Triterpenoids isolated from apple peels have potent antiproliferative activity and may be partially responsible for apple's anticancer activity

Bioactivity-guided fractionation of apple peels was used to determine the chemical identity of bioactive constituents. Thirteen triterpenoids were isolated, and their chemical structures were identified. Antiproliferative activities of the triterpenoids against human HepG2 liver cancer cells, MCF-7 breast cancer cells, and Caco-2 colon cancer cells were evaluated. Most of the triterpenoids showed high potential anticancer activities against the three human cancer cell lines. Among the compounds isolated, 2alpha-hydroxyursolic acid, 2alpha-hydroxy-3beta-{[(2E)-3-phenyl-1-oxo-2-propenyl]oxy}olean-12-en-28-oic acid, and 3beta-trans-p-coumaroyloxy-2alpha-hydroxyolean-12-en-28-oic acid showed higher antiproliferative activity toward HepG2 cancer cells. Ursolic acid, 2alpha-hydroxyursolic acid, and 3beta-trans-p-coumaroyloxy-2alpha-hydroxyolean-12-en-28-oic acid exhibited higher antiproliferative activity against MCF-7 cancer cells. All triterpenoids tested showed antiproliferative activity against Caco-2 cancer cells, especially 2alpha-hydroxyursolic acid, maslinic acid, 2alpha-hydroxy-3beta-{[(2E)-3-phenyl-1-oxo-2-propenyl]oxy}olean-12-en-28-oic acid, and 3beta-trans-p-coumaroyloxy-2alpha-hydroxyolean-12-en-28-oic acid, which displayed much higher antiproliferative activities. These results showed the triterpenoids isolated from apple peels have potent antiproliferative activity and may be partially responsible for the anticancer activities of whole apples.     

Phytochemicals of apple peels: isolation, structure elucidation, and their antiproliferative and antioxidant activities

Bioactivity-guided fractionation of Red Delicious apple peels was used to determine the chemical identity of bioactive constituents, which showed potent antiproliferative and antioxidant activities. Twenty-nine compounds, including triterpenoids, flavonoids, organic acids and plant sterols, were isolated using gradient solvent fractionation, Diaion HP-20, silica gel, and ODS columns, and preparative HPLC. Their chemical structures were identified using HR-MS and 1D and 2D NMR. Antiproliferative activities of isolated pure compounds against HepG2 human liver cancer cells and MCF-7 human breast cancer cells were evaluated. On the basis of the yields of isolated flavonoids (compounds 18- 23), the major flavonoids in apple peels are quercetin-3-O-beta-D-glucopyranoside (compound 20, 82.6%), then quercetin-3-O-beta-D-galactopyranoside (compound 19, 17.1%), followed by trace amounts of quercetin (compound 18, 0.2%), (-)-catechin (compound 22), (-)-epicatechin (compound 23), and quercetin-3-O-alpha-L-arabinofuranoside (compound 21). Among the compounds isolated, quercetin (18) and quercetin-3-O-beta-D-glucopyranoside (20) showed potent antiproliferative activities against HepG2 and MCF-7 cells, with EC 50 values of 40.9 +/- 1.1 and 49.2 +/- 4.9 microM to HepG2 cells and 137.5 +/- 2.6 and 23.9 +/- 3.9 microM to MCF-7 cells, respectively. Six flavonoids (18-23) and three phenolic compounds (10, 11, and 14) showed potent antioxidant activities. Caffeic acid (10), quercetin (18), and quercetin-3-O-beta-D-arabinofuranoside (21) showed higher antioxidant activity, with EC 50 values of <10 microM. Most tested flavonoids and phenolic compounds had high antioxidant activity when compared to ascorbic acid and might be responsible for the antioxidant activities of apples. These results showed apple peel phytochemicals have potent antioxidant and antiproliferative activities.     

