on the alcohol, lipid, and antioxidant metabolism in ethanol-treated rats. Male Sprague-Dawley rats were randomly divided into six groups (n = 10) based on six dietary categories: ethanol and naringin-free, ethanol (50 g/L) plus low-naringin (0.05 g/L), ethanol plus high-naringin (0.125 g/L), and three corresponding pair-fed groups. The pair-fed control rats received an isocaloric diet containing dextrin-maltose instead of ethanol for 5 wks. Among the ethanol treated groups, the naringin supplements significantly lowered the plasma ethanol concentration with a simultaneous increase in the ADH and/or ALDH activities. However, among the ethanol-treated groups, naringin supplementation resulted in a significant decrease in the hepatic triglycerides and plasma and hepatic total cholesterol compared to that in the naringin-free group. Naringin supplementation significantly increased the HDL-cholesterol and HDL-C/total-C ratio, while lowering the AI value among the ethanol-treated groups. Hepatic lipid accumulation was also significantly reduced in the naringin-supplemented groups compared to the naringin-free group among the ethanol-treated groups, while no differences were found among the pair-fed groups. Among the ethanol-treated groups, the low-naringin supplementation resulted in a significant decrease in the levels of plasma and hepatic TBARS, whereas it resulted in higher SOD and GSH-Px activities and gluthathion levels in the liver. Accordingly, naringin would appear to contribute to alleviating the adverse effect of ethanol ingestion by enhancing the ethanol and lipid metabolism as well as the hepatic antioxidant defense system.

Anti-Inflammatory **

3. J Lipid Res. 2003 Feb;44(2):380-7. Epub 2002 Nov 04. Modulation by flavonoids of PAF and related phospholipids in endothelial cells during oxidative stress. Balestrieri ML, Castaldo D, Balestrieri C, Quagliuolo L, Giovane A, Servillo L. Department of Biochemistry and Biophysics, Second University of Naples, Italy.

PAF-dependent transacetylase (TA) modifies the functions of platelet-activating factor (PAF), a potent inflammatory lipid, either by transferring the acetyl group from PAF to lysophospholipids (TAL activity), or to sphingosine (TAS activity) or by hydrolyzing PAF (acetylhydrolase activity). In stimulated endothelial cells (EC), TAL activity contributes to the synthesis of acyl-PAF, an acyl analog of PAF, that antagonizes PAF functions and is regulated by the cellular redox state. In this study, we investigated the possible involvement of TA in the flavonoid antioxidant mechanism(s) during oxidative stress in EC induced by hydrogen peroxide. The treatment of EC with H2O2 resulted in 4-fold increase of the acetyl-CoA acetyltransferase activity (AT), that is responsible for PAF biosynthesis, while the TAL activity increased only by 53%. However, the preincubation of H2O2-treated EC with the flavonoids hesperedin, naringin, and quercetin strongly inhibited AT activity and activated TAL by 290%, 340%, and 250%, respectively. The induction of TAL activity resulted in enhanced biosynthesis of 1-acyl-2-[3H]acetyl-PAF in intact EC and was related to the flavonoid structure. These findings suggest that TAL is involved in the Anti-Inflammatory **

Cancer **

4. Biochem Pharmacol. 2003 Oct 1;66(7):1139-50. Rutinoside at C7 attenuates the apoptosis-inducing activity of flavonoids. Chen YC, Shen SC, Lin HY. Graduate Institute of Pharmacognosy, Taipei Medical University, 250 Wu-Hsing Street, ROC, Taipei, Taiwan

Rutinoside (rhamnoglucoside; rhamnose+glucose) addition has been examined extensively in the metabolism of flavonoids, however the effect of rutinoside on apoptosis-inducing activity of flavonoids is still unknown. In the present study, the two pairs of flavonoids of hesperetin (HT) and hesperidin (HD; HT-7-rutinose), and naringenin (NE) and naringin (NE-7-rutinose), were used to study their apoptosis-inducing activities in HL-60 cells. Both HD and NI are flavonoids which contain a rutinoside at the C7 of HT and NE, respectively. Results of the MTT assay showed that HT and NE, but not HD and NI, exhibited significant cytotoxic effect in HL-60 cells, accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including an increase in DNA ladder intensity, morphological changes, appearance of apoptotic bodies, and an increase in hypodiploid cells by flow cytometry analysis. HT and NE, but not HD and NI, caused rapid and transient induction of caspase-3/CPP32 activity, but not caspase-1 activity, according to the cleavage of caspase-3 substrates poly(ADP-ribose) polymerase and D4-GDI proteins, the appearance of cleaved caspase-3 fragments detected in HT- or NE-, but not in HD- or NI-treated HL-60 cells. A decrease in the anti-apoptotic protein, Mcl-1, was detected in HT- and NE-treated HL-60 cells, whereas other Bcl-2 family proteins including Bax, Bcl-2, Bcl-XL, and Bag remained unchanged. The caspase-3 inhibitor, Ac-DEVD-FMK, but not the caspase-1 inhibitor, Ac-YVAD-FMK, attenuated HT- and NE-induced cell death. Interestingly, neither HT nor NE induced apoptosis in the mature monocytic cell line THP-1 and primary human polymorphonuclear cells, as characterized by a lack of DNA ladders, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and Mcl-1 decrease, compared with those in HL-60 cells. In addition, the rutinoside group in HD and NI was removed by hesperidinase and naringinase, accompanied by the production of HT and NE, respectively, according to HPLC analysis. Accordingly, hesperidinase and naringinase digestion recovered the apoptosis-inducing activity of HD and NI in HL-60 cells. Our experiments provide the first evidence to suggest that rutinoside in flavonoids prevents the induction of apoptosis, and that activation of the traditional caspase-3 cascade participates in HT- and NE-induced apoptosis.

