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Recovery of bioactive phenolic compounds from olive mill waste

water, pomegranate peel, and european cranberrybush (viburnum
opulus l.) juice by preparative MPLC

Article  in  Journal of Liquid Chromatography & Related Technologies · April 2014

DOI: 10.1080/10826076.2013.825843

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RECOVERY OF BIOACTIVE PHENOLIC

COMPOUNDS FROM OLIVE MILL WASTE
WATER, POMEGRANATE PEEL, AND
EUROPEAN CRANBERRYBUSH (VIBURNUM
OPULUS L.) JUICE BY PREPARATIVE MPLC
a a a
Rasim Alper Oral , Mahmut Doğan & Kemal Sarioğlu
a
Department of Food Engineering, Faculty of Engineering , Erciyes
University , Kayseri , Turkey
Accepted author version posted online: 06 Sep 2013.Published
Click for updates online: 01 Apr 2014.

To cite this article: Rasim Alper Oral , Mahmut Doğan & Kemal Sarioğlu (2014) RECOVERY OF
BIOACTIVE PHENOLIC COMPOUNDS FROM OLIVE MILL WASTE WATER, POMEGRANATE PEEL, AND
EUROPEAN CRANBERRYBUSH (VIBURNUM OPULUS L.) JUICE BY PREPARATIVE MPLC, Journal of Liquid
Chromatography & Related Technologies, 37:13, 1827-1836, DOI: 10.1080/10826076.2013.825843

To link to this article: http://dx.doi.org/10.1080/10826076.2013.825843

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 Journal of Liquid Chromatography & Related Technologies, 37:1827–1836, 2014
Copyright © Taylor & Francis Group, LLC
ISSN: 1082-6076 print/1520-572X online
DOI: 10.1080/10826076.2013.825843

RECOVERY OF BIOACTIVE PHENOLIC COMPOUNDS

FROM OLIVE MILL WASTE WATER, POMEGRANATE PEEL,
AND EUROPEAN CRANBERRYBUSH (VIBURNUM OPULUS L.)
JUICE BY PREPARATIVE MPLC

Rasim Alper Oral, Mahmut Doğan, and Kemal Sarioğlu

Downloaded by [Erciyes University] at 08:05 17 February 2015

Department of Food Engineering, Faculty of Engineering, Erciyes University, Kayseri,

Turkey

◻ In this study, preparative medium pressure liquid chromatography (MPLC) was used for the
purification of some phenolic compounds including hydroxytyrosol, chlorogenic acid, and puni-
calagin from olive mill waste water (OMWW), European cranberrybush (Viburnum opulus
L.) juice, and pomegranate peel, respectively. While the polyphenols of OMWW and European
cranberrybush juice were extracted with ethyl acetate (EtOAc), methanol was used for pomegran-
ate peel extraction. At the end of the extraction process, the solvents were removed by rotary evapo-
rator and the residue passed into the water phase. After the centrifugation and microfiltration
procedure, the injection of samples was carried out by MPLC. The outflow of the colon was col-
lected at specific periods and the predetermined polyphenols for purification were identified with
High-Pressure Liquid Chromatography with UV Detector (HPLC-UV). After the collection of
defined fractions, chlorogenic acid, and punicalagin were concentrated with a rotary evaporator
and transformed into particle form by freeze drying. Hydroxytyrosol was produced as concentrated
liquid by using a rotary evaporator. The purities of the obtained compounds were determined as
90.2% for hydroxytyrosol, 92.5% for chlorogenic acid, and 97.1% for punicalagin. Moreover,
the inhibition percentages of these compounds on 2,2-diphenyl-1-picrylhydrazyl (DPPH) were
established, respectively, as 76.01, 84.90, and 94.64.

