Irbic acid, a dicaffeoylquinic acid derivative from Centella asiatica cell cultures

Article abstract of DOI:10.1016/j.fitote.2011.05.008

3,5-O-dicaffeoyl-4-O-malonilquinic acid (1) (irbic acid) has been isolated for the first time from cell cultures of Centella asiatica and till now it has never been reported to be present in the intact plant. Evidence of its structure was obtained by spectroscopic analyses (MS/NMR). Besides 1, cell cultures produce also the known 3,5-O-dicaffeoylquinic acid, chlorogenic acid, and the triferulic acid 2 (4-O-8′/4′-O-8″-didehydrotriferulic acid). Biological activities were evaluated for compound 1, which showed to have a strong radical scavenging capacity, together with a high inhibitory activity on collagenase. This suggests a possible utilization of this substance as a topical agent to reduce the skin ageing process.

Full text of DOI:10.1016/j.fitote.2011.05.008

Fitoterapia 82 (2011) 950–954
Contents lists available at ScienceDirect
Fitoterapia
journal homepage: www.elsevier.com/locate/fitote
Irbic acid, a dicaffeoylquinic acid derivative from Centella asiatica
cell cultures
Fabiana Antognoni a, Nicoletta Crespi Perellino b, Sergio Crippa c, Roberto Dal Toso b,
b
a
b
Bruno Danieli c, , Anacleto Minghetti , Ferruccio Poli , Giovanna Pressi
⁎
a
Università di Bologna, Dipartimento di Biologia Evoluzionistica Sperimentale, Via Irnerio, 42, 40126, Bologna, Italy
Istituto di Ricerche Biotecnologiche (IRB) s.r.l., Via Lago di Tovel, 7, 36077 Altavilla Vicentina, Italy
Università degli Studi di Milano, Dipartimento di Chimica Organica e Industriale, Via Venezian, 21, 20133 Milano, Italy
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 April 2011
Accepted in revised form 9 May 2011
Available online 23 May 2011
3,5-O-dicaffeoyl-4-O-malonilquinic acid (1) (irbic acid) has been isolated for the first time from
cell cultures of Centella asiatica and till now it has never been reported to be present in the
intact plant. Evidence of its structure was obtained by spectroscopic analyses (MS/NMR).
Besides 1, cell cultures produce also the known 3,5-O-dicaffeoylquinic acid, chlorogenic acid,
and the triferulic acid 2 (4-O-8′/4′-O-8″-didehydrotriferulic acid). Biological activities were
evaluated for compound 1, which showed to have a strong radical scavenging capacity,
together with a high inhibitory activity on collagenase. This suggests a possible utilization of
this substance as a topical agent to reduce the skin ageing process.
Keywords:
Centella asiatica
Cell culture
Apiaceae
Antioxidant activity
Collagenase inhibitory activityy
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
acetylenes, triterpenoid saponins (asiaticoside, madecasso-
side) and their respective sapogenins (asiatic and madecassic
Centella asiatica (L.) Urban, previously also named
Hydrocotyle asiatica L., and commonly referred to as Indian
pennywort and gotu kola, is an herbaceous plant belonging
to the Apiaceae family with great medicinal value. It has been
used in Traditional Medicine as an antipyretic, diuretic and
antibacterial drug and in the treatment of skin disease, vein
insufficiency and mental disorders [1–3]. It is native to Asia and
mainly found in India, Pakistan and Madagascar, but the plant
also grows in tropical and equatorial Africa, America and the
tropical regions of Oceania. It has been widely cultivated as a
vegetable or spice in China, Southeast Asia, India, Sri Lanka,
Africa and Oceanic countries.
acid).
Due to the great demand for these major compounds, many
researchers have attempted to overproduce them through the
in vitro culture technique [5–7].
In our laboratory, Centella cell cultures were prepared
with the aim of producing asiaticoside and madecassicoside,
but besides low quantities of these products, unexpected
considerable amounts of caffeoyl derivatives were found.
After extraction, four caffeoyl derivatives were isolated and
identified.
The main product corresponded to 3,5-O-dicaffeoyl-4-O-
malonilquinic acid (1), a new compound for which we pro-
pose the name of irbic acid. The minor ones were the known
3,5-O-dicaffeoylquinic acid, chlorogenic acid and triferulic
acid (2). The structures of all these compounds have been
elucidated by means of HR-MS and NMR and by comparison
with known samples.