Antioxidant activity of grains

Epidemiological studies have shown that consumption of whole grains and grain-based products is associated with reduced risk of chronic diseases. The health benefits of whole grains are attributed in part to their unique phytochemical composition. However, the phytochemical contents in grains have been commonly underestimated in the literature, because bound phytochemicals were not included. This study was designed to investigate the complete phytochemical profiles in free, soluble conjugated, and insoluble bound forms, as well as their antioxidant activities in uncooked whole grains. Corn had the highest total phenolic content (15.55 +/- 0.60 micromol of gallic acid equiv/g of grain) of the grains tested, followed by wheat (7.99 +/- 0.39 micromol of gallic acid equiv/g of grain), oats (6.53 +/- 0.19 micromol of gallic acid equiv/g of grain), and rice (5.56 +/- 0.17 micromol of gallic acid equiv/g of grain). The major portion of phenolics in grains existed in the bound form (85% in corn, 75% in oats and wheat, and 62% in rice), although free phenolics were frequently reported in the literature. Ferulic acid was the major phenolic compound in grains tested, with free, soluble-conjugated, and bound ferulic acids present in the ratio 0.1:1:100. Corn had the highest total antioxidant activity (181.42 +/- 0.86 micromol of vitamin C equiv/g of grain), followed by wheat (76.70 +/- 1.38 micromol of vitamin C equiv/g of grain), oats (74.67 +/- 1.49 micromol of vitamin C equiv/g of grain), and rice (55.77 +/- 1.62 micromol of vitamin C equiv/g of grain). Bound phytochemicals were the major contributors to the total antioxidant activity: 90% in wheat, 87% in corn, 71% in rice, and 58% in oats. Bound phytochemicals could survive stomach and intestinal digestion to reach the colon. This may partly explain the mechanism of grain consumption in the prevention of colon cancer, other digestive cancers, breast cancer, and prostate cancer, which is supported by epidemiological studies.     

Antioxidant activity of Glossogyne tenuifolia

Glossogyne tenuifolia is a native traditional anti-inflammatory herb in Taiwan. It has previously been shown that the ethanol extract of G. tenuifolia (GT) inhibited the LPS-induced inflammatory mediator release from murine macrophage cell line and human whole blood. In the present work, the ethanol extracts of G. tenuifolia and its major constituent, luteolin-7-glucoside, were shown to be scavengers of 1,1-diphenyl-2-picrylhydrazyl, superoxide, and hydroxyl radicals. Moreover, copper-induced low-density lipoprotein oxidation was suppressed by GT and luteolin-7-glucoside as measured by decreased formation of malondialdehyde and conjugated diene as well as reduced electrophoretic mobility. GT and luteolin-7-glucoside were also against N-formyl-methionyl-leucyl-phenylalanine-induced reactive oxygen species (ROS) production in human polymorphonuclear neutrophils and peripheral blood mononuclear cells. In summary, these data indicated that GT is a potential ROS scavenger and may prevent atherosclerosis via inhibiting LDL oxidation or ROS production in human leukocytes. Moreover, luteolin-7-glucoside may serve as the active principal of GT.     

Antioxidant activity of dodecyl gallate

Dodecyl (C12) gallate exhibits both potent chain-breaking and preventive antioxidant activity. The pyrogallol moiety is responsible for both activities. Dodecyl (lauryl) gallate prevents generation of superoxide radicals by xanthine oxidase, and this activity comes from its ability to inhibit the enzyme. The inhibition kinetics analyzed by Lineweaver-Burk plots found that dodecylgallate is a noncompetitive inhibitor for the generation of superoxide anion. Dodecyl gallate also inhibits formation of uric acid. The inhibition kinetics analyzed by Lineweaver-Burk plots found that dodecyl gallate is a competitive inhibitor for this oxidation. Mitochondrial lipid peroxidation induced by Fe(III)-adenosine 5'-diphosphate/reduced nicotinamide adenine dinucleotide was inhibited by dodecyl gallate while its parent compound, gallic acid, did not show this inhibitory activity. Dodecyl gallate protected mitochondrial functions and human red blood cells against oxidative stresses, but gallic acid showed little effect. The hydrophobic dodecyl group is largely associated with the preventive antioxidative activity.     

Effect of heat treatment on the antioxidant activity of extracts from citrus peels

