Food Chemistry 124 (2011) 271–277

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Food Chemistry
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Phenolic acid content and radical scavenging activity of extracts from medlar
(Mespilus germanica L.) fruit at different stages of ripening
Jiri Gruz a, Faik Ahmet Ayaz b, Hülya Torun b, Miroslav Strnad a,*
a
Laboratory of Growth Regulators, Palacký University and Institute of Experimental Botany ASCR, Šlechtitelů 11, CZ-783 71 Olomouc, Czech Republic
b
Department of Biology, Karadeniz Technical University, 61080 Trabzon, Turkey

a r t i c l e i n f o a b s t r a c t

Article history: The medlar fruit (Mespilus germanica L.) has been gaining commercial importance in recent years, attract-
Received 20 October 2009 ing research on its chemical composition. In this study, we have investigated how the degree of ripeness
Received in revised form 22 April 2010 affects the concentrations and proportions of phenolic acids. The DPPH scavenging activity and the total
Accepted 8 June 2010
phenolic content (TPC) were determined. Five stages of fruit maturity were studied and eight phenolic
acids (protocatechuic, 4-hydroxybenzoic, syringic, 3-hydroxybenzoic, caffeic, salicylic, 4-coumaric and
sinapic) were determined by HPLC–MS. The concentrations of phenolic acids mostly decreased as the
Keywords:
fruit ripening progressed, except for insoluble ester-bound phenolics, which increased at the early stages
Phenolic acids
HPLC
of maturity and decreased only during the ripe to over-ripe stage of maturity. The DPPH scavenging activ-
Mass spectrometry ity also decreased during fruit maturation, suggesting a decrease of natural antioxidants in fruit. A strong
Fruit correlation between TPC and antioxidant capacity measured by the DPPH method was found. The data
Ripening presented demonstrates the influence of ripening on the phenolic acid content and antioxidant properties
Mespilus germanica of medlar fruit.
Medlar Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction
The phenolic compounds are a structurally diverse class of plant
secondary metabolites. Generally they possess an aromatic ring
bearing one or more hydroxyl substituents (Robards, Prenzler,
Tucker, Swatsitang, & Glover, 1999). As widely distributed non-
nutrient biologically active compounds, phenolics are also reported
to have multiple effects on tissue maturation processes, defence
mechanisms (Kubo & Matsumoto, 1984) and sensory qualities of
plant-derived food products, including astringency, bitterness
and aroma (Macheix, Fleuriet, & Billot, 1990).
In plants, the phenolic compounds are mainly produced by phe-
nylpropanoid pathway. The early metabolites of the phenylpropa-
noid pathway are hydroxycinnamic acids (HCA), which form
together with the structurally related hydroxybenzoic acids
(HBA) a specific group of plant metabolites called phenolic acids.
This group of phenolic compounds was found to be abundant in
many foods and beverages of plant origin, e.g. 200 ml of instant
coffee contains 50–150 mg of chlorogenic acid (Clifford, 1999).
Researchers and food manufacturers are interested in phenolic
acids because of their strong antioxidant properties, abundance
Abbreviations: PHA, phenolic acids; TPC, total phenolic content; HBA, hydroxy-
benzoic acid derivatives; HCA, hydroxycinnamic acid derivatives; nd, not detected.
* Corresponding author. Tel.: +420 585634850; fax: +420 585634870.
E-mail address: strnad@prfholnt.upol.cz (M. Strnad).
in the human diet, and their probable role in the prevention of var-
ious diseases associated with oxidative stress, such as cancers, car-
diovascular diseases and inflammation (Scalbert & Williamson,
2000). Phenolic acids are regarded as one of the functional food
components in fruits, and are thought to contribute to the health
effects of plant-derived products by scavenging free radical spe-
cies, inhibiting free radical formation, and preventing oxidative
damage to DNA (Lodovici, Guglielmi, Meoni, & Dolara, 2001). The
phenolic composition of plant foods is dependent on the plant
genotype (Harborne, 1984) and on environmental factors during
growth and postharvest. For instance, the content of fruit phenolics
and antioxidant capacity of two pawpaw fruit clones (Kobayashi,
Wang, & Pomper, 2008) and of a sour cherry (cv. Marasca) cultivar,
showed an apparent decrease during maturation (Pedisic, Levaj,
Dragovic-Uzelac, & Kos, 2007).
Medlar (Mespilus germanica L., fam: Rosaceae) fruit, for long
overlooked, has been gaining commercial importance as a food-
stuff for human consumption, stimulating the research into its
chemical and nutrient content during fruit maturation and ripen-
ing (Ayaz, Demir, Torun, Kolcuoglu, & Colak, 2008; Ayaz, Huang,
Chuang, VanderJagt, & Glew, 2002; Glew, Ayaz, Sanz, et al., 2003;
Glew, Ayaz, VanderJagt, et al., 2003). The plant is a spiny shrub
or small (2–3 m) tree, growing to a height of 4–6 m in cultivated
forms. The brown, sometimes reddish tinged, pear and apple-
shaped fruit of medlar with five stony seeds are subglobose or

