Structure-antioxidant Activity Relationship Research Articles

Structure-antioxidant activity relationship of methoxy, phenolic hydroxyl, and carboxylic acid groups of phenolic acids

The antioxidant activities of 18 typical phenolic acids were investigated using 2, 2′-diphenyl-1-picrylhydrazyl (DPPH) and ferric ion reducing antioxidant power (FRAP) assays. Five thermodynamic parameters involving hydrogen atom transfer (HAT), single-electron transfer followed by proton transfer (SET-PT), and sequential proton-loss electron transfer (SPLET) mechanisms were calculated using density functional theory with the B3LYP/UB3LYP functional and 6–311++G (d, p) basis set and compared in the phenolic acids. Based on the same substituents on the benzene ring, -CH2COOH and -CH = CHCOOH can enhance the antioxidant activities of phenolic acids, compared with -COOH. Methoxyl (-OCH3) and phenolic hydroxyl (-OH) groups can also promote the antioxidant activities of phenolic acids. These results relate to the O-H bond dissociation enthalpy of the phenolic hydroxyl group in phenolic acids and the values of proton affinity and electron transfer enthalpy (ETE) involved in the electron donation ability of functional groups. In addition, we speculated that HAT, SET-PT, and SPLET mechanisms may occur in the DPPH reaction system. Whereas SPLET was the main reaction mechanism in the FRAP system, because, except for 4-hydroxyphenyl acid, the ETE values of the phenolic acids in water were consistent with the experimental results.

Structure-Antioxidant Activity Relationships of Luteolin and Catechin.

Antioxidant activity of the natural flavonoids luteolin and catechin, which shows inconsistent comparative results in literature, was extensively evaluated as affected by the 1,4-pyrone moiety and 3-OH group. Catechin and luteolin were compared to the synthetic antioxidant-butylated hydroxytoluene for their 2,2-diphenyl-1-picrylhydrazyl (DPPH● ) scavenging activity in polar protic and aprotic solvents and ferric reducing-antioxidant power (FRAP). Moreover, their effect on lipid oxidation kinetics of canola and olive oil triacylglycerols as well as their oil-in-water (O/W) emulsions, in addition to oil stability indices (OSIs), was evaluated. In the DPPH● assay, catechin's 3-OH group led to lower IC50 values than luteolin's 1,4-pyrone moiety in acetone (3.4µM compared with 9.4µM), while there was no significant difference in methanol (IC50 = approximately 18.3µM) or tetrahydrofuran (IC50 = approximately 27.2µM). The FRAP test indicated a higher reducing power for catechin than for luteolin (689.4µM compared with 573.1µM). The antioxidants showed various performances in the oil triacylglycerols and their O/W emulsions due predominantly to the interfacial phenomena. A better OSI was found for catechin than for luteolin (20.0 to 52.7 hr compared with 3.5 to 4.2 hr) in Rancimat test.

Protection of natural antioxidants against low-density lipoprotein oxidation.

This chapter reports essential information about the protective action of antioxidants against LDL oxidation. The activity of individual compounds (tocopherols, vitamin C, phenolic compounds) as well as extracts obtained from plant material (cereals, fruits, legumes, nuts, mushrooms, by-products of food industry) is reported. The structure-antioxidant activity relationship of phenolic compounds is discussed. This article summarizes the findings to date of both in vitro and in vivo studies using foods or phenolic extracts isolated from foodstuffs at inhibiting the incidence of LDL oxidation. This chapter summarizes also the reportings to date of in vivo studies using foods or beverages at inhibiting the incidence of LDL oxidation.

Structure-antioxidant activity relationship of ferulic acid derivatives: Effect of ester groups at the end of the carbon side chain

Differences in the antioxidant activity of ferulic acid and its nine naturally occurring derivatives with the presence of different ester groups at the end of their carbon side chains were investigated using the density functional theory (DFT) method. The thermodynamics parameters related to the double H+/e− free radical scavenging mechanisms were calculated in the gas, benzene and water phases. In the investigated phases, ferulic acid with the COOH group or COO− group and its different ester derivatives show similar antioxidant capacities. They preferentially undergo the first H+/e− reaction from the 4−OH group and then proceed to the second H+/e− reaction from the 3−H group, forming a benzodioxole. The energy cost of the second H+/e− reaction is considerably reduced. In the gas and benzene phases, ferulic acid and its derivatives have the highest probability of using the HAT mechanism in the first H+/e‒ reaction. In water, the most possible mechanism is SPLET. In the studied phases, HAT is the most likely second H+/e‒ reaction mechanism for ferulic acid and its ester derivatives trap free radicals.

