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SHORT COMMUNICATION
Antioxidant and radical scavenging activities of chamazulene
Andrea Capuzzo
a
, Andrea Occhipinti
ab
and Massimo E. Maffei
ab
*
a
Biosfered S.r.l., Academic Spin-Off of the University of Turin, Via G. Quarello 15/A, 10135 Turin, Italy;
b
Department of Life Sciences and Systems Biology, University of Turin, Via G. Quarello 15/A, 10135 Turin,
Italy
(Received 12 May 2014; final version received 1 June 2014)
Essential oils (EOs) of chamomile contain several bioactive compounds, including
monoterpenes, sesquiterpenes, triterpenes and fatty acids. Hydrodistillation of
chamomile EO induces the formation of chamazulene, a bioactive compound.
Chamazulene was isolated from the EO by column chromatography. The total
antioxida nt cap acity confirmed a higher antioxidant activity of chamazulene
(IC
50
¼ 6.4 mgmL
21
) than of ascorbic acid (IC
50
¼ 12.8 mgmL
21
), a-tocopherol
(IC
50
¼ 20.5 mgmL
21
) and of butylated hydroxytoluene (BHT) (IC
50
¼ 30.8
mgmL
21
). Chamazulene was unable to react with DPPHz. However, when
chamazulene was assayed with ABTSz, a strong and significantly (P , 0.05) higher
free radical scavenging activity was observed (IC
50
¼ 3.7 mgmL
21
), with respect to
BHT (IC
50
¼ 6.2 mgmL
21
) and a-tocopherol (IC
50
¼ 11.5 mgmL
21
). The results of
this work show that chamazulene is an important factor for the antioxidant power of
chamomile oil.
Keywords: Matricaria chamomilla L; Asteraceae; chamazulene; antioxidant activity
1. Introduction
Bioactive compounds of chamomile (Matricaria spp.) are widely used by the pharmaceutical
and cosmetic industries for their antispasmodic, anti-inflammatory and antimicrobial properties
and also as a natural hair dye and fragrance (Singh et al. 2011; Ghavimi et al. 2012; Tschiggerl &
Bucar 2012). Among the most important constituents are a-bisabolol and bisabolol oxides,
spiroethers and b-farnesene. Another important compound is chamazulene, a bicyclic
sesquiterpene blue coloured decomposition product of matricin (prochamazulene), which is
formed at high temperatures during steam-distillation processing of essential oils (EOs)
(Jakovlev et al. 1983). It has been suggested that chamazulene possessing anti-inflammatory
activity (Safayhi et al. 1994) is involved in radical scavenging activity (Rekka et al. 1996) and
general antioxidant activity (Sizova 2012). We isolated chamazulene from chamomile EO and
tested its antioxidant activity by using DPPH, ABTS, the phosphomolybdenum and the reducing
power assays. Here we show that the presence of chamazulene is required for the antioxidant
activity of chamomile extracts.
2. Results and discussion
Since EOs and plant extracts containing chamazulene have been demonstrated to exert
antioxidant and radical scavenging activity (Sizova 2012; Ornano et al. 2013), we isolated
chamazulene from chamomile EO by column chromatography (see Supplementary material S1
q 2014 Taylor & Francis
*Corresponding author. Email: massimo.maffei@unito.it
Natural Product Research, 2014
Vol. 28, No. 24, 2321–2323, http://dx.doi.org/10.1080/14786419.2014.931393
Downloaded by [Universita degli Studi di Torino] at 05:52 24 November 2014
for materials and methods). The content (98%) and identity of chamazulene were confirmed by
GC-FID and GC–MS, respectively (Supplementary Figure S1).
Electron-donating capacity reflects the reducing power of bioactive compounds and is
associated with their antioxidant activity (Vladimir-Knezevic et al. 2011). Pu rified chamazulene
was then assayed for its reducing power and its activities were compared with the reference
antioxidants ascorbic acid, a-tocopherol and butylated hydrox ytoluene (BHT). Chamazulene
showed a higher reducing power (IC
50
¼ 7.6 ^ 0.4 mgmL
21
)thana-tocopherol
(IC
50
¼ 238.9 ^ 3.6 mgmL
21
)withvaluesclosertobutylated hydroxytoluene (BHT)
(IC
50
¼ 6.5 ^ 0.2 mgmL
21
) and ascorbic acid (IC
50
¼ 3.5 ^ 0.2 mgmL
21
).
The total antioxidant capacity of chamazulene was assayed by the phosphomolybdenum
method and compared with the reference antioxidant compounds. Antioxidant activity of
chamazulene (IC
50
¼ 6.4 ^ 0.1 mgmL
21
) was higher than the reference antioxidant
compounds: ascorbic acid (IC
50
¼ 12.8 ^ 0.1 mgmL
21
), BHT (IC
50
¼ 30.8 ^ 0.3 mgmL
21
)
and a-tocopherol (IC
50
¼ 20.5 ^ 0.2 mgmL
21
).
Free radical scavenging activities of extracts were assayed by using the stable DPPH radical.
Chamazulene was found to be inactive to DPPHz. This result depends on the ability of
chamazulene to interact with the stable, nitrogen-centred DPPHz, which is indicative of its
reduced potency in an iron-free system (Rekka et al. 1996). The generation of the ABTS radical
cation forms the basis of one of the spectrophotometric methods that have been applied to the
measurement of the total antioxidant act ivity of solutions of pure substances (Re et al. 1999).
