Antioxidant activity and mechanism of the abietane-type diterpene ferruginol

Article abstract of DOI:10.1080/14786419.2014.997233

The antioxidant activity of the abietane-type diterpene ferruginol was evaluated by comparison with that of carnosic acid, (±)-α-tocopherol and dibutylhydroxytoluene using 2,2-diphenyl-1-picrylhydrazyl, β-carotene bleaching and linoleic acid assays. Ferruginol had the lowest antioxidant activity of this group using the 2,2-diphenyl-1-picrylhydrazyl and β-carotene methods in polar solvent buffer. However, ferruginol exhibited stronger activity than carnosic acid and α-tocopherol for linoleic acid oxidation under non-solvent conditions. Five peaks corresponding to ferruginol derivatives were detected through GC-MS analysis of the reaction between ferruginol and methyl linoleate. The three reaction products were identified as dehydroferruginol, 7β-hydroxyferruginol and sugiol, and the other two peaks were assumed to be 7α-hydroxyferruginol and the quinone methide derivative of ferruginol. The time course of the reaction suggests that the quinone methide was produced early in the reaction and reacted further to produce dehydroferruginol, 7-hydroxyferruginol and sugiol. Thus, we inferred that quinone methide formation was a key step in the antioxidant reaction of ferruginol.

Full text of DOI:10.1080/14786419.2014.997233

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Antioxidant activity and mechanism of
the abietane-type diterpene ferruginol
a
a
ab
ab
H. Saijo , H. Kofujita , K. Takahashi & T. Ashitani
a
The United Graduate School of Agricultural Sciences, Iwate
University, 18-8, Ueda-3 chome, Morioka 020-8550, Japan
b
Faculty of Agriculture, Yamagata University, 1-23, Wakaba-
machi, Tsuruoka 997-8555, Japan
Published online: 14 Jan 2015.
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To cite this article: H. Saijo, H. Kofujita, K. Takahashi & T. Ashitani (2015): Antioxidant activity and
mechanism of the abietane-type diterpene ferruginol, Natural Product Research: Formerly Natural
Product Letters, DOI: 10.1080/14786419.2014.997233
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Natural Product Research, 2015
http://dx.doi.org/10.1080/14786419.2014.997233
SHORT COMMUNICATION
Antioxidant activity and mechanism of the abietane-type diterpene
ferruginol
a
H. Saijo *, H. Kofujita , K. Takahashi and T. Ashitani
a
ab
ab
a
The United Graduate School of Agricultural Sciences, Iwate University, 18-8, Ueda-3 chome, Morioka
20-8550, Japan; Faculty of Agriculture, Yamagata University, 1-23, Wakaba-machi, Tsuruoka 997-
555, Japan
b
0
8
(
Received 6 October 2014; final version received 6 December 2014)
OH
OH
Free radical
L
OH
LOO
O
H
H
OH
Dehydroferruginol
Quinone methide
Sugiol
H
Ferruginol
LH
OH
-Hydroxyferruginol
H
LOOH
7
Antioxidation step of ferruginol
The antioxidant activity of the abietane-type diterpene ferruginol was evaluated by
comparison with that of carnosic acid, (^)-a-tocopherol and dibutylhydroxytoluene
using 2,2-diphenyl-1-picrylhydrazyl, b-carotene bleaching and linoleic acid assays.
Ferruginol had the lowest antioxidant activity of this group using the 2,2-diphenyl-1-
picrylhydrazyl and b-carotene methods in polar solvent buffer. However, ferruginol
exhibited stronger activity than carnosic acid and a-tocopherol for linoleic acid
oxidation under non-solvent conditions. Five peaks corresponding to ferruginol
derivatives were detected through GC-MS analysis of the reaction between ferruginol
and methyl linoleate. The three reaction products were identified as dehydroferruginol,
7b-hydroxyferruginol and sugiol, and the other two peaks were assumed to be 7a-
hydroxyferruginol and the quinone methide derivative of ferruginol. The time course of
the reaction suggests that the quinone methide was produced early in the reaction and
reacted further to produce dehydroferruginol, 7-hydroxyferruginol and sugiol. Thus,
we inferred that quinone methide formation was a key step in the antioxidant reaction
of ferruginol.
Keywords: word; ferruginol; abietane-type diterpene; antioxidant mechanism;
quinone methide
1
. Introduction
The abietane-type diterpene ferruginol is a component of herbaceous and woody plants and is
considered a useful natural product because of its various biological effects, including anti-
oxidant (Wang et al. 2002; Cheng & Chang 2014), anti-fungal (Kofujita et al. 2006; Kusumoto
et al. 2010), anti-termite (Chang et al. 2001; Kusumoto et al. 2009) and anti-ulcerogenic
*
Corresponding author. Email: abe18136@tds1.tr.yamagata-u.ac.jp
q 2015 Taylor & Francis

