Radical Scavenging Activity of Natural Anthraquinones: a Theoretical Insight.

Anthraquinones (ANQs) isolated from Paederia plants are known to have antidiarrheal, antitussive, anthelmintic, analgesic, anti-inflammatory, antihyperlipidemic, antihyperglycaemic, and antimicrobial activities. The antioxidant properties were also noted but not confirmed thus far. In this study, the superoxide and hydroperoxide radical scavenging activities of six ANQs were evaluated using a computational approach. The results suggest that the ANQs exhibit low HOO• antiradical activity in all environments, including the gas phase (k < 102 M-1 s-1). In contrast, the ANQs might exert excellent O2 •- radical scavenging activity, particularly in aqueous solution. The rate constants of the superoxide anion scavenging in water (at pH = 7.4) range from 3.42 × 106 to 3.70 × 108 M-1 s-1. Compared with typical antioxidants such as ascorbic acid and quercetin, the superoxide anion scavenging activity of ANQs is significantly higher. Thus, the ANQs are promising O2 •- radical scavengers in polar media.

Introduction

Reactive <span class="Chemical">oxygenpan> species (<span class="Chemical">ROS) play an important role in the normal metabolism of humans. They participate in the bacterial ingestion of phagocytes as well as the regulation of signaling pathways, and thus cellular processes related to, for example, metabolism and proliferation.[1] However, excess <span class="Chemical">ROS production is harmful to the native biochemistry of the human body, indicted as a common pathway to some incurable conditions including cardiovascular disease, cancer, and diabetes.[2−6] The adverse effects of ROS are kept in check by dietary antioxidant intake; several recent studies showed that the antioxidant constituents from natural plants are vital factors in disease prevention.[7,8]

Species of Polygonaceae, Leguminosae, Rubiaceae, and some 30 other families contain an abundance of natural antioxidants, including but are not limited to phenolic compounds.[9,10] <pan class="Chemical">span class="Chemical">Anthraquinones (<sppan>an class="Chemical">ANQs) belong to this group but mostly have been used as colorants in food, drugs, and cosmetics.[11,12] ANQ derivatives with acidic or polar, hydrophilic substitution such as aloe-emodin, emodin, rhein, chrysophanol, and physcion were described with antiangiogenic activities.[13,14] Experimental antioxidant activity data of ANQs that were collected by the 2,2-diphenyl-1-picrylhydrazyl, total reducing power ability, oxygen radical absorbance capacity ,and low-density lipoprotein methods showed that ANQs could exhibit good antioxidant activity.[11,15−17]

<span class="Species">Paederiapan> plants i.e.,<span class="Species">P. foetida and P. scandens are distributed widely in temperate and tropical East Asia and tropical South-East Asia.[18] These plants are well known not only as traditional food ingredients in daily meals but also as traditional medicinal plants to treat <span class="Disease">toothache, tumors, rheumatoid arthritis, inflammation, diarrhea, and hemorrhoids.[19,20]Paederia extracts showed antidiarrheal, antitussive, anthelmintic, analgesic, anti-inflammatory, antihyperlipidemic, antihyperglycaemic, antimicrobial, and antioxidant activity.[21−29] From these extracts, a number of ANQs were isolated and characterized including 1,3-dihydroxy-2,4-dimethoxy-9,10-anthraquinone (1), 2-hydroxy-1,4-dimethoxy-9,10-anthraquinone (2), 1-methoxy-2-methoxymethyl-3-hydroxy-9,10-anthraquinone (3), 1-hydroxy-2 hydroxymethyl-9,10-anthraquinone (4), 1-methyl-2,4-dimethoxy-3-hydroxyanthraquinone (5), and 1-methoxy-3-hydroxy-2-ethoxymethylanthraquinone (6) (Figure ).[30,31] ANQ derivatives were also described as photosensitizers to create singlet oxygen and superoxide anions in UV environments,[32−34] whereas some studies indicated that these compounds could exhibit the O2•– scavenging activity, particularly in polar environments.[35,36] Hence, it is desirable to gather further insights into the radical scavenging activity of ANQs.

