Comparison of antioxidant abilities of magnolol and honokiol to scavenge radicals and to protect DNA

Biochimie 93 (2011) 1755e1760

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Research paper

Comparison of antioxidant abilities of magnolol and honokiol to scavenge radicals and to protect DNA Chao Zhao, Zai-Qun Liu* Department of Organic Chemistry, College of Chemistry, Jilin University, Changchun 130021, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 23 February 2011 Accepted 10 June 2011 Available online 23 June 2011

The antioxidant properties of magnolol and honokiol were evaluated in the experimental systems of reducing ONOO
and 1O2, bleaching b-carotene in linoleic acid (LH) emulsion, and trapping 2,20 -azinobis(3-ethylbenzothiazoline-6-sulfonate) cationic radical (ABTSþ) and 2,20 -diphenyl-1-picrylhydrazyl radical (DPPH), and then were applied to inhibit the oxidation of DNA induced by Cu2þ/glutathione (GSH) and 2,20 -azobis(2-amidinopropane hydrochloride) (AAPH). Magnolol and honokiol were active to reduce ONOO
and 1O2. Honokiol showed a little higher activity to protect LH and to inhibit Cu2þ/GSH-induced oxidation of DNA than magnolol. In addition, honokiol exhibited higher activities to trap ABTSþ and DPPH than magnolol. In particular, honokiol trapped 2.5 radicals while magnolol only trapped 1.8 radicals in protecting DNA against AAPH-induced oxidation. The obtained results suggested that low antioxidant ability of magnolol may be related to the intramolecular hydrogen bond formed between di-ortho-hydroxyl groups, which hindered the hydrogen atom in hydroxyl group to be abstracted by radicals. Therefore, the antioxidant capacity of magnolol was lower than that of honokiol. Ó 2011 Elsevier Masson SAS. All rights reserved.

Keywords: Magnolol Honokiol Antioxidant Free radical Oxidation of DNA

1. Introduction Honokiol and magnolol are major components in Magnolia officinalis reed that has been used in traditional Chinese medicine for thousands of years [1]. The pharmacological mechanism of the Magnolia family has also been summarized based on a large amount of therapeutic applications [2]. In addition, honokiol and magnolol possess some special abilities to inhibit endotoxin shock [3], protein kinase, NF-kB pathway [4], NADPH oxidase [5], bacteria in mouth [6], and tumor promotion [7], and to activate retinoid receptor [8], detoxifying enzymes, glutathione-S-transferase, and antioxidative enzymes [9]. Honokiol and magnolol have a biphenyl structure containing two allyl groups, which are beneficial for increasing the affinity of honokiol and magnolol toward endothelial cells. For example, magnolol shows high activity toward eicosanoid metabolism in neutrophils [10], and other allyl-substituted biphenyl compounds exert cytotoxic activity against K562, HeLa, and A549 cancer cell lines as well [11]. Honokiol and magnolol act as antioxidants to suppress the oxidation of low-density lipoprotein [12] and glucose-induced human endothelial cell apoptosis [13]

* Corresponding author. Department of Organic Chemistry, College of Chemistry, Jilin University, No. 2519 Jiefang Road, Changchun 130021, China. Tel.: þ86 431 88499174. E-mail address: [email protected] (Z.-Q. Liu). 0300-9084/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2011.06.012

because of high radical-scavenging activities [14]. The antiinflammatory effect of honokiol on neutrophils is directly related to the inhibition of reactive oxygen species production [15]. In addition, novel pharmaceutical technique is used to improve the action of honokiol and magnolol. For example, nanoparticles are employed to treat honokiol in order to improve therapeutic effects on malignant pleural effusion [16]. Some methods have been developed to prepare magnolol-type compounds [17] and to isolate them from herbs [18]. Honokiol and magnolol usually form a mixture, and it is difficult to isolate honokiol and magnolol by column chromatography because they are dihydroxyl isomers with similar polarities. A facile purification method as shown in Scheme 1 was employed to isolate honokiol and magnolol by forming a ketal of magnolol with 2,2-dimethoxypropane under acidic condition, while honokiol cannot form ketal under the same experimental condition. The polarity of the ketal of magnolol is lower than that of honokiol because ketal of magnolol does not contain hydroxyl group, leading to ketal of magnolol can be conveniently isolated from honokiol by column chromatography. Then, magnolol can be obtained after the hydrolysis under acidic condition [19]. Magnolol contains two hydroxyl groups both at ortho-position, and honokiol also includes two hydroxyl groups at ortho- and paraposition. Although many reports have dealt with the biological actions of them, the comparison of their antioxidant actions and the influence of the position of hydroxyl group on the antioxidant capacity are still worthy to explore in detail. Presented here is