0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.06.030
272 J. Gruz et al. / Food Chemistry 124 (2011) 271–277
pyriform, and is crowned by foliaceous sepals, ranging from 1.5 to
3 cm in diameter and weighing from about 10 g to more than 80 g
(Bignami & Tsipouridis, 1998; Browicz, 1972).
The astringency of the fruit is well known. It has been reported
that the bletted (bletting: the ripening of fruit, especially of fruit
stored until the desired degree of decay and softness is attained)
pulp or syrup of the fruit was a popular remedy for enteritis (Ayaz
et al., 2002; Bignami & Tsipouridis, 1998). Modern medicine recog-
nises medlar’s ability to act as a diuretic, and as a treatment for
kidney and bladder stones (Baytop, 1999).
The medlar fruit is widely consumed in Turkey. In northeast
Anatolia (Turkey) both wild and cultivated forms are grown, and
their fruit is used in different ways. In October, the hard fruit is
harvested from the medlar trees and stored in cold, dark and ven-
tilated places. However, a substantial part of the crop at different
stages of maturity is left on the trees, and is harvested later after
fruit softening has started. The fruit is often consumed or sold in
the local markets and stores. To our knowledge, information
regarding the changes in particular phenolic constituents and their
free radical scavenging activity throughout the fruit maturation is
limited. Here, we have studied how the accumulation of phenolic
acids in the medlar fruit is altered during maturation and fast rip-
ening. The present study also provides information about the typ-
ical phenolic acids in medlar fruit and their modifications during
fruit development, specifically at the immature, ripe and over-ripe
stages of maturity.
2. Materials and methods
2.1. Chemicals and reagents
Analytical grade standards of 3,5-dihydroxybenzoic, gallic, pro-
tocatechuic, 4-hydroxybenzoic, 3-hydroxybenzoic, gentisic, 2-cou-
maric, 4-coumaric, caffeic, ferulic, syringic, sinapic, chlorogenic,
salicylic, trans-cinnamic and 3-coumaric acids, were purchased
from Sigma–Aldrich Fine Chemicals (St. Louis, MO, USA). Deute-
rium-labelled standards of 4-hydroxybenzoic acid (2,3,5,6-D4)
and salicylic acid (3,4,5,6-D4) were purchased from Cambridge Iso-
tope Laboratories (Andover, MA, USA). Formic acid and acetonitrile
for HPLC were purchased from MERCK (Darmstadt, Germany), and
deionised water was prepared using Simplicity 185 deioniser (Mil-
lipore, Bedford, MA, USA).
2.2. Plant material
The medlar (M. germanica L., wild type, fam: Rosaceae) fruit was
randomly harvested from fourteen 20-year-old trees from various
single genotypes of bulk populations, located on the hillsides in
Trabzon (500–600 m over sea level), northeast Anatolia (Turkey).
The flowers were considered to be in full bloom on May 10, 2003
and the fruit were sampled on five occasions, after 129, 159, 191,
206 and 216 days. Based on the days, the fruit was divided into
three distinct maturity classes: immature (129 and 159), ripe
(191 and 206) and over-ripe (216), respectively. One kilogram of
medlar fruit was gathered in triplicate at each sampling time.
The harvested fruit was stored at 80 °C and freeze-dried. After
lyophilisation, the hard, dried fruit was broken up with a steel
hammer and then ground to a fine powder using a stainless steel
mill (125 lm particle size, Cole-Parmer Analytical Mill, USA).
2.3. Extraction of phenolic acids from medlar fruit
Phenolic acids were extracted and isolated according to Cvik-
rová, Hrubcová, Vágner, Machácková, and Eder (1994). Triplicate
160 g fruit samples were frozen in liquid N2 and ground in 80%
methanol in a high-speed blender. The homogenate was filtered