2
H. Saijo et al.
activities (Rodrıguez et al. 2006) and algal growth inhibition properties (Saijo et al. 2013).
Several plantation trees are known to produce ferruginol. Japanese cedar (Japanese name ‘Sugi’;
Cryptomeria japonica) is one of the primary forested trees in the Japanese wood industry. Sugi
contains ferruginol in its heartwood (Shibutani et al. 2007) and bark (Ashitani et al. 2001;
Kofujita et al. 2006); its bark contains a substantial amount of ferruginol. Sugi bark is discarded
in large quantities during the production of woody material; thus, a large amount of a potential
ferruginol source is wasted.
Various natural resources derived from plants are used as antioxidants (Brewer 2011),
particularly sage (Salvia officinalis) and rosemary (Rosmarinus officinalis), which are used as
antioxidants in the food industry worldwide (Tena et al. 1997). Masuda et al. (2001, 2002)
reported the antioxidant activity of sage extracts and demonstrated the antioxidant mechanism of
the primary active component, carnosic acid, and its derivatives. Carnosic acid is a commercially
available antioxidant and, similar to ferruginol, is an abietane-type diterpene. However, the
antioxidant mechanisms of ferruginol have not yet been examined. To efficiently utilise this
resource, the detailed characterisation and examination of the antioxidant activity of ferruginol
from plantation trees are needed.
2
2
. Results and discussion
.1. Assays for antioxidant activity
a-Tocopherol and carnosic acid exhibited the highest activities in the DPPH and b-carotene
methods, respectively. Ferruginol, as reported by Kolak et al. (2009), showed the lowest activity
than any commercial reagent (Table S1). However, Wang et al. (2002) and Cheng and Chang
(
2014) reported that ferruginol had comparatively high antioxidant activity in diterpene
components. Although the reference compounds dissolved completely in the buffer solution at
every concentration, an emulsion was observed at high concentrations (greater than 200 mM) of
ferruginol. Thus, the solubility of ferruginol in the buffer solution for both methods was less than
the solubility of the other three compounds. Figure 1 shows the antioxidant activities from the
linoleic acid method. This experiment was performed to directly estimate the ability of the
1
1
20
00
8
6
4
2
0
0
0
0
0
0
5
10
Time (h)
15
20
Linolic acid
BHT+Linolic acid
α-Tocopherol+Linolic acid
Carnosic acid+Linolic acid
Ferruginol+Linolic acid
Figure 1. Antioxidant activities of ferruginol and reference compounds by the linoleic acid method. Note:
The antioxidant activity was evaluated based on the amount of residual linoleic acid.

