Food and Chemical Toxicology Phytochemical Profile, Antioxidant

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Food and Chemical Toxicology 49 (201 1) 1549-1555

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Phytochemical profile, antioxidant, anti-inflammatory and hypoglycemic potential of hydroalcoholic extracts from Citrus medica L. cv Diamante flowers, leaves and fruits at two maturity stages a, Federica Menichini ", Monica R. Loizzo", Marco Bonesi', Filomena Conforti Damiano De Luca b Giancarlo A. Statti", Bruno de Cindio', Francesco Menichini", Rosa Tundis,'*

" Department of Phamaceutical Sciences, Faculty of Pharmacy and Nutrition dnd Health srjtences, lJniversity of Calabia, I-87036 Arcavacata di Rende (CS), ltaty b CAIAB, Laboratorio Chimico Merceologico della Catabna, |-BZO4T Montalto Ufugo (CS), Italy 'Department of Engineering Modeling, University of CalabrÌa, I-87036 Arcayacata di Rende (CS), Italy

ARTICLE INFO ABSTRACT

Article history: Since the past decade consumption ofcertain foods has been reported to have a positive effect on health. Received 1 7 February 20.1 1 The object of the study was to determine for the first time the chemical composition and the antioxidant, Accepted 26 March 2011 anti-inflammatory and hypoglycemic potential of Cftrus medicaL. cv Diamante flowers, leaves and fruits Available online 3O March 201 1 (endocarp and mesocarp) at two maturity stages. Flowers and leaves were characterized by the highest total phenols and flavonoids content. A declining trend was observed during maturity of fruits for both Keywords: phenols and flavonoids. The antioxidant activity evaluated by the p-carotene bleaching test showed a Citrus medica L. cv Diamante strong Flavonoids activity for flowers and endocarp of mature fruits with [C56 values of 2.8 pg/ml and 3.5 pg/ml, Antioxidant activify respectively, after 30 min of incubation. Interestingly, the mature fruits endocarp (lCso value of Anti-in fl ammatory activity 426.0 pglmL) could inhibit a-amylase with an IC56 value 2-fold higher than immature fruits. None of Hypoglycemic activity the tested extracts affected the proliferation ofhuman skin fibroblasts 142BR. The obtained results suggest a potential use of C. medica L. cv Diamante as new valuable Citrus species with functional properties for food or nutraceutical product on the basis of high content of phytochemicals. O 201 1 Elsevier Ltd. All rights reserved.

1. Introduction far been conducted on the chemical composition and biotogical activity of flowers, leaves and fruits endocarp and mesocarp. Citrus medica cv Diamante (Diamante citron) is a cultivar of In traditional societies nutrition and health care are strongly C. medica, which is best cultivated in the warm and moderate- interconnected and many plants have been consumed both as food warm climates of Calabria (ltaly) where the cultivation of citron and medicine (Pieroni, 2000). The consumption of edible plants extends along the coast of high Thyrrenium. This plant is of ancient plays a central role in the diet, but very few ethnopharmacological origin. The more accredited provenance is from India but it proba- and phytopharmacological studies have dealt exhaustively with bly arrived in Italy through the Hebrews who introduced the culti- the potential health benefits of such diets. On the other hand, stud- vation of the Diamante citron on the Calabrian coasts (Fersini et al., ies on the antioxidant properties of specific plant foods and their t 973). The main products obtained from Diamante citron are can- phenolic constituents have become very impressive (lorizzi et al., dy and liqueurs, while the oil is a minor product, used as flavouring 2001; Martinez-Tomé et al., 2001 ; Jagtap et al., 2010). Oxidative in sweets and beverages (Cutuli et al., 1985). The tree is of small stress play an important role in the pathogenesis ofvarious serious dimensions, the leaves are large and oblong, and the flowers are diseases such as neurodegenerative disorders, cancer, cardiovascu- of a red-purple colour. The ripe fruits are big, with a thin, smooth, lar diseases, inflammation, and diabetes (Yangthong et al., 2009). and lemon-yellow peel and the pulp does not yield much juice. The In diabetes, oxidative stress plays an important role since diabetic peel of Citrus fruits has been used in traditional Asian medicine for monocytes produce increased superoxide anion (Venugopal et al., centuries (Wichtl and Bisset, 1994; Blumenthal et al., 1998). The 2002). Diabetes is an increasing risk to the population and is due chemical composition and the biological properties of C. medica to a disorder of carbohydrate uptake (Zheng et al., 2007). Therefore cv Diamante peel extract and essential oil has been reported there is a need for novel strategies that could regulate the sugar (Conforti et al., 2007; Gabriele et al., 2009). No studies have so uptake and blood sugar levels. One therapeutic approach in the early stage diabetes is to decrease post-prandial hyperglycaemia. * Corresponding author. Tel.: +39 984 493246; fax: +39 984 493298. This is done by retarding the absorption of glucose through the Ennail oddress: [email protected] ( R. Tundis). inhibition of the carbohydrate-hydrolyzing enzymes, cr-amylase

