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Critical Reviews in Food Science and Nutrition, 41(5s):387–412 (2001)
The Health Effects of Tea and Tea Components: Opportunities for Standardizing Research Methods Report of an International Workshop organized by the ILSI International Subcommittee on the Health Effects of Tea Components, November 17–18 1999, Washington, DC, USA Dr. Sheila Wiseman,1 Dr. Andrew Waterhouse,2 and Dr. Onno Korver 3 1
Unilever; 2Workshop Chair; 3ILSI International Subcommittee on the Health Effects of Tea Components Chair
BACKGROUND AND OBJECTIVES Tea polyphenols are reported to be among the most potent antioxidants that occur in foods and beverages. These polyphenols — the monomeric and polymeric flavonoids of black and green tea, respectively — define the chemical complexity and are the active components of tea. The scientific description of tea composition, however, is often unclear due to an inconsistent nomenclature and the use of varying analytical methods to quantify components and their antioxidant activity. This lack of clarity in the scientific community confuses the media as well as consumers. Therefore, a goal of this workshop was the improvement of our scientific understanding of the health effects of tea and its components. Specifically, the workshop brought together key researchers in the field to: • Discuss preferred nomenclature for the active components in green and black tea, • Identify preferred methods for quantifying active components in tea, • Select the preferred method(s) for quantifying antioxidant activity of tea and tea components in vitro, • Assess the quality of methods currently used for examining antioxidant activity in vivo, identifying gaps and weaknesses, and • Review opportunities for establishing compositional data for flavonoids in foods.
The workshop’s objective was not to provide solutions to all of these issues, but rather to identify strategies for progress. Experts from the workshop proposed these strategies as recommendations for various groups (analytical chemists, nutritionists) working in this area.
Workshop issue #1: An Unambiguous, Simple, and Informative Nomenclature for the Phenolic Compounds in Green and Black Teas Professor Mike Clifford (University of Surrey, UK) proposed a nomenclature for phenolic compounds that is based mostly on existing, wellestablished nomenclatures (e.g., IUPAC), and that also accommodates the many phenols that are present in other commodities (see Appendix 1). Professor Clifford also made the following recommendations for distinguishing between ‘tannins’ (polymeric materials that at appropriate concentrations are able to produce leather from hides) and ‘derived polyphenols’ (substances not found in healthy, intact plant material but characteristic of some processed foods and beverages such as black tea, coffee, matured red wine, etc). Derived polyphenols, although sometimes called tannins, are chemically distinct from true tannins and are not able to produce leather from hides.
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For derived polyphenols: 1.
2.
3.
4.
5.
Indicate derivation of major contributor in terms of mass (e.g., cinnamate derived, flavonoid derived) with ‘flavanol derived’ if greater precision is required. If there are precursors of two structurally distinct classes use ‘flavanol and gallate derived’ or ‘flavanol and flavonol derived’ as required. In situations where the precursors have undergone little structural change, their identity is obvious, and they are directly linked (C–C or C–O–C), describe the derived polyphenols using a simple but unambiguous series of abbreviations to identify the precursors. Also use numbers to indicate the position of new bonds. In situations where the precursors have undergone little structural change and their identity is obvious, but they are linked by some ‘bridging unit’, describe the derived tannin as, for example, ‘methylene-bridged flavanol adducts’ (homo-bis-flavans, acetaldehyde-bridged flavanol adducts). Use the same unambiguous series of abbreviations to identify the precursors, and numbers to indicate the position of new bonds (as in 2 above). In situations where the precursors have undergone greater transformation (e.g., ring opening or elimination of CO2) use a systematic name for the new structural feature (e.g., benztropolone [BzT]; phenylindan) followed by the names of the precursors (or convenient unambiguous abbreviations). In situations where comparatively little information is available, use a generic term for a fraction, supported by a reference to its preparation. Describe the origin as precisely as possible.
Discussion: The workshop participants agreed that these recommendations would be useful in promoting a common language between analytical chemists and nutritionists. The participants were virtually unanimous in supporting recommendations that all references to polymeric phenolic material should indicate details of the extraction for duplication in experiments. Participants discussed ways to clarify
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how ‘catechin’ and ‘flavanol’ are used. ‘Catechin’ is the nomenclature commonly used in Southeast Asia. It only defines a single compound, and additional information is required to define the stereochemistry. However, ‘catechin’ is widely used to define the monomeric group. The more standard term ‘flavanol’ was criticized by some participants as being a source of potential ambiguity in scientific literature due to its similarity to the term ‘flavonol’. ‘Flavanol’ was generally preferred because it is clearly linked with the flavonoid family of phenolics, but additional usage of the terms ‘monomeric’, ‘dimeric’, or ‘polymeric’ may be necessary to avoid ambiguity. The use of the term ‘tannin’ provoked significant discussion because it is frequently confused with tannic acid and other crude chemical preparations used to produce leather from hides, which have negative health connotations. The use of the term ‘tannin’ was, however, seen to have value in certain areas, such as in reference to wine. One participant proposed using ‘derived polyphenols’ for the reasons given above. No consensus emerged from this discussion, but publication of the nomenclature proposal was encouraged in order to stimulate further debate.
Workshop Issue #2: Methodologies for Measuring Active Tea Components in Groups and as Individual Components Dr. Ulrich Engelhardt (Technical University of Braunschweig, Germany) and Dr. Xiaochun Wan (Anhui Agricultural University, P.R. China) proposed the Folin-Ciocalteau (FC) method as the best analytical tool for estimating the total flavonoid content of tea beverages (see also Appendices 2 and 3). Gallic acid was proposed as the preferred standard because it is relatively stable and commercially available, unlike tea catechins. The precise reactivity of both monomeric and polymeric flavonoids (e.g., thearubigins) in the Folin-Ciocalteau assay is not well defined and should be determined to improve current methods. The total FC response of tea extracts decreases increased fermentation times, suggesting that the polymeric flavonoids have a lower mass response to the Folin reagent. Further analysis should be pursued as part of an ongoing process involving academia, the tea industry, and the
International Standards Organisation (ISO). A major hurdle in standardizing analytical methods is the natural variability in thearubigin type and content. The monomeric flavonoids, theaflavins, and most significant nonflavonoid phenolics in tea — gallic acid, theogallin, and a number of chlorogenic acids — can be readily quantified by liquid chromatography (LC) methods. LC methods are preferred for quantification. The best strategy available for estimating the total flavonoid content of teas is to (1) determine the monomeric and simple polymeric flavonoids by HPLC methods, and (2) estimate the complex polymeric flavonoids using the FolinCiocalteau assay (see equation A below). To arrive at the latter, subtract the known monomeric and polymeric flavonoids and the known nonflavonoid phenolics that are determined by HPLC from the total polyphenolics determined by the FolinCiocalteau assay. This needs to be corrected with an appropriate response factor to be determined (see equation B below). An outline of the determination is: A. Total flavonoids = catechins (HPLC) + theaflavins (HPLC) + flavonols (HPLC) + thearubigins (FC) B. Thearubigins = [Factor (to be determined) × total polyphenols (Folin-Ciocalteau)] – catechins (HPLC) – theaflavins (HPLC) – flavonols (HPLC) – non-flavonoids (HPLC) When identifying, characterizing, and quantifying polymeric phenolic material, the key issue is to obtain fractions free of other monomeric or dimeric material. Good HPLC-based analytical methods are available for the separation and quantification of the main monomeric flavonoids and nonflavonoids phenolic components in tea. Discussion: Participants recognized the need for a standard tea polyphenol extract to be used when calibrating results and considered how a stable, standardized tea could be prepared and made available. This tea extract could be applied in different laboratories to compare methods and eventually could become a certified reference material.
It is currently not possible to compare results because of the variability in tea preparations. A candidate for this extract could be the “World Blend” prepared by the UN Food and Agricultural Organization (FAO) for use in feeding trials. The group also noted that a more pragmatic approach to thearubigin analysis should be pursued. All participants recognized that although broad polyphenol analyses are not ideal, they may be considered adequate. They also agreed on cooperating with ISO on the use of the Folin-Ciocalteau assay and clarifying the conversion factor for thearubigins. Unilever and Tetley agreed to coordinate activities in this area. The group also suggested that future work should focus on methodology to characterize black tea components, as well as defining one analytical system that would be applicable to both green and black tea.
Workshop Issue #3: Methodologies for Measuring Active Tea Components in Biological Matrices Dr. Peter Hollman (State Institute for Quality Control of Agricultural Products [RIKILT], The Netherlands) proposed an analytical approach to this issue (see Appendix 4). Methods for catechin (flavanoid) analysis in plasma and urine are primarily HPLC based. They use reversed phase columns with either isocratic or gradient elution, and eluents consisting of mixtures of low-pH buffers and methanol or acetonitrile. Plasma and urine are generally extracted using ethyl acetate, methanol, acetonitrile, solid phase adsorbents (alumina), or bonded hydrocarbons. However, little data are available on the most effective extraction methods. Electrochemical (coulometric) or fluorescence detection systems are most applicable for determining catechins due to the high sensitivity required. A number of papers have described ultraviolet (UV) detection, but this technique can only be applied at catechin concentrations higher than about 200 ng/ml. The literature has also described a sensitive spectrophotometric method based on reaction with dimethylaminocinnamaldehyde. Techniques based on gas chromatography–mass spectrometry (GC-MS) and LC-MS are now gaining attention. Currently there are no
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published reports on estimating theaflavin or thearubigin in biological fluids. The uncertain structure of thearubigins makes determining them a technical challenge worthy of attention. It is likely that the tea flavonoids undergo extensive metabolism in the gastrointestinal tract. Therefore, identifying and determining them will require powerful separation and detection techniques, such as GC-MS. A universal method that is suitable for all forms — native, conjugates, and metabolites — is needed. This method should also be independent of standards, as in the case of metabolites, where the exact structural forms may not be known. LC-MS and LC-MS-MS are good candidates, but sensitivity remains an issue. Discussion: In relation to determining tea flavonoids in plasma, workshop participants addressed whether to apply acid or enzymic hydrolysis in determining flavonoid conjugates. The group preferred enzymic hydrolysis because of the potential for loss of flavonoids (particularly flavanols and anthocyanins) under the harsh conditions of acid hydrolysis. The glucuronide conjugates require even harsher acid conditions to hydrolyze than the glycosides. The group also addressed the problems with identifying potentially active metabolites arising from tea flavonoid ingestion. NMR methods would identify unknown structures and would be ideal if limited to more specialized laboratories. A more pragmatic approach would be to link existing data from animal studies. This would allow some prediction of expected molecular structures and identification using conventional standard-based approaches. Another approach would be to use isotopically labeled compounds, but C14-labeled tea flavonoids are still not available. Participants questioned whether a total polyphenol analysis could be developed for plasma and/or urine. The consensus was that this kind of assay would be very useful, but a number of complex technical issues still need to be resolved (especially related to method calibration and in vivo conjugation of the reactive hydroxyl groups).