5. J Pharmacol Sci. 2003 Jun;92(2):166-70. Effects of naringin on hydrogen peroxide-induced cytotoxicity and apoptosis in p388 cells. Kanno S, Shouji A, Asou K, Ishikawa M. Department of Pharmacology and Toxicology, Cancer Research Institute, Tohoku Pharmaceutical University. syu-kan@tohoku-pharm.ac.jp

Flavonoids are widely recognized as naturally occurring antioxidants. Naringin (NG) is one of the flavonoid components in citrus fruits such as grapefruit. Hydrogen peroxide (H2O2) causes cytotoxicity through oxidative stress and apoptosis. In this paper, we examined the effects of NG on H2O2-induced cytotoxicity and apoptosis in mouse leukemia P388 cells. Cytotoxicity was determined by mitochondrial activity (MTT assay). Apoptosis and DNA damage were analyzed by measuring chromatin condensation and Comet assay (alkaline single cell gel electrophoresis), respectively. H2O2-induced cytotoxicity was significantly attenuated by NG or the reduced form of glutathione (GSH), a typical intracellular antioxidant. NG suppressed chromatin condensation and DNA damage induced by H2O2. These results indicate that NG from natural products is a useful drug having antioxidant and anti-apoptopic properties.

6. Anticancer Res. 2000 Sep-Oct;20(5A):3323-9. Effects of citrus phytochemicals on liver and lung cytochrome P450 activity and on the in vitro metabolism of the tobacco-specific nitrosamine NNK. Bear WL, Teel RW. Department of Physlology and Pharmacology, Loma Linda University, School of Medicine, CA 92350, USA.

NNK is a potent environmental carcinogen to which both smokers and nonsmokers are exposed. The response to NNK may be affected by factors including nutrition. We investigated the effects of five citrus phytochemicals on the in vitro metabolism of the tobacco-specific nitrosamine NNK and on the dealkylation of methoxyresorufin (MROD) and pentoxyresorufin (PROD) in liver and lung microsomes of the Syrian golden hamster. In the NNK metabolism experiments in vitro incubations contained 3 microCi [5-H3] NNK, 0.5 mg microsomal protein and 0.5 mumole of the citrus phytochemical diosmin, naringin, naringenin, quercetin or rutin. In the dealkylation studies incubations contained 0.5 microM methoxyresorufin or pentoxyresorufin, 0.5 mg microsomal protein and 0.5 mumole of citrus phytochemical. The major NNK metabolism pathway in hamster liver microsomes was NNK-reduction while in lung microsomes it was alpha-hydroxylation. The alpha-hydroxylation pathway produces metabolic products that methylate and pyridyloxobutylate DNA. Naringenin, a metabolite of naringin, and quercetin were the most potent inhibitors of alpha-hydroxylation of NNK in both liver and lung microsomes. This inhibition correlated with a potent inhibition of MROD and PROD activity in liver but not in lung microsomes. The metabolic activation of NNK is associated with cytochrome P450 isoforms 1A1, 1A2, 2B1, 2D6 and 2E1. Our results suggest that naringenin and quercetin from citrus fruits inhibit the activity of cytochrome P450 (CYP) isoforms that activate NNK and may afford protection against NNK-induced carcinogenesis.

Antioxidant properties / ROS **

7. Pharmazie. 2003 Aug;58(8):564-6. Naringin and naringenin inhibit nitrite-induced methemoglobin formation. Kumar MS, Unnikrishnan MK, Patra S, Murthy K, Srinivasan KK. Department of Pharmacology, College of Pharmaceutical Sciences, Manipal, India.

Naringin and naringenin protect hemoglobin from nitrite-induced oxidation to methemoglobin. The protection is not observed when naringin and naringenin are added after the autocatalytic stage of the oxidation of hemoglobin by nitrite. The ability of naringin and naringenin to scavenge oxygen free radicals may be responsible for the action because superoxide, hydroxyl and other free radicals are implicated in promoting the autocatalytic stage of oxidation of hemoglobin by nitrite. Both compounds showed less ability to protect intact erythrocytes suggesting that they may not cross the erythrocyte membrane in sufficient amounts. Naringenin was more effective than naringin, probably because of the extra phenolic group in the aglycone.