0278-691 5/S - see front matter Cr 201 1 Elsevier Ltd. All rights reserued. doi: I 0. I 0l bil.tcr.20 | 1.03.048 I 550 F. Menichini et al./Food and ChemicalToxicologr 49 (2011) 1549 1555 and d-glucosidase, in the digestive tract (Tundis et al., 2007). was mixed with 0.2 mL Folin-Ciocalteau reagent, 2 mL of distilled Consequently, inhibitors of these enzymes could reduce the rate water and 1 mL of 15% Na2CO3. The absorbance was measured at of glucose absorption and blunt the post-prandial plasma glucose 765 nm (using a W-Vis Jenway 6003 spectrophotometer) after rise. 2 h incubation at room temperature. The levels of total phenolics The aim of the present study was to evaluate the chemical com- content were determined in triplicate. Chlorogenic acid was used position and the biological properties of C. medica cv Diamante. For as a standard and data are expressed as chlorogenic acid equiva- this purpose the flowers, leaves and fruit portions (endocarp and lents in mg per 100g of fresh materials. mesocarp) at two maturity stages were analyzed. The antioxidant activity (assessed by DPPH assay and bleaching of B-carotene), the anti-inflammatory activity (vio the inhibition of nitric oxide 2.5. Determination of total flavonoids content N( ) production in RAW 264.7 cells), the cytotoxic activity against skin fibroblasts 142BR and the inhibition of cr-amylase and The flavonoids content was determined spectrophotometrically u-glucosidase were investigated and were related to the different using a method based on the formation of a flavonoid-aluminium chemical composition. complex (Yoo et al., 2008). One mL of the extract was added to a 10 ml volumetric flask. Distilled water was added to make a vol- 2. Experimental ume of 5 mL. At time zero, 0.3 ml of 5% (wlv) sodium nitrite was added to the flask. After 5 min, 0.6 mL of 10% (w/v) AlCl3 was added 2.1. Chernicals and then at 6 min 2 mL of 1 M NaOH were also added to the mixture, followed by the addition of 2.1 mL distilled water. Absorbance at 510 nm was read immediately. Methanol, ethanol, DMSO, sodium carbonate, sodium nitrite, and Quercetin was chosen as a stan- quercetin NaOH were obtained from VWR International s.r.l. (Milan, Italy). dard and data are expressed as equivalents in mg per 100 g fresh materials. Ascorbic acid, B-carotene, propyl gallate, linoleic acid, Tween 20, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Griess reagent (1% sulfana- mide and 0.1% N-(1-naphtyl) ethylenediamine dihydrochloride in 2.5% H3PO4), MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet- 2.6. HPLC analysis of C. medica cv Diamante flavonoid-ich fractton razolium bromide), Dulbecco's Modified Eagle's Medium (DMEM), r-glutamine, foetal bovine serum (FBS), antibiotic/antimicotic solu- The analysis of C. medica cv Diamante flavonoid-rich fraction was (SPE) tion (penicillin/streptomycin), LPS, indomethacin, potato starch, carried out by solid-phase extraction method and HPLC sys- performed sodium phosphate, sodium chloride, o-amylase from porcine pan- tem. Extraction wad by solid-phase extraction (SPE) method, SPE 12-Position Vacuum (15 creas (EC 3.2.1.1), u-glucosidase from Sacchoromyces cerevisiae using Manifold Set mm Hg) (33 (EC 3.2.1.20), maltose, sodium acetate, sodium potassium tartrate, and a Phenomenex strata-X pm) C18 cartridge. The cartridge 3,5-dinitrosalicylic acid, o-dianisidine color reagent (DIAN), glucose was conditioned with 20 mL of methanol, followed by 24 mL of 5% methanol. oxidase peroxidase enzyme solution (PGO), naringenin, naringiq hes- Then the sample was eluted through the cartridge under passing (24 peretin, hesperidin, rutin, nobiletin, tangeretin, quercetin, diosmin, vacuum by through 5% methanol mL) and the flavonoid- rich fraction was obtained with 10mL of methanol. Naringenin, apigenin, Folin-Ciocalteau reagent, AlCl3, sulforodamine B (SRB), trichloroacetic acid (TCA), HOAc and chlorogenic acid were obtained naringin, hesperetin, hesperidin, rutin, nobiletin, tangeretin, quercetin, diosmin were identified HPLC from Sigma-Aldrich S.p.A. (Milan, Italy). All other reagents were of and apigenin in system and their peak analytical grade and were from Carlo Erba products (Milan, ltaly). content was calculated from the integrated area of the sample and the corresponding standard. HPLC analyses were realized using an HPLC system HP 1100 equipped with a pump, UV-vis detector 2.2. Plant mateial (280 nm), column oven, injector, and a Cl8 RP column (Phenomenex Luna 5 pm C18,250 x 4.60 mm). The mobile phase was H2O/formic The flowers and leaves of C. medica L. cv Diamante used in this acid (0.1%)(A)and methanol (B)witha flowrate of 'l ml/min (2 min study were collected in October 2006 in Calabria (Southern ltaly), 1OO% A:8 min 80% A; 55 min 100% B; 65 min 100% A). loc. Santa Maria del Cedro (CS). The fruits were harvested at the same time but at two successive maturity stage on the basis of their colour as immature (1, green colour fruits) and mature (M, 2.7. Determination of hydrogen donation ability (DPPH test) yellow colour fruits). Samples were randomly harvested from 20 plants in order to obtain a sample representative. The authentica- Free radical scavenging activity was determined using a rapid tion was carried out at Natural History Museum of Calabria and TLC screening method based on the reduction ofa methanolic solu- Botanic Carden, Universis of Calabria, Italy. All samples were tion of the coloured free radical DPPH. After developing and drying examined for integrity and absence of dust and insect contamina- TLC plates (VW& silica gel plates, CH2CI2/MeOH B:2) were sprayed tion, and were freeze-dried and stored at until analysis. -20.C with a O.2% DPPH radical solution in MeOH. The plates containing the extracts and lipophilic fractions were examined 30 min after 2. Extr crctio n pro ce dure i. spraying. The samples with antioxidant activity appeared as yellow spots against a purple background. In order to determine the rad- quality Cood fruits, flowers and leaves were exhaustively ex- ical scavenging potency the samples, which exhibited antioxidant tracted 70% aqueous (96 with alcohol h x 5 times). The extract activi§, were investigated (Loizzo et al., 2009a). In an ethanol solu- was concentrated pressure under reduced to obtain a dry residue. tion of DPPH radical (final concentration was 1.0 x 10 4M), test The yield percentage extraction was reported in Table 1. samples were added at different concentrations. The reaction mix- tures were shaken vigorously and then kept in the dark for 30 min. 2.4. Determinotion of total phenols content The absorbance of the resulting solutions was measured ìn 1 cm cuvettes using a Perkin Elmer Lambda 40 W/VIS spectrophotome- The amount of total phenolics of C. medica cvDiamante peel ex- ter at 517 nm against blank (without DPPH radical). All tests were tracts was determined using Folin-Ciocalteau reagent according to run in triplicate and the mean values calculated. Ascorbic acid was the method previously reported by Gao et al. (2000). The extract used as a positive control. ,r; ì