Structure-antioxidant activity relationships, QSAR, DFT calculation, and mechanisms of flavones and flavonols

DFT calculations were performed on nine flavones and flavonols to explain their high antioxidant activity and variations in their activity. Conformational analysis showed that only flavonols with 3-OH directed toward the B-ring are nonplanar; however, flavones and resulted radicals are planar. Hydroxyl group eligible for dissociation is the one with ortho OH directed toward it; otherwise, one of B-ring (in 2′ or 4′-position) hydroxyl group. There are two main factors responsible for stabilizing the resulted radicals and lowering the bond dissociation energy and hence there were found well correlated with the experimental activity. First, driving force resulted from the conversion of nonplanar flavonols to planar radicals accompanied by resonance toward the carbonyl group and H-bond formation with 3-OH and second, radical stabilization by H-bond with ortho hydroxyl group with resonance toward carbonyl or pyrone oxygen. All resonance and H-bonds were confirmed by spin density, bond length, and molecular orbital calculations.

Chemical Structure-Antioxidant Activity Relationship of Water-Based Enzymatic Polymerized Rutin and Its Wound Healing Potential.

The flavonoid rutin (RU) is a known antioxidant substance of plant origin. Its potential application in pharmaceutical and cosmetic fields is, however, limited, due to its low water solubility. This limitation can be overcome by polymerization of the phenolic RU into polyrutin (PR). In this work, an enzymatic polymerization of RU was performed in water, without the addition of organic solvents. Further, the chemical structure of PR was investigated using 1H NMR, and FTIR spectroscopy. Size-exclusion chromatography (SEC) was used to determine the molecular weight of PR, while its acid/base character was studied by potentiometric charge titrations. Additionally, this work investigated the antioxidant and free radical scavenging potential of PR with respect to its chemical structure, based on its ability to (i) scavenge non biological stable free radicals (ABTS), (ii) scavenge biologically important oxidants, such as O2•, NO•, and OH•, and (iii) chelate Fe2+. The influence of PR on fibroblast and HaCaT cell viability was evaluated to confirm the applicability of water soluble PR for wound healing application.

Relationships Between Chemical Structure and Antioxidant Activity of Isolated Phytocompounds from Lemon Verbena.

Over the last few years, people have been concerned about the narrow relationship between nutrition and health leading to an increasing demand of nutraceutical products and functional food. Lemon verbena (Lippia citriodora Kunth) has been traditionally used for respiratory, digestive, and muscular diseases, showing effects that are promoted by the antioxidant activity of its phytoconstituents. The antioxidant power of several lemon verbena extracts has been tested but its isolated compounds activity has not been described. The aim of the present work was to isolate phytochemicals from a commercial lemon verbena extract through a semi-preparative high-performance liquid chromatography approach for further evaluation of its individual antioxidant activity using three different methods. The structure-antioxidant activity relationships revealed the influence of substitutions in the strong antioxidant power exerted by glycosylated phenylpropanoids, in contrast to the low antioxidant capacity showed by iridoids. Development of enriched extracts in these compounds could lead to greater antioxidant effects and improved functional ingredients to prevent chronic diseases.

Structure-antioxidant activity relationship of active oxygen catalytic lignin and lignin-carbohydrate complex

Lignin, as a natural antioxidant, has a great potential to replace the chemosynthetic ones due to this material benefits of being biodegradable, eco-friendly, abundant and low-cost. However, the commonly-used lignins, such as acid, kraft and alkali lignins, have the poor antioxidant ability, and their antioxidative mechanism and kinetics remain poorly understood. Moreover, the understanding of the effect of polysaccharides in lignin-carbohydrate complex (LCC) on lignin antioxidant activity is also extremely insufficient. Herein, we isolated lignin and LCC from rice straw and its alkali-oxygen spent liquor to investigate their structure-antioxidant activity relationship and antioxidative mechanism. Experimental results illustrated that the alkali-oxygen treatment can significantly enhance the antioxidant activity of lignin. Demethoxylation, ring-opening and the cleavage of aryl ether bonds occur to lignin structure leading to the decrease of molecular weight, while the more stable condensed β-1′, 5–5′ and β-5′ linkages maintain the thermostability of this antioxidant. The synergistic effect of adjacent methoxyl with phenolic hydroxyl is the key factor that endows lignin with the outstanding antioxidant activity. In contrast, oligosaccharides formed by alkali-oxygen treatment have a negative influence on the antioxidant activity of lignin. This work demonstrates that alkali-oxygen lignin is a promising antioxidant to replace chemosynthetic ones for polymeric materials.