and used either directly for analysis of antioxidant activity in pri-
mary extract (fraction F0) or it was further processed. To analyse
the phenolic acids, the F0 fraction was concentrated under vacuum
in a rotary evaporator. The concentrate was acidified with 1 M HCl
to pH 2 and then extracted four times with 100 ml diethyl ether.
The ether phase was evaporated to dryness under vacuum at
40 °C, the residue was dissolved in methanol, filtered using a
0.45 lm microfilter (Whatman No. 1), and then used for the anal-
ysis of free phenolic acids (fraction F1). The aqueous phase, i.e. the
F0 fraction after extraction with diethylether, was hydrolysed with
2 M NaOH for 4.5 h under nitrogen at room temperature, then acid-
ified with 6 M HCl to pH 2, extracted with diethylether, evaporated,
dissolved in methanol, filtered, and then used for the analysis of
methanol soluble ester-bound phenolic acids (fraction F2). The so-
lid residue obtained after filtration of the primary homogenate was
hydrolysed with 2 M NaOH for 4.5 h under nitrogen at room tem-
perature, then acidified with 6 N HCl to pH 2, extracted with dieth-
ylether, evaporated, dissolved in methanol, filtered, and then used
for the analysis of methanol insoluble ester-bound phenolic acids
(fraction F4).
2.4. HPLC–MS instrumentation and conditions
Phenolic acids were analysed as described earlier (Ayaz,
Hayirlioglu-Ayaz, Gruz, Novak, & Strnad, 2005). Briefly, internal
standards of deuterium-labelled salicylic (3,4,5,6-D4) and
4-hydroxybenzoic (2,3,5,6-D4) acids were added to extracted and
filtered sample solutions to a final concentration of 10 5 M. Ten
microlitre of sample solutions were injected onto a reversed phase
column (Luna Phenyl-Hexyl, 5 lm, 250  2 mm, Phenomenex, Tor-
rance, CA). Solvent (A) consisted of 5 mM formic acid and solvent
(B) consisted of methanol. HPLC–MS analyses were performed on
an Alliance 2690 Separations Module (Waters, Milford, MA, USA)
linked simultaneously to a PDA 996 (Waters, Milford, MA, USA)
and a ZMD 2000 single quadrupole mass spectrometer, equipped
with an electrospray interface (Micromass, Manchester, UK). Data
were processed by a MassLynx software (Data Handling System
for Windows, version 4.0, Micromass, Altrincham, UK).
2.5. Determination of DPPH scavenging activity and total phenolic
content (TPC)
The radical scavenging activity of extracts was determined
using a modified assay for DPPH (1,1-diphenyl-2-picrylhydrazol)
scavenging (Joyeux, Mortier, & Fleurentin, 1995). A 0.1 ml aliquot
of the extract solution (0.5–1 mg ml 1), 2 ml of 0.05 M acetate buf-
fer (pH 5.5), 1.9 ml of methanol and 1 ml of 0.5 mM DPPH were
mixed. The mixture was shaken immediately after adding DPPH
and was allowed to stand at room temperature in darkness. The
decrease in absorbance at 517 nm was measured after 30 min until
the reaction reached a plateau. These experiments were run in
duplicate. The inhibitory percentage of DPPH was calculated as fol-
lows: Scavenging effect (%) = [1 (A Ab)/A0]  100%, where A0 is
the A517 of DPPH without sample (control), A is the A517 of sample
and DPPH and Ab is the A517 of sample without DPPH (blank).
The total phenolic content (TPC) was determined in fractions F0,
F1, F2 and F4 according to Moyer, Hummer, Finn, Frei, and Wrols-
tad (2002). One-hundred times diluted fruit extracts were mixed
with a reaction mixture containing Folin–Ciocalteu reagent, incu-
bated at 40 °C, and the absorbance of the mixture was determined
at 755 nm on a UV–vis spectrophotometer (Techcomp 8500 II,
South Korea). TPC was expressed as mg of gallic acid equivalents
per g of fresh weight (mg of GAE/g fw).
 J. Gruz et al. / Food Chemistry 124 (2011) 271–277 273