F. Menichini et aI./Food and ChemicalToxicologl 49 (2011) 1549-1555

Table 1 Extraction yield (%) of different parts of C medica cv Diamante, and the content of total phenols and flavonoids therein,

Extraction yield (%) Phenols (mg/.100 g FW) Flavonoids (mg/l00 g FW)

Flowers 4.7 398.0 I 3.2 266.9 ! 7.2 Leaves 8.2 401.6 t 5.1 97.5 ! 2.8 lmmature fruits Mesocarp 6.1 181.3 I 3.1 95.7 !3_2 Endocarp 6.3 262.6 !3.7 64.9 !3.2 Mature fruits Mesocarp 5.7 723.7 !6.5 43.1 !1.2 Endocarp t 1.3 709.4 !2.9 37.5 I 1.6

Data are expressed as mean t S.D. (n = 3); Fly'y', fresh weight.

produce different concentrations. 2.8. ft-Carotene bleaching test further diluted in medium to One hundred microliters/well of each dilution were added to the Antioxidant activitv was determined using a B-carotene bleach- plates in six replicates to obtain the final concentrations ranging ing test (Shahsavari et al., 2008). Standard (propyl gallate) at the from 2.5 to 200 pg/ml The final mixture used for treating the cells same concentration as the samples was used for comparison. The contained not more than 0.5% of the solvent (DMSO), the same as measurement was carried out at initial time (t= 0) and succes- in the solvent-control wells. After 48 h of exposure 100 pL of ice- sively at 30 and 60 min. All samples were assayed in triplicate cold 40% trichloroacetic acid (TCA) was added to each well, left and averaged. for t h at 4'C, and washed with distilled water. The TcA-fixed cells The antioxidant activity (AA) was measured in terms of success- were stained for 30 min with 50 ytL of O.4% (w/v) SRB in 1% acetic ful bleaching of B-carotene by using the following equation: acid. The plates were washed with 1% HOAc and air dried over- night. For reading plate, the bound dye was solubilised with 100 AA - {1 l(Ao - At)/(A"o - A',)l} x 100 pL of 10mM tris base (tris[hydroxymethyl]aminomethane)' each well was read on a Molecular Devices where As and A'o are the absorbance values measured at the initial The absorbance of (Molecular Devices, Celbio, Milan, incubation time for samples/standard and control, respectively, SpectraMax Plus Plate Reader 490 nm. Cell survival was measured as the percentage while A1 and A"r are the absorbance values measure in the sam- Italy) at untreated control. Vinblastine sulfate ples/standard and control respectively at f= 30 and 60 min. absorbance compared to the salt was used as positive control.

