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      Analysis of Flavonoids in Lotus ( Nelumbo nucifera) Leaves and Their Antioxidant Activity Using Macroporous Resin Chromatography Coupled with LC-MS/MS and Antioxidant Biochemical Assays

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          Abstract

          Lotus ( Nelumbo nucifera) leaves, a traditional Chinese medicinal herb, are rich in flavonoids. In an effort to thoroughly analyze their flavonoid components, macroporous resin chromatography coupled with HPLC-MS/MS was employed to simultaneously enrich and identify flavonoids from lotus leaves. Flavonoids extracted from lotus leaves were selectively enriched in the macroporous resin column, eluted subsequently as fraction II, and successively subjected to analysis with the HPLC-MS/MS and bioactivity assays. Altogether, fourteen flavonoids were identified, four of which were identified from lotus leaves for the first time, including quercetin 3- O-rhamnopyranosyl-(1→2)-glucopyranoside, quercetin 3- O-arabinoside, diosmetin 7- O-hexose, and isorhamnetin 3- O-arabino- pyranosyl-(1→2)-glucopyranoside. Further bioactivity assays revealed that these flavonoids from lotus leaves possess strong antioxidant activity, and demonstrate very good potential to be explored as food supplements or even pharmaceutical products to improve human health.

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          Flavonoids as antioxidants.

          Pier-Giorgio Pietta (2000)
          Flavonoids are phenolic substances isolated from a wide range of vascular plants, with over 8000 individual compounds known. They act in plants as antioxidants, antimicrobials, photoreceptors, visual attractors, feeding repellants, and for light screening. Many studies have suggested that flavonoids exhibit biological activities, including antiallergenic, antiviral, antiinflammatory, and vasodilating actions. However, most interest has been devoted to the antioxidant activity of flavonoids, which is due to their ability to reduce free radical formation and to scavenge free radicals. The capacity of flavonoids to act as antioxidants in vitro has been the subject of several studies in the past years, and important structure-activity relationships of the antioxidant activity have been established. The antioxidant efficacy of flavonoids in vivo is less documented, presumably because of the limited knowledge on their uptake in humans. Most ingested flavonoids are extensively degraded to various phenolic acids, some of which still possess a radical-scavenging ability. Both the absorbed flavonoids and their metabolites may display an in vivo antioxidant activity, which is evidenced experimentally by the increase of the plasma antioxidant status, the sparing effect on vitamin E of erythrocyte membranes and low-density lipoproteins, and the preservation of erythrocyte membrane polyunsaturated fatty acids. This review presents the current knowledge on structural aspects and in vitro antioxidant capacity of most common flavonoids as well as in vivo antioxidant activity and effects on endogenous antioxidants.
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            Assessment of antioxidant capacity in vitro and in vivo.

            Etsuo Niki (2010)
            The role and beneficial effects of antioxidants against various disorders and diseases induced by oxidative stress have received much attention. Many types of antioxidants with different functions play their role in the defense network in vivo. The free radical scavenging antioxidants are one of the important classes of antioxidants and the assessment of their capacity has been the subject of extensive studies and argument. Various methods have been developed and applied in different systems, but many available methods result in inconsistent results. There is no simple universal method by which antioxidant capacity can be assessed accurately and quantitatively. In this review article, the available methods are critically reviewed on the basis of the mechanisms and dynamics of antioxidant action, and the methods are proposed to assess the capacity of radical scavenging and inhibition of lipid peroxidation both in vitro and in vivo. It is emphasized that the prevailing competition methods such as oxygen radical absorption capacity (ORAC) using a reference probe may be useful for assessing the capacity for scavenging free radicals but that such methods do not evaluate the characteristics of antioxidants and do not necessarily show the capacity to suppress the oxidation, that is, antioxidation. It is recommended that the capacity of antioxidant compounds and their mixtures for antioxidation should be assessed from their effect on the levels of plasma lipid peroxidation in vitro and biomarkers of oxidative stress in vivo. Copyright (c) 2010 Elsevier Inc. All rights reserved.
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              Antioxidant activity of a flavonoid-rich extract of Hypericum perforatum L. in vitro.

              Y. J. Lu, Y. L. Zou, Dongzhi Wei (2004)
              A flavonoid-rich extract of Hypericum perforatum L. (FEHP) was prepared by adsorption on macroporous resin and desorption by ethanol. Total flavonoid content of FEHP was determined by a colorimetric method. The major constituents of FEHP, including rutin, hyperoside, isoquercitrin, avicularin, quercitrin, and quercetin, were determined by HPLC analysis and confirmed by LC-MS. Different antioxidant assays were utilized to evaluate free radical scavenging activity and antioxidant activity of FEHP. FEHP was an effective scavenger in quenching DPPH and superoxide radical with IC50 of 10.63 microg/mL and 54.3 microg/mL, respectively. A linear correlation between concentration of FEHP and reducing power was observed with a coefficient of r2 = 0.9991. Addition of 150 microg of FEHP obviously decreased the peroxidation of linoleic acid during 84 h incubation, but the amount of FEHP over 150 microg did not show statistically significant inhibitory effect of peroxidation of linoliec acid (p > 0.05). FEHP exhibited inhibitory effect of peroxidation of liposome induced both by hydroxyl radical generated with iron-ascorbic acid system and peroxyl radical and showed prominent inhibitory effect of deoxyribose degradation in a concentration-dependent manner in site-specific assay but poor effect in non-site-specific assay, which suggested that chelation of metal ion was the main antioxidant action. According to the results obtained in the present study, the antioxidant mechanism of FEHP might be attributed to its free radical scavenging activity, metal-chelation activity, and reactive oxygen quenching activity.
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                Author and article information

                Contributors
                Isabel C. F. R. Ferreira: Role: Academic Editor
                Journal
                Journal ID (nlm-ta): Molecules
                Journal ID (iso-abbrev): Molecules
                Journal ID (publisher-id): molecules
                Title: Molecules
                Publisher: MDPI
                ISSN (Electronic): 1420-3049
                Publication date (Electronic): 08 June 2015
                Publication date Collection: June 2015
                Volume: 20
                Issue: 6
                Pages: 10553-10565
                Affiliations
                [1 ]Key Laboratory of Plant Germplasm Enhancement and Specialty Agriculture, Wuhan Botanical Garden, Chinese Academy of Sciences, Wuhan 430074, China; E-Mails: mzzhucn@ 123456hotmail.com (M.-Z.Z.); yangpf@ 123456wbgcas.cn (P.-F.Y.)
                [2 ]Changchun University of Chinese Traditional Medicine, Changchun 130000, China; E-Mails: weiwutian@ 123456hotmail.com (W.W.); jiaoaj@ 123456hotmail.com (L.-L.J.)
                Author notes
                [* ] Author to whom correspondence should be addressed; E-Mail: guomq@ 123456wbgcas.cn ; Tel./Fax: +86-27-8751-8018.
                Article
                Publisher ID: molecules-20-10553
                DOI: 10.3390/molecules200610553
                PMC ID: 6272455
                PubMed ID: 26060918
                SO-VID: 0ee82200-8995-499b-bd38-16aa1bcd05ef
                Copyright © © 2015 by the authors.
                License:

                Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution license ( http://creativecommons.org/licenses/by/4.0/).

                History
                Date received : 27 March 2015
                Date accepted : 29 May 2015
                Categories
                Subject: Article

                Keywords: lotus leaves,antioxidant activity,macroporous resin chromatography,mass spectrometry
                Data availability:
                Keywords: lotus leaves, antioxidant activity, macroporous resin chromatography, mass spectrometry

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