2.6. Statistical analysis
A random experimental design was run in triplicate for each
developmental stage. The data presented are the means of three
separate extractions and determinations. Data on phenolic acids,
total phenolic contents and radical scavenging activity were evalu-
ated by analysis of variance, using the general linear procedure, a
package program of the Statistical Analysis System (SAS Institute
Inc., Cary, NC, USA). Duncan’s Multiple Range Test was employed
to determine the statistical significance of differences among the
means. All comparisons were made at 5% (P = 0.05) level of
significance.
3. Results
The variations in the colour of the skin, pulp and state of matu-
rity during fruit maturation of medlar are summarised in Table 1.
The mean fruit weights were 4.68 ± 0.36 and 8.16 ± 0.66 g at
immature (between 129 and 159 days), 8.51 ± 0.26 and
8.62 ± 0.83 g at ripe (between 191 and 206 days) and 8.92 ± 0.86
at over-ripe (216 days) stages of maturity. In short, the initial fast
growth beginning (129–159 days) was followed by a slow and
gradual increase of fruit weight throughout ripening.
3.1. Changes in free phenolic acids (F1) during fruit ripening
Free phenolic acids were determined by an HPLC–MS method,
which allows up to 16 phenolic compounds to be identified and
quantified simultaneously (see Section 2). Table 2 shows the pro-
file of free phenolic acids determined in the fruit harvested at the
five maturation stages. Four hydroxybenzoic acids (HBA), including
protocatechuic, 4-hydroxybenzoic, syringic and salicylic acids, as
well as two hydroxycinnamic acids (HCA), including caffeic and
sinapic acids, were identified and quantified in the fruit (Table 2,