2.9. Cell culture 2.11. lnhibition of nitnc oxide (NO) production in LPS-stimulated RAW The murine monocytic macrophage RAW 264.7 cell line 264.7 celb (European Collection ofCell Cultures, London, UK) and skin fibro- product blasts 1428R (ECACC No. 90011806) were grown in plastic culture The presence of nitrite, a stable oxidized of NO, was flask in DMEM with L-glutamine supplemented with 10% FBS and determined in cell culture media by Criess reagent (1% sulfana- 1% penicillin/streptomycin solution under 5% COz at 37'C. After mide and 0.1% N-(1-naphtyl) ethylenediamine dihydrochloride in 4-5 days cells were removed from the culture flask by scraping 25%H3POò (Prakash et al., 2005). 100 pL of cell culture superna- and centrifuged for 10 min under 1500 rpm. The medium was then tant was removed and combined with 100 pL of Griess reagent in removed and the cells were re-suspended with fresh DMEM. Cells a 96-well plate followed by spectrophotometric measurement at (GDV counts and viability were performed using a standard trypan blue 550 nm using a microplate reader DV 990 B/V, Roma, Italy). cell counting technique. The cell concentration was adjusted to Nitrite concentration in the supernatants was determined by com- 1 x 106 cells/ml in the same medium. 100 pL of the above concen- parison with a sodium nitrite standard curve. lndomethacin tration were cultured in 96-wetl plate for one days to become (72.5-2OO pg/ml) was used as positive control. Cytotoxicity was nearly confluent. Concentrations ranging from 10 to 200pg/ml determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl- of the samples were prepared from the stock solutions by serial tetrazolium bromide (MTT) assay reported by Loizzo et al. per- dilution in DMEM to give a volume of 100pL in each well of a (2009b). The assay for each concentration of samples was microtiter plate (96-well). Then cells were cultured with the vehi- formed in triplicate and the culture plates were kept at 37 "C with cle and extracts in the presence of 1 pg/ml LPS for 24 h. 5% (vlv) CO2 for one day. After 24 h of incubation, 100 pL of medium was removed from each well. Subsequently, 100 pL of 0.5% w/v MTT, dissolved in phosphate buffer saline, was added to each 2.10. Assessment of cytotoxicity (SRB assay) well and allowed to incubate for a further 4 h. After incubation, pL DMSO was added to each well to dissolve the formazan The cytotoxic assay was performed as previously described 100 of crystals. Absorbance values at 550 nm were measured with a (Loizzo et al., 2005). The protein-staining sulforodamine B (SRB) microplate reader (GDV DV 990 B/V, Roma, Italy). Cytotoxicity assay was used for measurement of cell proliferation. The test is was expressed as CD5s which is the concentration to reduce the based on the estimation of cell number indirectly by providing a of treated cells by 50% with reference to the control sensitive index of total cellular protein content which is linear to absorbance (untreated cells). cell densi§. Cells were trypsinized, counted and placed in 96-well plates at optimal plating density of each cell line was determined ovef a range from 5 x 104 to 15 x 104 to ensure- exponential 2.12. Bioassay for d.-amylose inhibition growth throughout the experimental period and to ensure a linear relationship between absorbance at 490 nm and cell number The u-amylase inhibition assay method was performed using where analysed by the SRB assay and incubated to allow for cell the method previously described (Tundis et al., 2007). C. medica attachment. After 24 h the cells were treated with serial dilutions cv Diamante extracts were dissolved in buffer to give final concen- of the samples. Each sample was initially dissolved in DMSO and tration from 1mg/ml to 72.5 pg/mL Control and samples were 1552 F. Menichini et al. I Food and Chemical Toxicologt 49 (201 1 ) 1 549-1 555 added to starch solution and left to react with s-amylase solution flowers to 58.0 mglkg of fresh weight for mature fruits endocarp. at 25 "C for 5 min. The reaction was measured over 3 min. The gen- The flowers extract was characterized by the highest content ofdif- eration of maltose was quantified by the reduction of 3,5-dinitro- ferent flavonoids. Besides apigenin, quercetin (580.8 mg/kg) and salicvlic acid to 3-amino-5-nitrosalicylic acid, the product being diosmin (372.5mglkg) were also found in significant quantities. detectable at 540 nm. In the presence of an ct-amylase inhibitors The content of quercetin was much lower in leaves (7.9 mg/kg) less maltose will be produced and the absorbance value would de- and immature fruits mesocarp (11.0mglkg). The flavone diosmin, crease. The u-amvlase inhibition was expressed as percentage of abundant in the flowers extract, was detected only in the mature inhibition and calculated by the following equation: fruits mesocarp (18.2mC/kC). Naringin was identified only in mesocarp of immature fruits (556.0 mC/kg). Hesperidin, unlike of lMaltolel test ,./ rnhibirion : 100 - I tool r. s.o. correspondent aglycone hesperetin, was identified in immature \lMaltose] control' / fruits extracts with values of 26.0 mg/kg for mesocarp and 18.4mg/kg for endocarp. Flavanones, hesperetin and hesperidin were abundant in the flowers extract with values of 203.8 and 2.13. Bioassay for u-glucosidase inhibition 224.3 mglkg. Rutin was abundant in immature fruits endocarp ex- (484.7 ms/kg), followed by leaves and flowers extracts with The a-glucosidase inhibition was measured using a modified tract values of 264.2 arrd 156.5 mg/kg. The polymethoxylated flavones, Sigma-Aldrich bioassay method (urvrruv.sigmaaldrich.com). Ex- nobiletin and tangeretin were not identified in any of the C. medica tracts were dissolved in DMSO to give final concentration from cv Diamante extracts. 