Workshop Issue #4: Identifying Methods to Quantify the Antioxidant Activity of Tea and Tea Extracts In Vitro Professor Catherine Rice-Evans (Guy’s Hospital Medical School, London, UK) provided writ390
ten input describing the three main methodological approaches (see Appendix 5) for measuring antioxidant activity of pure phenolic compounds as well as plant or food extracts. They are chemical (e.g., TEAC and DPPH assays), biological (e.g., TRAP assays such as the ORAC assay and oxidation of low-density lipoprotein), and a novel approach applying reduction of ferrous iron (e.g., the FRAP assay). Assays of this nature are not expected to indicate efficacy of antioxidants in biological systems, but are useful in screening compounds and beverages for relative antioxidant potential. Antioxidant activity methodologies must be validated and compared in order to identify the most appropriate methods for determining antioxidant activity in aqueous and lipophilic phases. Professor Junji Terao (University of Tokushima School of Medicine, Japan) also discussed a “biological” approach based on oxidation of liposomes (see Appendix 6). It is a heterogeneous system mimicking biomembranes in which lipophilic and hydrophilic antioxidant activities can be determined. Both speakers emphasized that comparing antioxidant activity from different assays will give different absolute values, but in many cases similar relative rankings. Discussion: The group expressed confusion over the wide variety of assays available for performing “antioxidant activity” measurements, and the lack of clarity in what is actually being measured (e.g., radical scavenging activity or redox activity). Because a number of approaches had been presented, participants questioned whether one particular in vitro assay could be recommended in order to provide a “total antioxidant activity” number. This number could be included in food composition databases. Some workshop participants had applied a number of these assays (e.g., ABTS, FRAP, DPPH) to the same beverage samples and found similar antioxidant activity rankings and excellent correlations between the methods. Participants agreed that having a published comparison would be useful. Concerns about carotenoid antioxidant activity were raised because their reactivity may be substantially lower in assays dependent on redox activity. It was, again, emphasized that these antioxidant activity assays should not be used to imply health benefits.
Workshop Issue #5: Identifying Methods to Demonstrate Antioxidant Activity In Vivo in Intervention Trials with Tea/Tea Components Dr. Myron Gross (University of Minnesota, USA) presented the current status of markers of oxidative damage — that is, markers indicating the level of in vivo oxidation. Consideration was given to a wide range of protein, lipid, and DNA oxidation products and the available methods for their measurement (a detailed discussion of several possible markers is given in Appendix 7). Although numerous potential indicators of lipid, protein, and DNA oxidative damage have been developed, they generally remain to be validated — either in analytical terms, as oxidative damage markers, or as indicators of disease risk. Many of the available methods for the measurement of oxidative damage are currently under development. Major analytical concerns include the availability of appropriate standards, the recovery of oxidation products, and, most importantly, the possibility of artifactual oxidation. Some of these concerns have been addressed recently in several reports, and new methodologies have been proposed in each of the major types of damage, including lipid, protein, and DNA oxidation. Adoption and further development of these new methodologies is likely to provide reliable and accurate methods for the measurement of oxidative damage. For urinary analytes, an additional concern is the metabolic source of oxidation products. Generally, it is assumed that urinary oxidation products are produced in tissues, equilibrate with blood, and reflect overall oxidative stress. However, recent reports have suggested the possibility of the formation of oxidation products by the kidney and the possibility of a significant modification of urinary concentrations. Although this possibility requires verification, it may influence the interpretation of currently available data. Several of the markers can be modulated in human subjects by the administration of antioxidant supplements indicating the formation of oxidative damage by antioxidant-modifiable pathways. The modulation of oxidative damage has been shown most clearly for the products of lipid peroxidation. Few reports are available for the markers of protein and DNA oxidation; it remains an area for active investiga-
tion. Also, some markers, particularly those of DNA oxidation, have not shown modulation by antioxidant supplementation. These finding suggest the possibility of mechanisms for oxidative damage that will require alternative approaches or unique antioxidants for modification. Another important area of investigation is the association of oxidative damage with the risk of disease. Oxidative damage clearly occurs in advanced disease states. However, the temporal relationship between oxidative damage and various stages, particularly early stages, is not known. Various types of indirect evidence suggest a relationship between markers of oxidative damage in human subjects, but prospective human studies are necessary for characterization of the relationship between the markers and various disease endpoints. One promising marker in this area is the F2isoprostanes. They are products of free radical attack on arachidonic acid and, once formed, are quite stable. However, few studies have looked at the effect of dietary contribution to F2-isoprostane levels. This kind of information on dietary confounding factors should be combined with mechanistic and clinical data in order to fully establish such compounds as true markers of endogenous oxidative damage. Discussion: Discussion focused on the different options for measuring oxidative damage in vivo. The unavailability of prospective data linking elevated marker levels to disease end-points makes it difficult to recommend any one particular assay. However, on the basis of other criteria, the F2-isoprostanes remained the most promising candidates. Another issue related to markers of compliance in tea intervention studies is that blood catechin levels only give an indication of consumption over a small time frame. This is due to the short half-life of elimination from the bloodstream. A potential alternative marker for black tea consumption is n-ethyl glutamic acid, which is an amino acid specific to tea.
Workshop Issue #6: Establishing Compositional Data for Flavonoids in Foods Dr. Gary Beecher (USDA, Beltsville, Maryland, USA) described the rationale behind setting
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up a U.S. compositional database for flavonoids in foods (see also Appendix 8). Currently, analytical data are being retrieved from refereed, published literature sources. The data are being evaluated and reviewed prior to entry into the database. A major issue in determining the quality of data is the availability of certified reference material (CRM) that can be used to validate analytical procedures. Currently, there is no such reference material for tea flavonoids. It would be a major step forward if a characterized, stable tea preparation could be made available to the analytical community for method validation and quality control. Validating sample extraction procedures is frequently not performed, or at least not reported, and reference material would significantly improve this. It is anticipated that the database will contain data on the 15 to 20 most prominent flavonoids in the U.S. food supply. All data are presented as aglycones, apart from the gallate derivatives of tea. Data are aggregated according to the USDA Nutrient Databank Food Descriptions and Food Codes so that data can be integrated into other databases and can be used with large food intake surveys (e.g., NHANES). USDA is also doing flavonoid analyses using an HPLC system, which separates up to 20 flavonoid aglycones in a single, 60-min run. Cleavage of glycosides using mineral acids at high temperature induces losses, and enzyme hydrolysis systems are recommended. Discussion: The data quality criteria, which are being used for the flavonoid database, are similar to those that have already been used and published for the carotenoid database (J. Am. Diet. Assoc. 1993; 93:284). In the case of insufficient data on particular flavonoids (e.g., the anthocyanins), these data should be entered into a separate table to be reviewed. This will enable access to the data, which can then be reviewed as more data becomes available. There are currently no other flavonoid databases on par with
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the USDA’s. There is only a limited amount of published data available on flavonols and flavones in the Netherlands and Denmark. Data are drawn from foods that are prepared according to how they are consumed. In the case of tea, data are drawn from water, not methanol extractions. This raises the issue of large variations in tea infusion habits, both intra- and intercountry, and the need for a critical review process that excludes outlying data. The USDA’s analytical data must be published in refereed journals prior to being included in the database. Participants raised the issue of reference material for tea again, particularly because tea is a highly consumed product. This workshop seemed the appropriate forum to facilitate obtaining such material. In relation to previous discussions on a “world tea blend,” the group agreed that the ISO would be the most appropriate organization to take this matter further. It is anticipated that the flavonoid database will be complete by the end of 2001.
CONCLUSION The workshop was valuable for participants. They mapped out strategies for standardizing the nomenclature and methods used in researching the health effects of tea and identified major research gaps. A number of the strategies that were recommended could be taken forward by ISO. Also, the workshop format is a good model for exploring issues and reaching consensus on other natural, plant-derived “healthy” components.
ACKNOWLEDGMENTS The authors thank Drs. Gary Beecher, Mike Clifford, Ulrich Engelhardt, Myron Gross, Peter Hollman, Catherine Rice-Evans, Junji Terao, and Wan Xiaochun for their contribution.
Appendix 1 A Nomenclature for Phenols with Special Reference to Tea Dr. Mike Clifford University of Surrey
Objective To propose a nomenclature for phenols in green and black teas that will • Be unambiguous. • Simple and informative. • Take account of existing formal nomenclature where this exists, e.g., IUPAC. • Accommodate phenols found in other commodities but absent from tea.
• If more than one hydroxycinnamate residue is present (e.g., dicaffeoylquinic acids) they should strictly be viewed as polyphenols (q.v.), but for convenience this distinction often not made. • If three or more such residues are present (especially in the case of the galloylquinic acids) they would commonly be viewed as tannins (q.v.). Polyphenols — compounds containing at least two aromatic rings each bearing at least one aromatic hydroxyl.
Existing Terminology Phenol — a specific compound (C6H5OH). Simple Phenols — compounds containing a single aromatic ring and bearing one or more aromatic hydroxyl groups. • Other substituents present in most compounds, e.g., –OCH3, –COOH, etc. • Commonly a sidechain of several carbons — frequently 1–3, but may be up to 17. • Simple phenols usually occur as conjugates, for example with sugars (esters and glycosides), hydroxy acids, etc. Phenylpropanoids — An important subclass of simple phenols having a three carbon side chain (C6–C3 compounds) • Include cinnamates (phenyl-propenoates) and associated conjugates, including chlorogenic acids (hydroxycinnamoyl quinates) for which IUPAC nomenclature available. 1 • The galloylquinic acids (including theogallin) are covered by the same nomenclature and can be viewed as honorary chlorogenic acids.
Flavonoids — An important subclass of polyphenols having a C6–C3–C6 skeleton. • Term first used by Geissman.2 • Further classified by oxidation state of the C3 element. • If C3 element undergoes ring closure then these compounds are benzpyrans. • If C 3 element saturated sometimes called flavans. • If both the foregoing apply, then one (flavanones) or two (flavanols) asymmetric carbons are present, which should be described using the rules for absolute stereochemistry. • Further variation introduced by pattern of aromatic hydroxyls, pattern of conjugation (sugar(s) and acids, especially gallic acid). • Oligomers and polymers are known and generally are treated as tannins (q.v.). Tannins — compounds defined by function,3 that is, the ability to produce leather from hides. That function associated with • Mass in excess of 500 Da. • 12 to 16 phenolic groups and 5–7 aromatic rings per 1000 units of relative molecular mass.