The antioxidant activity and active sites of delphinidin and petunidin measured by DFT, in vitro chemical-based and cell-based assays.

A computational DFT B3LYP method with 6-311G (d,p) basis set, the in vitro chemical-based and cellular antioxidant activity (CAA) assays were applied in this study to explain the structure-antioxidant activity relationships of delphinidin and petunidin. The compound molecular structures, spectral properties, frontier orbital energy, and transition state of delphinidin and petunidin were compared. In transition state, the result of the active site (O21-H32 and O22-H33) was consistent with the result of bond length. The frontier orbital theory results indicated that the probable antioxidant activity order was petunidin (0.09126 a.u.) > delphinidin (0.09175 a.u.), which agreed well with the cell-based antioxidant activity determined by CAA. However, the order of ABTS•+ and DPPH radical scavenging activity was delphinidin>petunidin. Our study could help to provide a rational approach for the investigation of antioxidant activity of phytochemicals. PRACTICAL APPLICATIONS: As anthocyanins, delphinidin and petunidin with great antioxidant activity are widely found in various fruits and vegetables. However, there are many kinds of methods used to measure their antioxidant activity and the antioxidant mechanism which are not concrete and clear. Therefore, it is crucial to study the antioxidant actvity of anthocyanins utilizing the DFT method combined with in vitro chemical-based and cell-based assays. Our study could contribute not only to the elucidation of chemical mechanism of antioxidants and exploration the structural features in essence, but also to promote the further development of phytochemicals in the field of food chemistry and pharmacy.

Pengiraan Teori Fungsi Ketumpatan (DFT) bagi Struktur-Aktiviti Anti-oksida untuk Beberapa Sebatian Fenolik dan Flavonoid yang didapati dalam Madu di Malaysia

Phenolic acids and flavonoids exist naturally in Malaysian honey and contribute significantly to antioxidant contents. Antioxidants plays an important role in scavenging free radicals and prevent health deterioration. Total antioxidant content is measured using DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity. The phenolic acids such as gallic, caffeic, syringic and hydroxybenzoic acids and flavonoids like naringenin, apigenin, kaempferol, catechin and luteolin previously have been identified in Malaysian honey of tualang, gelam and borneo type using high-performance liquid chromatography (HPLC). In order to investigate the structure-antioxidant activity relationships of these phenolic compounds using hydrogen atom transfer (HAT) mechanism, density functional theory (DFT) calculation at B3LYP/6-311++G(d,p) levels of theory was performed. In this work, optimization of the compounds chemical structure and radical forms in gas-phase has been calculated with computation of bond dissociation enthalpy (BDE) as antioxidant descriptor. The finding showed that abstraction of H at different OH groups in the structure of the compound, led to a different scavenging free radical activity thus contribute to the overall variation in the antioxidant properties. Besides that, B ring of flavonoids and unsaturated bond in pyran ring are proposed factors that could lower the BDE values and consequently influence the antioxidant properties of the antioxidant compounds. Hence, DFT calculation with BDE descriptor had been successfully applied to investigate the relationship between structure of phenolic acids and antioxidant activity of Malaysian honey and the interesting results could contribute in future development of antioxidant compound.