Recently, cosmetic preparations obtained from this plant
are worldwide on the market for the treatment of cellulite [4].
The main bioactive compounds of C. asiatica include poly-
Surprisingly, the presence of phenolic compounds, strongly
absorbing in the UV range between 300 and 330 nm has never
⁎
Corresponding author. Tel.: +39 02 50314077; fax: +39 02 50314078.
E-mail address: bruno.danieli@unimi.it (B. Danieli).
0367-326X/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2011.05.008

F. Antognoni et al. / Fitoterapia 82 (2011) 950–954
951
been observed in C. asiatica plant extracts, even if the plant is
present in the herbal market since at least two decades.
secondary OHs with two 3-(3′,4′-dihydroxyphenyl)-propenoic
acid (caffeic acid) units and with another acyl group. The 1H-
NMR spectrum exhibited signals for two caffeoyl moieties (see
Experimental), and signals for the quinic acid protons at
5.63 ppm (H-3, ddd , J=3.3 Hz), 5.23 ppm (H-4, dd, J=10.2
and 3.4 Hz), 5.70 ppm (H-5, ddd, J=11.5, 10.2 and 4.5 Hz),
2.40 ppm (H-2eq, dd, J=15.3 and 3.46 Hz), 2.09 ppm (H-2ax,
dd, J=15.5 and 3.1 Hz) and 2.21 ppm (H-5eq and H-5ax,
apparent broad d, J=11 Hz).
Considering the molecular formula, the presence of a
quinic acid moiety and of two caffeic residues, a C3H2O3 unit
was missing, pointing for a malonic acid residue for the
remaining acil substituent.
However, in spite of careful inspection of 1H- and 13C-NMR
spectra, no evidences for an additional methylene signal could
be detected. By chance, changing the NMR solvent from CD3OD
to Py-D5 we could find in the 1H spectrum a broad singlet at
3.62 ppm and in the 13C spectrum a signal at 42.87 ppm that
were clearly attributed to the methylene of the malonic acid
residue.
A plausible explanation of the lack of methylene signal in
CD3OD is a rapid exchange of the active methylene protons
with the mobile deuterons of the solvent.
The clue that allowed to determine unambiguously the
position of the caffeoyl moieties at C-3 and C-5 was the observa-
tion that a sample of 1 in CD3OD suffered a spontaneous hy-
drolysis of the malonic unit to give an almost equimolar mixture
of 1 and 3,5-O-dicaffeoylquinic acid. Subsequently, a mixture of
3,5-O-dicaffeoylquinic acid and 4,5-O-dicaffeoylquinic acid was
formed as a result of an internal migration process.
Thus, after one week, the 1H-NMR spectrum showed in
addition to the signals of 1, new signals at 3.92 ppm (H-4, dd,
J = 10.1 and 3.5 Hz), 5.40 ppm (H-3, ddd, J = 3.2 Hz),
5.55 ppm (H-5, td, J=10.3 and 5.4 Hz), These values are in
complete agreement with those of the quinic acid moiety of
3,5-O-dicaffeoylquinic acid [8]. After 6 weeks the signals of 1
completely disappeared and together with those of 3,5-O-
dicaffeoylquinic acid, signals appeared at 4.32 ppm (H-3, ddd,
J=3.0 Hz), 5.12 ppm (H-4, dd, J=10.3 and 3.0 Hz), 5.66 ppm
(H-5, td, J=10.5 and 6.2 Hz), due to 4,5-O-dicaffeoylquinic
acid [9]. Finally, complete hydrolysis with MeOH furnished
quinic acid methyl ester, identical to an authentic sample.
A plausible structure of a 3,5-O-dicaffeoyl-4-O-malonyl
quinic acid has been attributed to one component present in
a total of 53 compounds found in the whole plant extract of
Erigeron breviscapus on the basis of HPLC analysis, MSn data
and without isolation [10].This paper is therefore the first
report on the isolation and structural and chemical character-
ization of irbic acid.
3. Results and discussion
3.1. Structural studies
Before extraction, ascorbic acid was added to the cell
cultures of C. asiatica in order to prevent oxidation by air of
the caffeoyl derivatives causing browning of the solution.