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• Efficient precipitators of protein and some alkaloids, for example, caffeine, cinchonine. • Strictly, this definition excludes ECG (442 Da) and EGCG (458 Da) and theaflavins (>500 Da) that are good precipitators of caffeine and protein, although they would not be viewed as agents for producing leather. Tannins — Subdivided by origin and structure. Condensed tannins = proanthocyanidins = flavolans. • Oligomers and polymers of flavanols that may bear gallates. • Linked by 4→8 or 4→6 C–C bonds. Ether links (e.g., 2'→7) may also be present. • Acid catalyzed depolymerisation (not hydrolysis) converts all but ‘bottom’ unit to equivalent anthocyanidin, hence proanthocyanidin, propelargonidin, procyanidin, prodelphinidin, etc. • Three asymmetric carbons in all but one terminal residue — complex stereochemistry — an established system of nomenclature developed by Hemingway4 — needs some refinement better to describe the variation now known to occur in dimers. • In discussion, it is obviously often convenient to include simple monomers ((epi)afzelchin, (epi)catechin, and (epi)gallocatechin). Hydrolysable tannins • Esters of gallic acid (gallotannins) or ellagic acid (ellagitannins) and a nonaromatic polyol (sugar, quinic acid, etc.). Gallic acid derivatives (e.g., tergallic acid, gallagic acid) also occur. • Some gallate residues occur as depsides, that is, esterified to a gallate rather than the polyol core. • Individual gallo- and ellagitannins may be further linked by C–C or C–O–C bonds (to form dimers, trimers, or tetramers), and a shorthand nomenclature to describe these structures has been proposed by Okuda.5 • Although compounds such as 5-caffeoyl quinic acid, 5-galloylquinic acid, and glucogallin are
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appreciably less than 500 Da, there is an obvious structural affinity, and in some circumstances convenient to consider them together. • Some hydrolysable tannins (complex tannins) incorporate a C–C-linked flavanol unit. Phlorotannins • Phloroglucinol derivatives characteristic of algae. Derived tannins • Compounds formed during processing of plant material and virtually or completely absent from intact, healthy tissues. • Structures often unknown, thus precluding a more precise nomenclature. • Once structure elucidated, more appropriate naming can be introduced. Examples of derived tannins • Vitisins, vinylphenol–anthocyanin adducts, pyruvic acid–anthocyanin adducts, acetaldehyde, or glyoxylic acid-bridged flavanol–flavanol (= homo-bis-flavans) and flavanol–anthocyanin (= homo-bis-flavonoids) adducts of matured red wines and ports that are absent from grapes. • Tetrahydroxyphenylindans of roasted coffee that are absent from green coffee beans. • Flavanol–chalcan adducts, homo-bis-flavans, oolongtheanins, theacitrins, theaflagallins, theaflavins, theaflavonins, theafulvins, theogallinins, thearubigins, theasinensins, etc. of commercial green, semifermented, or black teas, that are present in fresh leaf at no more than trace levels.
Naming Derived Tannins Background • Many of the derived tannins characterized so far have been given trivial names. • In some cases, for example, theogallinin and theaflavonins, the same name has been applied to two quite unrelated substances.
• Although sometimes giving an indication of the commodity with which they are associated (e.g., thea, theo = tea; vitis = wine) or their color (citrin = yellow; flavin = orange; fulvin = tawny; rubigin = brown), these names give little or no structural information, even when it is available. Strategy 1.
2.
3.
4.
5.
Indicate derivation of major contributor in terms of mass, for example, cinnamate derived, flavonoid derived, etc. with flavanol derived, etc. if greater precision is required. In the event that there are precursors of two structurally distinct classes, this can be accommodated by saying, for example, flavanol and gallate-derived, flavanol and flavonol derived, etc. as required. In the situation where the precursors have undergone only minimal change in structure, their identity is obvious, and they are directly linked (C–C or C–O–C), describe the derived tannin using a simple but unambiguous series of abbreviations to identify the precursors and numbers to indicate the position of new bonds. In the situation where the precursors have undergone only minimal change in structure, their identity is obvious, but they are linked by some ‘bridging unit’, describe the derived tannin as, for example, methylenebridged flavanol adducts (= homo-bisflavans, acetaldehyde-bridged flavanol adducts). Use the same unambiguous series of abbreviations to identify the precursors and numbers to indicate the position of new bonds, as in 2 above. In the situation where the precursors have undergone greater transformation (for example, ring opening, elimination of CO2, etc.) if possible use a systematic name for the new structural feature (e.g., benztropolone (BzT); phenylindan, etc.) followed by the names of the precursors (or convenient unambiguous abbreviations). In the situation where comparatively little information is available, use a generic term for a fraction, supported by a reference to its preparation, and describe the origin in as precise a manner as the evidence permits.
Application of the Proposed Strategy to Derived Tannins in Commercial Teas 1. Flavonoid-derived 1.1. Flavanol-derived 1.1.1. Flavanol-derived dimers (2'→2') 1.1.1.1. Flavanol-derived dimers (2'→2') (EGCG + EGCG) R (absolute stereochemistry) = theasinensin A 1.1.1.2. Flavanol-derived dimers (2'→2') (EGC + EGC) R (absolute stereochemistry) = theasinensin C 1.1.1.3. Flavanol-derived dimers (2'→2') (EGCG + EGC) S (absolute stereochemistry) = theasinensin D 1.1.1.4. Theogallinin (2'→2') (EGCG + 5GQA) (as used by Hashimoto et al.6) 1.1.1.5. etc. 1.1.2. Flavanol-derived dimers (8→8, or 8→6) 1.1.2.1. Methylene-bridged flavanol-derived dimers (8→8) (EGCG + EGCG) = Oolong-homo-bisflavan A. 1.1.2.2. Methylene-bridged lavanol-derived dimers (8→6) (EGCG + EGCG) = Oolong-homo-bisflavan A. 1.1.2.3. etc. 1.1.3. Flavanol and chalcan-derived dimers (2→8) 1.1.3.1. Flavanol and chalcan-derived
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dimer (2→8) (EGCG-chalcan + EGCG) = Assamaicin A 1.1.3.2. etc. 1.1.4. Flavanol-derived benztropolones 1.1.4.1. Flavanol-derived benztropolone (EC + EGC) = theaflavin 1.1.4.2. Flavanol-derived benztropolone (ECG + EGCG) = theaflavin-3,3'digallate 1.1.4.3. etc. 1.1.5. Flavanol and gallate-derived benztropolones 1.1.5.1. Flavanol and gal late-derived benztropolone (ECG + ECG) = theaflavate A 1.1.5.2. Flavanol and gal late-derived benztropolone (EC+5GQA) = Theogallinin (as used by Harbowy and Balentine7) 1.1.5.3. Flavanol gallate and gallate-derived benztropolone (GA+ EGCG) = Epitheaflagallin 3gallate 1.1.5.4. etc. 1.1.6. Flavanol and flavonol-derived benztropolones 1.1.6.1. Flavanol and flavonol-derived benztropolones (EC + Myr-3-glu) = Theaflavonin as used by Takino and Imagawa.8 1.1.6.2. etc. 1.1.7. Flavanol-derived hydroxycyclopentanones (HCP) 396
1.1.7.1.
Flavanol-derived oolongtheanin 3'gallate (EGCG + EGC) (Hashimoto et al6) 1.1.7.2. etc. 1.1.8. Flavanol-derived hydroxycyclopentanones (HCP) 1.1.8.1. Flavanol-derived theacitrin 3'-gallate (EGCG + EGCG) = Theacitrin A 1.1.8.2. etc. 2. Flavonoid-derived crude fractions 2.1. Flavonoid-derived thearubigins as prepared by Roberts and Smith (1963)9 2.2. Flavonoid-derived theafulvins as prepared by Bailey et al. (1992)10 2.3. Flavonoid-derived theafulvins as prepared by Powell (1995)11 2.4. etc.
REFERENCES 1. IUPAC, Nomenclature of cyclitols. Biochem. J. 153: 23–31 (1976). 2. Chemistry of Flavonoid Compounds, Geissman, TA, Ed., Pergamon Press, Oxford, UK, (1962). 3. Haslam E, Practical Polyphenolics: From Structure to Molecular Recognition and Physiological Action, Cambridge University Press, Cambridge, UK, pp. 438 (1998). 4. Hemingway RW and Foo LY, Linkage isomerism in trimeric and polymeric 2,3-cis-procyanidins. J. Chem. Soc. Perkin Trans. 1 1209–1216 (1982). 5. Okuda T, Yoshida T and Hatano T, Review Article Number 73. Classification of oligomeric hydrolysable tannins and specificity of their occurrence in plants. Phytochemistry 32: 507–521 (1993). 6. Hashimoto F, Nonaka G-I and Nishioka I, Tannins and related compounds. CXIV. Structures of novel fermentation products, theogallinin, theaflavonin and desgalloyltheaflavonin from black tea, and changes of tea leaf polyphenols during fermentation. Chem. Pharmaceut. Bull. 40: 1383–1389 (1992). 7 Harbowy ME and Balentine DA, Tea Chemistry. CRC Crit. Rev. Plant Sci. 16: 415–480 (1997). 8 Takino Y and Imagawa H, Studies on the oxidation of myricetin glycosides by tea oxidase. Formation of erycitrin a new red coloured glycoside having a
benzotropolone nucleus. Agric. Biol. Chem. 27: 666– 667 (1963). 9 Roberts EAH and Smith RF, The phenolic substances of manufactured tea. IX. The spectrophotometric evaluation of tea liquors. J. Sci. Food Agric. 14: 689–700 (1963).
10 Bailey RG, Nursten HE, and McDowell I, The isolation and analysis of a polymeric thearubigin fraction from black tea. J. Sci. Food Agric. 59: 365–375. (1992). 11 Powell C, The polyphenolic pigments of black tea, Ph.D. Thesis, University of Surrey (1995).
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Appendix 2 Flavonoids — Analysis Dr. Ulrich Engelhardt Technical University of Braunschweig
Methods to Measure Active Tea Components as a Group There are a number of methods available to measure the total polyphenols content in tea and other foods or beverages, such as Folin-Denis, FolinCiocalteu,1 Prussian Blue, and others. When using those methods, in fact, a change of the reagent is measured. Older methods relied on the interaction of polyphenols with skin powder, but those proved not to be convenient enough. Currently, the FolinCiocalteu method—against gallic acid as a standard—is the best analytical means. The standard is quite stable and commercially available.