8. Prostaglandins Leukot Essent Fatty Acids. 2003 Jul;69(1):73-7. Effects of flavonoids on the susceptibility of low-density lipoprotein to oxidative modification. Safari MR, Sheikh N. Department of Biochemistry and Nutrition, School of Medicine, Hamadan University of Medical Sciences and Health Services, Hamadan, Iran. safari@umsha.ac.ir

Dietary flavonoid intake has been reported to be inversely associated with the incidence of coronary artery disease. To clarify the possible role of flavonoids in the prevention of atherosclerosis, we investigated the effects of some of these compounds on the susceptibility of low-density lipoprotein (LDL) to oxidative modification. In this study, six flavonoids, "apigenin, genistein, morin, naringin, pelargonidin and quercetin", were added to plasma and incubated for 3h at 37 degrees C. Then, the LDL fraction was separated by ultracentrifugation. The oxidizability of LDL was estimated by measuring conjugated diene (CD), lipid peroxides and thiobarbituric acid-reactive substances (TBARS) after cupric sulfate solution was added. We showed that among flavonoids used, quercetin and morin significantly (P<0.01 by ANOVA) and dose-dependently prolonged the lag time before initiation of oxidation reaction. Also, these two flavonoids suppressed the formation of lipid peroxides and TBARS more markedly than others. Their ability to prolong lag time and suppression of lipid peroxides and TBARS formation resulted to be in the following order: quercetin>morin>pelargonidin>genistein>naringin>apigenin. LDL exposed to flavonoids in vitro reduced oxidizability. These findings show that flavonoids may have a role in ameliorating atherosclerosis.

9. Mol Cell Biochem. 2003 Apr;246(1-2):193-6. Anti-oxidant effect of flavonoids on the susceptibility of LDL oxidation. Naderi GA, Asgary S, Sarraf-Zadegan N, Shirvany H. Department of Biochemistry, Isfahan Cardiovascular Research Center, Amin Hospital, Isfahan University of Medical Sciences, Isfahan, Iran. isfcarvasrc@hotmail.com

In vitro studies have demonstrated increased atherogenicity of oxidized low-density lipoprotein (ox-LDL) compared to native LDL. Oxidative modification of LDL alters its structure allowing LDL to be taken up by scavenger receptors on macrophage, endothelial, and smooth muscle cells, leading to the formation of lipid-laden foam cells, the hallmark of early atherosclerotic lesions. The susceptibility of LDL to in vitro oxidation was assessed essentially by the technique described by Esterbauer et al. LDL oxidation were monitored by change in 234-absorbance in the presence and absence of pure flavonoids. Morin, genistein, apigenin and biochanin A, naringin and quercetin were used at different concentration. These flavonoids significantly inhibit in vitro LDL oxidation, genistein, morin and naringin have stronger inhibitory activity against LDL oxidation than biochanin A or apigenin. This study show that flavonoids prevent in vitro LDL oxidation and probably would be important to prevent atherosclerosis.

HEMATOCRITS – NORMALIZATION **

10. Int J Vitam Nutr Res. 1988;58(4):414-7. Ingestion of grapefruit lowers elevated hematocrits in human subjects. Robbins RC, Martin FG, Roe JM. Food Science and Human Nutrition Department, IFAS, University of Florida, Gainesville.

This study was based on in vitro observations that naringin isolated from grapefruit induced red cell aggregation and evidence that clumped red cells are removed from the circulation by phagocytosis. The effect on hematocrits of adding grapefruit to the daily diet was determined using 36 human subjects (12 F, 24 M) over a 42-day study. The hematocrits ranged from 36.5 to 55.8% at the start and 38.8% to 49.2% at the end of the study. There was a differential effect on the hematocrit. The largest decreases occurred at the highest hematocrits and the effect decreased on the intermediate hematocrits; however, the low hematocrits increased. There was no significant difference between ingesting 1/2 or 1 grapefruit per day but a decrease in hematocrit due to ingestion of grapefruit was statistically significant at the p less than 0.01 level.

ANIMAL **

11. Drug Metabol Drug Interact. 2000;17(1-4):351-63. Effect of the grapefruit flavonoid naringin on pharmacokinetics of quinine in rats. Zhang H, Wong CW, Coville PF, Wanwimolruk S. Natural & Complementary Medicine Research, New Zealand 's National School of Pharmacy, University of Otago, Dunedin. hu.zhang@stonebow.otago.ac.nz

The effect of the grapefruit flavonoid naringin, an inhibitor of CYP3A4, on the pharmacokinetics of quinine in rats after oral or intravenous (i.v.) dosing of quinine was investigated. Female Wistar rats (wt 190-220 g) were used in two separate studies, i.e. oral and i.v. administration of quinine. The animals were divided into two groups, one served as control and the other group was pretreated with 25 mg/kg naringin once a day for 7 consecutive days before the pharmacokinetic study. On the study day, quinine (25 mg/kg) was administered to the rats by either the oral or i.v. route. Blood samples were collected at different times, up to 6 h after quinine administration. Plasma quinine concentration was assayed by HPLC. Pretreatment with naringin did not cause any significant change in the pharmacokinetics of quinine after the i.v. dose. However pretreatment with naringin led to a 208% increase in peak plasma concentration (Cmax), a 93% increase in time to reach Cmax (tmax), and a 152% increase in the area under the plasma concentration-time curve (AUC) of quinine after oral administration. Consequently, the oral bioavailability of quinine was significantly increased (p < 0.05) from 17% (control) to 42% after pretreatment with naringin. There was no significant difference in the elimination half-life (t(1/2)beta) of quinine between the two groups. These results suggest that pretreatment with the grapefruit flavonoid naringin is associated with increased oral bioavailability of quinine in rats.

ALCOHOL Induced ULCER and ULCERS **

12. Pharmacology. 1994 Sep;49(3):144-50. Antiulcer effect of naringin on gastric lesions induced by ethanol in rats. Martin MJ, Marhuenda E, Perez-Guerrero C, Franco JM. Departamento de Farmacia y Tecnologia Farmaceutica, Facultad de Farmacia, Universidad de Sevilla, Espana.