1 mg/m[ to 5 pg/ml. The reaction was started by adding cr-gluco- In agreement with several published results for Cfrrus fruits sidase solution and tubes were left to incubate at 37'C for 30 min. (Ortufro et al., 1997), the highest flavanones contents were found After that time perchloric acid solution (4.2% w/v) was added to in fruits, with most synthesis taking place during the early stop reaction. ln the second step the generation of glucose was voung stages of fruit growth. Hesperidin represents 49% (immature) and quantified by the reduction of DIAN. The supernatant of tube of 18% (mature) of the dry weight of the C. reticulata Blanco ("Calleta" step I was mixed with DIAN and PGO and was left to incubate at mandarin) fruits. The levels for immature fruits are similar to those 37 "C for 30 min. The absorbance of DIAN was measured spectro- found in immature fruits of "Nova" hybrid (C. reticulata x C. paradise) photometrically at 500 nm. The u-glucosidase inhibition was ex- (48%), those for mature fruit far exceeds those found in pressed as percentage of inhibition and calculated by the but C. reticulata (2%). Previously, the distribution of naringin has been following equation: analvzed during the development of C. aurantium leaves, flower ,,4 buds, and fruits. These flavanones are at maximum concentration rnhibition: r00 /Jg!,.gTI§:l- roo\ -r s o. - \lclucosel control^ ) during the logarithmic phase of growth, gradually decreasing until the organs reach maximum development. However, this decrease in the concentration in leaves, flower buds and fruits is due to a 2. 1 4. Statistical analysis dilution ofthe flavonoid caused by cell growth, because total content per organ continues to increase (Castillo et al., 1992). Data were expressed as means t SD. Statistical analysis was performed by using Student's t test. Differences were considered significant at P < 0.05. The inhibitory concentration 50% (lcso) 3.2. Antioxidant properties was calculated from the Prism dose-response curve (GraphPad, Prism version 4.0 for Windows, CraphPad Software, San Diego, The effect of antioxidants on DPPH radical scavenging was CA, USA) obtained by plotting the percentage of inhibition versus thought to be due to their hydrogen-donating abili§. The scaveng- the concentrations. ing effects of the C. medica cv Diamante extracts on DPPH were examined at different concentrations. The IC56 values for DPPH as- 3. Results and discussion say ofthe samples are given in Table 3. The best free radical (DPPH) scavenging activity was exerted by mesocarp of immature fruits 3.7. Extracts composition (lC5s of 382.0 pglml) followed by flowers and leaves extracts with IC56 values of 425.0 and 502.0 pg/ml, respectively. The established health benefits of phenolics, due to their biolog- All C. medica cv Diamante extracts were able to inhibit the dis- ical activities, necessitates their quantification in foods. The total coloration of B-carotene. The results of the antioxidant effect of the soluble phenols and flavonoids of C. medica cv Diamante flowers, samples on the auto-oxidation of linoleic acid are shown in Table 3 leaves, and fruits (mesocarp and endocarp) at two maturity stages and Fig. 1. Flowers showed the highest and interesting inhibition of extracts were determined by Folin-Ciocalteau assay and using a linoleic acid oxidation (1C56 value of 2.8 pg/ml) at 30 min of incu- method based on the formation of a flavonoid-aluminium complex, bation. These results are important if compared to the reference respectively. As evidenced in data are reported in Table 1, flowers drug. and leaves were characterized by the highest total phenols and Previously, extracts of 13 commercially available Cifrus spp. flavonoids content. The phenols total content decreased during peels and edible parts were investigated for their antioxidant activ- fruits (mesocarp and endocarp) maturity. The flavonoids content ity by DPPH method with lC56 values ranged from 0.6 to 3.8 mg/ml followed the same trend of phenols. ln fact, for example values of (Ghasemi et al., 2009). Total phenolics and flavonoids content var- 95.7 and 43.1 mg/100 g of fresh weight were evidenced for meso- ied from 66.5 to 396.8 mg gallic acid equivalent/g of extract and carp extract of immature and mature fruits, respectively. from 0.3 to 31.1 mg quercetin equivalent per gram of extract, The health benefits of Cilrus fruit have mainly been attributed to respectively. the presence of bioactive compounds, among them flavonoids [22]. Moreover, the seeds of Citrus junos were investigated for their For this reason the content of ten flavonoids, namely naringenin, total polyphenol and flavonoid content and antioxidant activity naringin, hesperetin, hesperidin, rutin, nobìletin, tangeretin, (Lee et al., 2009). The hull and embryo of seeds were extracted with quercetin, diosmin, and apigenin, has been analyzed in this study n-hexane and 70% ethanol. DPPH radical scavenging activity of (Table 2). Apigenin was identified in all extracts, except for meso- hydroalcoholic extract was higher than that of n-hexane extract carp of mature fruits with values ranging from 941.0mg/kg for with ICso values of 3.18-8.43 mg/ml. 1 553 49 (2011) 1549-1555 F. Menichini et at./ Food and Chemical Toxicolog