How Does Polymeric Material, Which is Formed During Fermentation, React in the Folin-Reactions? This question is not easy to answer. We analyzed five fermentation series (green tea, and 30-, 60-, 90-, 120-min fermented samples from the same leaf). The amounts of total polyphenols decreased during fermentation (e.g., from 23% in the green sample to 16% in the 120-min fermented sample). This could mean that the conversion factor decreases with increasing fermentation time due to, for example, steric hindrance. Another reason might be the oxidative degradation of nonpolyphenolic components. On the other hand, the total soluble solids extractable by 70% methanol also decreased. This could mean that some of the polymer polyphenols are bound to insoluble leaf proteins.2 Further analytical work is in progress.
How to Deal with Non-flavonoid Polyphenols? Tea contains a number of well-known non-flavonoid polyphenols, such as gallic acid and 398
theogallin, as well as a number of chlorogenic acids.3 It is possible to quantify all these compounds in tea by LC methods. There are certainly some other polyphenolic species present, for example, strictinin in green teas.4 Using the FolinCiocalteu data for total polyphenols and subtract the known non-flavonoid polyphenols seems to be the best approximation to calculate the total flavonoid content.
Methods to Measure Active Tea Compounds as Individual Components In the case of some groups of polyphenols, such as catechins,1,5 theaflavins,6 flavonol glycosides,7 flavone C glycosides,8 proanthocyanidins,9,10 and chlorogenic acids,3 methods for the HPLC separation and quantification exist at least for the main compounds, an overview can be found in Refs. 11,12. LC-MS methods have also been published and the behavior of the catechins, flavonol, and flavone glycosides, and theaflavins is also well established.13-15 Capillary electrophoresis has been used for example, for the separation of catechins, but it is not widespread in use.16,17 The limited availability of calibration standards is still a problem, but in most cases this can be overcome. For example, in the case of the flavonol glycosides one standard curve set up by a glycoside of each aglycone can be used to determine the other glycosides by mass correction. In case of the proanthocyanidins the problem of getting suitable standards is more evident. Using high-speed countercurrent chromatography might be one future tool to get standards in suitable purity and quantity.18 Another analytical approach in case of flavonol glycosides is the hydrolysis of the glycosides and determine the aglycones.11,19 In this case it is self-evident no information about the intact glycosides can be obtained. To summarize, methods for the determination of individual monomers to trimers do exist.
How to Proceed with Polymeric Material Most of the work in the past relied on definitions of groups of the so-called thearubigins set up in the early work of Roberts.20 Some authors also include bisflavanols in this group, which are present in green tea and also formed during the fermentation. Some work has been published regarding the chromatographic separation of TR, often yielding a hump with some discrete peaks on top.21 Some work was carried out on chemical characterization of TR (e.g., Refs. 22,23), but to summarize we have not succeeded as yet. One of the problems is probably due to the fact that the “TR” fractions have not been well characterized as regards the content of known monomeric and dimeric compounds. Moreover, in the case of the isolation of TR using gel chromatography or other column chromatographic methods, the adsorption might be critical. The key issue nowadays is to get the complete TR material free of other components. It is at least possible to check whether the material contains well-known monomers or dimers. LITERATURE 1. ISO TC 34 SC8 (1997) Document N 463 2. Maiwald, B., Heckeroth, L., Engelhardt U.H. (1999) unpublished. 3. Engelhardt, U. H., Finger, A., Herzig, B. (1989): Lebensmittelchem. Gerichtl. Chem. 43, 58–59. 4. Nonaka G., Sakai R., Nishioka I. (1984) Phytochemistry, 23, 1753–1755. 5. Kuhr, S., Engelhardt, U.H. (1991) Z. Lebensm. Unters. Forsch. 192, S. 526–529.
6. Steinhaus, B., Engelhardt, U. H(1989) Z. Lebensm. Unters. Forsch. 188, S. 509–511. 7. Engelhardt, U.H.; Finger, A., Herzig, B., Kuhr, S. (1992) Dtsche Lebensmittel-Rundschau 88: 69–73. 8. Engelhardt, U.H., Finger, A., Kuhr, S. (1993) Z. Lebensm. Unters. Forsch. 197: 239–244. 9. Kiehne, A., Lakenbrink, C., Engelhardt, U.H. (1997) Z. Lebensm. Unters. Forsch. 205: 153–157. 10. Lakenbrink, C., Engelhardt, U.H. (1999) unpublished. 11. Engelhardt, U.H (1998): Analytical methods in polyphenol analysis — an overview. In: Amado, R., Andersson, H. Bardocz, S., Serra, F. (Hrsg) COST 916: Polyphenols in Food. pp 49–55. 12. Engelhardt, U.H., Lakenbrink, C. Lapczynski, S. Antioxidative phenolic compounds in green/black tea and other methylxanthine containing beverages. In: Parliment, T.H., Ho, C.-T., Schieberle, P.: Caffeinated Beverages, ACS Symposium Series 754, 111–118 . 13. Lin, Y.Y.; K.J. Ng ,S. Yang, S. (1993) J. Chromatogr. 629: 389–93. 14. Kiehne, A., Engelhardt, U.H. (1996) Z. Lebensm. Unters. Forsch. 202: 48–54. 15. Kiehne, A., Engelhardt, U.H. (1996): Z. Lebensm. Unters. Forsch. 202: 299–302. 16. Larger, P.J., Jones, A.D., Dacombe, C. (1998) J. Chromatogr. 799: 309–320. 17. Hoehne, H., Engelhardt, U.H. (1996) Lebensmittelchemie 50: 161–162. 18. Degenhardt, A., Knapp, H., Winterhalter. P. (1999). In: Ames, J.M., Hofmann, T. (Eds) Chemsitry and Physiology of Food Colors. ACS Symp. Ser. (in press). 19. Hertog M.G., Hollman P.C., Katan M.B., Kromhout, D. (1993) Nutr Cancer; 20(1):21–9. 20. Roberts, E.A.H. (1958) J. Sci. Food Agric. 9 (7): 381– 390. 21. Opie, S.C., Robertson, A., Clifford, M.N. (1990) J. Sci. Food Agric. 50: 547–61. 22. Kuhr, S.; Herzig, B., Engelhardt, U.H. (1994) Z. Lebensm. Unters. Forsch. 199: 13-16. 23. Bailey, R.G.; Nursten, H.E., McDowell, I. (1994) J. Sci. Food Agric. 64: 203–8.
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Appendix 3 Methods to Quantitatively Measure Active Components in Tea/Tea Extracts Dr. Xiaochun Wan Anhui Agricultural University
Tea is a kind of nutritious beverage that has a great deal of active components, such as polyphonls, caffeine, theanine, tea polysaccharides, etc. At present, however, there is no agreeable standard of method to quantitatively measure active components in tea/tea extracts as a group, as well as individual components, which bring about many difficulties with communicating among countries in science and economy.
Methods for Determination of Total Polyphenols Methods for determination of total polyphenols are abundant, of which colorimetric methods are preferred. This paper gives a comprehensive comment on these methods (e.g. Folin-Ciocaleau, Ferricyanide/Prussian Blue, Ferrous tartrate). With these kinds of methods, reagents are easy to get, results are given quickly, and experiments are convenient. However, we also realize their common weakness, that is, they are imprecise. Polyphenols are a kind of complex polyphenic mixture in which the total quantities and the percentage of individual components are varied with the varieties and the change of tea plant living conditions. Moreover, every individual component has different extent of reactions with analytical reagents, which make a light or deep color. So it is obvious that with different methods the results would be different even if total polyphenols in a sample are the same. So, great deviation will happen unavoidably if only a conversion coefficient is taken up. Finding out a conversion coefficient that can respond to all the tea plant living conditions is impossible. No matter which conversion coefficient and methods are used, the result is just a relative one. This is the unconquer-
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able weakness of colorimeter methods. Other methods, such as oxidimetry, UV-spectrophotometric methods, FIA, etc. have the same shortages that are the lack of standard substances. In our lab, we manage to gain a suitable method to chose HPLC to measure total polyphenols. Still, methods we perform to measure polyphenols need further development.
Methods of Measure Active Components in Tea The advantages and disadvantages of some methods such as HPLC, GPC, CE, etc. are assessed when these methods are used to analyze individual components. The HPLC method has the advantage of high precision, convenience, good separation, and the results can be repeated. The GPC method has the same advantage as HPLC, but these two methods can only separate catechins and caffeine in one procedure. Other important components, such as amino acids, have to be separated and analyzed by several methods. The CE method can make up for the weakness of HPLC and GPC, which is a useful separation technique in food analysis and element speciation. Special advantages are a short time of analysis and a high sensitivity without interaction between the analyzed components and the solidliquid phase interface. Meanwhile, compared with HPLC and GPC, parameters such as pH, temperature, and concentration can be optimized and controlled better in the CE analytical process. The CE method has some disadvantages. It deserves to be mentioned that Joule heat occurs, caused by current in the capillary, which can be overcome by cooling with air or water. This is very important for obtaining good reproducibility.
Some new techniques have been mentioned in this paper, such as FIA, ISFET, etc.
Methods to Separate and Measure the Polymeric Compounds in Tea There are many active polymeric compounds in tea, such as TFs, TRs, TPs, etc. Compared with the Flavognost method, the Roberts method is an improved method, which is used to measure the content of TFs. There are still some weaknesses. Factors such as the temperature of the extracting water and pH value affect the results. Especially, the Flavognost reagent can only be combined with the two cis ortho-hydroxyl of TFs, not the side hydroxyl of gallic acid. So the measured results are relatively lower. The Flavognost method has the weakness of bad reproducibility, which had notable diversity in the different laboratory, while less diversity in the same laboratory. The orthodox method — Roberts method — has bad reproducibility, and the results are also relative lower as there are no good methods to separate the TFs from the TRs. We should make better separation in the TRs1 compounds to improve the accuracy of the Roberts method. TRs are polymeric compounds that have various molecular weights and are different in characters. With the absence of a clear knowledge of the components, the physical and the chemical character of TRs, it is difficult to separate, analyze, and quantify. The preferred methods we used are the spectrometer method such as the Roberts method and HPLC (Bailey method, 1991), which was developed recently. More time is needed to establish a systematic and precise method to measure TRs.