Figure 1

Structures of the six ANQs in this study.

Structures of the six <span class="Chemical">ANQspan> in this study.

Previous studies confirmed that the quantum chemical calculation offers a useful and reliable pathway to evaluate the radical scavenging as well as the antioxidant activity of natural products.[8,37−42] Thus, in this study, the antioxidant activity of six <span class="Chemical">ANQspan> (Figure ) was investigated by using thermodynamic and kinetic calculations. The antioxidant properties of the <span class="Chemical">ANQs were investigated in the <span class="Chemical">HOO• and O2•– radical scavenging reactions in the gas phase as well as physiological environments.

Results and Discussion

Thermodynamic Study

Previous studies indicated that conformation plays an important role in the antioxidant activity of molecules that have multiple stable conformers.[8,37] Thus, the studied <span class="Chemical">ANQspan> were first screened to find the conformers with low electronic energy.[43] The five lowest electronic energy conformers were then analyzed to find the most stable conformers, and these forms were used in further computations. For example, for compounds 3 and 6 that contain both methoxy and ethoxy groups, the five lowest electronic energy conformers were analyzed by the M06-2X/6-311++G(d,p) method (Figure S1, Supporting Information) and the most stable forms (3 and 6, >99%) were achieved by using the Maxwell–Boltzmann distribution to estimate the relative populations of conformers.[44,45]

In the initial step of the study, the thermodynamic parameters, including bond dissociation energies (<span class="Chemical">BDEpan>s), ionization energies (IEs), and proton affinities (<span class="Chemical">PAs) were evaluated for all the studied compounds following the method reported previously.[38,40] To save computing time, the <span class="Chemical">BDE and PA values of all possible bonds were first calculated at the M06-2X/3-21G level of theory and the lowest values were then re-computed with a more accurate method (M06-2X/6-311++G(d,p) level of theory). The results are presented in Table .

Table 1

Calculated BDEs, PAs, and IEs of Studied Compounds and ΔGo (kcal/mol) for the ANQs + HOO• Reaction Following the FHT, SP, and SET Mechanisms in the Gas Phase

	FHT			SP			SET
comp.	positions	BDE	ΔG	positions	PA	ΔG	IE	ΔG
1	O3	88.7	1.6	O3	335.1	257.0	180.9	234.2
2	O2	90.6	2.1	O2	334.1	255.7	183.3	237.1
3	C2	83.8	–4.0	O3	334.4	255.8	191.7	244.9
4	C2	79.6	–7.1	O1	347.2	268.3	192.7	245.7
5	O2	88.2	1.1	O2	335.7	258.1	185.9	239.9
6	C2	82.7	–4.4	O3	338.6	260.3	189.1	242.8

As shown in Table , the lowest <span class="Chemical">BDEpan>s (X–H, X = O, C) of the <span class="Chemical">ANQs range from 79.6 to 90.6 kcal/mol. The lowest calculated <span class="Chemical">PAs are observed at (O–H) bonds with PA = 334.1–347.2 kcal/mol, which are significantly higher than the IE values of the ANQs (IE = 180.9–192.7 kcal/mol). Notably, the thermochemical parameters are generally higher than those of natural hydroanthraquinone derivatives (BDE = 76.2–88.6 kcal/mol; PA = 313.8–331.8 kcal/mol; and IE = 172.7–180.3 kcal/mol).[46] This result suggests that reducing the carbonyl group (C=O) of the ANQ system to the alcohol group (hydroanthraquinones) may decline the BDE, PA, and IE values and that the antioxidant activity of ANQs may be lower than the activities of the hydroanthraquinones.