Natural Product Research
3
samples to suppress the oxidation of unsaturated oil. The sample was added directly into the oil.
Suppression of oil oxidation was evaluated over a short time period of 1 day at 858C. The activity
of ferruginol was weaker than dibutylhydroxytoluene (BHT) but stronger than carnosic acid and
a-tocopherol in this method. Thus, the hydrophobic compound ferruginol was able to protect
linoleic acid under non-solvent conditions. This result suggests that ferruginol possesses
antioxidant activity against the oxidation of linoleic acid.
2
.2. Analysis of ferruginol derivatives in methyl linoleate by GC-MS
The mixture of ferruginol and methyl linoleate was analysed by GC-MS. Peaks 1–5 were
detected in the MS spectra and were presumed to be ferruginol derivatives (Figure S1). Other
peaks were regarded as fragments formed from methyl linoleate by its MS spectra (data not
shown). From the time course data of the five peaks (Figure S2a), peak 2 increased from the
initial reaction time to the intermediate time in this reaction but then disappeared in the later
reaction time. Peaks 1, 3 and 4 also increased from the initial to intermediate reaction stages, and
these three peaks then decreased in the late reaction stage. However, peak 5 increased gradually
during the final stages of the reaction. The reaction with a high concentration of methyl linoleate
OH
H
L
Ferruginol
OH
LOO
LH
LOOH
O
O
H
OH
Sugiol
H
L
L
LH
LOOH
LOO
LOO
LH
LOOH
H
O
O
Dehydroferruginol
H O
H O
2
2
OH
H
H
OH
OH
Ferruginol quinone methide
L
LOO
LH
LOOH
OH
H
H
H O
2
OH
H
H
7
α-Hydroxyferruginol 7β-Hydroxyferruginol
Figure 2. Proposed mechanism of ferruginol antioxidant activity against methyl linoleate oxidation. Note: []
is the postulated chemical structure.

4
H. Saijo et al.
(
Figure S2b) proceeded more rapidly and showed the same tendency. Peaks 1, 4 and 5 were
identified as dehydroferruginol, 7b-hydroxyferruginol and sugiol, respectively. Peak 3 had a
very similar spectrum pattern to peak 4 (Figure S3). Thus, peak 3 was considered likely to be a
diastereomer, 7a-hydroxyferruginol, of the peak 4 component. We considered peak 2 to be the
first product derived from ferruginol in the above reaction. We explored the isolation of the
compound from this reaction solution; however, this compound was very unstable and could not
þ
be isolated. The compound showed m/z ¼ 284.2203 (M ) (284.2140 calculated for C H O) by
2
0 28
HR-MS analysis. Peaks 1, 3, 4 and 5 were all compounds oxidised at the 7-position of ferruginol.
Therefore, it was estimated that peak 2 had the quinone methide structure derived from
ferruginol. Otani and Sumimoto (1983) also indicated the quinone methide as an intermediate in
the formation of dehydroferruginol by the chlorine treatment of ferruginol. Thus, this result
matches our hypothesis of quinone methide formation in the reaction of ferruginol with oxygen
radicals. The antioxidant mechanism (Figure 2) was analysed based on the above results. In the
first reaction, ferruginol lost two hydrogen atoms by radical species produced from unsaturated
oil to give the ferruginol quinone methide as an intermediate. Dehydroferruginol could be
formed from the ferruginol quinone methide by isomerisation. As dehydroferruginol is more
stable than the quinone methide, the formation of dehydroferruginol might be predominant in the
isomerisation. In an alternative pathway, the ferruginol quinone methide was attacked by H O
2
produced from oil oxidation to form 7-hydroxygerruginols. 7-Hydroxyferruginols could also be
produced by the addition of H O to dehydroferruginol, and 7-hydroxyferruginols were
2
dehydrated to produce dehydroferruginol. Finally, the radicals of the oxidative oil abstracted two
hydrogen atoms from 7-hydroxyferruginols to give sugiol as the final product. From the above
mechanism, it can be concluded that ferruginol inhibited oxidation of the oil by trapping free
radicals and preventing propagation. Sugiol has weaker antioxidant properties than ferruginol
(Wang et al. 2002). Thus, the key step in the antioxidant mechanism of ferruginol is the
formation of quinone methide as the initial step.
Supplementary material
Experimental details relating to this paper are available online, alongside Table S1 and
Figures S1–S3.
Funding
This study was supported by the 2013 UGAS-IU Student Research Project and Grant-in-Aid for JSPS
Fellows.
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