Table 1
Variation in the colour of the skin and pulp of medlar (Mespilus germanica L.) fruit at different stages of ripening.

Harvest date Days Fruit ripeness Fruit skin and pulp colour
15 September 2003 129 Immature Skin greenish (40%) and brownish (60%), pulp white, fruit very hard
15 October 2003 159 Immature Skin brownish, pulp white, fruit hard
15 November 2003 191 Ripe Skin completely brown, pulp white, fruit half soft
30 Nov 2003 206 Ripe skin completely dark brown, pulp whitish (50%)-brownish (50%), fruit soft and juicy
10 December 2003 216 Over-ripe Skin completely dark brown, pulp fully dark brown, fruit fully soft

Table 2
Content of phenolic acids (ng/g fw) in the free fraction (F1), the ester-bound methanol soluble fraction (F2), and the ester-bound methanol insoluble (F4), isolated from the medlar
(Mespilus germanica L.) fruit during ripening. Values represent the mean ± SD of three separate extractions and determinations. For comparison of means, analysis of variance was
used. Values with the same letter are not significantly different at P < 0.05. Means were compared within each row of the data.

Phenolic acid Immature Ripe Over-ripe

129 days* 159 days 191 days 206 days 216 days
F1
Protocatechuic 224.1 ± 15.1c 282.9 ± 19.4d 186.0 ± 19.9b 181.9 ± 8.9b 112.6 ± 21.4a
4-Hydroxybenzoic 32.0 ± 6.1a 33.1 ± 6.2a 97.2 ± 11.8b 280.3 ± 12.2d 215.7 ± 41.8c
Syringic 28.8 ± 7.1c 39.9 ± 1.9d 15.1 ± 1.0b nda nd
Salicylic 42.6 ± 3.9c 20.2 ± 4.6b 19.7 ± 3.1b 3.0 ± 0.3a 1.3 ± 0.5a
Caffeic 73.6 ± 8.6c 10.5 ± 1.5a 12.9 ± 4.2a,b 20.7 ± 4.8b 9.3 ± 3.8a
Sinapic 24.8 ± 0.9c 8.2 ± 0.6b 4.1 ± 0.8a 3.6 ± 0.4a 3.7 ± 0.8a
RHBAb 327.5 376.1 318 465.2 329.6
RHCAc 98.4 18.7 17 24.3 13
RPHAd 425.9 394.8 335 489.5 326.6
F2
Protocatechuic 911.4 ± 55.7c 695.8 ± 144.0b 423.4 ± 4.1a 402.0 ± 11.4a 307.3 ± 5.0a
4-Hydroxybenzoic 260.1 ± 27.2a 339.0 ± 9.2b 394.9 ± 19.1c 318.7 ± 6.8b 289.1 ± 6.8a
3-Hydroxybenzoic 131.8 ± 2.3d 136.4 ± 3.9d 95.9 ± 8.0c 27.2 ± 1.1b 6.7 ± 0.6a
Syringic 4.4 ± 0.8b nd nd nd nd
Caffeic 3398.2 ± 555.1c 3348.5 ± 215.6c 2646.7 ± 86.3b 744.0 ± 35.9a 353.4 ± 27.9a
Sinapic 25.4 ± 4.2b 28.0 ± 3.5b 47.7 ± 4.0c 6.4 ± 1.1a 2.3 ± 0.3a
RHBA 1307.7 1171.2 914.2 747.9 603.1
RHCA 3423.6 3376.5 2694.4 750.4 355.7
RPHA 4731.6 4547.7 3608.6 1498.3 958.8
F4
Protocatechuic 210.3 ± 16.8b 301.4 ± 21.4c 421.4 ± 5.7d 191.4 ± 7.2ab 182.5 ± 3.0a
4-Hydroxybenzoic 46.5 ± 2.5a 211.4 ± 2.5c 253.7 ± 21.7d 184.7 ± 19.7b 61.2 ± 3.5a
3-Hydroxybenzoic 6.6 ± 1.2b 7.5 ± 2.2b 12.8 ± 1.9c 5.6 ± 1.3b 1.9 ± 0.9a
Salicylic 8.0 ± 0.6a 8.0 ± 1.1a 16.3 ± 2.6b 8.5 ± 1.4a 6.6 ± 0.8a
4-Coumaric 12.5 ± 2.1b 57.0 ± 3.4d 30.7 ± 2.3c 15.2 ± 8.4b 2.6 ± 0.7a
Caffeic 136.0 ± 11.1b 423.5 ± 90.6d 214.2 ± 3.2c 155.2 ± 99.9b,c 15.1 ± 3.7a
RHBA 272.3 527.4 704.2 390.2 252.2
RHCA 148.5 480.5 244.9 170.4 17.7
RPHA 420.8 1007.9 949.1 560.6 269.9
F1 + F2 + F4
RPHA 5578.3 5950.4 4892.7 2548.4 1555.3
a
Not detected.
b
Sum of benzoic acid derivatives.
c
Sum of trans-cinnamic acid derivatives.
d
Sum of phenolic acids.
274 J. Gruz et al. / Food Chemistry 124 (2011) 271–277

a Benzoic acid derivates Functional group

R1 R2 R3 R4
R1 R2 4-Hydroxybenzoic acid H H OH H
Protocatechuic acid H OH OH H
HOOC R3
Salicylic acid OH H H H
R4 Vanillic acid H OCH 3 OH H
Syringic acid H OCH3 OH OCH3

b
R5 COOH
Trans-cinnamic acid derivates Functional group
R5 R6
HO 4-Coumaric acid H H
R6
C af f ei c ac i d H OH
Sinapic acid OCH3 OCH3

Fig. 1. Structures of benzoic (a) and trans-cinnamic (b) acid derivatives found in the medlar (Mespilus germanica L.) fruit.