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O

OH

O CH3OH

HO

1 M HCl reflux

(CH3)2C(OCH3)2

intramolecular hydrogen bond H O

H O

OH CH3

OH

SO3H OH

OH

room teperature magnolol honokiol

mixture of magnolol and honokiol

These two compounds can be isolated by column chromatography.

Scheme 1. Magnolol and honokiol can be isolated from their mixture by reacting with 2,2-dimethoxypropane, leading to the formation of magnolol ketal. Then, magnolol ketal and honokiol are isolated by column chromatography. Finally, magnolol is obtained by hydrolyzing magnolol ketal.

a comparison of honokiol and magnolol on reducing peroxynitrite (ONOO
) and singlet oxygen (1O2), scavenging 2,20 -azinobis(3ethylbenzothiazoline-6-sulfonate) cationic radical (ABTSþ) and 2,20 -diphenyl-1-picrylhydrazyl radical (DPPH), and bleaching b-carotene in linoleic acid (LH) emulsion. Moreover, the antioxidant capacities of honokiol and magnolol are compared in Cu2þ/glutathione (GSH)- and 2,20 -azobis(2-amidinopropane hydrochloride) (AAPH, ReN]NeR, R ¼ eCMe2C(]NH)NH2)-induced oxidation of DNA. 2. Materials and methods 2.1. Materials and instrumentation Diammonium salt of 2,20 -azinobis(3-ethylbenzothiazoline-6sulfonate) (ABTS salt) and 2,20 -diphenyl-1-picrylhydrazyl radical (DPPH) were purchased from Fluka Chemie GmbH, Buchs, Switzerland. 2,20 -Azobis(2-amidinopropane hydrochloride) (AAPH), the naked DNA sodium salt (DNA), linoleic acid (LH), 4-nitroso-N,Ndimethylaniline (NDMA), and glutathione (GSH) were purchased from ACROS ORGANICS, Geel, Belgium. Other agents were of analytical grade and used directly. Magnolol and honokiol were a mixture and can be isolated following a previous description [19]. Their structures can be identified by 1H NMR (Varian Mercury 300 NMR spectrometer), and the purities of magnolol and honokiol (>99.0%) were analyzed by HPLC.

hypochlorite, 10 mM H2O2, and 50 mM NDMA were mixed with 45 mM sodium phosphate buffer (pH ¼ 7.1) in a test tube. 1O2 was produced by the reaction between hypochlorite and H2O2 and can decolorized NDMA (lmax ¼ 440 nm). The absorbance at 440 nm was recorded and assigned as A0 and was measured once again after 40 min and assigned as Aref. Meanwhile, in other test tubes for the generation of 1O2, various concentrations of magnolol or honokiol were contained. The absorbance was recorded after the addition of magnolol or honokiol of 40 min and assigned as Adetect. The percentage of 1O2 quenched by magnolol or honokiol was calculated by (Adetect
Aref)/(A0
Aref)  100. The above experiment was carried out at 30  C. 2.4. ABTSþ and DPPH assays The experiments of magnolol and honokiol to trap ABTSþ and DPPH were carried out following a previous description [22]. The 4.0 mM ABTS salt and 1.41 mM K2S2O8 were mixed in 2.0 mL of water for 16 h to generate ABTSþ, and then diluted by 100 mL of ethanol. The absorbance at 734 nm (3734 ¼ 1.6  104 M
1 cm
1 [23]) was around 0.70. DPPH was dissolved in ethanol directly, and the absorbance at 517 nm (3517 ¼ 4.09  103 M
1 cm
1 [24]) was around 1.00. The ethanolic solution of magnolol or honokiol was added to the aforementioned radical solutions. The final concentration of magnolol or honokiol was 100 mM in trapping ABTSþ and 300 mM in trapping DPPH. The decay of the absorbance of the radical solutions was recorded at room temperature.