Irbic acid (1) was isolated from cell cultures by extraction
with EtOH and purification by column chromatographies on
LH20 and RP-C18 resins.
Compound 1, obtained as a white amorphous powder,
showed a UV spectrum having its maximum absorption at
328 nm and a typical shape pointing out a caffeoyl derivative.
A second peak at 220 nm, absent in the phenylethanoid
structures (e.g. verbascoside) and characteristic of the quinic
acid derivatives, was present.
The HRESIMS spectra in the positive and negative modes
obtained with a FT-ICR instrument (see Experimental) showed
a very complex cluster of molecular ions due to extensive cat-
ionization, accompanied by peaks due to the loss of CO2 and of
a C-3 fragment.
However, from the observation of the peaks at m/z 601
in the negative mode and of cationated peaks at m/z 663 and
647 in the positive mode, it was possible to attribute to com-
pound 1 the MW of 602 corresponding to the molecular for-
3,5-O-Dicaffeoylquinic acid and chlorogenic acid were
identified by comparison of their NMR spectra with those of
authentic samples, respectively.
Compound 2 showed UV maxima at 294 and 314 nm char-
acteristic of ferulic acid structures. The HR-ESI-MS spectrum
in the negative mode showed a [M-H]− ion at m/z 577.13446,
corresponding to a MW of 578 and to a molecular formula of
C30H26O12 [calc 577.13515]. This was confirmed by the positive
mula C28 H 26 O15
.
spectrum which showed sodium adducts at m/z 601.13204, [for
The homo-, heteronuclear and J-correlated NMR spectra in
CD3OD suggested the presence of a 1,3,4,5-tetrahydroxycy-
cloesan-1-carboxylic acid (quinic acid) substituted at the
C ,
30H26O12Na+, calc 601.13208] , 623.11354 [for C30H25O12Na2+
calc 623.11359] and 645.09560 [for C30H24O12Na3+, calc
645.09571].

952
F. Antognoni et al. / Fitoterapia 82 (2011) 950–954
100
80
60
40
20
0
as positive controls. Thus, compound 1 turned out to have a
IC50=2.29 µg/ml
very high radical scavenging efficacy. It can result to be even
more effective than ascorbic acid, since the antioxidant activity
of caffeic acid derivatives is maintained at very low concentra-
tions and virtually no pro-oxidant activity has been found for
the quinic acid esterified derivatives of caffeic acid even in the
presence of copper ions, as reported by Grace and Logan [12].
Skin anti-aging activity, as measured by the collagenase
inhibition assay, was also evaluated in the presence of
increasing concentrations of irbic acid. In this assay, a type
1 collagen gel is incubated with the enzyme collagenase and
with a specific test compound. If the test substance inhibits
the activity of collagenase, the collagen gel will not be
degraded, and the amount of this non-degraded form is
estimated through the spectrophotometric analysis of the
hydroxyproline content, which represents the 11–12% of total
amino acids content in collagen. Thus, the amount of
hydroxyproline is indirectly proportional to collagenase
activity.
0.01
0.1
1
10
Irbic acid concentration (µg/ml)
Fig. 1. DPPH radical scavenging capacity (expresses as % DPPH reduction) in
the presence of different doses of irbic acid.
The 1H-NMR spectrum (see Experimental) showed an AB
system for the vinilic protons of one ferulic acid residue, three
ABX systems for the benzene protons of three unsubstituted
ferulic acids and two singlets for the β-vinilic protons of α-
substituted ferulic acids. In spite of some differences due to the
solvent used, these data are in agreement with those reported
for a triferulic acid (4-O-8′/4′-O-8″-didehydrotriferulic acid)
previously isolated from saponified maize bran [11]. Therefore
this structure is attributed to compound 2.
As shown in Fig. 2, irbic acid exerts a clear and significant
inhibitory effect on collagenase activity. The inhibition is
strictly dose-dependent in the range 50–250 μg/ml, thereaf-
ter remaining constant.
The degradation of the collagen matrix in the dermal skin
layers by external agents such as UV light or as a consequence
of physiological ageing is one of the major factors bringing to
wrinkle formation. Thus, the ability of irbic acid to significantly
inhibit collagenase activity, together with the small size and
the hydrophobicity of the molecule, represents an important
finding, since it suggests a possible utilization of this substance
as a topical agent to reduce the skin ageing process.