Fig. 1). In contrast to the phenolic esters (F2), free HBA were found
at several fold higher amounts (3–25 times) than free HCA. The
most abundant compounds detected were protocatechuic and 4-
hydroxybenzoic acids, which are both HBA (Table 2). Although
the total contents of hydroxybenzoic (RHBA), hydroxycinnamic
(RHCA) and phenolic acids (RPHA) changed significantly
(P < 0.05) during maturation, they did not exhibit a consistent
decreasing trend, due to the specific increase in 4-hydroxybenzoic
acid at 206 days. The highest content of RPHA was found at the
late ripe stage of maturity, towards the end of November
(206 days, 489.5 ng/g fw).
protocatechuic, 3-hydroxybenzoic and caffeic acids showed a grad-
ual decrease from immature to over-ripe fruit, the contents of 4-
hydroxybenzoic and sinapic acids increased to their maximum at
191 days and then, their concentrations were gradually decreasing
during the final stages of fruit maturation. Syringic acid, a HBA, was
determined only at the initiation of the immature stage of maturity
(129 days). In contrast to the free fraction, a statistically significant
(P < 0.05) decreasing trend of RHBA, RHCA and RPHA was found
(Table 2). Thus, at the over-ripe stage of maturity, the concentra-
tions of RHBA, RHCA and RPHA decreased to 50%, 20% and 10%
of their initial state, respectively.

3.2. Changes in the methanol soluble ester-bound phenolic acids (F2) 3.3. Changes in methanol insoluble ester-bound phenolic acids (F4)
during fruit ripening during fruit ripening

Six phenolic acids, four of which were HBA and two were HCA
(Table 2), were identified and quantified in the soluble phenolic es-
ter fraction. The major component of this fraction was caffeic acid,
with a highest concentration of 3398.1 ng/g fw in the immature
fruit (129 days). This fraction contained the highest amount of phe-
nolic acids, being from 3 to 10 times higher than the respective
contents of fractions F1 and F4. Although the concentrations of
Four HBA (protocatechuic, 4-hydroxybenzoic, vanillic and sali-
cylic acids) and two HCA (4-coumaric and caffeic acids) were quan-
tified in this fraction (Table 2). The concentrations of all insoluble
(cell wall-bound) phenolic acids increased at the early stages of
maturity but they began to decrease during the ripe to the over-
ripe stage of maturity. All individual HBA reached maximum con-
centrations at 191 days, while both HCA reached the maximum at

120
F1 Free acids F2 Soluble esters
F4 Insoluble esters F0 Primary extract
100
DPPH scavenging (%)

80

60

40

20

0
129 159 191 206 216
Days

Fig. 2. DPPH scavenging activity of free (F1), methanol soluble ester-bound (F2), and methanol insoluble ester-bound (F4) fractions, as well as of the primary extract (F0),
isolated from the medlar (Mespilus germanica L.) fruit during ripening.
 J. Gruz et al. / Food Chemistry 124 (2011) 271–277 275

9
F1 Free acids F2 Soluble esters
8 F4 Insoluble esters F0 Primary extract

TPC (mg GAE/g FW)

7
6
5
4
3
2
1
0
129 159 191 206 216
Days

Fig. 3. Total phenolic content (TPC) of free (F1), methanol soluble ester-bound (F2) and methanol insoluble ester-bound (F4) fractions, as well as of the primary extract (F0),
isolated from the medlar (Mespilus germanica L.) fruit during ripening.