Structure–antioxidant activity relationship of β-cyclodextrin inclusion complexes with olive tyrosol, hydroxytyrosol and oleuropein: Deep insights from X-ray analysis, DFT calculation and DPPH assay

Olives and olive oil, a key food type of the Mediterranean diets, are packed with various important polyphenols including oleuropein (OLE), hydroxytyrosol (HTY) and tyrosol (TYR). OLE and HTY are highly powerful antioxidants and play a prime role in the therapeutics of free radical-related diseases. Their molecular stabilities and antioxidant properties can be improved by cyclodextrin (CD) encapsulation. Here, we present a systematic investigation on the inclusion complexes of β-CD–TYR (1), β-CD–HTY (2) and β-CD–OLE (3) by combined single-crystal structure determination, DFT complete-geometry optimization and DPPH antioxidant assay. X-ray analysis and DFT calculation reveal the preference of inclusion geometry with deep protrusion of the aromatic ring moieties of TYR, HTY and OLE from the β-CD O6–H-side, and the common host-guest stabilization scheme via intermolecular O–H⋯O hydrogen bonding interactions. No polyphenol OH group is shielded in the β-CD cavity, in contrast to the structures of β-CD–tea catechins complexes. The established host-guest O–H⋯O hydrogen bonds help to elevate antioxidant capacities of the olive polyphenols upon β-CD encapsulation. The order of antioxidant activity 2 >3 ≫ 1 based on the DPPH measurement is in fair agreement with their relative thermodynamic stabilities derived from DFT calculation.

The structure-antioxidant activity relationship of dehydrodiferulates

In this study, 11 dehydrodiferulic acids (DFAs) and 8 diethyl dehydrodiferulates (DEFs) were synthesized and evaluated by Trolox equivalent antioxidant capacity (TEAC) and 2,2′-diphenyl-1-picrylhydrazyl (DPPH) assays for their antioxidant properties to understand the Structure-Antioxidant Activity Relationship (SAR) of these dehydrodiferulates. In both assays, the order of antioxidant activity for all tested ferulic acid dimers were consistent except for 3-(4-Hydroxy-3-methoxy-benzylidene)-5-(4-hydroxy-3-methoxy-phenyl)-3H-furan-2-one (2, 8-8-lactone DC DFA, not occurred naturally) being the best antioxidant by TEAC test. The order of antioxidant activity of diferulic acid ethyl esters, evaluated by both assays, was not consistent; however, TEAC and DPPH assays provided consistent results for certain set of ethyl diferulates. In this study most of dimeric ferulates, with three exceptions, showed higher radical-scavenging efficacy than the monomers. Comparing the antioxidant activities of the tested diferulates suggested that the phenolic hydroxyl group, electron donating methoxyl group, and stable conjugated transient structures dictate the antioxidant activity of diferulates.

Studies of the structure-antioxidant activity relationships and antioxidant activity mechanism of iridoid valepotriates and their degradation products.

Oxidative stress has been associated with diverse diseases, including obesity, cancer and neurodegeneration. In fact, Valeriana jatamansi Jones (valerian) and its extracts possess strong antioxidant activities that extend their application in clinical practice to the treatment of these illnesses, even though the underlying mechanisms are not well understood. Iridoid valepotriate, a characteristic iridoid ester in valerian with poor chemical stability, possesses considerable antioxidant components. The original compounds and their degradation products have been found to exhibit strong antioxidant activities. However, the relationship between their structure and antioxidant effects and the mechanism underlying their oxidation resistance remain unclear. A forced degradation study using three iridoid valepotriates (valtrate, acevaltrate and 1-β acevaltrate) was performed in this work, and the structures of their degradation products were estimated by TLC-MS and LC-MS. Comparison of the antioxidant activities of the iridoid valepotriates before and after forced degradation revealed that degradation reduced the activities of the iridoid valepotriates in free radical scavenging and cytotoxic and cell apoptosis tests. The results suggested that the oxirane nucleus is important for defining the antioxidant profile of iridoid valepotriate. We uncovered possible mechanisms that could explain the antioxidant activities, including the generation of two hydroxyl groups through intramolecular transfer of an H• from an oxirane ring and a reduction in ROS levels through interactions with GABAergic signalling pathways.