TPs (tea polysaccharides) are a kind of glycoprotein. The content of either protein or sugar used to represent total TPs is unsuitable. Considering that there are no standards in the world at present, this work need to be completed with cooperation.
REFERENCES 1. Zhang Kainong et al., China Tea, 1995,(5): 28–29. 2. Xiao Chun et al., Tea Communication, 1996, (4): 27– 29. 3. Xiao Chun et al., Tea Communication, 1995, (3): 29– 31. 4. Zhong Luo et al., Shanghai Science Press, 1989. 5. Ruan Yuchen et al., China Tea , 1995, (3): 20–21. 6. Zhang Kainong et al., China Tea Process, 1995, (2): 32–36. 7. Guo Bingying, China Tea, 1988, (4) 5. 8. Zhou Weinong et al., China Tea Process, 1990, (4): 45–47. 9. Horie. H/ et al., J. Chromatogr. A, 1997, 758(2): 332– 335. 10. Horie, H. et al., J. Chromatogr. A, 1998, 817(12): 139–144. 11. Harms, J. et al., Fresenius’ J. Anal. Chem., 1994, 350: 93–100 12. Horie, H. et al., Food Sci. Technol. Int, Tokyo, 1997, 3(1): 27–30. 13. Watanabe, T. et al., Anal. Sci., 1998, 14(2): 435–438 14. Mcdowell, I., Secretariat of ISO TC34 SC8 1986,7– 10P. 15. Xia Tao et al., China Tea Process, 1992, (2): 42–45 16. Mcdowell, I. et al., J. S. Food Agric , 1990, 53: 411– 414. 17. Bailey, R.G. et al., J. S. Food Agric., 1990, 52: 509– 525. 18. Bailey, R.G. et al., J. Chromatogr., 1991, 542: 115– 128. 19. Bailey, R.G. et al., J. S. Food Agric., 1992, 59: 365– 375 . 20. Bailey, R.G. et al., J. S. Food Agric., 1994, 64: 231–238. 21. Opie, S.C. et al., J. S. Food Agric., 1990, 50: 547–561. 22. Bailey, R.G. et al., J. S. Food Agric., 1994, 66: 203–208.
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Appendix 4 Methods to Quantify Tea Components in Biological Matrices Dr. Peter C.H. Hollman State Institute for Quality Control of Agricultural Products, The Netherlands
Catechins Many spectrophotometric methods are available for the analysis of catechins in foods and plants, but most of them lack sufficient sensitivity and specificity to enable analyses in biological fluids for pharmacokinetic and metabolic studies.3,13 However, a simple and rapid spectrophotometric for total catechins in plasma achieved an acceptable limit of detection of 15 ng/ml.15 This method includes solid phase extraction of catechins with alumina, followed by the formation of a colored complex (λ = 637 nm) with dimethylaminocinnamaldehyde. It has been applied successfully to determine the pharmacokinetics of green and black tea in volunteers.26 Although the method has equimolar responses for (+)-catechin (CAT), (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg), it is not clear to what extent methoxylated catechins, and glucuronides and sulfates of catechins and methoxylated catechins are included. Generally, individual catechins have been determined with high-performance liquid chromatography (HPLC); only one research group employed gas chromatography with mass spectrometry (GC-MS). 17 All HPLC methods used reversed phase columns with either isocratic or gradient elution; eluents consisted of mixtures of low-pH buffers and methanol or acetonitrile. Some methods only determined free catechins, 5,11,17,25 whereas sulfate or glucuronide conjugates were included in the other methods. 6,7,8,10,16,18,21,22,24,27 These authors described enzymatic hydrolysis with β-glucuronidase/sulfatase mixtures for the determination of total catechins in plasma or urine; however, Maiani18 hydrolyzed with hydrochlo-
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ric acid. The distinction between sulfates and glucuronides has been achieved by applying sulfatase and glucuronidase treatments separately or by chromatographic separation of glucuronides. 10 Data on optimization of these enzymatic procedures are lacking; only Lee 16 mentioned that enzyme concentrations and incubation times had been optimized; however, these data were not shown. Lee 16 warned that unwanted enzyme activity in some of the βglucuronidase preparations was capable of coverting EGCg to EC. Methoxylated catechins, liver metabolites of catechins, have been determined with HPLC 10,21 and GC. 9 Plasma or urine has been extracted with ethyl acetate, methanol, acetonitrile ore solid phase adsorbents (alumina11,15 or bonded hydrocarbons25). Tsuchiya24 exploited the catechol structure of catechins and extracted catechins and gallocatechins and their gallates after complexation with diphenylborate. Some authors applied multiple extractions.6,16,18,21 Surprisingly, no data are available to demonstrate that optimal extraction had been achieved. Recoveries of catechin standards added to plasma were sometimes rather low (about 70%). As a remedy, plasma contents were determined from standard curves obtained by spiking of blank plasma with known amounts of catechins.11,16,17,25 In HPLC, fluorescence and electrochemical (coulometric) detection have been used most frequently. Ultraviolet (UV) detection has been described in a number of papers,10,18,21 but is only applicable if concentrations are higher than about 200 ng/ml. In addition, UV detection lacks specificity, which may easily lead to overestimation because of interfering compounds in plasma.9 Arts1 found that detection limits ( signal/noise ratio = 3) of catechin standards (CAT, EC, ECg, EGC, and EGCg)
ranged from 30 to 300 ng/ml with UV detection, but decreased to 8 ng/ml with fluorescence detection. Only CAT and EC show fluorescence. Donovan9 also compared various detection techniques this time for CAT, EC, and their methoxylated metabolites and found somewhat higher detection limits for fluorescence and UV detection; the sensitivity of GCMS (Single Ion Mode, SIM) was comparable to that of HPLC fluorescence detection. This GC-MS work has not been expanded to the gallocatechins and gallates. No data on LC-MS determination of catechins in plasma or urine are as yet available. Detection limits of catechins, gallocatechins, and their gallates achievable with coulometry ranged from 0.5 to 1.5 ng/ml.16,20 However, methoxylated metabolites will have increased detection limits. Chemiluminescence has been used as a very sensitive detection technique for EGCg in HPLC with detection limits of 0.5 ng/ml.20 EGC is expected to have similar sensitivity, whereas EC, and probably also CAT, will have very reduced responses of only 0.5% of that of EGCg.19 This technique requires a postcolumn oxidation reaction.
not feasible that these polymeric condensation products are absorbed in humans. Thus, their determination in plasma or urine is not sensible. Several spectrophotometric methods are available for the analysis of these condensation products in feces, but these methods lack specificity.13
Metabolites Lee16 found that only 2% of the monomeric catechins from green tea were excreted with an intact flavonoid structure into urine. Although Lee did not determine methoxylated catechins, Donovan8 showed that they only play a minor role. This suggests that extensive metabolism occurs starting with ring fission and leading to all kinds of phenolic acids.12 Microorganisms in the colon cleave the flavonoid ring and also metabolize the unabsorbed condensation products. To study these metabolites methods a powerful separation method connected to a detector that gives structural information of the compounds is needed, for example, GC-MS.
Theaflavins REFERENCES Theaflavin, theaflavin gallates, and the digallate in tea have been separated with HPLC and UV detection.2,23 Capillary electrophoresis failed as a separation method because of irreversible adsorption of theaflavins onto the capillary wall.4 However, there are no papers on the determination of theaflavins in plasma or urine. Detection techniques with a higher sensitivity than UV probably will be needed, and extraction from proteins in these biological samples will need special attention. Thermospray LC-MS has been used for tea,14 but probably still lacks sensitivity for biological samples. No data have been published on coulometric detection of theaflavins. Chemiluminescence emission of theaflavin digallate appeared to be about 50% of that of EGCg and emission of theaflavin was only 2% of that of EGCg.19
Thearubigins The structure of thearubigins is uncertain, which makes their determination difficult. It is
1. Arts, I.C.W. and Hollman, P.C.H. (1998) Optimization of a quantitative method for the determination of catechins in fruits and pulses. J. Agric. Food Chem., 46, 5156–5162. 2. Bailey, R.G. and Nursten, H.E. (1991) Comparative study of the reversed-phase high-performance liquid chromatography of black tea liquors with special reference to thearubigins. J. Chromatogr., 542, 115– 118. 3. Beecher, G.R., Warden, B.A. and Merken, H. (1999) Analysis of tea polyphenols. Proc. Soc. Exp. Biol. Med., 220, 267–270. 4. Begoña Barroso, M. and van de Werken, G. (1999) Determination of green and black tea composition by capillary electrophoresis. J. High Resol. Chromatogr., 22, 225–230. 5. Carando, S., Teissedre, P.-L., and Cabanis, J.-C. (1998) Comparison of (+)-catechin determination in human plasma by high-performance liquid chromatography with two types of detection: fluorescence and ultraviolet. J. Chromatogr.B, 707, 195– 201. 6. Chen, L., Lee, M.J., Li, H., and Yang, C.S. (1997) Absorption, distribution, and elimination of tea polyphenols in rats. Drug Metab. Dispos., 25, 1045– 1050.
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7. Da Silva, E.L., Piskula, M., and Terao, J. (1998) Enhancement of antioxidative ability of rat plasma by oral administration of (-)-epicatechin. Free Radic. Biol. Med., 24, 1209–1216. 8. Donovan, J.L., Bell, J.R., Kasim-Karakas, S., German, J.B., Walzem, R.L., Hansen, R.J., and Waterhouse, A.L. (1999a) Catechin is present as metabolites in human plasma after consumption of red wine. J. Nutr., 129, 1662–1668. 9. Donovan, J.L., Luthria, D.L., Stremple, P., and Waterhouse, A.L. (1999b) Analysis of (+)-catechin, (-)-epicatechin and their 3′- and 4′-O-methylated analogs — a comparison of sensitive methods. J. Chromatogr.B, 726, 277–283. 10. Harada, M., Kan, Y., Naoki, H., Fukui, Y., Kageyama, N., Nakai, M., Miki, W. and Kiso, Y. (1999) Identification of the major antioxidative metabolites in biological fluids of the rat with ingested (+)-catechin and (-)-epicatechin. Biosci. Biotech. Biochem., 63, 973– 977. 11. Ho, Y., Lee, Y.L. and Hsu, K.Y. (1995) Determination of (+)-catechin in plasma by high-performance liquid chromatography using fluorescence detection. J. Chromatogr.B, 665, 383–389. 12. Hollman, P.C.H. and Katan, M.B. (1998) Absorption, metabolism, and bioavailability of flavonoids. In RiceEvans, C. and Packer, L., Eds. Flavonoids in Health and Disease, Marcel Dekker Inc., New York, pp. 483–522. 13. Hollman, P.C.H., Tijburg, L.B.M., and Yang, C.S. (1997) Bioavailability of flavonoids from tea. Crit. Rev. Food Sci. Nutr., 37, 719-738. 14. Kiehne, A. and Engelhardt, U.H. (1996) ThermosprayLC-MS analysis of various groups of polyphenols in tea II. Chlorogenic acids, theaflavins and thearubigins. Z. Lebensm. Unters. Forsch., 202, 299-302. 15. Kivits, G.A.A., van der Sman, F.J.P., and Tijburg, L.B.M. (1997) Analysis of catechins from green and black tea in humans: a specific and sensitive colorimetric assay of total catechins in biological fluids. Int. J. Food Sci. Nutr., 48, 387-392. 16. Lee, M.-J., Wang, Z.-Y., Li, H., Chen, L., Sun, Y., Gobbo, S., Balentine, D.A., and Yang, C.S. (1995) Analysis of plasma and urinary tea polyphenols in human subjects. Cancer Epidemiol. Biomark. Prev., 4, 393–399.