3.2. Biological activities
Triferulic acid is also a previously described and structurally
identified molecule, but has been little tested for its biological
properties. In the DPPH antioxidant test, triferulic acid has a
much lower antioxidant capacity compared with irbic acid
(data not shown) and this result appears to be correlated to the
covalent links of the phenolic hydroxy moieties of the caffeic
acids.
An initial screening for the biological activities associated
to the isolated irbic acid was the antioxidant and radical
scavenging activity evaluated by the DPPH assay. In Fig. 1 the
percentage of DPPH reduction is reported as a function of
increasing concentrations of this compound in the range from
0.05 μg/ml to 10 μg/ml. From the plotted graph, IC50 values
were obtained. Irbic acid showed an IC50 value of 2.29 μg/ml,
which is comparable to that of ascorbic acid and caffeic acid
(2.1 μg/ml and 1.15 μg/ml, respectively), and much lower than
that of resveratrol (10.96 μg/ml) and rutin (16.47 μg/ml), used
4. Experimental
4.1. General methods
120
100
80
NMR spectra were recorded at 400 MHz (1H) and 100 MHz
(
13C) on a Bruker instrument. Chemical shifts are quoted in δ
(ppm) and coupling constants are in Hz.
HR-ESI-MS spectra were acquired with a FT-ICR Bruker
Daltonics Apex II instrument equipped with a 4.7 Tesla cryo-
magnet. Compounds were dissolved in MeOH/H2O.
HPLC analyses were carried out using an Agilent Technol-
ogies mod HP1100 instrument equipped with a diode array
detector. Separations were performed on a Luna C18(2) column
(3 μm particles, 4.6×250 mm; Phenomenex) in the following
conditions: solvent A: H3PO4 0.1% added with 0.5% solvent B.
Solvent B: H3PO4 0.1% in CH3CN added with 0.5% solvent A.
Gradient from 20 to 40% solvent A in B. Flow 0.8 mL/min.
Column chromatographies were performed on medium pres-
sure Büchi preparative columnsfilled with lypophilic Amberlite
XAD-4 (Rohm and Haas), Sephadex LH-20 (Pharmacia) or RP-
C18 (Merck).
60
40
20
0
0
CTR
g/ml
g/ml
g/ml
g/ml
g/ml
g/ml
µ
µ
µ
µ
µ
µ
50
100
250
500
750
1000
Fig. 2. Percent of collagenase inhibition by different concentrations of irbic
acid. CTR=negative control.

F. Antognoni et al. / Fitoterapia 82 (2011) 950–954
953
4.2. Cell cultures
4.3.1. 3,5-O-dicaffeoyl-4-O-malonylquinic acid (1)
White-grey amorphous powder; [α]D28—220 (c 0.18, MeOH);
UV (MeOH) λmax 245sh, 299, 327 nm; + NaOH 265sh, 309, 373;
1H NMR (CD3OD) 7.64 and 6.26 (AB system, J=15.1, H-3′ and
H-2′ or H-3″ and H-2″), 7.55 and 6.39 (AB system, J=15.9, H-3″
and H-2″ or H-3′ and H-2′), 7.10 and 7.07 (each 1 H, J=1.96, H-5′
and H-5″), 6.98 and 6.90 (each 1 H, dd, J=7.8, 2.0, H-6′ and
H-6″), 6.80 and 6.78 (each 1 H, d, J=7.9, H-5′ and H-5″), 5.70
(1 H, ddd, J=11.5, 10.2, 4.5, H-5), 5.63 (1 H, apparent quartet,
J=3.3, H-3), 5.23 (1 H, dd, J=10.2, 3.4, H-4), 2.40 (1 H, dd,
J=15.3, 3.46, H-2eq), 2.21 (1 H, apparent d, H-5eq and H-5ax),
2.09 (1 H, dd, J=15.5, 3.1, H-2ax); 13C NMR (CD3OD), selected
data: 76.40 (C-1), 75.17 (C-4), 71.53 (C-3), 69.85 (C-5).
Cell cultures of C.asiatica were obtained from plants grown
in a glass house of the Botanical Garden of the University of
Bologna in June 2008. A voucher specimen (no. BOLO 0500914)
is kept in the Herbarium of the Department of Biology of
University of Bologna (BOLO).