Phenolic compounds reduce formation of Nε-(carboxymethyl)lysine and pyrazines formed by Maillard reactions in a model bread system

This study had the objective of determining the antiglycation activity of phenolic compounds (PCs) ((+)-catechin, quercetin, gallic, ferulic, and caffeic acids) added to a model bread with regards to the inhibition of Nε-(carboxymethyl)lysine (CML) formation. PCs were found to significantly reduce CML (31.77%−87.56%), even at the lowest concentration, with the exception of ferulic acid (FA). The strongest inhibitory effect of FA (∼62%) appeared when concentration was increased to 1.0g/100g of flour. The available lysine losses (0.00%−90.51%) showed a significant correlation (0.853–0.990) with effectiveness of CML inhibition, except in the case of samples with FA. (+)-Catechin reduced CML levels the most, probably due to its structure–antioxidant activity relationship, its thermal stability (∼51% loss), and its reactivity with ε-lysine side chains (∼40.77% loss). Although the bread supplemented with PCs contained low levels of CML, this process may adversely affect bread flavor, reducing the formation of pyrazines (1.10%–80.77%).

Synthesis and biological evaluation of hydroxylcoumarin derivatives as antioxidant agents

In this study, esculetin(1) was chosen as a lead compound and some structural modifications were designed to explore the antioxidant activities of esculetin derivatives. Meanwhile, a convenient method for selective methylation of catechol coumarins with different bases was developed. Furthermore, a few 5,7-dihydroxylcoumarins were synthesized and 7-hydroxylcoumarins were employed in order to explore the potential structure-antioxidant activity relationships. The antioxidant activities of these compounds were evaluated and compared with standard antioxidant Trolox by the 2,2′-diphenyl-1-picrylhydrazyl(DPPH) assay, 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) cation(ABTS+) assay and ferric reducing antioxidant power(FRAP) assay. The results show that the catechol group is the key pharmacophore. Meanwhile, introducing electronegative groups at the C4 position of esculetin(1) may enhance the antioxidative capacity, while introducing a group containing nitrogen as a hydrogen bond acceptor at the C8 position may slightly reduce the antioxidative capacity. Among them, the most powerful antioxidants are com-pounds 5 and 7, which exhibit higher antioxidant activity than esculetin(1) in all assays.

Metabolomic tool to identify soybean [Glycine max (L.) Merrill] germplasms with a high level of shade tolerance at the seedling stage

The isoflavone profiles of seeds of various soybean genotypes with different levels of shade tolerance at the seedling stage were investigated. High-performance liquid chromatography (HPLC) was used to quantify 12 isoflavones, and the data were analyzed using a multivariate statistical analysis. Combined with field experimental data and an orthogonal partial least-squares discriminant analysis (OPLS-DA), several aglycones (genistein (GE), daidzein (DE), and glycitein (GLE)) were selected and identified as key compounds involved in the shade tolerance of soybean seedlings. Additional correlation analysis and laboratory shading stress experiments with soybean seedlings also confirmed the function of these selected isoflavones, especially GE, in the discrimination of soybean seedlings with different levels of shade tolerance. Furthermore, the structure–antioxidant activity relationships between a range of isoflavones and the plant shade-tolerance mechanism are discussed. Targeted metabolomic analyses of isoflavones could reveal the diversity of shade tolerance in soybean seedlings, thus contributing to the breeding of excellent soybean varieties.

Antioxidant capacity and identification of the constituents of ethyl acetate fraction from Rhus verniciflua Stokes by HPLC-MS

Ethyl acetate fraction (EAF) from Rhus verniciflua Stokes is an important source of bioactive compounds. The aim of this study was the tentative identification and quantification of phenolic compounds, comparison of the phenolic structure–antioxidant activity relationships. Twelve compounds of EAF belonging to polyphenol types were detected by high performance liquid chromatography and analysed on line with negative ion electrospray ionisation tandem mass spectrometry, which were ethoxy 3-hydroxy benzoic acid, gallic acid (GA), 3,4-dihydroxy amygdalic acid, gallic acid cetyl ester, protocatechuic acid (PA), fustin, ethyl gallate (EG), garbanzol, fisetin, sulfuretin, butin and 3,7-dihydroxyflavanone-4′-rhamnoside. The antioxidant activity were evaluated based on the different types of radical scavenging capacities, i.e. DPPH·, ABTS·+ and OH. The antioxidant capacity of EAF mainly depended on the GA, EG, PA, fisetin, sulfuretin and butin. The phenolics exhibited a dose-dependent behaviour and high antioxidant ability.