Keywords chlorogenic acid, hydroxytyrosol, MPLC, punicalagin, polyphenol,

purification

INTRODUCTION
Oxygen derived free radicals are known as reactive oxygen spe-
cies (ROS).[1] ROS may act as initiators of degenerative events, such
as the damaging of DNA and mutation, some kinds of cancer, aging,
and heart diseases. Many plant polyphenols such as chlorogenic acid,
Address correspondence to Rasim Alper Oral, Department of Food Engineering, Faculty of
Engineering, Erciyes University, 38039 Kayseri, Turkey. E-mail: [email protected]
 1828 R. A. Oral et al.

pomegranate peel extracts, and hydroxytyrosol have been proposed

as potential antimutagenic and anticarcinogenic agents.[2,3] Moreover,
some polyphenols can inhibit/reduce the formation of carcinogenic/
mutagenic heterocyclic aromatic amines and potential carcinogenic/
neurotoxic acrylamides.[4,5]
Chlorogenic acid, punicalagin, and hydroxytyrosol are the most
abundant polyphenols in European cranberrybush (Viburnum opulus)
juice, pomegranate peel, and olive mill waste water (OMWW), respec-
tively.[6–8] Hydroxytyrosol can inhibit the degeneration of retinal pig-
ment epithelial cells resulting from oxidative stress, pro-inflammatory
cytokines, increasing influenza A virus (H9N2), replication and infec-
tion of HIV-1, and phytopathogens.[9–13] Furthermore ester, which has
α-lipoic acid is reported to have prevented multiplication of colon can-
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cer cells (HT 29), was found to have more effective antitumor activity
than hydroxytyrosol and α-lipoic acid.[14] Chlorogenic acid can prevent
the formation of mutagenic/carcinogenic N-nitroso compounds due to its
inhibiting N-nitrosation reaction in vitro. Moreover, chlorogenic acid can
inhibit damage to DNA in vitro.[15] Punicalagin has been used pharma-
cologically due to its anti-inflammatory, hepatoprotective, and antigeno-
toxic properties.[16]
Therefore, the present paper reported the preparative separation
and purification of hydroxytyrosol from OMWW, chlorogenic acid from
European cranberrybush, and punicalagin from pomegranate peel by
medium pressure liquid chromatography (MPLC). The production of
these polyphenols from natural sources allows their use as dietary supple-
ments, as stabilizers in foods, and in cosmetic preparations and nutraceu-
tics. And European cranberrybush is first time used as chlorogenic acid
source in the present studies.

MATERIALS AND METHODS

Reagents, Materials and Apparatus
Oleuropein was obtained from ABCR GmbH & Co. Methanol, HCl,
ethyl acetate, and chlorogenic acids were obtained from Merck. Punicalagin
was obtained from the laboratories of Ege University. Adsorbent for use in
MPLC was procured from LiChroprep RP-18 (15–25 µm). The glass colon
for MPLC (4.9087 cm2 × 40 cm) was obtained from Biochemfluidics. The
extracts were prepared with an extraction shaker (Simsek Laborteknik,
Turkey), an ultrasonic bath (Bandelin Sonorex, Germany), and a mag-
netic mixer (Heidolph MR Hei-Standard, Germany). Ultra pure water
was provided by Millipore-Simplicity 185. Centrifugation was performed
by a Hettich-320 centrifuge.
 Recovery of Bioactive Phenolic Compounds 1829

Preparative analysis was performed on an Agilent 1100 series MPLC

system with a 5 mL injection loop (Rheodyne, USA), glass colon (4.9087
cm2 × 40 cm, RP-18), and a diode array detector (DAD). Chromatographic
analysis was carried out on a Shimadzu (Japan) Prominence HPLC system
with a C18 colon (15 × 4.6 mm, 5 µm), a SIL-20A HT auto sampler, and UV
detector. Freeze drying was performed by a Labconco (USA) freeze dryer.

Preparation of Crude Punicalagin

The pomegranate peel was obtained from a fruit juice processing
plant located in the city of Denizli in Turkey. It was dried to constant
weight at room temperature and then ground into powder. Thirty grams
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of the powder was put into a 100 mL flask and methanol was added up to
the 100 mL level. The mixture was processed in an extraction shaker for
1 hr. After filtration using Whatman 4 paper, the methanol was removed
under reduced pressure by rotary evaporation. Then, 15 mL distilled
water was added and incubated in an ultrasonic bath for 5 min. Finally, the
suspension was centrifuged at 5000 rpm for 5 min and the supernatant
was filtered by using a syringe filter (0.22 µm) and analyzed by MPLC.