This study was designed to determine the gastroprotective properties of naringin on and the involvement of endogenous prostaglandins in mucosal injury produced by absolute ethanol. Oral pretreatment with the highest dose of naringin (400 mg/kg), 60 min before absolute ethanol was the most effective antiulcer treatment. Subcutaneous administration of indomethacin (10 mg/kg) to the animals treated with naringin (400 mg/kg) partially inhibited gastric protection, but the prostaglandin E2 determination did not show any increase in prostanoid levels. The contents of gastric mucus and total proteins were not significantly modified. Naringin-treated rats showed a marked increase in hexosamine levels, but this increase was less in animals pretreated with indomethacin. These results show that naringin has a 'cytoprotective' effect against ethanol injury in the rat, but this property appears to be mediated by non-prostaglandin-dependent mechanisms.

13. Int J Tissue React. 1983;5(4):415-20. The gastric anti-ulcer activity of naringenin, a specific histidine decarboxylase inhibitor. Parmar NS.

The gastric anti-ulcer activity of a specific histidine decarboxylase inhibitor naringenin, the aglycone of naringin, a naturally occurring flavanone glycoside obtained from kino and grapefruits, has been studied on the various types of ulcers experimentally induced in rats, viz., pylorus-ligated (Shay method) and restraint ulcers, and on the gastric mucosal damage induced by aspirin, phenylbutazone or reserpine. Naringenin possessed significant anti-ulcer activity in all these models, manifesting a dose-dependent anti-ulcer effect on the pylorus-ligated and restraint ulcers. However, the ED50 value against ulcers in the pylorus-ligated rats (132 mg/kg) was significantly greater than that against ulcers in the restraint rats (42 mg/kg). Amongst all the models used, naringenin was found most effective against the restraint rats. It is suggested that a mechanism involving the inhibition of formation and release of endogenous histamine in the gastric mucosa of rats is implicated in the protective effect of naringenin.

ANTI-OXIDANT – ROS

14: Clin Chim Acta. 2002 Mar;317(1-2):181-90.

Comparison of antioxidant effects of naringin and probucol in cholesterol-fed rabbits.

Jeon SM, Bok SH, Jang MK, Kim YH, Nam KT, Jeong TS, Park YB, Choi MS.

Department of Food Science and Nutrition, Kyungpook National University, 1370 Sankyuk Dong Puk-ku, 702-701, Taegu, South Korea.

BACKGROUND: Due to the strong evidence on the involvement of active oxygen species in a variety of disorders, the role of antioxidants against oxidative stress has recently received increased attention. METHODS: Twenty male rabbits were served a high-cholesterol (HC, 5 g/kg diet) diet or high-cholesterol diet supplemented with naringin (0.5 g/kg diet) or probucol (0.5 g/kg diet) for 8 weeks to compare the antioxidative effects of the citrus bioflavonoid (naringin) and antioxidative cholesterol-lowering drug (probucol). RESULTS: The plasma thiobarbituric acid-reactive substances (TBARS) concentration was not significantly different between the groups, whereas the hepatic TBARS concentration was significantly lower in the probucol group than in both normal and HC control or naringin group. Probucol and naringin supplementation led to an increase in the hepatic superoxide dismutase (SOD) and catalase (CAT) activities, and a decrease in the hepatic mitochondrial hydrogen peroxide (H(2)O(2)) content compared to the HC-control group. However, there was no difference in the cytosolic H(2)O(2) content or cytosolic glutathion peroxidase (GSH-Px) activity in the liver between the groups. Both naringin and probucol supplements significantly increased the plasma vitamin E concentration compared to the HC-control group. As regards the antioxidant enzyme gene expressions, naringin significantly increased the expression of three antioxidant enzyme mRNAs compared to the HC-control group, whereas probucol significantly increased the only SOD mRNA expression. CONCLUSIONS: The probucol supplement was very potent in the antioxidative defense system, whereas naringin exhibited a comparable antioxidant capacity based on increasing the gene expressions in the antioxidant enzymes, while also increasing the hepatic SOD and CAT activities, sparing plasma vitamin E, and decreasing the hepatic mitochondrial H(2)O(2) content.

ANTI-VIRAL **

15. Biol Pharm Bull. 2003 Jan;26(1):108-9. Anti-Sindbis activity of flavanones hesperetin and naringenin. Paredes A, Alzuru M, Mendez J, Rodriguez-Ortega M. Biomedical Institute, Venezuelan Central University, PO BOX 4043, Caracas 1010A, Venezuela.

The effect of hesperetin, naringenin and its glycoside form on the Sindbis neurovirulent strain (NSV) replication in vitro was studied. All flavanones tested were not cytotoxic on Baby Hamster cells 21 clone 15 (BHK-21). Antiviral effect was evaluated by a colorimetric assay using MTT (3-(4,5 dimethylthiazol-2-yl)-2,5-dipheyl-tetrazolium bromide) and by plaque reduction assay. Hesperetin and naringenin had inhibitory activity on NSV infection. The 50% inhibitory doses (ID(50%)) of both compounds were 20.5 and 14.9 microg/ml respectively, as established by plaque assay. However their glycosides, hesperidin and naringin did not have inhibitory activity. Implying that the presence of rutinose moiety of flavanones blocks the antiviral effect. Oxygenation on the 3' positions at the B rings on the hesperetin skeleton decrease the anti viral activity at 25 microg/ml.