Table 2 C' medica cv Diamante extracts' ,iirc .-n"fyri, of flavonoids (mglkg of fresh weight) from QUE 372.5 r6.4 941.0 i 8.0 156.5 1 3.3 580.8 ! 3.1 224.3 !3.2 203.8 r 3.1 432.7 !4.3 Flowers 264.2 !3.6 7.9 ! 0.6 Leaves 284.0 !2.5 lmmature fruits 1 1.0 r 1.1 26.0 ! 0.8 108.0 r 3.7 Mesocarp 556.0 !7.4 !3.'l 18.4!06 484.7 Endocarp - 78.2 !2.6 Mature fruits 58.0 I 3.9 Mesocarp 9.0 i 0.9 Endocarp rutin:QUE,quercetiniDSM,diosminlAPt,apigenin.

Table 3 RadicalscavengingandantioxidantactivitiesandinhibitionofNoproductioninR,{w264.7LPs-stimulatecellsofC.medicocvDiamanteextracts.

IC5o (pg/mL) Inhibition of NO Production p-Carotene bleaching test

30 min 60 min 10.0.10' 525.0 r 1.9' 2.8 0.002" 5.7 425.0 !2.95' r 57 4.O Flowers 5.1 i 0.009" r2.6' 3.01' >100 Leaves 502.0 t >1000 lmmature fruits 4.1 r 0.009' 3.7 r 0.007" >1000 MesocarP 382.0 !2.45' 4.s i o.oo9' >1000 4.1 i 0.009" Endocarp >1 000 Mature ffuits 0.075 45.4 !0."175 >1 000 36.6 ! >1 000 MesocarP 7.1 i 0.015- >1 3.s ! 0.008" 000 1.0 0.01 Endocarp 1.0 1 0.01 i Propyl gallate 1.2 Ascorbic acid 2.0 i 0.01 53.0 r lndomethacin d indomethacin are used as positive controls'

" p < 0.05 Dunnett's test' p > 0.05 Dunnett's test. ' p < 0.01 Dunnett's test' "t- p < 0.000.1 One-way ANOVA test'

production properties 3.3. lnhibition of nitnc oxide Xu et al. (2008) previously reported the antioxidant of quality param- ,rrrougn Àirè."nt m;thods and the quantification as a mediator and regulator in sweet Nitric oxide (NO) is recognized 15 Citrus varieties (seven mandarins' four responses ii;ri*t from pathological reactions, especially in acute inflammatory "ì.., one grapefruit' and two pummeloes) from oranges,-Évutia one lemon, (Terao, 2009). qzs c' sinensis) achieved the highest ''i;;;;;1" éil.3. G. reti;ilam x ' inhibition of No production activitv' the murine and the highest total the antioxidant and radical scavenging activity cell line RAW 264'7 was employed as a phenols and flavonoids *oroiyti. macrophage pr',"nori.r. n r"cent study evalualedlhe total òf inflammation mediators such as No' once peel and edible i.t ibition H;;;;à',il'antioxidant activitv of c' sulcata -"J"ii"i stimulation, macroph,ages produce a ultrasound ir","a by inflammatory obtained by high-piessure extraction' molecules' Flowers and leaves extracts i*iii the edible "ih.d;;É, of cytotoxic "r,a.r.ttand Soxhìet In the DPPH model of LPS-induced NO extraction of C. medica cv Diamante showed inhibition "*it"àon'showed the highest radical scavenging with i.ui r-"*a.".,"d by Soxhlet o.Ju.tion in RAW 264.7 cells in a dose-dependent manner, activity (wang et al., 2010) (Table 4)' and 574'0 pg/ml' respectivelv (Table 3)' between anti- ilrc;;t; oi szs.o Several studies have evaìuated the relationships extracts on the inhibition of produc- phenolic content' it UÉn"n.i.f effect of tested oxidant activity of plant products and their " correlation between total tionoftheinflammatorymediatorsinmacrophagesmaybedueto v"ii"gf, et al. i1998) reported a high of phenols and flavo-noids and is proba- vegeta- il'p;;; or nign t"u"t and antioxidant activity in selected fruits' products of phago- ;;;;;il.on*nì i',r, throigh oxidative degradation of extract was characterized by api- lrJor.a in bles and grain products. Flower òi: Jna Hocl' rhe cvtotoxic errect or all extracts constituents' Both flavonoids are ;i;;:;;h ; g"*, .nà quercetin as main (1 pg/ml) was also evaluated' c- medica cv mechanisms of action àiLpS ir.ii r.no*., às antioxidant through different "'.àr"n* show anv cvtotoxicity up to 1mg/ml most po- ;ffi;;" .*tt.ct'aià-not and I(atan, 1999: Terao, 2009)' Quercetin is the iH"ifÀm antioxidative concentration. i"n, ,."u"ng"r of ROS (Boots et al" 2008)' These Citrus (peel and juice extracts) were of two anti- Previously, thirty-one ."p".iti", oiquercetin aie attributed to the presence activities toward NO generation in that have the opti- screened for their suppressive pharmacophores within the molecule RAW264'7 cells stimulated with LPS and i'e' the catechol ;;;il macrophage ".iJ""t*"r ì"r:rù"*tion for free radical scavenging' (rvruàtami et a1', 2000)' Generallv' the inhibitory group at position-3 of the AC rins' il;;;"; t 6. B ring and the oH were largely found to than those -rraoiàor"r,;;il1; empower the activities of peel parts -b9-h-igher qu"r..iin is suggestÉd tò substantially juice pitti' choi et al' (2007) investigated total àìin".*i"t'p"nding ttt tiléta au" to its contribution to the and NO production correlation between the flàvonoid content "naog"nort'untioxidant which is 6'24 times higher than the the study the plasma antioxidant capacity activity of Citrus fruit peel extracts' In this whereas the contribution of both ,.friUiaw corre- reference antioxidant tiolJx, ptoOr.tion inhibitory activities of the peel extracts were similar to that of trolox (Boots Nò data ;;i; C and uric acid is almost i";eà-;rh both the noÉiletin and tangeretin contents. Our et al., 2008). 1554 F. Menichini et al. I Food and Chemical Toxicologt 49 (2011) 1549-1555

