Phytochemical Composition, Antioxidant Activity And Anti-inflammation Activity Of Lentils (Lens Culinaris)

Lentils are considered to be an excellent source of macronutrients,micronutrients and phytochemicals. Epidemiological and interventional studies haveshown that the consumption of legumes including lentil was associated with a lowerincidence of chronic diseases, such as coronary disease, type2diabete, cardiovasculardisease, cancer and aging. The phytochemicals existed in lentil including carotenoids,tocopherols and phenolic compounds are native antioxidants, and are believed to playa critical role in human health. Incorporation of lentil into diets has been highlyrecommended due to its health benefits. Increasing studies have reported thatphytochemicals originated from fruits, vegitable and legumes exhibited strongactivities in anti-inflammation, anti-oxidative stress, lowering cholesterol and anti-tumor. However, available information on the phytochemical component in lentils,antioxidant activities and anti-inflammation activity are still limited. In addition, theeffects of cooking process and gastrointestinal digestion after consumption onphytochemical compositions, antioxidant activities and anti-inflammatoru activitieshave not been reported as well.In order to better understanding the nutrient value of lentil, the present studyextracted lipophilic and hydrophilic fractions (including extractable and boundphenolics) from lentils, respectively, and quantitively and quantatively analysed thephytochemicals in lentils by GC, HPLC and LC-MS. Their antioxidant activities weredetermined by both chemical-based antioxidant activities assay and cell-basedantioxidant activity model in caco-2cell line. In addition, the present studyinvestigated the bioaccessibility of phenolics compounds in cooked lentil by in vitrodigestion. The oxidative stress and inflammatory cell model were established todetermine their bioactivities in anti-oxidative stress and anti-inflammation. The mainresults are shown as follows:1. Quantitively and quantitatively analysed the fatty acids, carotenoids andtocopherols composition in lipophilic extracts of20cultivars of lentil. Lentilscontained40.73to47.06%of18:2n-6linoleic acid,20.11to28.00%of18:1oleic acid and12.67to14.82%of16:0palmitic acid, as well as rich in18:3n-3(9.00-13.28%) linolenic acid. The total carotenoids content (TCC), total carotenoids indexand tocopherols content among20lentil cultivars were significant difference. Luteinand zeaxanthin are the major carotenoids in the lentil. TCC ranged from5.32-28.13μg/g DW, and TCI ranged from4.64-19.63μg/g DW in20lentils. Total tocophenolsranged from37-64μg/g DW, predominantly γ-tocopherol (96-98%of the tocopherolcontent), followed by δ-and α-tocopherol. Trans-lutein was the primary and majorcarotenoid (64-78%) followed by trans-zeaxanthin (5-13%). Cultivar Greendlandcontained highest γ-tocopherol, while cultivar Impact has the highest TCC and TCI.2. The antioxidant activities of lipophilic extracts from20lentils weredetermined by96-well plate protocol. DPPH values varied from3.61-4.48μmol TE/gDW, in which the strongest was found in Imvincible, followed by Impact andGreenland. Antioxidant activities determined by PCL varied from2.73-6.23μmolTE/g DW, in which the highest value was found in Impact, followed by Imperial andRedcliff.3. Antioxidant activities measured by DPPH and PCL showed positivecorrelation with carotenoids and tocopherols contents in20lentil cultivars.Carotenoids and tocopherols very likely act synergistically in DPPH assay, whilecarotenoids (mainly lutein) was the major contributor to the PCL activity. Totalphenolic content (TPC), total flavonoids content (TFC) and condense tannin content(CTC) in extractable fraction of lentils were determined by established96-well plateassay. Results showed that TPC varied from4.56to8.34mg GAE/g DW, while TFCand CTC varied from0.60-1.98mg CE/g DW and3.00-7.80mg CE/g DW,respectively, among20lentil cultivars. Cultivar Asterix had the highest TPC, TFCand CTC, followed by Greenland and Redcliff. Phenolic composition in extractablephenolics fraction were identified and quantitatived by HPLC and LC-MS, in whichflavonols including kaempferol glycosides dominated the phenolic profile of lentils,with the tetraglycoside and triglycoside having the highest concentrations