When chamazulene was assayed with ABTSz, a strong and significantly higher free radical
scavenging activity was observed (IC
50
¼ 3.7 ^ 0.7 mgmL
21
)thana-to copherol
(IC
50
¼ 11.5 ^ 1.1 mgmL
21
) and with intermediate values between BHT (IC
50
¼ 6.2 ^ 0.5
mgmL
21
) and ascorbic acid (IC
50
¼ 1.6 ^ 0.6 mgmL
21
). The observed IC
50
values of
chamazulene were also comparable to those observed for other strong radical scavenging
compounds such as quercetin, delphinidin and lycopene (Re et al. 1999).
The antiradical activity characterises the ability of the compounds to react with free radicals (in a
single free radical reaction), whereas the antioxidant activity represents the ability to inhibit the
process of oxidation. Consequently, test systems using DPPH and ABTS give information on the
radical scavenging or antiradical activity, although in many cases this activity doesnot correspond to
the antioxidant activity (Tirzitis & Bartosz 2010). Our results are in agreement with recent findings
where the ABTS
zþ
-scavenging activity of the chamazulene-rich Artemisia arborescens EO was
found to be much higher than its capacity to scavenge the DPPH radical (Ornano et al. 2013).
3. Conclusions
The results of this work show that chamazulene is an important contributor to the antioxidant and
radical scavenging activity of chamomile EOs. The isolation of chamazulene from chamomile
EO allowed evaluat ing its bioactive properties showing both a strong radical scavenger and a
powerful antioxidant ability along with a strong reducing power. Our data confirm the need to
avoid DPPH assay to test chamazulene radical scavenging activity owing to its interference with
the nitrogen-centred DPPHz and suggest the use of ABTS.
Supplementary material
Experimental details relating to this article are available online, alongside Figure S1.
Acknowledgement
This work was supported by the Doctorate School of Pharmaceutical and Biomolecular Sciences of the
University of Turin, Italy.
2322 A. Capuzzo et al.
Downloaded by [Universita degli Studi di Torino] at 05:52 24 November 2014
References
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Supplementary Figure S1. Chamazulene was isolated from EO by column chromatography. 5 ml of
EO were loaded on a glass column (45 × 1.5 cm) packed with silica gel 60 (0.040–0.063 mm) (Merck,
Germany). n-Hexane was used as the solvent and fractions were collected in 1 ml tubes. The content and
identity of chamazulene was assessed by GC-FID and GC-MS, respectively, as reported above, and the
fractions containing the highest chamazulene percentage and content were pooled together and
concentrated under N2. The figure shows the GC-FID analysis of the chamazulene fraction used in this
work (purity 98%) as well as the mass spectrum and chemical structure of chamazulene.
3. Experimental
3.1 Plant material
Essential oil (EO) of Matricaria chamomilla L. (Chamomile) was provided by the company
Chialva Nicolao di Dr Franco Chialva Co. (S.A.S.), Pancalieri, Italy with batch number 20120101.
3.2. Purification of Chamazulene
Chamazulene was isolated from chamomile EO by column chromatography. Five ml of EO were
loaded on a glass column (45×1.5 cm) packed with silica gel 60 (0.040–0.063 mm) (Merck,
Germany). n-Hexane was used as the solvent and fractions were collected in 1 ml tubes. The
content and identity of chamazulene was assessed by GC-FID and GC-MS, respectively, as reported
below, and the fractions containing the highest chamazulene percentage and content were pooled
together and concentrated under N
2
.
3.3. GC-MS and GC-FID analysis of chamazulene
Chamazulene content was determined with a gas chromatograph (Agilent Technologies 6890)
equipped with a capillary (HP5-MS) column (30 m x 0.25 mm; film thickness of 0.25 µm) coupled
with a Flame Ionization Detector (FID) as previously described (Occhipinti et al., 2014).
Chamazulene was identified by gas chromatography (Agilent Technologies 6890) equipped with a
capillary (HP5-MS) column (30 m x 0.25 mm; film thickness of 0.25 µm) coupled with an Agilent
EI-quadrupole mass spectrometer detector (MSD) model 5973 as detailed elsewhere (Arceusz et al.,
2013). The data reported are the mean of at least three replicates.
3.4 Reducing power assay
Reducing power of chamazulene, and reference compounds [ascorbic acid (99.0%, Sigma, Milan,
Italy), α-tocopherol (98.0%, Sigma), and butilated hydroxytoluene (BHT) (99.0%, TCI Europe)]
was determined by the method of Oyaizu (Oyaizu, 1986).
3.5 Total antioxidant capacity assays
The total antioxidant capacity assay was based on the reduction of Mo(VI) to Mo(V) by the sample
analyte according to Prieto and co-workers (1999).
3.6 Free radical scavenging assays:
Free radical scavenging activities of samples was determined by using two different radical species:
the 2,2-Diphenyl-1-picrylhydrazyl radical (DPPH
•
) performed by following the procedure of
Vladimir-Knezevic and co-workers (2011), and the 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic
acid) radical cation (ABTS
•+
) assay (Tirillini, Menghini, Leporini, Scanu, Marino, & Pintore, 2013)
as described by Re and collaborators (1999).
3.7 Statistical analysis
All experiments were carried out in triplicate, and the results are expressed as mean ± standard
deviation (SD). Differences were estimated by ANOVA and Student's t-test and the values p < 0.05
were considered statistically significant. Antioxidant activities of DPPH and ABTS assays were
calculated by interpolation from linear regression analysis and expressed as IC
50
, the concentration
required in order to observe a reduction of 50% of the initial radical concentration. For total
antioxidant capacity and reducing power assays, IC
50
values were calculated as the concentrations at
which absorbance was 0.5 (Vladimir-Knezevic et al., 2011). All statistical analyses were carried out
using SYSTAT 10.0
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