The lowest <span class="Chemical">BDEpan> is calculated for the 4–C2–H bond (<span class="Chemical">BDE = 79.6 kcal/mol), while the lowest for PA and IE are observed at compound 2 with PA = 334.1 kcal/mol and 1 with IE = 180.9 kcal/mol. The <span class="Chemical">BDEs of ANQs are higher than those of typical natural antioxidants, such as Trolox, ascorbic acid (BDE < 78 kcal/mol);[8,47] thus, the radical scavenging activity of these ANQs following the FHT mechanism is expected to be weaker than the reference antioxidants.

As an initial assessment of the <span class="Chemical">HOOpan>• radical scavenging ability of the <span class="Chemical">ANQs, Gibbs energies (ΔGo) of the reactions between <span class="Chemical">HOO• radicals and the ANQs were calculated for each of the three typical radical scavenging mechanisms: formal hydrogen transfer (FHT),[48] sequential proton [SP, first step of SP loss electron transfer mechanism (SPLET)],[49−52] and single electron transfer [SET, first step of SET followed by the proton transfer mechanism (SETPT)];[7,53] the results are summarized in Table . The results showed that the HOO• radical scavenging was only spontaneous (ΔGo < 0) for the FHT mechanism and not for the other two mechanisms (SP and SET) in the gas phase. Thus, these mechanisms can be disregarded in the following kinetic modeling.

Kinetic Study

HOO• Radical Scavenging of ANQs in the Gas Phase

As shown in the thermodynamic section, the FHT pathway is the favored mechanism for the <span class="Chemical">HOOpan>• radical scavenging of <span class="Chemical">ANQs. Thus, in this section, the kinetic calculations were performed for the H-abstraction of the X–H (X = C, O) bonds with the lowest <span class="Chemical">BDEs (Table ). The results are shown in Table and Figure .

Table 2

Calculated ΔG⧧ (kcal/mol), κ, and kEck (M–1 s–1) for the HOO• Scavenging of the ANQs in the Gas Phase

reaction	ΔH	ΔG⧧	κ	kEck
1–O3–H + HOO•	7.7	18.0	39.5	1.63 × 101
2–O2–H + HOO•	9.1	19.1	48.5	3.13
3–C2–H + HOO•	8.8	18.2	84.8	2.23 × 101
4–C2–H + HOO•	8.4	17.9	50.6	2.47 × 101
5–O2–H + HOO•	7.0	17.4	61.3	6.63 × 101
6–C2–H + HOO•	7.2	17.1	30.8	5.24 × 101

Figure 2

Optimized geometries of TSs between the studied compounds and HOO• radical in the gas phase following the FHT mechanism.

Optimized geometries of TSs between the studied compounds and <span class="Chemical">HOOpan>• radical in the gas phase following the FHT mechanism.

It is clear from Table that the <span class="Chemical">HOOpan>• radical scavenging of <span class="Chemical">ANQs is slow with kEck = 3.13–66.3 M–1 s–1 and ΔG⧧ = 17.1–19.1 kcal/mol. This is in logical agreement with the high <span class="Chemical">BDE values obtained from the thermodynamic calculations. Previous studies showed that the HOO• antiradical activity of most model lipid media (i.e., in pentyl ethanoate solvent) mainly occurred following the FHT pathway, and the rate constant was usually lower than in the gas phase.[39,40,54] Thus, this suggests that the AQNs are not good antioxidants in the lipid media.

HOO• and O2•– Scavenging of ANQs in Aqueous Solution

Acid–Base Equilibria

As a requirement of antioxidant activity evaluation in aqueous solution,[8,37,48] the molar fractions (f) of the deprotonated state of <span class="Chemical">ANQspan> were calculated at physiological pH. The <span class="Chemical">alcohol moiety has a dissociation equilibrium as per reaction , and the pKa values are calculated according to eq (Table ).[37,40,55]

Table 3

Calculated pKa and f at pH = 7.4

			f
comp.	positions	pKa	HA	A–
1	O3–H	6.94	0.253	0.747
	O1–H	10.76a
2	O2–H	7.00	0.285	0.715
3	O3–H	7.72	0.676	0.324
4	O1–H	8.66	0.948	0.052
5	O2–H	7.53	0.574	0.426
6	O3–H	7.75	0.691	0.309

pKa.