rangingfrom210.05to297.15μg/g DW and99.96to181.81μg/g DW, respectively, followedby flavanols including catechin glucoside, catechin gallate, epicatechin glucoside,with the concentration ranging from99.27-130.10、44.92-83.55和42.87-73.93μg/g DW, respectively.4. The antioxidant activities of extractable extracts from20lentils weredetermined by96-well plate protocol. The antioxidant activity as assessed by theDPPH assay ranged from23.83to35.03μmol TE/g DW, the FRAP value which is ameasurement of the reducing power ranged from18.75to34.52AAE/g DW with thehighest antioxidant activity being nearly double of the lowest. The ORAC values ofthe lentil extracts also varied from105.06to168.03μmol TE/g DW. The overallhighest antioxidant activity was found in cultivar Asterix for all three antioxidantassays.5. Antioxidant activities measured by DPPH, FRAP and ORAC showedsignificant positive correlation with phenolics contents in20lentil cultivars. Theantioxidant activity as measured by the FRAP assay exhibited strongest correlationwith TPI, TPC, TFC and CTC. The inhibitory ability of extractable phenolics in lentilon α-glucosidase and lipase activity were tested by established96-well plate assay, inwhich sensitive fluorescent substrate4-MUG and4-MUO were employed. Theinhibitory IC50values of the20extracts (de-sugared) ranged from23.08-42.15mg/mLand6.26-9.26mg/mL for α-glucosidase and lipase, respectively, in which cultivarAsterix possessed highest inhibitory activities against both α-glucosidase and lipase.The inhibitory ability of extractable extracts on both enzymes was positive correlatedwith the phenolics contents significantly. Neither the catechins nor procyanidin B1showed inhibitory activity against α-glucosidase or pancreatic lipase. The flavonolsincluding kaempferol and quercetin glycoside are strong inhibitors of α-glucosidaseand pancreatic lipase.6. The hydrophilic fractions in lentils contributed much more to the antioxidantactivity than the lipophilic fraction. The phenolic content of lentils was mainlycomprised of extractable compounds, with bound phenolics only account for roughly5percent of the extractable phenolics content. Significant changes (P<0.05) incarotenoids, tocopherols, total phenolic and condensed tannin contents of bothextractable and bound phenolics fractions, as well as in antioxidant activities werefound in lentils before and after cooking processing. More specifically, cooking wasfound to favour the release of carotenoids and tocopherols and flavonols (kaempferol glycosides), but lead to losses of flavanols (monomeric and condensed tannin). Whilereduced flavonols and other phenolic compounds may have negatively affected theantioxidant activity, other components, especially the lipophilic antioxidants wereincreased.7. The bioaccessibility of phenolics compounds in cooked lentil Greenland wasinvestigated by in vitro digestion. The present results indicated that the flavonoids hasthe highest bioaccessibility, which is66.67%, while condense tannin showed lowesebioaccessibility, which is26.54%, after gastrointestinal digestion in vitro. Morespecificly, flavanols compounds (catechin and epicatechin glycosides) exhibitedhighest around50%of bioaccessibility after gastric digestion, followed by21-45%for phenolic acids and20%for flavonols compounds (kaempferol and quercetinglycosides). After further intestinal digestion in vitro, phenolic acids can not bedetected in digestates, and the bioaccessibility of flavanols compounds were alsosignificant decreased (p <0.05), while the bioaccessibility of flavonols compoundsmarkedly increased to55-65%(p <0.05). The extractable phenolic fraction of cookedlentil also possessed strong antioxidant ability in established caco-2cell-basedantioxidant activity assay before and after in vitro digestion. In addition, theextractable phenolic compounds exhibited protective effects on H2O2-induced caco-2cell oxidative injury, and positive correlated with dosage.8. TNF-α was employed to induce inflammatory response in caco-2cell line.Results showed that extractable phenolic fraction of cooked lentil Greenlandsignificant inhibited the release of proinflammatory factor IL-8determined by ELISA,and significant down-regulate the mRNA expression of IL-6、IL-1β and COX-2determined by qRT-PCR. The anti-inflammatory activities improved with dosageincrease.