Evaluation of Antioxidant Activity of Tea Polyphenols by a Quantum Chemistry Calculation Method - PM6

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Figure 1. Structures of the tea catechins

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Anthocyaninsmainly distributed in shoots of tea plant and account for the bitter taste of tea. Previous studies showed that pelargonidin, cyanidin and delphinidinwere the major anthocyanins in tea ^[27]. As shown in Table 2, the HOF value order was delphinidin < cyanidin < pelargonidin, and the antioxidant potential sequence of anthocyanins determined by SRSA and TEAC assays was delphinidin>cyanidin>pelargonidin ^{[28, 29]}. These results suggested that higher antioxidant ability of anthocyanins is associated with lower HOF value. The structure difference of the three anthocyanins is the number and position of hydroxyl at B-ring. Delphinidin possesses three hydroxyls at C3′, 4′ and 5′ positions, cyanidinhas two hydroxylsat C3′ and 4′ positions, and pelargonidin possesses onehydroxyl at C4′ position in the B-ring (Figure 2). These result indicated HOF could serve as a theoretical measure for ranking anthocyanins as antioxidants.

Table 2. Antioxidant activities of various anthocyanins in different assays and their HOF values

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Figure 2. Structures of the anthocyanins

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Tea contains more than 20 kinds of flavonols, which are considered to account for tea infusion color ^[30]. Our data showed the HOF values of kaempferol, quercetin and myricetin were -179.36, -220.23 and -262.53 kcal/mol, respectively. Figure 3 showed that the three flavonols have different amount of hydroxylin the B-ring, indicating that more hydroxylin the B-ring, lower HOF value. It was reported the activity of scavenging hydroxyl (^.OH) radicals decreases in the order: myricetin > quercetin > kaempferol ^[31], which meant the negative correlation existed between HOF and antioxidant activity of flavonol.

Figure 3. Structures of oligomericproanthocyanidins in tea and their HOF values

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Flavanones are another type of flavonoids which contribute to the antioxidant activity of tea ^[32]. In order to explore the relationship between antioxidant ability and structure except for hydroxyl, EGC and dihydromyricetin were selected because they possess the same number and location of hydroxyl substitute. The HOF values of EGC and dihydromyricetin calculated in this study are -262.07 and -286.22kcal/mol, respectively (Figure 4). Furthermore, we used frontier molecular orbital theory to analyze the energy difference △E (△E = E_LUMO-E_HOMO) of the two compounds. The △E of EGC and dihydromyricetin is 12.99 and 7.16 eV, respectively, indicating dihydromyricetin is less stable and easier deprotonated than EGC ^[33]. Active electron orbital of EGC mainly focuses on B-ring, whereas that of dihydromyricetin distributes in the C4-carbonyl moiety, C2-3 bond, C3-OH group and adjacent hydroxyl substitute on the A and B-rings (Figure 5). Previous study showed that dihydromyricetin had stronger antioxidant potential than EGC ^[34]. Compared with EGC, dihydromyricetin contains a carbonyl moiety at C4, which is considered to be an essential structural requirement for flavonoid aroxyl radical stabilization ^{[35, 36]}. The chromone structure in C-ring can enhancethe dissociation of hydroxyl of C-ring, and promote hydrogen generation of the C3-alcohol hydroxide ^[12]. In addition, the altered bonding in C ring allows delocalization between the A and B rings, thus stabilize the aryloxyl radical after hydrogen donation ^{[37, 38, 39]}. All these results may explain for the lower HOF value and higher antioxidant potential of dihydromyricetin compared with EGC.

Figure 4. Structures of the major flavonols in tea

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Figure 5. Structures of EGC and dihydromyricetin and their HOF values

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In addition to the polyphenol monomers, the relevance between HOF value and antioxidant activity of polymers was studied in this work.Our data showed the HOF value sequence was quercetin-Fe complex (3:1)<quercetin-Zn complex (2:1)<quercetin, indicating the order of antioxidant activity was quercetintrimer>dimer >monomer. This prediction was consistent with the previous antioxidant data determined by SRSA assay in Table 3 ^[40].

Table 3. The inhibition rates of quercetin and quercetin-metal complexes on O₂^.-radical (Liet al., 2006)

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Proanthocyanidins (PAs) are a class of colorless phenolics characterized by an oligomeric or polymeric structure based on flavan-3-ol units. Flavan-3-ol units are connected via C4→C8, C4→C6, C2→O7 or C2→O5 linkages ^[41]. EC is considered to be one of most common constitutive units of Pas ^[27]. Our results showed the HOF value of PAs decreased with the rise ofdegree of polymerization (Figure 7). It was reported that the antioxidant activity of PAs largely depends on the degree of polymerization. Although the antioxidant activity of monomers was inferior to that of oligomers, the antioxidant ability of polymers reduced with the increased degree of polymerization. High polymerization degree may attenuate the dissociation of hydroxyl due to the existence of steric hindrance ^{[42, 43]}. These results indicated the HOF value may only applies to oligomeric proanthocyanidins.

Figure 6. Highest Occupied Molecular Orbital (HOMO) and Lower Unoccupied Molecular Orbital (LUMO) of EGC and dihydromyricetin

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Figure 7. Structures of quercetin-metal complexes and their HOF values

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Black tea is the most popularlyconsumed tea in the world, and has excellent antioxidant activity (Tuet al., 2007). During the production of black tea, catechins are enzymatically oxidized to produce polymerized polyphenols including theaflavins and thearubigins. Theaflavins possess a benzotropolone skeleton that is formed from co-oxidation of appropriate pairs of catechins ^[27]. The major theaflavins in black tea are theaflavin (TF1), theaflavin-3-gallate (TF2A), theaflavin-3-gallate (TF2B) and theaflavin-3,3-digallate (TF3). On the other hand, thearubigins are largerand less well-characterized polymers. (Figure 8) ^[45]. Table 4 showed theHOF valueorderwas TF1>TF2>TF3. TF2A and TF2B had the similar HOF. The antioxidant dataof the four theaflavins determined by TEAC, HRSA and DPPH assays suggested that lower HOF value, higher antioxidant activity ^{[46, 47, 48]}. In addition, all the HOF values of theaflavinsis lower than that of EGCG (-401.82 kcal/mol), which is the strongest antioxidant among catechins. This result was in agreement with the previous report that theaflavins possess stronger antioxidant activity than catechins ^{[49, 50]}. Theaflavins have two A-rings of flavanols linked by a fused seven-member ring. These structural features may provide more interaction sites with radicals. In addition, the benzotropolone moiety of theaflavins plays an important role in affording antioxidant protection for the preferred oxidation site, and might be responsible for electron donation because of the existence of resonanceforms ^[51].

Table 4. Antioxidant activities of various TFs in different assays and their HOF values

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Figure 8. Structures of the maintheaflavins

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Figure 9. Scatter plot of calculated HOF value versus experimental TEAC values of 11 tea polyphenols (one outlier was omitted)

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