Recent Advances in Polyphenol Research. Группа авторов
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href="#ulink_b4a1d76f-f67a-5aea-9034-0fe437279d71">eq. (31) with eq. (34), and eq. (32) with eq. (35) the following relations are obtained:
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1.3.2 Reverse pH Jumps from Equilibrium
From eq. (36) to eq. (38) all equilibrium constants except those regarding the trans‐chalcones can be obtained. Moreover, the cis‐trans isomerization constants can be calculated from a reverse pH jump from the equilibrated solutions. Considering that formation of the flavylium cation from the trans‐chalcones is very slow, this kinetics should be followed by a standard spectrophotometer. The quinoidal bases, hemiketals, and cis‐chalcones are transformed to flavylium cation much faster than the trans‐chalcones and appear as an initial absorption.2 From this point all the equilibrium constants have been calculated. The mole fraction of trans‐chalcone is thus obtained from the ratio of the absorbance of the trace amplitude/total absorbance. The mole fractions of the other species at equilibrium are obtained from those at pseudo‐equilibrium, calculating the respective proportion.3 For example, if at pseudo‐equilibrium A=0.3, B=0.2, and Cc=0.5 and the mole fraction of Ct at equilibrium is 0.5, the mole fractions of A, B, and Cc at equilibrium are the following: A=0.15, B=0.1, and Cc=0.25.
1.4 The Kinetic Processes
Scheme 1.5 represents the four kinetic processes of anthocyanins and related compounds in acidic medium. It is worth noting that, like in the case of the formation of the quinoidal base from flavylium cation, all the other anionic species are formed as in Scheme 1.3, from proton transfer. This reaction represents step 1 in the kinetic process and takes place in microseconds during the mixing time of the stopped flow. Only using special techniques such as temperature jumps (Brouillard and Dubois 1977), and in some favourable cases flash photolysis, are these constants obtained.4 This fact makes the kinetics reported in Scheme 1.5 the only relevant ones upon direct pH jumps, since the formation of the anionic species is immediate when compared with hydration, tautomerization, and isomerization. Moreover, the first process after a direct pH jump (from flavylium cation) is the formation of the quinoidal base, which equilibrates with the flavylium cation. In the subsequent kinetic steps these two species behave as a single one.
Scheme 1.5 Energy level diagram of the relative thermodynamic level of the five species of pelargonidin‐3‐glucoside appearing in acid medium. The three distinct kinetic steps taking place in very different time scales are observed, allowing for separation of the kinetics into three kinetic equations.
The following kinetic step is the hydration followed by tautomerization (Scheme 1.5). Except in very acidic solutions (not accessed by direct pH jumps), the tautomerization reaction is faster than hydration and by consequence this last one is the rate‐determining step of this kinetic process. This kinetic step can thus be considered as in eq. (39).
During the hydration both AH+/A and B/Cc can be considered as a single species.
where XAH+ is the mole fraction of AH+ in its equilibrium with A, and XB is the mole fraction of A in its equilibrium with Cc.
In eq. (39) the forward reaction takes place only from the reaction of AH+ to form B, because, as mentioned above, the quinoidal base A does not hydrate in acidic medium (Brouillard and Dubois 1977).
For anthocyanins and many of the flavylium derivatives, the last step is controlled by the isomerization of chalcones, which is by far the slowest process of the kinetics. A similar reasoning used for step 2 can be made for step 3. In this case all species except Ct can be considered equilibrated.
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1.4.1 Heavenly Blue Anthocyanin
The experimental procedure above reported was used to rationalize the multistate of heavenly blue anthocyanin and two derivatives (Scheme 1.6).
Heavenly blue anthocyanin, HBA1, has attracted the attention of the scientific community due to its peculiar properties, specifically the fact that the same anthocyanin is used by the plant to confer purplish color to the buds and blue color to the petals (Yoshida et al. 1995; Goto and Kondo 1991; Kondo et al. 1992). Moreover, in vitro the blue color is persistent in neutral and moderately basic solutions (Kondo et al. 1992; Yoshida et al. 2009). Structural information regarding HBA1 fully supports the intramolecular stacking shown in Scheme 1.7.
The system was studied up to the mono‐anionic forms because at higher pH values a slow decomposition takes place and the data does not have sufficient accuracy. In spite of equilibrium being reached in one to two weeks, the neutral and mono‐anionic species are relatively stable. Table 1.1 summarizes the data.
Scheme 1.6 Heavenly blue anthocyanin HBA1 and their derivatives bis‐deacyl‐HBA2 and tris‐deacyl‐HBA3.
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