CANCER **

16. Mutagenesis. 2003 Jul;18(4):337-43. Naringin, a citrus flavonone, protects against radiation-induced chromosome damage in mouse bone marrow. Jagetia GC, Venkatesha VA, Reddy TK. Department of Radiobiology, Kasturba Medical College, Manipal 576119, India. gc.jagetia@kmc.manipal.edu

Free radicals are responsible for the induction of damage to the cellular DNA that leads to the formation of chromosome aberrations. Antioxidants are known to scavenge free radicals, thereby decreasing the degree of such effects. Radiation is a well-known inducer of free radicals and compounds that can scavenge free radicals may reduce radiation-induced DNA damage. Naringin, a bioflavonoid predominant in grapefruit and other citrus fruits, has been found to scavenge free radicals, therefore it may also reduce radiation-induced damage. The aim of the present study was to evaluate the radioprotective action of 2 mg/kg naringin in the bone marrow of mice exposed to different doses of (60)Co gamma-radiation by scoring the frequency of asymmetrical chromosomal aberrations. The irradiation of mice resulted in a dose-dependent elevation in the frequency of aberrant cells, acentric fragments, chromatid and chromosome breaks, dicentrics and exchanges. All these aberrations were elevated with scoring time up to 24 h post-irradiation and declined thereafter, except chromatid breaks, which were maximum at 12 h post-irradiation. Treatment of mice with 2 mg/kg body wt naringin before exposure to various doses of gamma-radiation resulted in a significant reduction in the frequencies of aberrant cells and chromosomal aberrations like acentric fragments, chromatid and chromosome breaks, centric rings, dicentrics and exchanges. The evaluation of free radical scavenging activity of naringin revealed a dose-dependent scavenging of hydroxyl, superoxide and 2,2 equal to or precedes -diphenyl-1-picryl hydrazyl radical. Naringin at 5 microM scavenged the 2,2-azino-bis-3-ethyl benzothiazoline-6-sulphonic acid cation radical very efficiently, where a 90% scavenging was observed. Our study demonstrates that naringin can protect mouse bone marrow cells against radiation-induced chromosomal damage.

17. Mutat Res. 2002 Aug 26;519(1-2):37-48. The grapefruit flavanone naringin protects against the radiation-induced genomic instability in the mice bone marrow: a micronucleus study. Jagetia GC, Reddy TK. Department of Radiobiology, Kasturba Medical College, Manipal 576119, India. gc.jagetia@kmc.manipal.edu

The effect of various doses, viz. 0, 0.5, 1, 2, 4, 6 and 8 mg/kg body weight of naringin (NIN) (a citrus flavanone) was studied on the alteration in the radiation-induced micronucleated polychromatic (MPCE) and normochromatic (MNCE) erythrocytes in mouse bone marrow exposed to 2 Gy of 60Co gamma-radiation. The treatment of mice with various doses of NIN before exposure to 2 Gy resulted in a significant decline in the frequency of MPCE when compared to the non-drug-treated irradiated control. However, the greatest reduction in MPCE was observed for 2mg/kg body weight NIN, accompanied by a highest PCE/NCE ratio when compared with the non-drug-treated irradiated control. Therefore, further studies were carried out using this dose of NIN, where the animals were administered with 2mg/kg body weight of NIN before exposure to 0, 0.5, 1, 2, 3 and 4 Gy of gamma-radiation. The frequency of MPCE and MNCE increased in a dose-dependent manner in both the non-drug-treated irradiated control and NIN-pretreated irradiated groups up to a dose of 2 Gy, while a further increase in the irradiation dose resulted in a significant decline in MPCE and MNCE frequencies in both groups. Pretreatment of mice with 2mg/kg body weight of NIN resulted in a significant decline in the frequencies of MPCE and MNCE. NIN treatment not only reduced the frequency of MPCE with one micronucleus, but also of MPCE with multiple micronuclei (MN), indicating its ability to reduce complex chromosome aberrations. Conversely, the PCE/NCE ratio declined in a dose-dependent manner in both groups. The treatment of mice with NIN before exposure to different doses of gamma-radiation resulted in the inhibition in this decline in the PCE/NCE ratio. Our study demonstrates that NIN is able to protect mouse bone marrow cells against the radiation-induced DNA damage and decline in the cell proliferation as observed by a reduction in the micronucleus frequency and an increase in PCE/NCE ratio, respectively, in the NIN-pretreated irradiated group.