Isolation, Structure Elucidation of Ferulic and Coumaric acids from Fortunella japonica Swingle leaves and their Structure Antioxidant activity relationship

Objective: The present study deals with isolation, structure elucidation of ferulic and coumaric acids from Fortunella japonica Swingle leaves and their structure antioxidant activity relationship. Structural analysis was conducted using UV, IR, mass and Nuclear Magentic Resonance (NMR) spectroscopy. Methodology: In particular, completely assigned 1H and 13C-NMR data are presented. Also, the present study was extended to investigate the hepatoprotective activity of isolated compounds against paracetamol-induced toxicity in freshly isolated rat hepatocytes model. Freshly isolated rat hepatocytes were exposed to paracetamol (5 mM) along with/without various concentrations of the isolated compounds (40–80μg/ml). Results and Discussion: Exposure of isolated hepatocytes to paracetmol resulted in lipid hydroperoxides formation, depletion in protein thiols, superoxide dismutase (SOD), succinate dehydrogenase (SDH), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) levels as well as elevation of alanine transaminase (ALT), aspartate transaminase (AST), lactate dehydrogenase (LDH) and alkaline phosphatase (ALP). The isolated coumaric and ferulic acids have been found to efficiently inhibit paracetamol-induced biochemical alterations, namely oxidative stress biomarkers and protein oxidation. It also significantly prevented paracetamol-induced loss in the activity of antioxidant enzyme and the important endogenous antioxidant glutathione. Conclusion: The study suggests that coumaric and ferulic acids can act as an antioxidant and hepatoprotective in physiological systems.

Z-Isomers of (4α→6″, 2α→O→1″)-phenylflavan substituted with R′=R=OH. Conformational properties, electronic structure and aqueous solvent effects

Procyanidins are highly hydroxylated polymers known as antioxidant compounds, thereby exhibiting beneficial effects. These compounds are protective agents against oxidative stress and the damage induced by free radicals in membranes and nucleic acids. This paper describes a study of the conformational space of (4α→6″, 2α→O→1″)-phenylflavan substituted with R'=R=OH as part of a larger study of similar structures with different substitutions. The relationships between aqueous solution-vacuum variations of some properties were studied, as well as the stabilization and reactivity of (4α→6″, 2α→O→1″)-phenylflavan substituted with R'=R=H, R'=H, R=OH, R'=R=OH, and (+)-catechin. The variations in geometric parameters and electronic properties due to conformational changes, as well as the effects of substituents and polar solvents, were evaluated and analyzed. Bader's theory of atoms in molecules was applied to characterize intramolecular interactions, along with a natural bond orbital analysis for each conformer described. The molecular electrostatic potential was rationalized by charge delocalization mechanisms and interatomic intramolecular interactions, relating them to the structural changes and topological properties of the electron charge density. Molecular polarizability and permanent electric dipole moment values were estimated. The results show the importance of a knowledge of the conformational space, and values for each conformer. Based on our previous results, we showed the existence of electron charge delocalization mechanisms acting cooperatively as "delocalization routes", showing interactions between different rings not even sharing the same plane. These "delocalization routes" were more effective for (4α→6″, 2α→O→1″)-phenylflavan substituted with R'=R=OH than for (+)-catechin, and are proposed as adding insight into the structure-antioxidant activity relationship of flavans.

Enhancement of antioxidant activity of green tea epicatechins in β-cyclodextrin cavity: Single-crystal X-ray analysis, DFT calculation and DPPH assay

Green tea catechins are potent antioxidant for prevention of various free radical-related diseases. Their antioxidant properties can be improved by encapsulation in cyclodextrins (CDs). Four inclusion complexes of β-CD with (–)-epicatechin (EC), (–)-epigallocatechin (EGC), (–)-epicatechin gallate (ECG) and (–)-epigallocatechin gallate (EGCG) have been investigated using single-crystal X-ray diffraction analysis combined with full geometry optimization by DFT/B3LYP calculation and the DPPH assay, aiming to deepen the understanding on their structure–antioxidant activity relationship. Scrutinizing the inclusion structures and conformational changes of the four encapsulated epicatechins reveals the common host–guest stabilization scheme and the epicatechin conformational flexibility facilitating the enhancement of activity. Thermodynamic stability order derived from DFT calculation in vacuum fairly agrees with the order of improved antioxidant capacity deduced from the DPPH assay, β-CD–EGCG>β-CD–ECG>β-CD–EGC≈β-CD–EC.