2.2. Peroxynitrite assay ONOO
can be prepared by a chemical reaction as described in a previous report [20]. A solution containing 2.2 mL of 30% H2O2 and 50 mL of water was cooled in ice-water bath. Then, 4 mL of 5 M NaOH and 5 mL of 0.04 M diethylenetriaminepentaacetic acid (dissolved in 0.05 M NaOH) were added. The above mixture was diluted by water to the total volume of 100 mL, followed by adding 2.7 mL of isoamyl nitrite and vigorously stirring for 5 h at room temperature. The aqueous phase was washed by 200 mL of dichloromethane for six times to remove organic compounds and was then mixed with MnO2 to remove surplus H2O2. Consequently, ONOO
in the aqueous solution (3302 ¼ 1670 M
1 cm
1) was stable enough within one week [20]. Magnolol or honokiol was mixed with ONOO
in 0.1 M NaOH, in which the final concentrations of magnolol or honokiol and ONOO
were 8.0 mM and 0.57 mM, respectively. The absorbance of the mixture was scanned from 250 nm to 550 nm under ambient temperature at every 30 min. 2.3. Singlet oxygen assay 1 O2 can be prepared by a chemical reaction as described in a previous report [21]. The 10 mM histidine, 10 mM sodium

2.5. b-Carotene-bleaching test The 5.0 mg of b-carotene, 40 mg of LH, and 400 mg of Triton X-100 were dissolved in 5.0 mL of CHCl3. After CHCl3 was evaporated under vacuum pressure, 100 mL of oxygen-saturated water was added and shaken under ultrasonic vibration to form homogeneous b-carotene-LH emulsion (lmax ¼ 460 nm) [25]. The 0.1 mL of ethanolic solution of magnolol or honokiol were mixed with 1.9 mL of b-carotene-LH emulsion, and the final concentration of magnolol or honokiol were 300 mM. The decay of the absorbance at 460 nm was recorded at room temperature. 2.6. Cu2þ/GSH-induced oxidation of DNA assay The Cu2þ/GSH-induced oxidation of DNA was performed following a previous description [26] with a little modification. Briefly, DNA, CuSO4, and GSH were dissolved in phosphate buffered solution (PBS1: 6.1 mM Na2HPO4, 3.9 mM NaH2PO4) to the final concentrations of DNA, Cu2þ, and GSH at 2.0 mg/mL, 5.0 mM, and 3.0 mM, respectively. Magnolol or honokiol was dissolved in dimethyl sulfoxide (DMSO) as the stock solution and was added to the above mixture to the final concentration of 0.4 mM. Then, the

C. Zhao, Z.-Q. Liu / Biochimie 93 (2011) 1755e1760

Magnolol

Magnolol

0.3

3. Results and discussion 3.1. Reducing ONOO
and quenching 1O2 ONOO
and 1O2 produced in metabolism can oxidize DNA, proteins, lipids, and membranes to cause aging and some diseases [28]. In an in vitro experimental system, ONOO
and 1O2 are prepared by chemical reactions and then employed to evaluate the antioxidant abilities of phenolics [29]. ONOO
was synthesized by the oxidation of isoamyl nitrite with H2O2, and the organic byproduct, isoamyl alcohol, was removed by the extraction with dichloromethane, and the surplus H2O2 was removed by MnO2. ONOO
under basic condition can be preserved at least one week according to the literature [20]. The absorbance of ONOO
at 302 nm did not decrease within two days at room temperature, demonstrating that ONOO
was stable enough in the absence of organic compounds. Fig. 1 outlines the UV spectra of the mixture of ONOO
(0.57 mM) and magnolol or honokiol (8.0 mM) scanned at every 30 min. It can be found that the absorbance around 300 nm decreases with the increase of the measurement time, indicating that ONOO
was exhausted with the time increasing. Magnolol and honokiol are the only organic compounds in the solution of ONOO
, and thus, it is the addition of magnolol and honokiol that causes the decrease of the absorbance of ONOO
. Therefore, the decrease of the absorbance around 300 nm in UV spectra provides an evidence for magnolol and honokiol to exhaust ONOO
.