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17. Luthria, D.L., Jones, A.D., Donovan, J.L., and Waterhouse, A.L. (1997) GC-MS determination of catechin and epicatechin levels in human plasma. J. High Resol. Chromatogr., 20, 621-623. 18. Maiani, G., Serafini, M., Salucci, M., Azzini, E., and Ferro-Luzzi, A. (1997) Application of a new high performance liquid chromatographic method for measuring selected polyphenols in human plasma. J. Chromatogr.B, 692, 311–317. 19. Miyazawa, T. and Nakagawa, K. (1998) Structurerelated emission spectrometric analysis of the chemiluminescence of catechins, theaflavins and anthocyanins. Biosci. Biotechnol. Biochem., 62, 829–832. 20. Nakagawa, K. and Miyazawa, T. (1997) Chemiluminescence high performance liquid chromatographic determination of tea catechin, (-)-epigallocatechin-3gallate, at picomole levels in rat and human plasma. Anal. Biochem., 248, 41–49. 21. Okushio, K., Suzuki, M., Matsumoto, N., Nanjo, F., and Hara, Y. (1999) Identification of (-)-epicatechin metabolites and their metabolic fate in the rat. Drug Metab. Dispos., 27, 309-316. 22. Piskula, M.K. and Terao, J. (1998) Accumulation of (-)-epicatechin metabolites in rat plasma after oral administration and distribution of conjugation enzymes in rat tissues. J. Nutrit., 128, 1172–1178. 23. Temple, C.M. and Clifford, M.N. (1997) The stability of theaflavins during HPLC analysis of a decaffeinated aqueous tea extract. J. Sci. Food Agr., 74, 536–540. 24. Tsuchiya, H., Sato, M., Kato, H., Kureshiro, H., and Takagi, N. (1998) Nanoscale analysis of pharmacologically active catechins in body fluids by HPLC using borate complex extraction pretreatment. Talanta, 46, 717–726. 25. Unno, T., Kondo, K., Itakura, H., and Takeo, T. (1996) Analysis of (-)-epigallocatechin gallate in human serum obtained after ingesting green tea. Biosci. Biotech. Biochem., 60, 2066–2068. 26. van het Hof, K.H., Wiseman, S.A., Yang, C.S., and Tijburg, L.B.M. (1999) Plasma and lipoprotein levels of tea catechins following repeated tea consumption. Proc. Soc. Exp. Biol. Med., 220, 203–209. 27. Yang, C.S., Chen, L., Lee, M.-J., Balentine, D., Kuo, M.C., and Schantz, S.P. (1998) Blood and urine levels of tea catechins after ingestion of different amounts of green tea by human volunteers. Cancer Epidemiol. Biomark. Prev., 7, 351–354.
Appendix 5 Methods to Quantify Antioxidant Activity of Tea/Tea Extracts In Vitro Dr. Catherine Rice-Evans Guy’s Hospital, King’s and St Thomas’ Medical School, King’s College
There are three main methodological approaches that can be applied to the quantification of the antioxidant activities (or reducing properties) of tea/tea extracts in vitro that relate to methods established for the measurement of the antioxidant activity of pure phenolic compounds and plant or food extracts: 1.
2.
The chemical approach is based on the application of a model free radical to screen the relative abilities of such systems to scavenge or reduce the radical. The results depend on the relative rates of reaction of the components in question with the free radical and their hydrogen-donating properties. Thus, different methods will not give the same absolute values of antioxidant activity, but should provide the same relative rankings of the compounds or extracts. The antioxidant activity values measured should also show a broad inverse relationship with the reduction potentials of the compounds. Such screening methods include the TEAC (Trolox equivalent antioxidant capacity) assay based on the scavenging of the ABTS•+ radical cation,1-4 which can be applied in aqueous and lipophilic systems, and the DPPH• radical applied in lipophilic solvents. The trend of results obtained for the ABTS•+ assay for most flavonoids in general correlate broadly with the reduction potentials. The perceived disadvantage of this assay is that it is a chemical screening system for relative antioxidant biomarkers and does not provide a physiologically/pathologically relevant radical against which to screen the components of interest. The more biological approach involves the generation of a free radical of pathological
3.
significance and assessment of the relative abilities of the antioxidants in question as scavenging agents. The much explored approach here is the generation of peroxyl radicals, using azo initiators, and their reduction. Again, the outcomes of such analyses should be dependent on the rate of reaction of the relevant antioxidants with the peroxyl radical and, in the realistic biological milieu, requires considerations of the accessibility of the antioxidant to the radical, or the partition coefficient of the antioxidant components. The TRAP assay was the initial method developed for such studies,5,6 and this has been modified and developed in terms of endpoint and reactants by a number of researchers.7-11 The most common TRAP assay applied currently is the ORAC assay, the oxygen radical antioxidant capacity assay.9,10,12 This assay has its strengths in that it can involve a free radical of in vivo relevance, but its timescale and fluorescent technique make it rather less user-friendly.13,14 However, the ORAC value depends on the radical selected for assay, and thus due to varying rates of reaction of the antioxidants with different free radicals, different results will be obtained depending on the radical selected.14a This again creates limitations for the biological or in vivo relevance of the assay A more recent novel approach was devised mainly for plasma by Benzie and Strain,15 namely, the ferric reducing ability of plasma or FRAP assay. This method depends on the ability of plasma antioxidants collectively to reduce ferric iron to ferrous, which binds to chromophore forming a complex that is measurable spectrophotometri-
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cally. The FRAP assay has been less applied to in vitro measurement of the total antioxidant activities of pure compounds or food extracts, but the method shows promise in this direction. Published reports applying the TEAC method to food extracts and pure compounds (except quercetin in one case) show comparable results from lab to lab when carried out under identical conditions. Comparing antioxidant activities of black and green tea preparations, closely similar results for the two extracts are obtained from the TEAC assay4,16 (measured on extracts supplied by Unilever), but the ORAC assay shows about a factor of two in difference to the mean values obtained from measurement of a variety of different commercial preparations of the teas, the range of values for the black teas studied varying by a factor of 3, and that within the green teas varying by a factor of 5).14 The establishment of a league table of antioxidant activities of beverages relative to fixed volumes/concentrations might have its use in comparing the propensity for food extracts/beverages as dietary sources of antioxidants. However, different assays based on different chemistry seem to give different results, and within an assay the application of different radicals gives different results. Thus, due to the nature of the assays, the variety of radicals applied and the contrasting approaches to the expression of the results (and their units), it is not possible at the present time to make an absolute comparison of ORAC with TEAC, just of trends. However, the question as to what we are trying to achieve here must be addressed. If the requirement is the screening of compounds and beverages for their relative antioxidant potentials in vitro, then assays based on the pregeneration of a free radical (such as TEAC or ORAC assays) and the subsequent scavenging profile of antioxidants would seem quite appropriate. In order to demonstrate potential biological antioxidant activity in vivo and to gain some understanding from in vitro studies through interaction of tea phenolics with biologically relevant reactive species of pathological relevance, for example, peroxyl radicals as might occur in vivo, knowl-
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edge of the relevant circulating forms of the phenolics is also required. Thus, the definition of the interactions of the tea phenolics as they cross the GI tract is crucial to put this level of assay and interpretation into context. Others screen the efficacy of antioxidants in biological systems in vitro using the assessment of their relative abilities to inhibit the peroxida-tion of low-density lipoproteins. 4,17,18 This again provides the in vivo ‘mimic’ of lipid peroxyl radicals and takes into consideration the partitioning properties and hydrogen-donating abilities of the phenolics/phenolic extracts relative to α-tocopherol, all positive points. However, the form of tea extract/phenolic compound used in these in vitro assays is not necessarily the major form found in vivo, for example, methylated, glucuronidated, sulfated derivative, which would clearly influence the H-donating and the solubility properties of such constituents. Thus, although such approaches have taught us a lot about mechanisms of action, they cannot be extrapolated to the in vivo picture. This again underlies the importance of gastrointestinal studies.19 In conclusion there is a requirement for validation and comparison of methodologies, the first requirement possibly being to reach a consensus on the nature of the redox active systems used to screen for antioxidant activity — both for the aqueous and lipophilic phases. A concerted effort to measure the same material in different laboratories using a range of appropriately selected assays would resolve these issues and help to provide a clear statement for a consensus approach to such investigations. REFERENCES 1. R. Re, N. Pellegrini, A. Proteggente, A. Pannala, M. Yang and C Rice-Evans (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay, Free Rad Biol Med, 26, 1231–1237. 2. N. Pellegrini, R. Re, M. Yang and C. Rice-Evans. (1999) Screening of dietary carotenoids and carotenoid-rich fruit extracts for antioxidant activities applying the ABTS∑+ radical cation decolorisation assay. Methods Enzymol., 379–389. 3. C.A. Rice-Evans, N.J. Miller and G. Paganga (1996) Structure-antioxidant activity relationships of
4.
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6.
7.
8.
9.
10.