To obtain calli, leaves were surface-sterilized with a 15%
commercial bleach solution (final concentration of active
chlorine 5%), dissected in small fragments and placed on Petri
dish containing solidified Gamborg B5 (13) medium supple-
mented with with 20 g/L sucrose, 1 g/L plant peptone, 1 mg/L
kinetin, 1 mg/L naphthalenacetic acid and 0.2 mg/L indolacetic
acid at pH 6.5. Explants were incubated at 25 °C in the dark and,
after the callus induction, were subcultured every 30 days.
Calli grown on solid Gamborg B5 medium, as described
above, were subjected to subculture for at least 3 months and
subsequently were used to inoculate Erlenmeyer flasks of 1 L
volume, with the final liquid Gamborg B5 medium content
of 250 mL for each flask. After 14 days at 25 °C in the dark, the
cells were collected and the secondary metabolites extracted.
1H-NMR (Py-D5), selected data: 6.11 (2 H, m, H-3 and H-5),
5.79 (1 H, dd, J=7.03, 3.30, H-4), 3.62 (2 H, broad s, malonic
methylene); 13C NMR (Py-D5), selected data: 74.80 (C-1), 72.82
(C-4), 69.53 and 68.83 (C-3 and C-5), 42.87 (malonic methy-
lene), 38.69 (C-6), 36.90 (C-2).
HRESIMS (negative mode): m/z 639.08213 [calcd for C28
H24 O15 K1 (−1), 639.07578]; 633.04917 [calcd for C27 H23
O13 K2 (−1), 633.04183]; 623.10752 [calcd for C28 H24 O15
Na1 (−1), 623.10184]; 617.07414 [calcd for C27 H23 O13 K1
Na1 (−1), 617.06789]; 601.12362 [calcd for C28H25O15(−1),
601.11989]; 595.08991 [calcd for C27 H24 O13 K1 (−1),
595.08595]; 579.11503 [calcd for C27 H24 O13 Na1 (−1),
579.11201]; 575.05831 [calcd for C25 H21 O12 K1 Na1 (−1), calc
575.05733]; 557.13023 [for C27 H25 O13 (−1), calc 575.05733];
553.07632 [for C25 H22 O12 K1 (−1) calc 553.07538]; 537.10263
(calcd for C25 H22 O12 Na1 (−1), 537.10144), 515.11975 [calcd
for C25 H23 O12 (−1) 515.11975].
4.3. Extraction and isolation
15-days old cell cultures (15 L) were filtered and the
medium discarded. Cells were added with 15 g of solid ascorbic
acid, extracted with two volumes of EtOH and homogenized.
After centrifugation, the extract was concentrated under re-
duced pressure. The phenolic compounds present in the
aqueous residue were quantitatively recovered by solid phase
extraction using a column (7×50 cm) containing 1.5 kg of
XAD-4 resin suspended in 5% aqueous HCOOH. The column
was washed with H2O and eluted with 80% EtOH. Fractions
containing caffeic derivatives were concentrated under re-
duced pressure and the aqueous residue freeze dried, yielding
25.72 g of grey powder. By HPLC analyses, the material showed
the presence of 6.50 g of 1, 2.71 g of 3,5-O-dicaffeoylquinic acid,
2.98 g of 2 and 1.12 g of chlorogenic acid, all expressed as chlo-
rogenic acid.
In order to separate the different caffeic derivatives, the
powder was resuspended in H2O and completely dissolved
with a few drops of conc. NH4OH. Solution was submitted to
Sephadex LH20 CC (8×24 cm) suspended in aqueous 15% EtOH
added with 1% HCOOH. The column was first eluted with the
same solvent, and fractions (100 mL/each) were collected and
analyzed by HPLC. Fractions 45–53were pooled and lyophilized
obtaining mg 753 of pure chlorogenic acid. Fractions 63–71
gave 2.37 g of 3,5-O-dicaffeoylquinic acid. Elution was contin-
ued with aqueous EtOH 30% in HCOOH 1% and fractions 79–98
gave 9.53 g of material containing a mixture of compounds 1
and 2. In order to separate these products, the powder obtained
by freeze drying fractions 79–98 was submitted to a second
column chromatography in reverse phase on RP-C18 Lichro-
sphere (Merck). A column (5×40 cm), filled with resin
suspended in aqueous CH3CN 1% added with HCOOH 1%, was
used. The column was eluted with increasing amounts of
CH3CN added of 1% HCOOH and 150 mL fractions were
collected. Pure compound 1 (4.25 g) and pure compound 2
(1.95 g) were recovered in the fractions with 20% CH3CN and
50% respectively.