159 days. The contents of RHBA, RHCA and RPHA were altered
significantly (P < 0.05), exhibiting a gradual increase during the a 120
early stages of maturity (129–191 days), followed by a dramatic
decrease, in particular at the over-ripe stage of maturity.
100 y = 16.867x + 4.364
DPPH scavenging activity (%) R2 = 0.8865
3.4. Radical scavenging activity (DPPH) and total phenolic content
(TPC) of individual fractions isolated from the fruit 80

Both the DPPH scavenging activity and the TPC of the primary
extract (F0), the free (F1), the methanol soluble ester-bound (F2) 60
and the methanol insoluble ester-bound (F4) fractions exhibited
a decreasing trend during ripening (Figs. 2 and 3). The linear corre-
lation of DPPH activity with TPC was strong and significant in all 40
fractions tested (F0, r = 0.991; F1, r = 0.949; F2, r = 0.949; and F4,
r = 0.993; data not shown), being the highest in the primary extract
20
fraction (F0). The TPC and DPPH activity of the primary extract
fraction (F0) were also closely correlated (r = 0.953 and 0.941,
respectively) with the sum of the methanol-soluble phenolic acids,
0
i.e. F1 + F2 fractions (Fig. 4). 0 1 2 3 4 5 6
Sum of methanol soluble phenolic acids F1+F2
4. Discussion (µg/g FW)

Fruit tissues are able to synthesize phenolic compounds, and b 9

changes in this content can be induced by biotic and abiotic stress
conditions (Kataoka, Beppu, Sugiyama, & Taira, 1996). The phenolic 8
acids can be divided into two classes, derived from benzoic and y = 1.3604x + 0.6643
cinnamic acids, respectively (Fig. 1). The hydroxycinnamic acids, 7 R2 = 0.9081
whose major components are p-coumaric, caffeic, ferulic and sina-
TPC (µg GAE/g FW)