The effect of heat treatment on the antioxidant activity of extracts from Citrus unshiu peels was evaluated. Citrus peels (CP) (5 g) were placed in Pyrex Petri dishes (8.0 cm diameter) and heat-treated at 50, 100, or 150 degrees C for 10, 20, 30, 40, 50, and 60 min in an electric muffle furnace. After heat treatment, 70% ethanol extract (EE) and water extract (WE) (0.1 g/10 mL) of CP were prepared, and total phenol contents (TPC), radical scavenging activity (RSA), and reducing power of the extracts were determined. The antioxidant activities of CP extracts increased as heating temperature increased. For example, heat treatment of CP at 150 degrees C for 60 min increased the TPC, RSA, and reducing power of EE from 71.8 to 171.0 microM, from 29.64 to 64.25%, and from 0.45 to 0.82, respectively, compared to non-heat-treated control. In the case of WE from CP heat-treated at the same conditions (150 degrees C for 60 min), the TPC, RSA, and reducing power also increased from 84.4 to 204.9 microM, from 15.81 to 58.26%, and from 0.27 to 0.96, respectively. Several low molecular weight phenolic compounds such as 2,3-diacetyl-1-phenylnaphthalene, ferulic acid, p-hydroxybenzaldoxime, 5-hydroxyvaleric acid, 2,3-diacetyl-1-phenylnaphthalene, and vanillic acid were newly formed in the CP heated at 150 degrees C for 30 min. These results indicated that the antioxidant activity of CP extracts was significantly affected by heating temperature and duration of treatment on CP and that the heating process can be used as a tool for increasing the antioxidant activity of CP.     

Antioxidant activity of malt rootlet extracts

To improve the malt rootlet value, the antioxidant potentialities of this byproduct of the malting industry have been analyzed. Three extracts have been considered from the points of view of dry matter yield, level of antioxidant compounds, and efficiency and cost of the extraction protocols. They respectively contain rootlet oil, free phenolic compounds, and bound phenolic compounds. The rootlet oil contains only a low quantity of tocopherols (respectively, 20.6 and 4.2 microgram of alpha-tocopherol and gamma-tocopherol per gram of dry rootlets), and a weak antioxidant activity, evaluated in a stripped corn oil by following spectrophotometrically the conjugated dienes, has been pointed out. The bound compound extract presents a good antioxidant power mainly due to the presence of trans-ferulic and trans-p-coumaric acids, but the dry matter yield is low (2%). The free compound extract has a good antioxidant power, and the valuable dry matter, mainly composed of proteins (52%), sugars (33%), and reducing compounds (5.5%), has a yield of 12%. The mixing of bound and free compound extracts presents an antagonistic effect on the antioxidant power, but a synergistic effect has been pointed out for the mixing of alpha-tocopherol and free compound extract.     

Antioxidant activity of coffee model systems

Coffee model systems prepared from combinations of chlorogenic acid (CGA), N(alpha)-acetyl-1-arginine (A), sucrose (S), and cellulose (C) were roasted at 240 degrees C for 4 min prior to analysis by UV-visible spectrophotometry, capillary zone electrophoresis (CZE), and the ABTS radical cation decolorization assay. The A/CGA/S/C and A/S/C systems were also fractionated by gel filtration chromatography. Antioxidant activity of the systems showed a positive, nonlinear relationship with the amount of CGA remaining after roasting. Sucrose degradation was a major source of color in the heated systems. There was no relationship between antioxidant activity and color generation.     

Antioxidant activity of indigenous edible mushrooms

The current study was undertaken to measure the antioxidant potential from water and methanolic extracts of fruiting bodies of 23 species of mushrooms naturally grown in different geographic locations of India. The antioxidant ability of each species was analyzed for the total antioxidative status, employing multimechanistic antioxidative assays such as inhibition of lipid peroxidation, determination of reducing power, and free radical scavenging ability, in addition to determination of total phenolics and identification of phenolic acids by HPLC analysis, because the phenolics are known to contribute largely to antioxidant potential. The antioxidant potential of these varieties of mushrooms was determined by summing the antioxidative activity (AOA) of each variety by varied antioxidant assays followed by determining the relative percent of AOA defined as the "antioxidant index" (AI). On the basis of the AI, the mushroom species were graded as very high, high, moderate, and low. Termitomyces heimii was identified as the best variety, which showed 100% AI with 37 mg of phenolics/g of sample, 418 units of reducing power ability (RPA)/g, and an IC50 of approximately 1.1 mg (dry weight)/mL, free radical scavenging activity (FRS) in the water extract followed by 11.2 mg of phenolics/g, 275 units of RPA/g, and an IC50 of approximately 2.7 mg (dry weight)/mL of FRS in the methanolic extract. Following T. heimii, Termitomyces mummiformis exhibited an AI of 86% within the "very high" group. Potent inhibitions of lipid peroxidation of approximately 100 and 69% was also observed in T. heimii and T. mummiformis, respectively. Water extracts ranged from 34 to 49% and methanolic extracts varied from 20 to 32% on dry weight of mushroom fruiting body. Total phenolic compounds were higher in the water extracts (2-37 mg/g) than in methanolic extract (0.7-11.2 mg/g). The AOA measured in the water extract was better than that from the methanolic extract. HPLC analysis of phenolic acids in the two mushroom species, namely, T. heimii and T. mummiformis, displaying maximum AOA potential indicated a preponderance of tannic acid, gallic acid, protocatacheuic acid, and gentisic acid. Studies thus provide the precise antioxidant status of 23 indigenous species of mushrooms, which can serve as a useful database for the selection of mushrooms for the function of preparation of mushroom-based nutraceutics.     