{a}'oo rlq qi*fleffs cannot confirm this correlation since floral extract was character- #- I *+ * '.ry " ized by a high content of apigenin and quercetin. The effects of : _Y-' *#-'f.opyl i g€lla re both flavonoids on a variety of inflammatory processes -§'" and im- 1A i.'/ -*" laava mune functions have been extensively studied and it has been ,." :§ uo: demonstrated to inhibit several enzymes that are activated in cer- tain inflammatory conditions (Surh et al., 2001 al., 2007). È 'ura . *,.& ; lee et f ...{** " Apigenin, unlike quercetin, is non-toxic and non-mutagenic, and w.-..o,' blocks the development of mouse skin tumors and the proliferation tù, ,l L- ''' L t0 *' ," of human breast-cancer cells. Apigenin is a potent antioxidants, t0 J' cyclooxygenase inhibitor, cell cycle inhibitor, protein kinase C . / inhibitor, apoptosis inducer, and NO production inhibitor Also o " *^--,,-*-*1.- [34]. quercetin is known to possess strong anti-inflammatory 1,t5 :,5 5 :.0 :5 50 100 capacities (Wang et al., 2010). Severat in vifro studies have ttnrefitration p&lml shown that the fla* vonoid is capable of inhibiting LPS-induced cytokine production. Moreover, in glial cells quercetin can inhibit LPS-induced mRNA (b;*o +PropylEaìistè i-_- rylal;"§t..- levels of two cytokines, i.e. TNFu and IL-1 u. A possible explanation x.*Èl -;§ * -*-3 - -+'Mèsocatp for these anti-inflammatory effects of quercetin may be found 80 \.s/ in --à* Endmsrp the interplay between oxidative stress and inflammation. ROS are 7A § not only involved in the occurrence of oxidative stress, but also §60 ,.ìJ in the promotion of inflammatory processes via t,J;'! activation of tran- scription factors such as NF-KB and protein §unÉ activator (AP)-l which -40 induce the production of cytokines TNFcr. n§ like Consequently, scav- JO .$ .1 enging ROS would not only prevent the occurrence of oxidative t0- stress but also help mitigate inflammation. Indeed, it has already ì0 been shown that quercetin can inhibit the production as well as Ò the gene expression of TNFcr via modulation of NF-xB in human peripheral l,:s t,5 5 10 25 §0 100 blood mononuclear cells (Boots et al., 2008). Conrsiltration pg/rrrL 3.4. Hypoglycemic activity

| 7 -..é .&M€§mlrp {c}'oo . -,u The hypoglycemic potential of C. medica cv Diamante extracts 90 : ,q. F.osyl sallatÈ was evaluated by the cr-amylase and cr-glucosidase inhibition as- §0 .-L- t.r _, --** ffldoears says. Glucose can be readily absorbed from the gastro-intestinal x) .67 /--*4 tract into the blood stream after the hydrolysis of glycosidic tso .g /.rf, bonds in digestible carbohydrate foods containing starch by N/,,, ,/. t #,Èt u-amylase €r0 r and o-glucosidase. Inhibition of these enzymes could iao &/ reduce the high post-prandial blood glucose peaks in diabetics ,s (Tundis et al., 2007). The leaves extract exhibited a good activity against o-amylase with ICso value of 438.5 pg/ml. Interestingly, 10 the comparison between results of endocarp obtained from ma- s ture fruits and immature fruits revealed that mature fruits inhib- 2.3 ited the u-amylase with ICso value 2-fold higher that immature (lcso (oncentration fruits value of 426.0 pglmL} On the contrary the d-glucosi- F$lmL dase was inhibited more by the immature fruits with IC56 Fig. l. Antioxidant activity using fl-carotene bleaching test of C medica cv 472.9 pglmL. Correlation belween phenol and flavonoids content Diamante extracts from (a) aerial parts, (b) immature fruits, and (c) mature fruits revealed that carbohydrate-hydrolyzing enzyme inhibitory activ- after 30 min of incubation. gallate Propyl was used as positive control. Data are i§ could not be related with these phytochemicals since flowers exptessed as mean t S.D. (n = 3). that are characterized by the highest content were unable to inhibit the enzymes. Moreover, analysis of HpLC profile of endocarp in mature fruits revealed the presence of apigenin that previously Table 4 had not known to exert any activity on u-amylase and cr-glucosi- C medica cv Diamante extracts cr,-amylase and a-glucosidase inhibitory activity. dase. Among the selected flavonoids markers, only quercetin previously had demonstrated a strong enzyme inhibition activi§ Extract lC56 (pg/mL) (0.50 mM) on porcin pancreatic o-amylase. Both quercetin and ct-Amylase oa-Clucosidase hesperetin are able to inhibit cr-glucosidase from Saccharomyces Flowers >1000 >1000 cerevisiqe with ICsg values of 7 and 50 pM, respectively. On the Leaves 438.5 r 5.2* 777.8 ! 5.4* contrary, apigenin showed a 43% of enzyme inhibition percentage lmmature fruits at maximum concentration tested 200 trrM (Tadera et al., 2006). MesocÀrp 702.2 ! 5.7* 539..7 !6.4* In our previous work n-hexane Endocarp 844.5 t 3.6* 472.9 ! 4.7* C. medica cv Diamante peels Manffe fruits extract inhibited u-amylase with an IC56 value of 625 pg/ml Mesocarp 707.4 !5.6* 633.1.1 3.4* (Conforti et al., 2007). This activity was related to the content Endocarp 426.0 !4.4,* 574.1 i 5.8* of terpenoids considering that the lipophilicity of these phyro- Acarbose 50.0 r 0.9 35.5 ! 1.2 chemicals may facilitate access to the enzymatic site (Ali et al., ICso mean 1 S.D. (n = 3). Acarbose: positive control. 2006). Recently, the C. medica var. sarcodactylis *"p .-p essential oil was < 0.001 One-way ANOVA testi < 0. 01 Dunnett's test tested in vivo for its hypoglycaemic and antidiabetic effects 1 555 F. Menichini et at./Food and ChemicalToxicolog/ 49 (2011) 1549-1555