18. Life Sci. 1999;65(24):2591-602. In vitro and in vivo effects of naringin on cytochrome P450-dependent monooxygenase in mouse liver. Ueng YF, Chang YL, Oda Y, Park SS, Liao JF, Lin MF, Chen CF. National Research Institute of Chinese Medicine, Taipei, Taiwan, ROC. ueng@cma23.nricm.edu.tw

In vitro and in vivo effects of naringin on microsomal monooxygenase were studied to evaluate the drug interaction of this flavonoid. In vitro addition of naringin up to 500 microM had no effects on benzo(a)pyrene hydroxylase (AHH) activity of mouse liver microsomes. In contrast, the aglycone naringenin at 300 to 500 microM decreased AHH activity by 50% to 60%. Analysis of Lineweaver-Burk and Dixon plots indicated that naringenin competitively inhibited AHH activity with an estimated Ki of 39 microM. Naringenin at 100 microM also reduced metabolic activation of benzo(a)pyrene to genotoxic products as monitored by umuC gene expression response in Salmonella typhimurium TA1535/pSK1002. In the presence of equimolar naringenin and benzo(a)pyrene, umuC gene expression presented as beta-galactosidase activity was reduced to a level similar to the control value. Administration of a liquid diet containing 10 mg/ml naringin for 7 days caused 38% and 49% decreases of AHH and 7-methoxyresorufin O-demethylase activities, respectively. In contrast, the administration had no effects on cytochrome P450 (P450)-catalyzed oxidations of 7-ethoxyresorufin, 7-ethoxycoumarin, N-nitrosodimethylamine, nifedipine, erythromycin and testosterone. Microsomal P450 and cytochrome b5 contents and NADPH-P450 reductase activity were not affected. Immunoblot analysis using MAb 1-7-1, which immunoreacted with both P450 1A1 and 1A2, revealed that the level of P450 1A2 protein was decreased by 38%. These results demonstrate that naringenin is a potent inhibitor of AHH activity in vitro and naringin reduces the P450 1A2 protein level in vivo. These effects may indicate a chemopreventive role of naringin against protoxicants activated by P450 1A2.

19. Eur J Pharmacol. 1999 Mar 5;368(2-3):245-50. Suppression of lipopolysaccharide-induced tumor necrosis factor-release and liver injury in mice by naringin. Kawaguchi K, Kikuchi S, Hasegawa H, Maruyama H, Morita H, Kumazawa Y. Medicinal Plant Garden, School of Pharmaceutical Sciences, Kitasato University, Sagamihara, Japan.

Suppressive effects of naringin on lipopolysaccharide-induced tumor necrosis factor (TNF) release followed by liver injury were investigated. Intraperitoneal (i.p.) treatment with naringin prior to an intravenous (i.v.) challenge of lipopolysaccharide significantly reduced serum TNF levels in a dose-dependent manner and was the most effective when administered 60 min prior to lipopolysaccharide challenge. Treatment with naringin 3 h prior to lipopolysaccharide challenge resulted in complete protection from lipopolysaccharide lethality in D-galactosamine-sensitized mice. Histological estimation revealed that massive cell infiltration followed by severe injury developed in the livers of lipopolysaccharide-treated and D-galactosamine-treated mice unless they had been pretreated with naringin. Appearance of apoptotic cells was also found to decrease by treatment with naringin. Increases in serum levels of aspartate aminotransferase, alanine aminotransferase and creatine kinase, responsible for lipopolysaccharide-induced liver injury, blocked by naringin administration and the levels were nearly to the normal level. These results indicate that action of naringin is mediated through suppression of lipopolysaccharide-induced TNF production.

20. Carcinogenesis. 1989 Oct;10(10):1953-5. Modulating effect of plant flavonoids on the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine. Francis AR, Shetty TK, Bhattacharya RK. Biochemistry Division, Bhabha Atomic Research Centre, Bombay India.

Tests have been carried out with several plant flavonoids to detect their ability to suppress mutagenesis in Salmonella typhimurium strain TA100 NR induced by the direct-acting carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Among the most effective flavonoids are the isoflavone, biochanin A, the flavanone glycoside, naringin, and its aglycone, naringenin, and several flavonols, e.g. morin, fisetin, kaempferol, gossypetin and quercetin, including a flavonol glycoside, rutin. In particular, naringin possesses exceptional antimutagenic activity, in as much as, less than half the equimolar amount can reduce the mutagenic potency of this carcinogen by 50%. These flavonoids appear to act either by preventing passage of the carcinogen into bacterial cells or by altering some cellular processes.

CHOLESTEROL **

21. Arch Latinoam Nutr. 2001 Sep;51(3):258-64. [Hypocholesterolemic effect of naringin and rutin flavonoids] [Article in Portuguese] da Silva RR, de Oliveira TT, Nagem TJ, Pinto AS, Albino LF, de Almeida MR, de Moraes GH, Pinto JG. Universidade Federal de Vicosa, Minas Gerais, Brazil.

Flavonoids are pigments fenolics of plants that possess several biological activities, and many of these are associated with prevention of chronic diseases as cancer and hyperlipidemia. This work had as objective evaluates the effect of the flavonoids naringin and rutin on the metabolism lipidic of chicks hypercholesterolemic. In agreement with the results it can be observed that naringin and rutin reduced the levels of total cholesterol significantly, cholesterol-LDL, cholesterol-VLDL and triglycerols, not presenting, however, reductions in the levels of cholesterol-HDL.

22. Life Sci. 2001 Nov 2;69(24):2855-66. Antioxidative activity of naringin and lovastatin in high cholesterol-fed rabbits. Jeon SM, Bok SH, Jang MK, Lee MK, Nam KT, Park YB, Rhee SJ, Choi MS. Korea Institute of Bioscience and Biotechnology, KIST, Yusong, Taejon.