400

500 300 Wavelength(nm)

400

500

Fig. 1. The absorbance (from 250 to 550 nm) of 0.1 M NaOH aqueous solution containing 8.0 mM magnolol or honokiol and 0.57 mM ONOO
is scanned at 0, 30, 60, 90, 120, 150, and 180 min under room temperature. The obtained eight UVevisible spectra are presented in the same chart.

1

O2 formed by the reaction of NaClO and H2O2 can oxidize NDMA and cause the decrease of the absorbance at 440 nm. In the blank experiment, the decrease of the absorbance from 1.232 to 0.828 after 40 min indicates that the amount of 1O2 can be expressed by the decrease of the absorbance of NDMA and can be assigned to 100%. On the contrary, the absorbance decreases to 1.178 when 750 mM magnolol is added to the above mixture, indicating that magnolol are more active to react with 1O2 than NDMA, and thereby hinders the decrease of the absorbance of NDMA. So, in the presence of magnolol the difference of the absorbance between 1.178 and 0.828 is due to the reaction of 750 mM magnolol with 1O2. The percentage of 1O2 quenched by magnolol is expressed by (1.178
0.828)/(1.232
0.828)  100 ¼ 86.6%. Following the aforementioned method, the percentages of 1O2 quenched by various concentrations of magnolol and honokiol were measured and illustrated in Fig. 2. Fig. 2 outlines that more 1O2 can be quenched with the concentrations of magnolol and honokiol increasing. The slope of the line indicates the sensitivity of the quenched 1O2 to the increase of the concentration of antioxidants. It can be found that magnolol exerts higher efficiency to quench 1O2 than honokiol, which is in agreement with a previous report [30].

90 Magnolol Honokiol

60 Scavenged

All the experiments were carried out at least three times, and the presented data were the average value from three independent measurements with the standard deviations within 10%. The equations were analyzed by one-way ANOVA using Origin 8.0 professional Software, and p < 0.001 indicated a significance difference.

300

Percentage of 1O2

2.8. Statistical analysis

Honokiol

0.0

2.7. AAPH-induced oxidation of DNA assay AAPH-induced oxidation of DNA was carried out following a previous description [27]. Briefly, DNA and AAPH were dissolved in phosphate buffered solution (PBS2: 8.1 mM Na2HPO4, 1.9 mM NaH2PO4, 10.0 mM EDTA) to the final concentrations of DNA and AAPH at 2.0 mg/mL and 40 mM, respectively. Various concentrations of DMSO solution of magnolol or honokiol were added, and the mixture was delivered into some test tubes with each one containing 2.0 mL. The test tubes were incubated at 37  C to initiate the oxidation of DNA. Three tubes were taken out at every 120 min and cooled immediately. Then, 1.0 mL of TBA solution and 1.0 mL of 3.0% trichloroacetic acid aqueous solution were added in turn. The tubes were heated in boiling water for 15 min. After the test tubes were cooled to room temperature, 1.5 mL of n-butanol was added and shaken vigorously to extract TBARS, whose absorbance was measured at 535 nm.

Honokiol

0.6

Absorbance

mixture was delivered into some test tubes with each one containing 2.0 mL. The test tubes were incubated at 37  C to initiate the oxidation of DNA. Three tubes were taken out at every 30 min and cooled immediately. Then, 1.0 mL of PBS1 solution of 30.0 mM EDTA, 1.0 mL of thiobarbituric acid (TBA) solution (1.00 g TBA and 0.40 g NaOH dissolved in 100 mL PBS1), and 1.0 mL of 3.0% trichloroacetic acid aqueous solution were added in turn. The tubes were heated in boiling water for 30 min. After the test tubes were cooled to room temperature, 1.5 mL of n-butanol was added and shaken vigorously to extract thiobarbituric acid reactive substance (TBARS), whose absorbance was measured at 535 nm.