11.
flavonoids and phenolic acids, Free Rad Biol Med, 20, 933–956. N. Salah, N.J. Miller, G. Paganga, L. Tijburg, G.P. Bolwell, and C. Rice-Evans (1995) Polyphenolic flavonols as scavengers of aqueous phase radicals and as chain-breaking antioxidants. Arch Biochem Biophys, 322, 399–346. D.D.M. Wayner, G.W. Burton, K.U. Ingold, and S. Locke (1985). Quantitative measurement of the total peroxyl radical-trapping antioxidant capability of human blood plasma by controlled peroxidation. The important contribution made by human plasma proteins, FEBS Lett, 187, 33–37. D.D.M. Wayner, G.W. Burton, K.U. Ingold, L.R.C. Barclay, and S.J. Locke (1987) The relative contributions of vitamin E, urate, ascorbate and proteins to the total peroxyl radical-trapping antioxidant activity of human blood plasma, Biochim Biophys Acta, 924, 408–419. R.J. DeLange and A.N. Glazer (1989) Phycoerythrin fluorescence-based assay for peroxy radicals: a screen for biologically relevant protective agents. Anal Biochem, 177, 300–306. Ghiselli, M. Serafini, G. Maiani, E. Azzini, A. FerroLuzzi (1995) A fluorescence-based method for measuring total plasma antioxidant capability, Free Rad Biol Med, 18, 29–36. G. Cao, H.M.A. Lessio, and R.G. Cutler (1993) Oxygen radical absorbance capacity for antioxidants, Free Rad Biol Med, 14, 303–311. G. Cao, C.P. Verdon, A.H.B. Wu, H. Wang, and R.L. Prior (1995) Automated assay of oxygen radical absorbance capacity with the Cobas Fara II, Clin Chem, 41, 1738–1744. T.P. Whitehead, G.H.G. Thorpe, and S.R.J. Maxwell (1992) Enhanced chemiluminescent assay for antioxi-
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13.
14.
14a
15.
16.
17.
18.
19.
dant capacity in biological fluids, Anal Chim Acta, 266, 265–277. H. Wang, G. Cao, and R.L. Prior (1996) Total antioxidant capacity of fruits, J Agric Food Chem, 44, 701– 705. Y.L. Lin, I.M. Huan, Y.L. Chen, Y.C. Liang, and J.K. Lin (1996) Composition of polyphenols in fresh tea leaves and association of their oxygen radical absorbing capacity with antiproliferation actions in fibroblast cells. J Agric Food Chem, 44, 1387–1394. R.L. Pryor and G. Cao (1999) Antioxidant capacity and polyphenolic components of teas: implications for altering in vivo antioxidant status. Proc Soc Exp Biol Med, 220, 255–261. G. Cao, E. Sofic, and R.L. Prior (1996) Antioxidant capacity of tea and common vegetables. J Agric Food Chem, 44, 3426–3431. I.F.F. Benzie and J.J. Strain (1996) The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal Biochem, 239, 70–76. C. Rice-Evans (1999) Implications of the mechanism of action of tea polyphenols as antioxidants in vitro for chemoprevention in humans. Proc Soc Exp Biol Med, 220, 262–266. J.A. Vinson and Y.A. Dabbagh (1998) Antioxidant effectiveness of teas, tea components, tea fractions and their binding with lipoproteins. Nutr Res, 18, 1067–1075. N.J. Miller, C. Castelluccio, L. Tijburg, and C. RiceEvans (1996) The antioxidant properties of theaflavins and their gallate esters – radical scavengers or metal chelators? FEBS Lett, 392, 40–44. J.P.E. Spencer, G. Chowrimootoo, R. Choudhury, E.S. Debnam, S.K. Srai, and C. Rice-Evans (1999) The small intestine can both absorb and glucuronidate luminal flavonoids. FEBS Lett, 458, 224–230.
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Appendix 6 Methods to Quantify Antioxidant Activity of Tea and Tea Extracts In Vitro Dr. Junji Terao The University of Tokushima School of Medicine
Antioxidants can be classified as preventive antioxidants and chain-breaking antioxidants. Preventive antioxidants suppress the initiation of lipid peroxidation by scavenging chain-initiating reactive oxygen species (ROS) or inhibiting the generation of ROS. Tea and tea extracts can act as preventive antioxidants by scavenging ROS such as superoxide anion, perhydroxyl radical, and peroxynitrite. We emphasize that chelation of metal ion is also involved in the mechanism of tea and tea extracts as preventive antioxidants, because metal ion is mostly responsible for the generation of ROS. We improved 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay for radical scavenging activities of beverages with the use of HPLC. It seems to be a simple and convenient method for estimating ROS-scavenging activity of tea and tea extracts. Deoxyribose assay can be used as a conventional test for measuring metal ion-chelating activity of tea extracts. We already reported that the activity of tea catechins as chain-breaking antioxidants is much lower than that of alpha-tocopherol, a well-known lipophilic antioxidant in vivo. However, tea catechins seem to be concentrated in the interface between the lipid-phase and water-phase in cellu-
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lar and intracellular fluids because of their hydrophilicity. We adopted liposomal suspension as a biomembrane model and lipophilic or hydrophilic radical generator as a lipid peroxidation inducer. It is a suitable in vitro model for estimating antioxidant activity of tea catechins in biomembrane system because of their reproducibility. The result suggested that tea catechins can effectively prevent the progression of radical chain reaction in membrane lipid peroxidation by trapping chaininitiating ROS in the interface of the membranes. Oxidation of isolated low-density lipoprotein (LDL) is an in vitro model for estimating the antioxidant activity of tea catechins in blood circulation. We used lipoxygenase, azo radical generators, copper ion, photosensitizers, and peroxynitrite generators as inducers of LDL oxidation. Nonabsorbed tea catechins and those from enterohepatic circulation may exert an efficient antioxidant activity for protecting the membrane surface of epithelial cells in the digestive tract. Oxidation of intestinal mucosa homogenate may be an alternative in vitro model for the measurement of antioxidant activity of tea catechins. The principle and advantages of these methodology should be discussed.
Appendix 7 The Current Status of Markers of Oxidative Damage Dr. Myron Gross University of Minnesota
Oxidative damage has been associated with the development of numerous chronic disease conditions, including cancer and cardiovascular disease. Validation of these associations requires the establishment of objective measures of oxidative damage and the verification of oxidative damage–disease relationships in the context of human and epidemiologic studies. The identification of biomarkers and development of methodology for the measurement of oxidative damage has evolved rapidly in recent years, but it remains a challenging area of investigation. Numerous indicators have been examined and show potential as indicators of oxidative damage and disease risk. The potential indicators include oxidation products of lipids, protein, and DNA. However, in most cases gold standards against which to judge the assays do not exist. Furthermore, no single marker has been found that is a global indicator of oxidative damage. Even the most developed markers require careful consideration before application in human studies. Lipid peroxidation occurs readily under conditions of oxidative stress and yields numerous products.1,2 It is likely that lipids oxidize more readily than proteins or DNA. Lipid peroxidation therefore may be a more sensitive indicator of oxidative stress than oxidation products of proteins and DNA.3 The two major measures of products of lipid peroxidation are the commonly used thiobarbituric acid reactive substances (TBARS) assay and the recently developed F2-isoprostanes assay. The interpretation of results from the TBARS assay is limited by a well-recognized low specificity for products of lipid peroxidation4,5,6 and the possibility of artifactual oxidation.6 Both of these technical concerns have been addressed recently with the implementation of HPLC-based procedures and careful sample handling procedures. The new TBARS procedures require fur-
ther validation studies before application in human studies. The F2-isoprostanes are produced by free radical-mediated, cyclooxygenease-independent peroxida-tion of arachidonic acid and consists of several isomers of prostaglandin F2.7,8 In vitro formation of F2-isoprostanes and 8-epi prostaglandin F2, a major isomer, are correlated with the formation of other markers of oxidative damage9 and elevated in numerous in vivo conditions associated with oxidative stress.10 The F2-isoprostanes may be the most reliable and quantitative indicator available for the measurement of lipid peroxidation. F2-isoprostanes occur in esterified and free forms in plasma and the free form in urine. Commonly, total free F2-isoprostanes have been measured in plasma by gas chromatography–mass spectrometry and a single, predominant isomer, 8-epi prostaglandin F2, has been measured in urinary samples by antibody-based methods. Both approaches may provide useful information and can be applied with several caveats. Plasma free F2-isoprostanes may reflect the steady state concentrations of lipid peroxidation products, but measurements do involve the use of sophisticated and time-consuming methodology.11 Urinary 8-epi prostaglandin F2 measurements are an inexpensive and convenient measure of total free 8-epi prostaglandin F2 formation, but may be influenced by the renal formation of 8-epi prostaglandin F2. Further, their measurement may be variable because of poor specificity of available antibodies. Another significant relatively unexplored aspect of isoprostanes that should be addressed is the identification of the quantitative contribution, if any, of dietary F2-isoprostane intake to plasma and urinary concentrations of these compounds. Two additional measures of lipid peroxidation, exhaled pentane and ethane and the oxidation of low-density lipoproteins (LDL), may be useful in
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some situations, but currently have significant limitations for widespread application. The measurement of exhaled pentane and ethane is technically difficult and is accompanied by several unique and unresolved issues regarding quality assurance. These measurements may be utilized effectively in small populations, but application is currently difficult for large populations. Resistance of LDL particles to oxidation is measured using a nonphysiologic stressor and may have little physiological relevance to disease. It is difficult to interpret at this time. Several oxidation products of proteins are found commonly in skin and blood.12,13,14 They include protein carbonyl derivatives, stable amino acid derivatives (e.g., chlorotyrosine, nitrotyrosine, and dityrosine), glycation, and glycoxidation products.15,16,17,18,19 The structure of many of these products is unknown, poorly characterized, and the products are difficult to quantitate with the exception of stable amino acid modifications.12,13,20,21 The stable amino acid modifications can be detected reliably, but only by complex analysis procedures, generally involving chromatography and mass spectrometry. Thus, most of these oxidation products require additional methodologic development and further validation before application in human studies. In the near future, a possible exception may be a few products (pentosidine and some carbonyl proteins) wherein antibodybased procedures are under development and may become generally available. The oxidation of DNA produces over 20 products, only a few of which have been explored as biomarkers of oxidative damage. The most common oxidation products22 are 8-hydroxydeoxyguanosine (8OHdG) and 8-oxoguanine (8-oxoG). These products may be the most sensitive indicators of DNA oxidative damage.23 The products have been measured in white blood cells and urine, and presumably reflect steady state concentrations and overall oxidative damage, respectively. The amounts and modulation of DNA products in urine are dependent on the specificity and activity of repair pathways.24 Recent data suggest that oxidative damage involving guanine may be repaired primarily by the release of 8-oxoG rather than 8OHdG.25,26 This possibility may limit the effective use of urinary measurements in the assessment of oxidative damage as diet can be
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a significant contributor of 8-oxoG and may confound the interpretation of measurements of 8-oxoG, except in very well-controlled human experiments. Furthermore, recent evidence suggests that urinary 8OHdG may partially reflect kidney metabolism rather than systemic damage.27 On the other hand, the concentration of 8OHdG in white blood cells is influenced by oxidative stress and can be modulated by antioxidants.28 It may provide a useful indicator of oxidative damage, and further validation studies are warranted for this biomarker. The isolation and analysis of DNA from white blood cell DNA often involves lengthy procedures that are prone to the artifactual oxidation of DNA. In particular, the use of derivatization procedures may produce artifacts in the gas chromatographic analysis of DNA samples. Recent recognition of these problems has led to new procedures for the isolation and analysis of 8OHdG.29 These methods employ procedures for the prevention of artifactual oxidation and electrochemical detection. The new methods have been linked with the measurement of 8OHdG by HPLC and electrochemical detection. This combination of methods is promising and may detect 8OHdG in an effective manner. Another measure of DNA oxidative damage that has significant promise is the Comet assay. This assay has been described previously30 and incorporates the use enzymatic reagents for the detection of damaged DNA bases. Two concerns are the efficiency of enzymatic digestion and the methods of quantitation, both areas need additional development for the collection of quantitative data. Additional analytical methods, validation experiments, and epidemiologic experiments are necessary for the development of new biomarkers of oxidative damage and an evaluation of relationships between oxidative damage and disease associations. REFERENCES 1. Esterbauer, H. and Ramos, P. Chemistry and pathophysiology of oxidation of LDL. Rev. Physiol. Biochem. Pharmacol. 127:31–64; 1995. 2. Esterbauer, H. Estimation of peroxidative damage. A critical review. Pathol. Biol. 44: 25–28; 1996. 3. Dean, R.T., Fu, S., Stocker, R., and Davies, M.J. Biochemistry and pathology of radical-mediated protein oxidation. Biochem. J. 324: 1–18; 1997.