The consumption of a cholesterol-enriched diet increases the degree of lipid peroxidation, which is one of the early processes of atherosclerosis. The aim of this trial was to determine the antioxidative effects of the citrus bioflavonoid, naringin, a potent cholesterol-lowering agent, compared to the cholesterol-lowering drug, lovastatin, in rabbits fed a high cholesterol diet. Male rabbits were served a high-cholesterol (0.5%, w/w) diet or high-cholesterol diet supplemented with either naringin (0.5% cholesterol, 0.05% naringin, w/w) or lovastatin (0.5% cholesterol, 0.03% lovastatin, w/w) for 8 weeks to determine the plasma and hepatic lipid peroxide, plasma vitamin A and E levels, and hepatic hydrogen peroxide levels, along with the hepatic antioxidant enzyme activities and gene expressions. Only the lovastatin group showed significantly lower plasma and hepatic lipid peroxide levels compared to the control group. The naringin supplementation significantly increased the activities of both hepatic SOD and catalase by 33% and 20%, respectively, whereas the lovastatin supplementation only increased the catalase activity by 23% compared to control group. There was no difference in the GSH-Px activities between the various groups. Content of H2O2 in hepatic mitochondria was significantly lower in groups supplemented with lovastatin and naringin than in control group. However, there was no difference in cytosolic H2O2 content in liver between groups. The concentration of plasma vitamin E was significantly increased by the naringin supplementation. When comparing the antioxidant enzyme gene expression, the mRNA expression of SOD, catalase and GSH-Px was significantly up-regulated in the naringin-supplemented group. Accordingly, these results would appear to indicate that naringin, a citrus bioflavonoid, plays an important role in regulating antioxidative capacities by increasing the SOD and catalase activities, up-regulating the gene expressions of SOD, catalase, and GSH-Px, and protecting the plasma vitamin E. In contrast, lovastatin exhibited an inhibitory effect on the plasma and hepatic lipid peroxidation and increased the hepatic catalase activity in high-cholesterol fed rabbits.

23. J Cardiovasc Pharmacol. 2001 Dec;38(6):947-55. Naringin has an antiatherogenic effect with the inhibition of intercellular adhesion molecule-1 in hypercholesterolemic rabbits. Choe SC, Kim HS, Jeong TS, Bok SH, Park YB. Department of Internal Medicine, Soonchunhyang University Bucheon Hospital, Bucheon, Korea.

Naringin, a bioflavonoid found in citrus fruit peel, is known to have an antioxidative effect, but its effect on atherosclerosis has not been studied. This study evaluated the effect of naringin on blood lipid levels and aortic fatty streaks, and its action mechanism in hypercholesterolemic rabbits. Male New Zealand white rabbits were fed a 0.25% cholesterol diet and divided into an untreated group (n = 4), a naringin-treated group (n = 5; 500 mg/kg per day), and a lovastatin-treated group (n = 5; 20 mg/kg per day). After 8 weeks, blood was sampled and analyzed biochemically. Aorta and liver were harvested and examined histologically. Cholesterol level in rabbits fed the 0.25% cholesterol diet reached 17 times normal and decreased in the rabbits fed naringin and lovastatin, whose effects were not statistically significant (p > 0.05). However, both naringin and lovastatin effectively decreased the area of fatty streak in thoracic aorta on macroscopic analysis (p < 0.05) and significantly reduced subintimal foam cell infiltration on microscopic morphometry (p < 0.05). These foam cells were macrophages on immunohistochemical analysis. Naringin treatment inhibited hypercholesterolemia-induced intercellular adhesion molecule-1 (ICAM-1) expression on endothelial cells. Hypercholesterolemia caused fatty liver and elevation of liver enzymes, which was prevented by naringin but not by lovastatin. Naringin significantly reduced fatty streak formation and neointimal macrophage infiltration and also inhibited the expression of ICAM-1 in endothelial cells, suggesting that suppression of ICAM-1 contributed to the antiatherogenic effect. Naringin, unlike lovastatin, has a hepatoprotective action.

24. Ann Nutr Metab. 2001;45(5):193-201. Effect of naringin supplementation on cholesterol metabolism and antioxidant status in rats fed high cholesterol with different levels of vitamin E. Choi MS, Do KM, Park YS, Jeon SM, Jeong TS, Lee YK, Lee MK, Bok SH. Department of Food Science and Nutrition, Kyungpook National University, 1370 Yank-Suk Dong Pak-Ku, 702-701, Taegu, Korea. mschoi@knu.ac.kr

Some bioflavonoids are potent antioxidants and have pharmacological effects similar to those of vitamin E. The interactive effect of naringin and vitamin E was studied with respect to cholesterol metabolism and antioxidant status. Naringin supplementation (0.1%, wt/wt) with comparable levels of vitamin E was given to rats with a high-cholesterol (1%, wt/wt) diet for 5 weeks. The amount of vitamin E included in naringin-free and naringin diets was a low (low-E) and a normal (normal-E) level. The naringin supplementation significantly lowered the concentrations of plasma cholesterol and triglyceride compared to the naringin-free group in low vitamin E-fed rats. HMG-CoA reductase activity was significantly lowered by naringin supplementation within both the low-vitamin E group (794.64 +/- 9.87 vs. 432.18 +/- 12.33 pmol/min/mg protein, mean +/- SE; p < 0.05) and normal-vitamin E group (358.82 +/- 11.4 vs. 218.22 +/- 9.47 pmol/min/mg protein, mean +/- SE; p < 0.05) compared to each of the naringin-free group. The HMG-CoA reductase activity was also significantly lowered by increased dietary vitamin E when compared within the naringin and naringin-free group, respectively. Neither dietary naringin nor vitamin E did significantly change the activities of hepatic antioxidant enzymes and plasma thiobarbituric acid-reactive substance level. These data indicate that naringin lowers the plasma lipid concentrations when the dietary vitamin E level is low. The HMG-CoA reductase-inhibitory effect of naringin was more potent when dietary vitamin E was at a normal level. These data may contribute to understanding the interactive effect of naringin and vitamin E on cholesterol biosynthesis in high-cholesterol-fed rats. Copyright 2001 S. Karger AG, Basel