1757

30

0 0

200

400 600 Concentration (μM)

800

Fig. 2. The absorbance (at 440 nm) of 45 mM sodium phosphate buffer (pH ¼ 7.1) containing 10 mM histidine, 10 mM sodium hypochlorite, 10 mM H2O2, and 50 mM 4nitroso-N,N-dimethylaniline (NDMA) was measured after incubating at 30  C for 40 min and assigned as Aref. Meanwhile, the absorbance in the above mixture containing various concentrations of magnolol or honokiol was measured under the same experimental condition and assigned as Adetect. The absorbance of the mixture is also measured before incubation and assigned as A0. The relationships between (Adetect
Aref)/(A0
Aref)  100 and the concentration of magnolol or honokiol are presented.

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3.2. Trapping ABTSþ and DPPH The decrease of the absorbance of ABTSþ and DPPH is shown in Fig. 3 when 100 mM magnolol or honokiol is used to trap ABTSþ and 300 mM of them to trap DPPH. The reaction with ABTSþ indicates the ability of hydroxyl groups in an antioxidant to reduce radicals, and the reaction with DPPH indicates the ability to contribute its hydrogen atom to N-centered radicals. Recently, a chemical kinetic method was applied to express the interaction of an antioxidant with ABTSþ [31]. Following the expression in the literature [31], [ABTSþ]0 and [ABTSþ]N are used to stand for the concentration of ABTSþ at the beginning and the end of the reaction with the antioxidant. As shown in Equation (1), the variation of the concentration of ABTSþ during the reaction is divided by the concentration of the antioxidant to obtain a value, n, which can be regarded as the number of electrons in ABTSþ trapped by the antioxidant in the primary stage of the reaction with ABTSþ.

½ABTSþ 0
½ABTSþ N ½antioxidant

(1)

  ab ½ABTSþ  ¼ ½ABTSþ N þ bþt

(2)

Absorbance at 734 nm

A0

Honokiol Magnolol

0.9

A0

Honokiol Magnolol

0.9

B

A

0.6 0.6 0.3 A∞

A∞

20

0 50 40 Reaction period (min)

100

Absorbance at 517 nm

where a ¼ napp[antioxidant]0, b ¼ 1=ðkapp 2 ½antioxidant0 Þ, t is the reaction time, and napp and k2app refer to the number of electrons trapped by the antioxidant and the rate constant of the secondary reaction, respectively. Therefore, n þ napp is the total number of electrons trapped by the antioxidant. This method is also applied to treat the reaction of the antioxidant with DPPH. The data of [ABTSþ] and [DPPH] at the beginning and the end of the reaction together with the concentration of magnolol or honokiol are fitted into Equation (1) to obtain n of magnolol and honokiol. Then, [ABTSþ] and the corresponding time are fitted into Equation (2) to obtain napp of magnolol and honokiol. The numbers of electrons in ABTSþ and DPPH trapped by magnolol and honokiol are listed in Table 1. As can be seen from Table 1, in trapping ABTSþ the n of magnolol is 0.42, similar to that of honokiol (0.46). The napp of honokiol (0.55) is higher than that of magnolol (0.44), leading to the total number of electrons trapped by honokiol (n þ napp ¼ 1.01) is higher than that of magnolol (0.86). In trapping DPPH the n of honokiol (0.70) is higher than that of magnolol (0.44), indicating that H

0

Antioxidant

n

napp

n þ napp

Magnolol Honokiol

0.42  0.02 0.46  0.02

0.44  0.02 0.55  0.03

0.86  0.04 1.01  0.05

DPPH

Magnolol Honokiol

0.44  0.02 0.70  0.03

0.36  0.02 0.59  0.03

0.80  0.04 1.28  0.06

atoms of hydroxyl groups in honokiol are readily abstracted by N-centered radical. For magnolol, as can be seen in Scheme 1, hydrogen atoms in ortho-hydroxyl groups can form an intramolecular hydrogen bond that hinders the hydrogen atom in hydroxyl group to be abstracted by radicals. But hydrogen atoms in ortho- and para-hydroxyl group cannot form an intramolecular hydrogen bond within honokiol and can easily donate to radicals.