Preparation of Crude Hydroxytyrosol and Chlorogenic Acid

OMWW was obtained from a discontinued processing plant located in
the city of Mersin in Turkey. European cranberrybush juice was derived
from squeezed fruit obtained from a market located in the city of Kayseri
in Turkey. Extraction was carried out by using the batch procedure with
ethyl acetate. One liter of each of the samples was placed into a glass ves-
sel with a volume of 2 L. Then, 250 mL EtOAc was added and mixed with
a magnetic mixer for 30 min. The mix was transferred to a separation
funnel and rested until the end of the separation of two phases. After
removing the upper phase, this operation was repeated three times. The
gathered upper phases were evaporated under vacuum at 40°C in a rotary
evaporator. The residue was re-dissolved in 25 mL of ultra pure water and
centrifuged at 5000 rpm for 5 min. The precipitate of chlorogenic acid
was re-dissolved in 25 mL of water and centrifuged again. After filtration
by a syringe filter (0.22 µm), samples were analyzed by MPLC.

MPLC Separation Procedure

MPLC was conducted with an Agilent 1100 series equipped with a
5 mL injection loop (Rheodyne, USA), glass colon (4.9087 cm2 × 40 cm,
RP-18), and DAD. The phenolic compounds were detected at 280 nm and
 1830 R. A. Oral et al.

360 nm. Solvent gradients were formed by the dual pumping system by
changing the ratio of solvent A [water–acetic acid (99:1, (v/v)] to solvent
B (methanol). The solvent gradient elution program is demonstrated in
Table 1. The flow rate was 1.3 mL min−1.

Preparation of Standard of Hydroxytyrosol

15 mg oleuropein was heated with 1 mL of 3 N HCl at 100°C for 10
min in a water bath. 1 mol hydroxytyrosol, 1 mol elenolic acid, and 1 mol
glucose were formed by the hydrolyzing of 1 mol oleuropein.[17]
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HPLC Analysis and Identification of Chlorogenic Acid,

Punicalagin, and Hydroxytyrosol
The polyphenol composition of OMWW, European cranberrybush
juice, pomegranate peel, and each fraction from MPLC such as chloro-
genic acid, punicalagin, and hydroxytyrosol were analyzed/confirmed
by HPLC-UV. A Shimadzu Prominence LC system with C18 colon at a
colon temperature of 30°C was used for the analyses. Ultra pure water
with acetic acid –1% (solvent A) and methanol (solvent B) were used as
the mobile phase. Gradient elution was performed according to the fol-
lowing program: solvent B was 5% until 8 min and increased from 5% to
70% in 25 min then decreased to 5% in 7 min the analysis lasted for 45
min. Confirmation of identity was made by comparison of the retention
time against the pure chlorogenic acid, punicalagin, and hydroxytyrosol.
Sample calculations were made by comparison of the peak area with that
of the standard.

TABLE 1 MPLC Solvent Gradient

Elution Program

Time (min) Solvent B (%)

0 0
5 0
20 15
110 15
150 30
170 30
250 80
300 30
 Recovery of Bioactive Phenolic Compounds 1831

Total Antioxidant Capacity of Chlorogenic Acid, Punicalagin,

and Hydroxytyrosol
The total antioxidant capacity of phenolic compounds from prepara-
tive MPLC on DPPH was determined according to the procedure defined
by Boskou et al.[18] with some modifications. Reduction of DPPH due to the
polyphenols, antioxidant scavenging activity, was observed by a decrease in
absorbance at 517 nm. The color turned from purple to yellow when the
antioxidants blocked all of the free radicals. Solutions of chlorogenic acid,
punicalagin, and hydroxytyrosol (in concentrations of 500 ppm) were pre-
pared in methanol. 0.1 mL of each of the methanol solutions was added to
3.9 mL of DPPH solution (0.1 mM). After mixing, the solutions were incu-
bated in the dark for 15 min. The absorbance of the control (DPPH with-
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out any antioxidant) was measured daily and kept in the dark. Then, the
absorbance values of the polyphenol solutions were determined by compar-
ing them with the control. Percentage of inhibition was estimated as follows:

Inhibition% = (C–S)/C*100

where C: absorbance value of control at 517 nm; S: Absorbance value of

sample at 517 nm.