Antioxidant activity of capsaicinoid in canola oil

Interest in replacing synthetic antioxidants, namely, butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), with natural antioxidants is increasing. The present study examined the antioxidant activity of capsaicinoid from chili pepper in heated canola oil. The oxidation was conducted at 60, 90, 120, and 180 °C by monitoring oxygen consumption and the decrease in linoleic acid and α-linolenic acid in canola oil. At 60 °C, capsaicinoid was more effective against oxidation of canola oil compared with BHT. At higher temperatures of 90, 120, and 180 °C, capsaicinoid possessed an antioxidant activity similar to or slightly weaker that that of BHT. It was found that capsaicinoid prevented canola oil from oxidation in a dose-dependent manner. To study the structure-antioxidant relationship, it was found that the trimethylsiloxy (TMS) derivatives of capsaicinoid did not exhibit any antioxidant activity, suggesting the hydroxyl moiety was the functional group responsible for the antioxidant activity of capsaicinoid. It was concluded that capsaicinoid had the potential to be further explored as a natural antioxidant in foods, particularly spicy foods.     

Antioxidant activity of polyphenols in carob pods.

We extracted polyphenols from carob (Ceratonia siliqua L.) pods, and evaluated the in vitro antioxidant activity of the crude polyphenol fraction (CPP). The total polyphenol content in CPP determined by the Folin-Ciocalteu method was 19.2%. The condensed tannin content determined by the vanillin and proanthocyanidin assay systems was 4.37% and 1.36%, respectively. beta-Carotene bleaching, 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging, inhibition of lipid peroxidation by the erythrocyte ghost, and microsomal assay systems were used to evaluate the antioxidant activity. CPP showed a stronger inhibitory effect against the discoloration of beta-carotene than other polyphenol compounds such as catechins and procyanidins. CPP had weaker antioxidant activity in the DPPH free radical scavenging, the erythrocyte ghost, and microsomal systems than authentic polyphenol compounds at the same concentrations. The activity adjusted by the polyphenol concentration was, however, comparable to that of authentic polyphenol compounds. Considering most carob pods are discarded and not effectively utilized at present, these results suggested that carob pods could be utilized as a functional food or food ingredient.     

Antioxidant activity of Sempervivum tectorum and its components

The antioxidant properties of components of leaf extracts of the evergreen plant, Sempervivum tectorum (ST), have been evaluated using UV irradiated liposomal systems containing the spin trap 5-(diethoxyphosphoryl)-5-methyl-pyrroline-N-oxide. Decreases in free radical activity in the liposomal systems as measured by electron paramagnetic resonance (EPR) spectroscopy demonstrate that the lipophilic ST juice components, kaempferol (KA) and kaempferol-3-glucoside (KG) contribute significantly to the antioxidant properties of the juice. EPR spectral simulation established the presence of oxygen and carbon centered free radical adducts. The mixtures with low pH, citric and malic acid, and ST juice reveal increased EPR signals from oxygen centered radicals in comparison to the control, pointing to the important role of pH in oxygen radical formation. Parallel assays that measured thiobarbituric acid related substances confirm the antioxidant effects of KA and KG and explain the results of spin trapping experiments complicated by low pH's.     

Antioxidant activity and phenolic content of Oregon caneberries

Five types of caneberries [evergreen blackberries (Rubus laciniatus), marionberries (Rubus ursinus), boysenberries (Rubus ursinus x idaeus), red raspberries (Rubus idaeus), and black raspberries (Rubus occidentalis)] were analyzed for antioxidant activity by measuring their oxygen radical absorbance capacity (ORAC). In addition, the berries were analyzed for total phenolics, anthocyanins, procyanidins, and ellagic acid content. All of the berries had high ORAC activity ranging from 24 to 77.2 micromol of Trolox equiv/g of fresh berries. Anthocyanin content ranged from 0.65 to 5.89 mg/g, and phenolics ranged from 4.95 to 9.8 mg/g. Black raspberries had the highest ORAC and anthocyanin and phenolic contents. Only red raspberries had detectable amounts of procyanidin oligomers (monomer, dimers, and trimers). All berries had high levels of ellagic acid (47-90 mg/g), but boysenberries had the highest level prior to hydrolysis. The results from this study indicate that these caneberries were high in antioxidant activity and were rich sources of anthocyanins and phenolics.     