The calculated pKa values (Table ) range from 6.94 to 10.76; hence, at pH 7.4, the neutral species are present in 25.3–94.8% and the dissociated state makes up the remaining 5.2–74.7%. Among the studied compounds, 1 could also form a dianion with pKa2 = 10.76. However, at pH 7.4, this state only populates 0.033% of the total concentration of 1; thus, the dianion pan class="Chemical">species can be omitted in the kinetic calculations.where ΔGBAo was obtained from reaction following eq and m and C0 are fitted parameters directly obtained from ref (55).[55]

Kinetic Study

The study on the <span class="Chemical">HOOpan>• radical scavenging of <span class="Chemical">ANQs in the gas phase showed that the rate constants for this process according to the FHT pathway were low. Thus, the study on radical scavenging in the polar environment only focused on the SPLET mechanism (the second step as the SET mechanism) against typical radicals, that is, <span class="Chemical">HOO• and O2•–. It was already established that the SET mechanism is the main pathway for superoxide anion scavenging in polar media to form stable diradical 3O2.[56−58] Therefore, the HOO• and O2•– scavenging of ANQs in aqueous solution could occur via reactions –7, and the overall rate constants (koverall) were calculated according to eqs and 9. The results are presented in Table .

Table 4

Calculated ΔG⧧ (in kcal/mol), Nuclear Reorganization Energy (λ), and Rate Constants (kapp, kf, M–1 s–1) of the HOO• and O2•– Scavenging of the ANQs in Aqueous Solution Following the SPLET Mechanism

		HOO•					O2•–
comp.		ΔG⧧	λ	kapp	kfa	koverall	ΔG⧧	λ	kapp	kfa	koverall
1	HA	40.8	22.7	7.90 × 10–18	2.00 × 10–18	1.57 × 101	6.0	20.6	2.60 × 108	6.68 × 107	6.59 × 107
	A–	15.6	19.5	2.10 × 101	1.57 × 101		10.5	20.2	1.30 × 105	9.71 × 104
2	HA	33.2	30.3	2.90 × 10–12	8.27 × 10–13	2.50 × 10–1	7.8	20.2	1.20 × 107	3.42 × 106	3.42 × 106
	A–	18.1	15.5	3.50 × 10–1	2.50 × 10–1		13.4	19.8	8.60 × 102	6.15 × 102
3	HA	56.1	18.9	4.70 × 10–29	3.18 × 10–29	7.78 × 10–3	7.1	20.0	4.20 × 107	2.84 × 107	2.84 × 107
	A–	19.7	14.9	2.40 × 10–2	7.78 × 10–3		11.7	19.0	1.70 × 104	5.51 × 103
4	HA	53.4	17.9	4.50 × 10–27	4.27 × 10–27	4.63 × 10–1	5.7	20.5	3.90 × 108	3.70 × 108	3.70 × 108
	A–	16.2	15.4	8.90	4.63 × 10–1		13	18.7	1.90 × 103	9.88 × 101
5	HA	42.5	22.7	4.00 × 10–19	2.30 × 10–19	8.95 × 101	8.3	21.1	5.10 × 106	2.93 × 106	2.93 × 106
	A–	14.3	16.4	2.10 × 102	8.95 × 101		13.6	20.4	6.50 × 102	2.77 × 102
6	HA	49.3	20.9	4.30 × 10–24	2.97 × 10–24	5.56 × 10–3	7.1	20.7	3.70 × 107	2.56 × 107	2.56 × 107
	A–	19.8	14.6	1.80 × 10–2	5.56 × 10–3		11.5	19.3	2.30 × 104	7.11 × 103

kf = f·kapp.