Some products from the reaction of the antioxidant with ABTSþ may be able to trap ABTSþ, which is called the secondary stage of the reaction with ABTSþ. The variation of the concentration of ABTSþ ([ABTSþ]) can be expressed by Equation (2).

0.3

Free radical ABTSþ

0.0

Fig. 3. (A) The absorbances at 734 nm of a mixture of ABTSþ and 100 mM magnolol and honokiol are measured at some time intervals and plotted vs reaction period; (B) the absorbances at 517 nm of a mixture of DPPH and 300 mM magnolol and honokiol are measured at some time intervals and plotted vs reaction period.

3.3. b-Carotene-bleaching test

b-Carotene-bleaching test is usually used to screen the ability of an antioxidant to protect polyunsaturated fatty acid [25]. The antioxidant properties of magnolol and honokiol to protect linoleic acid (LH) are estimated by b-carotene-bleaching test. As shown as the blank line in Fig. 4, the decay of the absorbance of b-carotene indicates that peroxyl radical (LOO) derived from the autoxidation of LH depletes b-carotene with the incubation period increasing. The addition of 300 mM magnolol or honokiol retards the exhaustion of b-carotene, revealing that magnolol and honokiol can inhibit the oxidation of LH to form LOO. The absorbance in the presence of honokiol decreases slower than that of magnolol, indicating that ortho- and para-hydroxyl groups are more active to inhibit the autoxidation of LH than di-ortho-hydroxyl groups. This result suggests that the intramolecular hydrogen bond affects the antioxidant capacity of magnolol by hindering the hydrogen atom in hydroxyl groups to be abstracted by LOO. 3.4. Protecting DNA against Cu2þ/GSH-induced oxidation DNA can be damaged by the intracellular glutathione (GSH) and Cu(II) because GSH radical (GS) from the oxidation of GSH by Cu(II)

1.00

Absorbance at 460 nm



n ¼

Table 1 ABTSþ and DPPH are mixed with magnolol or honokiol, respectively, and the numbers of electrons in ABTSþ and DPPH trapped by magnolol and honokiol in the primary stage (n), the secondary stage (napp), and the total numbers (n þ napp) are obtained by using Equations (1) and (2).

Blank Magnolol Honokiol

0.75

0.50

0.25 0

300

600

Incubation period (min) Fig. 4. The 5.0 mg of b-carotene, 40 mg of linoleic acid (LH), 400 mg of Triton X-100, and 100 mL of oxygen-saturated water form homogeneous b-carotene-LH emulsion, and 0.1 mL of ethanolic solution of magnolol or honokiol were mixed with 1.9 mL of bcarotene-LH emulsion for detecting the absorbance at 460 nm at some time intervals.

C. Zhao, Z.-Q. Liu / Biochimie 93 (2011) 1755e1760

Absorbance at 535 nm

Table 2 The relationship between the concentration of magnolol and honokiol and the generated inhibition period (tinh), and the stoichiometric factor (n) of magnolol and honokiol is calculated accordingly.a

Blank Magnolol Honokiol

0.6

1759

0.4

Antioxidant

tinh (min) ¼ (n/Ri) [antioxidant (mM)] þ constantb

n

Honokiol Magnolol

tinh (min) ¼ 0.76 [honokiol]
9.80 tinh (min) ¼ 0.53 [magnolol] þ 31.56

2.5 1.8

a Ri ¼ Rg ¼ 1.4  10
6 [AAPH] s
1 ¼ 3.36 mM min
1 when 40 mM AAPH was employed, thus, n ¼ coefficient  3.36 mM min
1. b The constant was generated from the linear regression analysis.