Peroxidase activity and superficial scald development in apple fruit

The relationship between soluble peroxidase (EC 1.11.1.7; POX) activity and the development of a chilling-related disorder, superficial scald, was studied in three apple fruit (Malus x domestica Borkh.) systems: a White Angel x Rome Beauty population with progeny with different scald susceptibilities; Delicious from three harvests with progressively declining scald susceptibility; and the scald-resistant Idared and the scald-susceptible Law Rome. Differences in incidence and severity of scald in progeny from White Angel x Rome Beauty progeny tended to show relationships with POX activity at harvest, but, overall, associations were not consistent. However, greater scald incidence and lower POX activity were found in less mature Delicious fruit than in later harvested fruit. Also, the scald-resistant Iotadared had a much higher POX activity compared with the scald-susceptible Law Rome. A general hypothesis that POX activity is related to scald susceptibility was generally supported, but exceptions were observed.     

Antioxidant activity of betalains from plants of the amaranthaceae

Antioxidant activity of betalain pigments (seven pure compounds and four combined fractions) from plants of the family Amaranthaceae was evaluated using the modified DPPH(*) (1,1-diphenyl-2-picrylhydrazyl) method. All tested betalains exhibited strong antioxidant activity. Their EC(50) values ranged from 3.4 to 8.4 microM. Gomphrenin type betacyanins (mean = 3.7 microM) and betaxanthins (mean = 4.2 microM) demonstrated the strongest antioxidant activity, 3-4-fold stronger than ascorbic acid (13.9 microM) and also stronger than rutin (6.1 microM) and catechin (7.2 microM). Antioxidant activity of the tested betalains decreased in the following order: simple gomphrenins > acylated gomphrenins > dopamine-betaxanthin > (S)-tryptophan-betaxanthin > 3-methoxytyramine-betaxanthin > betanin/isobetanin > celosianins > iresinins > amaranthine/isoamaranthine. This study also investigated and discussed the relationship between the chemical structure and the activity of the betalains. The free radical scavenging activity of the betalains usually increased with the numbers of hydroxyl/imino groups and, moreover, depended on the position of hydroxyl groups and glycosylation of aglycones in the betalain molecules.     

Antioxidant activity of a combinatorial library of emulsifier-antioxidant bioconjugates

A combinatorial chemistry approach was employed for the design and systematic synthesis of antioxidant-emulsifier bioconjugates to improve antioxidant activity in the interface between oil and water. A combinatorial library of 12 bioconjugates was synthesized from the phenolic antioxidants Trolox (a water-soluble alpha-tocopherol analogue), dihydroferulic acid, dihydrocaffeic acid, and gallic acid in combination with serine ethyl ester, serine lauryl ester, and lauroyl serine by esterification of the serine side chain or amidation, respectively. The bioconjugates were characterized by NMR and mass spectrometry, and each was tested for antioxidant activity by measuring the radical scavenging rate of 2,2-diphenyl-1-picrylhydrazyl (DPPH (*)) in methanol, the radical scavenging rate of DPPH (*) in a heterogeneous solvent system, the rate of oxygen consumption in an oil-in-water emulsion with metmyoglobin initiated oxidation, and the lag phase for diene formation in unilamellar liposomes with free radical initiation in the aqueous phase; each case was compared to the antioxidant activity of the parent antioxidant. In general, the conjugates with longer chain derivatives exhibited improved antioxidative activity in heterogeneous systems, whereas no improvement was observed in homogeneous solution. The rate of oxygen consumption in oil-in-water emulsion was found to decrease with increasing octanol/water partition coefficient, which is suggested to correspond to a saturation of the water/oil interface with antioxidant bioconjugate to approach maximal protection.     

