SE-HPLC separation of myosin complex with tannins from bearberries (Arctostaphylos uva-ursi L. Sprengel) leaves - a short report.

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_Vol. 27, 2009, No. 5: 386–391_ _Czech J. Food Sci._

# SE-HPLC Separation of Myosin Complex with Tannins of Bearberry ( Arctostaphylos uva-ursi L. Sprengel) Leaves – A Short Report

_Ryszard AMAROWICZ_ _1_ _, Ronald B. PEGG_ _2_ _and Agnieszka KOSIŃSKA_ _1_

_1Institute of Animal Reproduction and Food Research of Polish Academy of Sciences,_

_Division of Food Science, Olsztyn, Poland;_ _2_ _Department of Food Science and Technology,_

_University of Georgia, Athens, USA_

Abstract

Amarowicz R., Pegg R.B., Kosińska A. (2009): SE-HPLC separation of myosin complex with tannins
from bearberries ( _Arctostaphylos_ _uva-ursi_ L. Sprengel) leaves – a short communication. Czech J. Food
Sci., 27 : 386–391.

Phenolic compounds were extracted from bearberry ( _Arctostaphylos uva-ursi_ L. Sprengel) leaves into 95% (v/v) ethanol. The tannin constituents were separated from the crude extract using Sephadex LH-20 column chromatography
with 95% (v/v) ethanol and 50% (v/v) acetone as the mobile phases. Myosin was isolated and purified from excised
pork knuckle muscles using the standard salt-solution extraction procedure followed by gel filtration chromatography. Myosin was precipitated from the solution with bearberry-leaf tannins at pH 5.0. The recovered complex was
washed, lyophilised, and subjected to size-exclusion high performance liquid chromatography (SE-HPLC). Based on
the basic conditions of the HPLC analysis, a portion of the tannin constituents was liberated from the complex. The
UV spectra of these compounds were characterized by a maximum at ~ 300 nm. A portion of the tannins was present
in the complex with myosin, and this was confirmed by UV spectra.

Keywords : myosin; tannins; SE-HPLC; bearberry; protein-tannins complex

Nutritionists define tannins as antinutrients of
plant origin because they can precipitate proteins,
inhibit the action of digestive enzymes, and decrease the utilisation/bioavailability of vitamins
and minerals (Lachman _et al_ . 1998). The presence
of tannins in foods can also be highly undesirable,
as they contribute to astringent or bitter tastes in
certain foods and beverages (Amarowicz _et al._
2004b). On the other hand, tannins have also been
considered as “health-promoting” components in
plant-derived foods and beverages. For instance,
they have been reported to possess anticarcinogenic and antimutagenic potentials as well as

386

antimicrobial properties (Cos _et al_ . 2003; Awika
_et al_ . 2005; Amarowicz _et al_ . 2000b, 2008). Several studies have reported on the antioxidant and
antiradical activities of tannins (Amarowicz 2007;
Amarowicz _et al_ . 2000a; 2004a, 2005).

The phenomenon of protein-tannins interaction is attributed to some combinations of hydrogen and/or hydrophobic bonding (Hagerman &
Butler 1981; Siebert 1999; Papadopoulou &
Frazier 2004). Tannins bind to salivary proteins
which leads to the precipitation of insoluble complexes and the perception of astringent flavour
(Baxter _et al_ . 1997). The findings of some studies

_Czech J. Food Sci._ _Vol. 27, 2009, No. 5: 386–391_

have shown that tannins are potent free-radical
scavengers, even when bound to protein, and that
the complexes formed may act as a radical sink
within the gastrointestinal tract (Riedl & Hagerman 2001).