pic acids, are observed more commonly than the hydroxybenzoic 6

acid derivatives (Hamauzu, 2006; Robards et al., 1999). These phe-
nolic acids normally exist as bound forms, such as glycosylated 5
derivatives or esters of quinic, shikimic, tartaric or other organic
acids. The hydroxybenzoic acids are generally minor phenolics in 4
edible plants, but are often observed as components of complex
structures, such as hydrolysable tannins (Clifford & Scalbert, 2000). 3
In the present study, hydroxycinnamic acids were quantified in
2
substantially higher amounts than hydroxybenzoic acids, but their
concentrations mostly decreased during ripening, with the excep-
1
tion of insoluble ester-bound phenolic acids. The simultaneous de-
crease of free esters of phenolic acids together with their
0
accumulation in insoluble form at the early stages of maturity 0 1 2 3 4 5 6
(129–191 days) suggests that they are progressively bound to the
Sum of methanol soluble phenolic acids F1+F2
cell walls. The integration of phenolic esters into cell walls is an
(µg/g FW)
important mechanism by which plants defend themselves against
pathogens and strengthen the cell walls (Dixon, Harrison, & Lamb, Fig. 4. The correlations of the content of methanol-soluble phenolic acids with
1994). Furthermore, the accumulation of these esters protects the either the DPPH scavenging activity (a) or the total phenolic content (b).
276 J. Gruz et al. / Food Chemistry 124 (2011) 271–277
cells against membrane damage caused by reactive oxygen species
(Tamagnone et al., 1998). The subsequent decrease in insoluble
phenolics throughout the maturation (191–216 days) probably oc-
curs by the transformation (polymerisation, oxidation and conju-
gation) of bound phenolic acids into states that are no longer
detectable by spectrophotometry and HPLC–MS. Apparently, the
decrease in phenolics is also related to the reduction of primary
metabolism in the over-ripe fruit, resulting in a lack of substrates
necessary for the biosynthesis of phenolic compounds. In accor-
dance with our findings, the concentrations of free and bound phe-
nolic acids in orange and grape fruits declined during growth, and
reached a minimum at maturity (Hanna, Michael, Russell, & Uri,
1991). In persimmon (Diosypros lotus L.) fruit, a steady amount of
phenolic acids, which was followed by a sharp decrease during fi-
nal stages of ripening, was also reported (Ayaz, Kadioglu, & Reuna-
nen, 1997). Furthermore, the phenolic acids in cherry laurel were
found at highest concentration 12 weeks after flowering, and con-
stituted 61.22 mg/100 g dw. Afterwards, their concentrations con-
tinually decreased to 2.94 mg/100 g dw in the ripest fruit (Ayaz,
2001). The previously described species-independent decrease in
phenolics during fruit ripening was further confirmed in the pres-
ent study on medlar by the spectrophotometric determination of
TPC, which showed the same decreasing trend as the phenolic
acids (Fig. 2).
Accordingly, we have shown previously that TPC, expressed as
catechin equivalents, also decreased during ripening of the medlar
fruit (Ayaz et al., 2008). Despite its name, TPC refers more to the
antioxidant capacity than to the phenolic composition of the plant
extracts. This results from the electron-transfer mechanism of Fo-
lin–Ciocalteu reaction (Huang, Ou, & Prior, 2005). Accordingly, we
have shown that the TPC of both individual fractions and the pri-
mary extract (F0) were strongly correlated (r > 0.949) with the
antioxidant capacity measured by the DPPH assay. In addition,
the sum of the methanol-soluble phenolic acids (F1 + F2), mea-
sured by HPLC–MS, was correlated with both the TPC (r = 0.953)
and the DPPH scavenging activity (r = 0.941) of the primary extract
(F0), indicating that the methanol-soluble phenolic acids and their
esters act as endogenous antioxidants in medlar fruit (Fig. 4). Alen-
Ruiz, García-Falcón, Perez-Lamela, Martinez-Carballo, and Simal-
Gandara (2009) also observed a strong correlation between TPC
and DPPH assays in wine. However, they found only a poor corre-
lation between the HPLC-determined phenolic compounds, (phe-
nolic acids, anthocyanins, flavonol and catechins) and the DPPH
scavenging activity of wine samples, suggesting that those pheno-
lics were not the active radical scavenging compounds. In this
study we also evaluated the impact of the extraction procedure
on the antioxidant capacity of extracts, and compared the DPPH
activity of the methanol soluble fractions (F1 and F2) with the pri-
mary extract fraction (F0). Surprisingly, the DPPH scavenging activ-
ity of the primary extract was found to be similar to that of the
fraction of soluble esters alone (F2; Fig. 2). The relatively high
activity of the fraction of soluble esters could be explained by the
increased radical scavenging activity of aglycones released after
alkaline hydrolysis compared to their respective esters.
As the ripening of the medlar fruit progresses to the over-ripe
stage of maturity, an apparent softening of the texture is inevitable
(Table 1). Both the ripe fruit (between 191 and 206 days) with a
brown skin and a pulp with a moderate soft texture and the
over-ripe fruit, (more than 206 days) are consumed, even if the
over-ripe fruit loses most of its nutritional value (Ayaz et al.,
2002, 2008; Glew, Ayaz, Sanz, et al., 2003; Glew, Ayaz, VanderJagt,
et al., 2003). In the present study, we have found that the over-ripe
fruit also lost its functional properties and antioxidant capacity. As
an illustration, the over-ripe medlar fruit (216 days) had more than
two times lower antioxidant capacity, expressed either as TPC or
DPPH, than the ripe fruit (206 days), harvested only 10 days later
(Fig. 2). From this point of view, it is extremely important to take
into account the stage of maturity of the fruit when evaluating
its health-promoting potential. Based on our experimental results,
it should be recommended that the fruit is consumed at the ripe
maturity stages (between 191 and 206 days) at the latest, in order
to provide more dietary antioxidants.
In summary, the data presented in the present study demon-
strate that the concentrations of phenolic compounds and the anti-
oxidative capacity are significantly altered by the degree of
maturation of the medlar fruit, especially during the ripe to over-
ripe periods. Consequently, the harvest time must be scheduled
carefully to gain the highest portion of bioactive compounds.
Acknowledgements
The present study was supported by the Grant of the Research
Fund of Karadeniz Technical University (Project No.
2003.111.004.5), Academy of Sciences of the Czech Republic
(KAN 200380801), and the Czech Ministry of Education (MSM
6198959216). Support by the Research Fund of Karadeniz Techni-
cal University is greatly appreciated.
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