25. Biochem Biophys Res Commun. 2001 Jun 15;284(3):681-8. Anti-atherogenic effect of citrus flavonoids, naringin and naringenin, associated with hepatic ACAT and aortic VCAM-1 and MCP-1 in high cholesterol-fed rabbits. Lee CH, Jeong TS, Choi YK, Hyun BH, Oh GT, Kim EH, Kim JR, Han JI, Bok SH. Genetic Resources Center, Korea Research Institute of Bioscience and Biotechnology, Taejon 305-600, Korea.

The anti-atherogenic effects of the citrus flavonoids, naringin and naringenin, were evaluated in high cholesterol-fed rabbits. At 3 months of age, 30 male New Zealand White (NZW) rabbits were divided into three groups (n = 10 per group). The rabbits were fed a 1% cholesterol diet alone (control group) or a diet supplemented with either 0.1% naringin or 0.05% naringenin for 8 weeks. The plasma lipoprotein levels, total cholesterol, triglyceride, and high-density lipoprotein showed no significant differences in the control and experimental groups. Hepatic acyl-CoA:cholesterol acyltransferase (ACAT) activity was slightly low in naringin (5.0%)- and naringenin (15.0%)-fed rabbits, compared to control group. The aortic fatty streak areas were significantly lower in both the naringin (19.2 +/- 5.6%)- and naringenin (18.1 +/- 6.5%)-supplemented groups than in the control group (60.4 +/- 14.0%). The expression levels of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1), by semiquantitative RT-PCR analysis of the thoracic aorta, were significantly lower in the flavonoids supplemented groups than in the control group. These results suggest that the anti-atherogenic effect of the citrus flavonoids, naringin and naringenin, is involved with a decreased hepatic ACAT activity and with the downregulation of VCAM-1 and MCP-1 gene expression. Copyright 2001 Academic Press.

26. Int J Vitam Nutr Res. 1999 Sep;69(5):341-7. Hypocholesterolemic effect of naringin associated with hepatic cholesterol regulating enzyme changes in rats. Shin YW, Bok SH, Jeong TS, Bae KH, Jeoung NH, Choi MS, Lee SH, Park YB. Korea Research Institute of Bioscience & Biotechnology, KIST, Yusong, Taejon, Korea.

The effects of the citrus bioflavonoid naringin were tested by using it as a supplement in a high-cholesterol diet. Male rats were fed for 42 days with a 1% (wt/wt) high cholesterol diet either with or without naringin-supplementation (0.1%, wt/wt) to study the effect on plasma lipid levels, hepatic lipid contents, hepatic enzyme activity, and the excretion of fecal neutral sterols. Naringin did not significantly alter the levels of plasma triglycerides, however, the levels of plasma cholesterol (3.80 +/- 0.31 mmol/L vs. 2.61 +/- 0.30 mmol/L, mean +/- SE; p < 0.05) and hepatic cholesterol (70.3 +/- 4.3 mg/g vs. 54.3 +/- 3.8 mg/g, mean +/- SD; p < 0.05) were significantly lowered compared to those of the control. HMG-CoA reductase (2487.0 +/- 210.0 pmole/min/mg vs. 1879.0 +/- 236.0 pmole/min/mg, mean +/- SE; p < 0.05) and ACAT (806.0 +/- 105.0 pmole/min/mg vs. 643.0 +/- 80.0 pmole/min/mg, mean +/- SE; p < 0.05) activities were both substantially lower in the naringin-supplemented group than in the control. The naringin supplementation markedly decreased the excretion of fecal neutral sterols (204.7 +/- 28.5 mg/day) compared to the control (521.9 +/- 53.9 mg/day). The combination of the inhibited HMG-CoA reductase (-24.4%) and ACAT (-20.2%) activities as a result of naringin supplementation could account for the decrease of fecal neutral sterols.

HISTAMINE **

27. Acta Physiol Pharmacol Bulg. 1980;6(2):70-5. Flavonoids with antioxidant action (naringin and rutin) and the release of mastocytic and nonmastocytic histamine. Lambev I, Belcheva A, Zhelyazkov D.

In view of the data about the antioxidant mechanism of the membrane-stabilizing action of the bioflavonoids, the participation of this mechanism in the release of histamine as well is assumed to be possible. Experiments are carried out on 35 male albino rats. The effect of the flavonoids naringin and rutin on the level of mastocytic and nonmastocytic histamine is studied, as well as on its release induced by compound 48/80 (2 mg/kg i. p.). The substances are applied intraperitonealy in doses of 200 mg/kg; corresponding to 10% DL50. The histamine content is determined fluorimetrically by the method of Schore et al. (1959). The results show that naringin and rutin have no effect on the levels of mastocytic and nonmastocytic histamine. They prevent the release of mastocytic histamine, induced by compound 48/80.