HRESIMS (positive mode): m/z 717.00445 [(for C28 H24 O15
K3 (+1) calc 717.00209]¸ 701.02885 [ for C28 H24 O15 Na1
K2 (+1) calc 701.02815]¸ 685.05479 [for C28 H24 O15 Na2
K1(+1) calc 685.05422] ; 669.08240 [for C28 H24 O15 Na3 (+1)
calc 669.08028], 663.07343 [for C28H25O15Na1K1(+1) calc
663.07227]; 647.10067 [for
C28H25O15Na2(+ 1) calc
647.09834]; 615.02881 [for C25 H22 O12 Na1 K2 (+1)
calc 615.02776]; 599.05572 [for C25 H22 O12 Na2 K1(+1) calc
599.05382]¸ 577.0730 [for C25 H23 O12 Na1 K1 (+1) calc
577.07188]; 561.09932 [for C25 H23 O12 Na2 (+1) calc
561.09794].
After one week. 1H NMR (CD3OD): in addition to the signals
of 1, selected signals due to 3,5-O-dicaffeoylquinic acid are
present at 5.56 (1 H, td, J=10.3 and 5.4, H-5), 5.39 (1 H, ap-
parent quartet, J=3.2, H-3), 3.94 (1 H, dd, J=10.0 and 3.6,
H-4), 2.31 (1 H, dd, J=14.5 and 3.1, H-2eq), 2.18 (2 H, apparent
d, H-5eq and H-5ax), 2.08 (1 H, dd, J=14.6 and 3.7, H-2ax); 13
C
NMR (CD3OD), selected data: 76.25 (C-1), 72.20 and 72.10 (C-3
and C-5), 69.80 (C-4).
After six weeks. 1H NMR (CD3OD): in addition to the
signals of 3,5-O-dicaffeoylquinic acid, selected signals due
to 4,5-O-dicaffeoylquinic acid are present at: 5.66 (H-5, td,
J=10.5, 6.2 Hz), 5.12 (H-4, dd, J=10.3, 3.0 Hz), 4.32 (H-3,
ddd, J=3.0 Hz); 13C NMR (CD3OD): selected data: 75.40
(C-4), 69.50 (C-5), 69.10 (C-3).
4.3.2. 3,5-O-dicaffeoylquinic acid
1H NMR (CD3OD) selected data: 5.54 (1 H, td, J=10.3, 5.3,
H-5), 5.40 (1 H, apparent q, J=3.15, H-3), 3.92 (1 H, dd,
J=10.0, 3.4, H-4), 2.31 (1 H, dd, J=14.6, 3.0, H-2 eq), 2.18
(2 H, apparent d, H-5eq and H-5ax), 2.09 (1 H, dd, J=14.7,

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F. Antognoni et al. / Fitoterapia 82 (2011) 950–954
3.6, H-2ax). 13C NMR (CD3OD), selected data: 76.30 (C-1),
72.18 and 72.09 (C-3 and C-5), 69.80 (C-4). For an authentic
sample of 3,5-O-dicaffeoylquinic acid: 5.52 (1 H, td, J=10.2,
5.5, H-5), 5.42 (1 H, apparent q, J=3.5, H-3), 3.92 (1 H, dd,
J=9.1, 3.4, H-4).
4.4.2. Collagenase inhibition assay
The test utilized was that described by Madhan et al. [14].
Specifically, 500 μL of a standard Type I collagen solution
(1 mg/mL) were plated on tissue culture dish (35×10 mm)
kept at room temperature for 10 min, until solidification of the
solution occurred.
4.3.3. Chlorogenic acid
The following samples were incubated with the collagen
gel:
1H NMR (CD3OD): 7.58 and 6.28 (each 1 H, d, J=15.9, H-7′
and H-6′), 7.03 (1 H, d, J=2.0, H-2′), 6.95 (1 H, dd, J=8.2, 2.0,
H-6′), 6.75 (1 H, d, J=8.3, H-5′), 5.36 (1 H, dt, J=10.4, 5.2,
H-5), 4.15 (1 H, apparent q, J=3.1, H-3), 3.75 (3 H, s, OMe),
3.70 (1 H, dd, J=7.7, 3.4, H-4), 2.2–2.0 (4 H, m. H2-6 and H2-2).