The antioxidative properties of tannin-rich extracts have been observed in pork model system
studies, as shown by the reduction in the formation of 2-thiobarbituric acid reactive substances
(TBARS) during the storage of cooked meat. Noteworthy is that the antioxidant activity was also
observed with the myosin-tannins complex so
prepared; the complex incorporated into a pork
model system at 500 and 750 ppm levels exhibited
an antioxidant efficacy equivalent to that of free
tannins from a bearberry-leaf ( _Arctostaphylos_
_uva-ursi_ ) extract added at 50 ppm (Pegg & Amarowicz 2004).

The objective of the present study was to examine SE-HPLC as a tool to separate and partially characterise myosin-tannins complexes. The
leaves of the bearberry ( _Arctostaphylos uva-ursi_ L.
Sprengel) plant were used as the source of tannin
constituents, due to the fact that a strong antioxidant activity has been reported for bearberry-leaf
polyphenolics (Amarowicz _et al_ . 1999; 2004a;
Pegg _et al_ . 2005).

MATERIAL AND METHODS

_Materials_ _._ All solvents used were of HPLC or analytical grade, unless otherwise specified. Bearberry
( _Arctostaphylos uva ursi_ L. Sprengel) leaves were
obtained as a gift from Dr. Branka Barl, formerly
of the Department of Plant Sciences, University of
Saskatchewan, Saskatoon, Canada. Excised pork
knuckle muscles were purchased from Mitchell’s
Gourmet Foods (Saskatoon, Canada).

_Extraction of phenolic compounds from bear-_
_berry leaves_ _._ The leaves were dried at 35°C, cutup, ground in a Moulinex coffee mill, transferred
to dark-coloured flasks, mixed with 95% (v/v)
ethanol at the material to solvent ratio of 15:100
(w/v), and then extracted in a shaking Magni
Whirl constant temperature bath (Blue M Electric Company, Model MSG-1122A-1, Blue Island,
IL) at 50°C for 30 min (Amarowicz _et al_ . 1999).
The extraction was repeated two more times,
the supernates were combined and ethanol was
evaporated under vacuum at 40°C using a Büchi
Rotavapor/Water bath (Models EL 131 and 461,

respectively, Brinkmann Instruments Ltd., Toronto,
Canada). The extracts obtained were stored at 4°C
until further analysed.

_Column chromatography_ _._ A 2-g portion of
the crude bearberry-leaf extract was suspended
in 20 ml of 95% (v/v) ethanol and applied onto a
chromatographic column (5 cm × 40 cm) packed
with Sephadex LH-20 and equilibrated with 95%
(v/v) ethanol. Low-molecular-weight phenolic
compounds were eluted from the column using
1000 ml of 95% (v/v) ethanol. To obtain the tannin
constituents, the column was washed with ~ 600 ml
of 50% (v/v) acetone (Strumeyer & Malin 1975).
Then, the organic portion was evaporated using
the Rotavapor and water was removed from the
aqueous solution by lyophilisation.

_Isolation of myosin_ _._ Myosin was isolated from
the pork knuckle muscles according to Dudziak
& Foegeding (1988) with slight modifications.
All steps involved are shown in Figure 1. For purification, gel filtration chromatography was employed. The separation conditions were as follows:
column was 3 cm × 60 cm packed with Toyopearl
WH-55F gel; the mobile phase was 20mM sodium
phosphate (pH 7.0) in 0.5M NaCl; 4-ml fractions
were collected using a fraction collector (Model
SC100, Beckman Coulter, Inc., Fullerton, Canada)
in the cold room at 4°C; and absorbance readings
at 280 nm were taken to identify the myosin elution. The collected fractions containing myosin
were lyophilised and subsequently used for the
preparation of myosin-tannins complex.