Antioxidant activity of diterpenes and polyphenols from Ophryosporus heptanthus

The antioxidant activity of 14 compounds (1-14) isolated from the ether and butanolic extracts of the aerial parts of Ophryosporus heptanthus has been assayed using a beta-carotene bleaching method and the DPPH technique. Compounds 1 and 13 showed the most potent antioxidant activity. Their structures have been established by spectroscopic techniques (mainly NMR). Compounds 7 and 12 are new natural products, and their structures have been confirmed by chemical synthesis.     

Antioxidant activity of methanol extracts obtained from Plantago species

Antioxidative activities of methanol extracts from five Plantago species (P. afra, P. coronopus, P. lagopus, P. lanceolata, and P. serraria) were characterized by the DPPH scavenging test and the inhibition of Fe2+/ascorbate-induced lipid peroxidation on bovine brain liposomes. All extracts showed antioxidant activity in both methods. Whereas P. serraria exhibited the strongest activity as a DPPH scavenger, P. lanceolata and P. serraria were found to be the most active in the lipid peroxidation inhibition assay. The extracts were investigated regarding their composition by different colorimetric techniques, such as the content of total phenolic compounds by the Folin-Ciocalteu assay, flavonoids by AlCl3 reagent, phenylpropanoid glycosides (PPGs) by Arnow reagent, and iridoids by Trim-Hill assay. A high correlation was found between the scavenging potency and the total phenolic and phenylpropanoid content of the extracts but not between the lipid peroxidation potency and the extract composition. P. serraria is presented as a possible new source of natural antioxidants.     

Polyphenolic compounds and antioxidant activity of new and old apple varieties

There is considerable evidence to show that a greater intake of apple contributes to improved health by reducing the risk of diseases, such as cardiovascular disease and some forms of cancer. Apple fruit is a major source of phenol compounds, because its consumption is widespread in many countries and it is available on the market for the whole year. The phenolic composition of 67 varieties of apple cultivars (new and old varieties) was examined for the concentration of some important phytochemicals and antioxidant activity. For the first time, we have looked at the correlation and compared polyphenolic coumpounds in Golden Delicious variety and new varieties grown from it. Up to 18 compounds, including catechin, procyanidin, hydroxycinnamates, flavonols, anthocyanins, and dihydrochalcones, were analyzed by high-performance liquid chromatography with diode array detection analysis of crude extracts and after thiolysis and LC-MS. The mean content of total polyphenols lay between 523.02 and 2723.96 mg/100 g dw and depending upon the apples variety. Flavanols (catechin and oligomeric procyanidins) are the major class of apple polyphenols, representing more than 80%, followed by hydroxycinnamic acids (1-31%), flavonols (2-10%), dihydrochalcones (0.5-5%), and in red apples, anthocyanins (1%). In this study, the best correlation was found for the total polyphenols and ABTS method, with a lower correlation for FRAP and DPPH methods ( r = 0.871, 0.839, and 0.804, respectively). The presented data clearly demonstrated that new varieties, i.e., Ozark Gold, Julyred, and Jester, of apple had the same or higher value of bioactive compounds in comparison to the old varieties, i.e., Golden Delicious, Idared, and Jonagold.     

Investigation of heat treating conditions for enhancing the anti-inflammatory activity of citrus fruit (Citrus reticulata) peels

In traditional Chinese medicine, dried citrus fruit peels are widely used as remedies to alleviate coughs and reduce phlegm in the respiratory tract. Induction of inducible nitric oxide synthase (iNOS) in inflammatory cells and increased airway production of nitric oxide (NO) are well recognized as key events in inflammation-related respiratory tract diseases. Despite the fact that the enhancing effect of heat treatment on the antioxidant activity of citrus fruit peels has been well documented, the impact of heat treatment on citrus peel beneficial activities regarding anti-inflammation is unclear. To address this issue, we determined the anti-inflammatory activities of heat-treated citrus peel extracts by measuring their inhibitory effect upon NO production by lipopolysaccharide-activated RAW 264.7 macrophages. Results showed that the anti-inflammatory activity of citrus peel was significantly elevated after 100 degrees C heat treatment in a time-dependent fashion during a period from 0 to 120 min. Inhibition of iNOS gene expression was the major NO-suppressing mechanism of the citrus peel extract. Additionally, the anti-inflammatory activity of citrus peel extract highly correlated with the content of nobiletin and tangeretin. Conclusively, proper and reasonable heat treatment helped to release nobiletin and tangeretin, which were responsible for the increased anti-inflammatory activity of heat-treated citrus peels.