RESULTS AND DISCUSSION

Separation and Quantification of Chlorogenic Acid, Punicalagin,
and Hydroxytyrosol
Chlorogenic acid was separated from Flos Lonicerae by high-speed coun-
ter-current chromatography. 16.9 mg chlorogenic acid of 94.8% purity was
obtained.[19] The amount of chlorogenic acid in European cranberrybush
juice was found by HPLC-UV to be 718 mg L–1. This purified fraction was
freeze dried and 512 mg of chlorogenic acid was obtained; the purity of
the chlorogenic acid after separation was 92.5%. The chromatograms of
European cranberrybush juice and purified chlorogenic acid are shown
in Figures 1 and 2. The α- and β-anomers of punicalagin were previously
reported and total pomegranate tannins contain 80–85% of punicalagin
anomers. Also, 1 kg of husk includes 58–60g total pomegranate tannins.[20]
105 mg of punicalagin of 92% purity was obtained from 5g of husk by high-
speed counter-current chromatography.[21] The punicalagin level of pome-
granate peel was determined as 30.765 g kg–1. 2170 mg of punicalagin of
97.1% purity was acquired from 90g of husk by MPLC. Figures 3 and 4 show
the results obtained from pomegranate husk by MPLC and the methanol
extract of husk. 0.091–1 g hydroxytyrosol was produced by using different
 1832 R. A. Oral et al.
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FIGURE 1 HPLC chromatogram of purified punicalagin from pomegranate peel at 370 nm.

separation processes from 1 L of OMWW.[22] One quantitative study has

demonstrated that hydroxytyrosol is the main component in OMWW and
in EtOAc extract (1.433 g L–1).[6] The amount of hydroxytyrosol in OMWW
was estimated as 1459 mg L–1. 1028 mg of hydroxytyrosol of 90.2% purity
was acquired from 1 L of by-product by MPLC. The hydroxytyrosol content
of OMWW and the purified part are shown in Figures 5 and 6.

Total Antioxidant Capacity of Chlorogenic Acid, Punicalagin,

and Hydroxytyrosol
Punicalagin is a high molecular weight (MW = 1108 Da) water solu-
ble phenolic compound which scavenges a variety of ROS due to its high
antioxidant properties (presence of 16 dissociable–OH groups) in vitro.[16]
Also chlorogenic acid and hydroxytyrosol are reported to have exerted

FIGURE 2 HPLC chromatogram of methanol extract of pomegranate peel at 370 nm.

 Recovery of Bioactive Phenolic Compounds 1833
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FIGURE 3 HPLC chromatogram of purified chlorogenic acid from European cranberrybush

(Viburnum opulus L.) juice at 370 nm.

FIGURE 4 HPLC chromatogram of European cranberrybush (Viburnum opulus L.) juice (10–1 dilution)
at 370 nm.

FIGURE 5 HPLC chromatogram of purified hydroxytyrosol from OMWW at 280 nm.

 1834 R. A. Oral et al.
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FIGURE 6 HPLC chromatogram of OMWW (10–1 dilution) at 280 nm.

strong antioxidant activity higher than 2,6-di-tert-butyl-hydroxytoluene

(BHT).[15,23] The inhibition percentages of hydroxytyrosol, chlorogenic
acid, and punicalagin on DPPH were established as 76.01, 84.90, and 94.64,
respectively.

CONCLUSION
The chromatographic purification of hydroxytyrosol, chlorogenic acid,
and punicalagin from OMWW, European cranberrybush juice, and pome-
granate peel, respectively, was carried out. It is well known that the prefer-
ence for natural food ingredients has increased due to the belief that they
are safer, healthier, and less hazardous than synthetic additives. In addition
they may be used instead of BHA and BHT owing to their antioxidant
properties. Finally, the purified polyphenols described above have been
included in foods, cosmetics, and pharmaceuticals. Furthermore, this study
may supply researchers with a rich source of polyphenols due to the bio-
logical properties of these polyphenols and their effects on diseases such as
cancer and other conditions related with viruses (HIV-1, influenza, etc.). In
addition, the by-products of food processing such as OMWW and pome-
granate peel, could be recycled by purification of phenolic compounds and
the environmental problem of wastewater may also be prevented.

FUNDING
The author would like to thank the Unit of Scientific Investigations in
Erciyes University for its financial support of this work.
 Recovery of Bioactive Phenolic Compounds 1835

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