As shown in Table , the neutral states (HA) have virtually no contribution in the <span class="Chemical">HOO• radical scavenging of <span class="Chemical">ANQs in aqueous solution (ΔG⧧ = 33.2–56.1 kcal/mol and kapp = 4.70 × 10–29 to 2.90 × 10–12 M–1 s–1). The <span class="Chemical">HOO• scavenging activity of the anions (A–) is somewhat higher; however, kapp and kf values range from 1.80 × 10–2 to 2.10 × 102 and 5.56 × 10–3 to 8.95 × 101 M–1 s–1, respectively. The highest overall rate constant for the HOO• antiradical activity is koverall = 89.1 M–1 s–1 that is much lower than that of typical natural antioxidants, that is, Trolox[8] and ascorbic acid.[37] As anticipated based on the energetic analysis above, ANQs exhibit significantly lower (∼105 to 106 times) HOO• radical scavenging activity than natural hydroanthraquinone derivatives,[46] particularly in the polar media, emphasizing the importance of the alcohol moieties. Consistently, the studied ANQs are poor HOO• radical scavengers in physiological environments.

However, <span class="Chemical">ANQpan> compounds exhibit excellent O2•– radical scavenging activity, particularly in polar environments. The anion states have moderate O2•– antiradical activity with kapp = 6.50 × 102 to 1.30 × 105 M–1 s–1, whereas the activities of the HA states fall range from 5.10 × 106 to 3.90 × 108 M–1 s–1. As a result, the koverall range from 3.42 × 106 to 3.70 × 108 M–1 s–1. That is in good agreement with the previous findings in <span class="Chemical">carotenoids.[59] Compound 4 exhibits the highest O2•– radical scavenging activity with koverall = 3.70 × 108 M–1 s–1. This is about 100 times higher than that of compound 5 (the lowest O2•– radical scavenger). Compared with typical antioxidants such as <span class="Chemical">ascorbic acid and quercetin (k = 0.1 × 103 to 3.0 × 103 M–1 s–1),[60] the superoxide anion scavenging of ANQs is much higher. Thus, ANQs are promising O2•– radical scavengers in polar environments.

Conclusions

In this study, the <span class="Chemical">superoxidepan> and <span class="Chemical">HOO• radical scavenging activity of six <span class="Chemical">ANQs was evaluated. It was found that the ANQs have very low HOO• antiradical activity in both gas phase and physiological environments. The rate constants of the HOO• radical scavenging of ANQs are less than 102 M–1 s–1, implying that they are not useful HOO• radical scavengers. However, ANQs were found to be excellent O2•– radical scavengers, particularly in aqueous solution. The rate constants of the superoxide anion scavenging of ANQs range from 3.42 × 106 to 3.70 × 108 M–1 s–1, which is much higher than that of typical antioxidants, such as ascorbic acid and quercetin. Thus, the ANQs are promising O2•– radical scavengers in polar environments.

Computational Methods

Thermochemical properties (<span class="Chemical">BDEpan>s, IEs, and <span class="Chemical">PAs) as well as kinetic parameters, activation energies ΔG⧧ (kcal/mol), tunneling corrections (κ), and rate constants (k) in the studied environments (the gas phase and in physiological environments) were computed using the M06-2X functional.[48,61−64] M06-2X/6-311++G(d,p) has been well established as a suitable method to compute both thermochemical and kinetic parameters due to its good accuracy compared with the more complex functionals [i.e., G3(MP2)-RAD] and experimental data.[8,48,61,64] The quantum mechanic-based test for the overall free radical scavenging activity protocol[8,37,61,65−67] was used to perform the kinetic calculations. The rate constant (k) was calculated by using the conventional transition-state theory and 1M standard state at 298.15 K.[40,54,62,67−72] The details of the method are shown in Table S3, Supporting Information.[54] All DFT calculations in this work were carried out using Gaussian 16 package.[73]