2022' demonstrating to be very beneficial to type 2 diabetes mellitus structure determination and biological activity. J. Agric. Food Chefi. 49' 2029. patients (Peng et al., 2009). and Jagtap, U.8., Panaskar, S.N., Bapat, V.A., 20'l O. Evaluation of antioxidant capaci§ Food phenol content in J ackfruit (Artocarpus heterophyllus lÀm') fruit pulp Plant Hum. Nutr. 65.99-104. 4. Conclusions Lee,J.H., Zhou, H.Y., Cho, S.Y., Kim, Y.S., Lee, Y.S.,Jeong, C.S., 2007. Anti-inf'lammatory mechanisms of apigenin: inhibition of cyclooxygenase-2 expression, adhesion This study reported for the first time the chemical composition, of monocytes to human umbilical vein endothelial cells' and expression of Arch. Pharm. Res.30, 13.18-1327. the antioxidant and anti-inflammatory activity, and the effect of cellular adhesion molecules. Lee, YJ., Hwang, 1.G., Joung, E.M., Kim, H.Y, Park, 8.S., Woo, K.S., Jeong, H.S.' 2009. C. medica cv Diamante extracts on glucose homeostasis related en- Physiological activity and antiproliferation effects of Citron seed extracts on zymes. For this purpose flowers, leaves and fruit portions (endo- cancer cells.J. Korean Soc. Food sci' Nutr' 38, 1672-1678. F., Tundis, R., Bonesi, M', saab, A., Statti, G., de carp and mesocarp) at two maturity stages were analyzed. The Loizzo, M.R., Menichini, F., Conforti, Cindio, 8., Houghton, PJ., Menichini, F., Frega, N.G., 2009a. Chemical analysis, clifferent chemical composition showed by the two maturity fruits antioxidant, antiinflammatory and anticholinesterase activities of OriSalum stages affect the in vilro biological activities. ln particular, imma- ehrenbergtl Boiss and oigaftum syriocum L essential oils. Food chem. 117, 174- ture fruits have an interesting antioxidant properties and inhibited 1 80. Loizzo, M.R., Said, A., Tundis, R., Hawas, U.W., Rashed, K., Menichini, F., Frega, N.C., u-glucosidase activity. Menichini, F., 2009b. Antioxidant and antiproliferative activity ofDiospyros ,ofxs C. medica cv Diamante was actually used for industrial purpose L extract and isolated compounds. Plant Food Hum. Nutr' 64,264-270. exclusively for candy and liqueur using peel without consideration Loizzo, M.R., Tundis, R., Statti, G.A., Menichini, F., Houghton, PJ., 2005. In vitro effects on human tumour cell lines of extracts and portions. This study would propose antiproliferative for other C. medica cv Diamante jacaranone from senecio leucanthemifolius Poiret. J. Pharm. Pharmacol. 57' the use of leaves, flowers and immature fruits as an accessible pos- 897-902. sible source of phenolic compounds useful for preparation of food Martinez-Tomé, M., Jiménez, A.M., Ruggieri, S., Frega, N., Strabbioli, R., Murcia, M.A , Antioxidant properties of Mediterranean spices compared witll common supplements or for nutraceutical products. 2001. food additives. J. Food Protect. 64,1412-1419. Further in vivo studies, in particular on insulin secretagogue Murakami, A., Nakamura, Y., Ohto, Y., Yano, M , Koshiba, T., Koshimiztt, K, Tokuda, activi§, are warranted to confirm the biological activity of the H., Nishino, H., Ohigash, H., 2000. Suppressive effects of cìtrus fruits on free generation and nobiletin, an anti-inflammatory polymethoxyflavonoid. C. medica cv Diamante and to investigate the molecular mecha- radical BioFactors 12, 787 -192. for the bioactivity of the most active extract' F., Lid6nc, G.4., nisms responsible Ortuflo, A, Reynaldo, t., Fustera, M.D., Botia,J ' Puiga, D.G., Sabatera, Porrasc, 1., Del Rio,J.A., 1997. Citrus cultivars with high flavonoid contents in the fruits. Science Horticulture 68, 231-236. Conflict of Interest Peng, C.H., Ker, Y.8., Weng, c.F., Peng, C.C., Huang, c.N.' Lin, L.Y., Peng, R.Y., 2009. Insulin secretagogue bioactivity of finger citron fruit (Citrils nedica L vat. 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