4. Bowen, P.E. and Mobarhan, S. Evidence from cancer intervention and biomarker studies and the development of biochemical markers. Am. J. Clin. Nutr. 62: 1403S–14095; 1995. 5. Loft, S. and Poulsen, H.E. Cancer risk and oxidative DNA damage in man. Am. J. Mol. Med. 62: 297–312; 1996. 6. Janero, D.R. Malondialehyde and thiobarbituric acidreactivity as diagnostic indices of lipid perodoxidation and peroxidative tissue injury. Free Radic. Biol. Med. 9: 515–540; 1990. 7. Morrow, J.D., Hill, K.E., Burk, R.F., Nammour, T.M., Badr, K.F., and Roberts, L.J., II. A series of prostaglandin F2- like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. Proc. Natl. Acad. Sci. USA 87: 9383–0387; 1980. 8. Fitzgerald, G.A., Tigges, J., Barry, P., and Lawson, J.A. Markers of platelet activation and oxidant stress in atherothrombotic disease. Thromb. Haemost. 264: 280–284; 1997. 9. Delanty, N., Reilly, M.P. Pratico, D., Lawson, J.A., McCarthy, J.F., Wood, A.E., Ohnishi, S.T., FitzGerald, D.J., and FitzGerald, G.A. 8-Epi PFG2a generation during coronary reperfusion. A potential quantitative marker of oxidant stress in vivo. Circulation 95: 2492– 2499; 1997. 10. Roberts II, L.J. and Morrow, J.D. Isoprostanes as markers of lipid peroxidation in atherosclerosis. In: Serhan, C.N. and Ward, P.A., Eds. Molecular and Cellular Basis of Inflammation. Totowa, NJ; Humana Press; 1999: 141ff. 11. Morrow, J.D. and Roberts II, L.J. Mass spectrometric quantification of F2-isoprostanes in biological fluids and tissues as measure of oxidant stress. Methods Enzymol. 300: 3ff; 1999. 12. Sell, D.R. and Monnier, V.M. Structure elucidation of a senescence cross-link from human extracellular matrix. Implication of pentoses in the aging process. J. Biol. Chem. 264: 21597–21602; 1989. 13. Ahmed, M.U., Brinkmann Frye, E., Degenhardt, T.P., Thorpe, S.R., and Baynes, J.W. Ne-(Carboxyethyl)lysine, a product of the chemical modification of proteins by methylglyoxal, increases with age in human lens proteins. Biochem. J. 324: 565– 570; 1997. 14. Requena, J.R., Ahmed, M.U., Fountain, A.W., Degenhardt, T.P., Reddy, S., Perez, C., Lyons, T.J., Jenkins, A.J., Baynes, J.W., and Thorpe, S.R. Carboxymethylethanolomine, a biomarker of phospholipid modification during the Maillard reaction in vivo. J. Biol. Chem. 272: 17473–17479; 1997. 15. Dean, R.T., Fu, S., Stocker, R., and Davies, M.J. Biochemistry and pathology of radical-mediated protein oxidation. Biochem. J. 324:1–18; 1997. 16. Berlett, B.S. and Stadtman, E.R.; Protein oxidation in aging, disease, and oxidative stress. J. Biol. Chem. 272: 20313–20316; 1997.
17. Hazen, S.L., Crowley, J.R., Mueller, D.M., and Heinecke, J.W. Mass spectrometric quantification of 3-chlorotyrosine in human tissues with attomole sensitivity: a sensitive and specific marker for myeloperoxidase-catalyzed chlorination at sites of inflammation. Free Radic. Bio. Med. 23: 909–916; 1997. 18. Huggins, T.G., Wells-Knecht, M.C., Detorie, N.A., Baynes, J.W., and Thorpe, S.R. Formation of o-tyrosine and dityrosine in proteins during radiolytic and meta-catalyzed oxidation. J. Biol. Chem. 268: 12341– 12347; 1993. 19. Wells-Knecht, M.C., Lyons, T.J., McCance, D.R., Thorpe, S.R., and Baynes, J.W. Age-dependent increase in ortho-tyrosine and methionine sulfoxide in human skin collagen is not accelerated in diabetes. Evidence against a generalized increase in oxidative stress in diabetes. J. Clin. Invest. 100: 839–846; 1997. 20. Viassara, H. Recent progress in advanced glycation end products and diabetic complications. Diabetes 46: S19–S25; 1997. 21. Nagaraj, R.H., Shipanova, I.N., and Faust, F.M. Protein cross-linking by the Maillard reaction. J. Biol. Chem. 271: 19338–19345; 1996. 22. Loft, S. and Poulsen, H.E. Cancer risk and oxidative DNA damage in man. J. Mol. Med. 74: 297-312; 1996. 23. Dizdaroglu, M. Oxidative damage to DNA in mammalian chromatin. Mutat. Res. 275: 331–342; 1992. 24. Park, E.M., Shigenaga, M.K.., Degan, P., Korn, T.S., Kitzler, J.W., Wehr, C.M., Kolachana, P., and Ames, B.N. Assay of excised oxidative DNA lesions: isolation of 8-oxoguanine and its nucleoside derivatives from biological fluids with a monoclonal antibody column. Proc. Natl. Acad. Sci. USA 89 :3375; 1992. 25. Shigenaga, M.K., Gimeno, C.J., and Ames, B.N. Urinary 8-hydroxy-2’-deoxyguanosine as a biological marker of in vivo oxidative DNA damage. Proc. Natl. Acad. Sci. USA 86: 9697; 1989. 26. Loft, S., Larsen, P.N., Rasmussen, A., Fischer-Nielsen, A., Bondesen, S., Kirkegaard, P., Rasmussen, L.S., Ejlersen, E., Tornoe, K., Bergholdt, R., and Poulsen, H.E. Oxidative DNA damage after transplantation of the liver and small intestine in pigs. Transplantation 59: 16; 1995. 27. Lindahl, T. Instability and decay of the primary structure of DNA. Nature 362: 709–715 (1993). 28. McCall, M.R. and Frei, B. Can antioxidant vitamins materially reduce oxidative damage in humans? Rad. Bio. Med. 26:1034–1053; 1999. 29. Collins, A., Cadet, J., Epe, B., and Gedik, C. Problems in the measurement of 8-oxoguanine in human DNA. Carcinogenesis 18: 1833–1836; 1997. 30. Collins, A. R., Dusinska, M., Gedik, C. M., and Stetina, R. Oxidative Damage to DNA: Do We Have a Reliable Biomarker? Environ Health Perspect 104 (Suppl 3): 465–469; 1996.
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Appendix 8 Establishing Compositional Data for Flavonoids in Foods Dr. Gary Beecher U.S. Department of Agriculture
Data for the development of a database for flavonoid values for foods are derived from a wide variety of studies, each of which have specific objectives and experimental designs not necessarily oriented toward the generation of food composition data per se. As a result, we have developed an evaluation procedure that critically reviews analytical data and ultimately assigns a confidence code or reliability score. Data are reviewed based on five categories: analytical method, analytical quality control, food sampling plan, number of samples, and sample handling during sampling and processing for analysis. Within each category, scores are assigned ranging from 0 to 3 based on specific critical factors. Relative to analytical method, the highest score can only be achieved if a certified reference material (CRM) is available that can be used to validate the analytical procedure. These materials usually are certified and made available by the government agency of a country or region responsible for metrology. In the absence of such materials, other procedures, such as methods of standards additions and recovery of pure compounds, must be used to validate the analytical method and as a result a correspondingly lower score is assigned this category. A CRM for flavonoids in tea is unavailable at this time. With the knowledge of tea processing and storage collectively vested in the tea industry, a major contribution to the improvement of the quality of analytical data for tea would be the development of a stable tea preparation that could be characterized
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for flavonoid components and ultimately provided to the analytical community for method validation and quality control. The category, analytical quality control, refers to periodic analysis of a material or food (quality control material) that is similar to the food being analyzed so that stability of the analytical process can be assessed. Data from the analysis of these materials also provide estimates of analytical precision throughout the analysis. Relative to food sampling plan and number of samples, higher scores are given if larger numbers of samples are selected from broad geographical areas which are linked to the demographics of the country. The category, sample handling, is scored based on documented handling of the sample to retain maximum nutrients and processing analogous to preparation for consumption. The existing flavonoid data for teas and other foods have begun to be critically reviewed. When the process is complete, a database of values will result that will have for each food: a brief description, a USDA food code, and for the content of each flavonoid of each food: mean, standard error of the mean, minimum and maximum values, number of values included in the calculation of statistics, and a confidence code. This database will provide the critical link in the association of the consumption of flavonoids with health and also will provide dieticians and other health professionals with estimates of the flavonoid content of various foods, including teas.