0.2

0

60

120

Comparing with the linear increase of the absorbance of TBARS in the blank experiment, the addition of magnolol or honokiol bends the absorbance lines, and the formation of TBARS actually slows down at the beginning of the oxidation and then recovers as the blank experiment. This fact implies that magnolol and honokiol can inhibit the oxidation of DNA for a period (designated as inhibition period, tinh). The tinh was measured at least three times with a certain concentration of magnolol or honokiol employed, as shown as panel B in Fig. 6, tinh correlates linearly with the increase of the concentration of magnolol or honokiol, and the quantitative relationships are listed in Table 2. The chemical kinetics deduces the quantitative relationship between tinh and the concentration of an antioxidant as shown as Equation (3) [34], which can be employed to evaluate the antioxidant efficacy in biological experimental systems [35].

180

Incubation period (min) Fig. 5. The absorbance of TBARS in the mixture of DNA (2.0 mg/mL), 5.0 mM Cu2þ, 3.0 mM GSH, and 0.4 mM magnolol or honokiol is measured at some time intervals.

converts DNA into carbonyl species. So, the process of Cu2þ/GSHinduced oxidation of DNA can be followed by detecting the oxidative products, which can react with thiobarbituric acid (TBA) to form colorful TBA reactive substance (TBARS, lmax ¼ 535 nm) [32]. As shown as the blank line in Fig. 5, the increase of the absorbance indicates that more carbonyl species are generated with the incubation period increasing. The lines of the absorbance in the presence of 0.4 mM magnolol or honokiol locate below but very approach to the blank experiment. It is necessary to apply some statistical methods for clarifying whether these lines exhibit significant difference. It is found that the lines of magnolol and the blank experiment do not exhibit significant difference, whereas the lines of honokiol and the blank experiment exhibit significant difference. Thus, honokiol rather than magnolol presents weak ability to protect DNA against GS-induced oxidation.

tinh ¼ ðn=Ri Þ½antioxidant

The n is stoichiometric factor to express the number of the radical-propagation terminated by one molecule of the antioxidant. Ri is the radical-initiation rate. Because the sodium salt of DNA and AAPH are both dissolved in water phase, it is safely to assume that Ri is equal to the radical-generation rate (Rg) from the decomposition of AAPH (Rg ¼ (1.4  0.2)  10
6 [AAPH] s
1) [35]. Thus, the n of magnolol or honokiol is the product of Ri ¼ Rg ¼ 1.4  10
6  40 mM s
1 ¼ 3.36 mM min
1 and the coefficient in the equation of tinh w [magnolol or honokiol] and is listed in Table 2. As can be seen in Table 2, the coefficient in the equation of tinh w [honokiol] (0.76) is larger than that in tinh w [magnolol] (0.53), indicating that tinh increases more rapidly with the concentration of honokiol than with that of magnolol. Moreover, the values of n reveal that honokiol can terminate 2.5 radical-propagations, and magnolol can only terminate 1.8 radical-propagations. The antioxidant efficacy of honokiol is higher than that of magnolol in protecting DNA against AAPH-induced oxidation.

3.5. Protecting DNA against AAPH-induced oxidation The decomposition of AAPH generates peroxyl radical (ROO) that abstracts H atom from the C-40 position of DNA and produces carbonyl species eventually. Thus, the process of AAPH-induced oxidation of DNA can also be followed by measuring TBARS [33]. As shown as the panel A in Fig. 6, in the blank experiment the linear increase of the absorbance of TBARS indicates an increase of the amount of carbonyl species with the incubation period.

Honokiol

Magnolol

B

300

1.2 Honokiol

250 tinh(min)

Absorbance at 535 nm

A

(3)

0.9

0.6

200 Magnolol

150 0.3

0

250

500

750

0

250

500

Incubation period (min)

750

200

300

400

Concentration (μM)

Fig. 6. (A) The absorbance of TBARS in the mixture of DNA (2.0 mg/mL), 40 mM AAPH, and various concentration of magnolol or honokiol is measured at some time intervals, and (B) the relationship between tinh and the concentration of magnolol and honokiol.

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C. Zhao, Z.-Q. Liu / Biochimie 93 (2011) 1755e1760

4. Conclusion Magnolol and honokiol are isomers and are difficult to be isolated directly. The di-ortho-hydroxyl groups in magnolol can react with 2,2-dimethoxypropane to form ketal, which can be readily isolated from honokiol. Both magnolol and honokiol can reduce ONOO
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