13C NMR (CD3OD), selected data: 76.0 (C-4), 73.6 (C-1), 72.6
(C-5), 67.5 (C-3). For an authentic sample of chlorogenic acid:
5.34 (1 H, dt, J=10.3, 5.3, H-5), 4.17 (1 H, apparent q, J=3.1,
H-3), 3.75 (3 H, s, OMe), 3.68 (1 H, dd, J=7.8, 3.5, H-4).
1. Type IA Collagenase 30 μg/mL (70 U/mL) in TES buffer
(positive control)
2. Irbic acid at different dilutions+collagenase 30 μg/mL
(70 U/mL), always in TES buffer
3. TES buffer (without enzyme and sample to be tested) as
negative control 1 mL of these solutions was added in each
culture dish and incubated for 15 h at 37 °C. After this time,
the excess medium was discharged and the non-cleaved
collagen washed twice with phosphate buffered saline (PBS)
1×. The remaining collagen was hydrolyzed in sealed hy-
drolysis tubes with 6 N HCl for 15–18 h. The hydrolyzed
products were evaporated to dryness with nitrogen gas flux
in an evaporator(PIERCE Reacti-VapIII, 18785). Theacid free
residue was redissolved in a defined volume of TES buffer
and the percentage of hydroxyproline was determined using
the method of Edwards and O'Brien [15] after preparing a
standard curve with this amino acid.
4.3.4. 4-O-8′/4′-O-8″-Didehydrotriferulic acid (Triferulic acid)
(2)
Amorphous solid; UV (MeOH) λmax 245sh, 294, 314 nm; 1H
NMR (CD3OD): 7.63 and 6.42 (each 1 H, d, J=16.1, H-7 and
H-8), 7.41 and 7.38 (each 1 H, s, H-7 and H-7′), 7.61, 7.37 and
7.34 (each 1 H, d, J=1.9, H-2, H-2′ and H-2″), 7.12, 7.10
and 7.07 (each 1 H, dd, J=8.3, 1.9, H-6, H-6′ and H-6″), 6.81 and
6.75 (2) (each 1 H, d, J=8.3, H-5, H-5′ and H,5″), 3.99, 3,79
and 3.60 (each 3 H, OMe); 13C NMR (CD3OD): 170.52, 166,76,
166.54, 150.68, 150.21, 149.87, 149.32, 148.97, 148.72, 145.97,
140.41, 138.90, 130.85, 128.97, 128.59, 127.94, 126.51,
125.70, 123.18, 117.97, 116.31, 115.17(3), 114.65, 114.12,
113.28, 56.99, 56.54, 56.15 HRESIMS (negative mode):
577.13446 [for C30H25O12(−1) calc 577.13515], 367.67631,
345.87728; HRESIMS (positive mode): m/z 601.13204, [for
The percentage of collagenase inhibition is expressed as:
ꢀh
ꢁ
μg= ml hydroxyprolinesample−μg = ml hydroxyprolinepositive control
h
ꢁi
ꢀ
μg = ml hydroxyprolinenegative control × 100
C30H26O12Na(+1), calc 601.13208], 623.11354 [for C30H25
O12Na2(+1), calc 623.11359], 645.09560 [for C30H24O12
Na3(1), calc 645.09571], 365.10574, 203.05255.
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4.4.1. Antioxidant activity
Free radical scavenging activity was measured using the
radical chromogen DPPH (2,2-diphenyl-1-pycryl-hydrazyl)
according to the method described by Molyneux [13]. Irbic acid
was diluted to different final concentrations of 0.05, 0.1, 0.5, 1.0,
2.5, 5.0, 10.0 μg/mL and was added to a 0.1 mM DPPH solution.
After 15 min, the absorbance values were spectrophoto-
metrically measured at 517 nm (Shimadzu UV-1601, Japan).
A negative control was prepared without the sample.
The percentage of radical reduction (quenching, Q) is ex-
pressed as:
Q ¼ 100ðAbsnegative ctrÞ–AbssampleÞ=Absnegative control
Results were expressed as IC50 (Inhibitory Concentration)
values, defined as the concentration of substrate that induces
50% of DPPH reduction (loss of colour).
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