_Preparation of myosin-tannins complex_ _._ A 50-mg
portion of tannins separated via Sephadex LH-20
column chromatography was dissolved in 50 ml
of deionised water. An equal mass of myosin was
dissolved in 50 ml of 0.20M acetate buffer (pH 5.0)
containing 0.17M sodium chloride (Hagerman
& Butler 1978). The two solutions were mixed,
followed by a 30-min quiescent period at room
temperature. The reaction mixture was transferred
to a 250-ml polypropylene centrifugal tube. The
tube was centrifuged for 10 min at 10 800 × g in a
Sorvall RC-6 Plus Superspeed Centrifuge (Thermo
Fisher Scientific Inc., Waltham, USA). The supernate was carefully decanted. The precipitate
adhering to the wall of the centrifuge tube was
scraped of and the product was mixed with _ca._
20 ml of deionised water and transferred into a
50-ml beaker. The beaker and its contents were
frozen at –30°C and then placed on a shelf in a
FreeZone 2.5 Liter Benchtop Freeze Dry System

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_Vol. 27, 2009, No. 5: 386–391_ _Czech J. Food Sci._

Excised knuckle muscles from pigs

Grind, 3/8� and then 1/8� plates

Wash: 20mM EDTA, pH 7.0, @ 1:2 (w/v, 15 min)

Centrifuge at 8300 × g, 30 min

Supernate A (discard) Pellet

Repeat EDTA wash twice more

Extract: modified Hasselbach-Schneider (H-S) solution

(1:2, w/v, 15 min). Centrifuge at 10 800 × g, 30 min

Figure 1. Isolation and purification of myosin from
porcine knuckle muscles

Supernatant B (H-S) Pellet (discard)

Dilute with deionised water to � = 0.03

Centrifuge at 10 800 × g, 30 min

Supernate C (discard) Pellet

Suspend in 1.5M KCl & 0.5M Tris, pH 7.0

Add 60% (NH4)2SO4 saturation
Myosin precipitation. Centrifuge

Supernate D (discard) Pellet

Suspend in 1.5M KCl & 0.5M Tris, pH 7.0
Dialyse against buffer to remove (NH4)2SO4

Crude porcine myosin solution

Gel filtration chromatography, 3 × 60 cm Toyopearl WH-55F

Mobile phase: 20mM Na2HPO4 in 0.5M NaCl, pH 7.0

Fractionation, UV280 detection and confirmation of myosin

by SDS-PAGE

(Labconco Corporation, Kansas City, USA) until
all water was removed.

_SE-HPLC analysis_ _._ The tannins fraction and
myosin-tannins complex were analysed using a
Shimadzu HPLC system (Shimadzu Corp., Kyoto,
Japan) consisting of a LC-10AD pump, SCTL 10A
system controller, and SPD-M 10A photodiode array detector. The separation was achieved with a
TSK G2000SWXL column (7.8 mm × 300 mm, 5 µ m;
Tosoh Bioscence LLC, Montgomeryvile, USA); the
mobile phase of borate buffer (0.2M, pH 8.3) was

388

delivered at a flow rate of 0.8 ml/min; the detection
was monitored at 220 nm; and the samples were dissolved in the mobile phase (i.e., 2 mg of complex/ml
and 0.25 mg of tannins/ml) before their injection
(20 μl) onto the column (Pegg _et al._ 2008).

RESULTS AND DISCUSSION

Figure 2 depicts a chromatogram of the myosin-tannins complex (A) and the tannins frac

_Czech J. Food Sci._ _Vol. 27, 2009, No. 5: 386–391_

Figure 2. SE-HPLC separation of myosin-tannins complex (A) and tannin fraction from bearberry ( _Arctostaphylos_
_uva ursi_ L. Sprengel) leaves

tion isolated from the ethanolic bearberry-leaf
extract (B). Size-exclusion chromatography of the
tannins-myosin complex (Figure 2A) was characterised by the presence of six peaks with the
retention times of 6.57, 7.37, 8.65, 9.25, 11.99, and
15.59 minutes. Peaks 1 and 2 originated from the
tannins complex of myosin, as confirmed by UV
spectra. Figures 3: 1 and 3: 2 show an absorption
band at 284 nm which is typical for proteins, and

a shoulder at 304 nm that originated from the
tannin constituents.

The SE-HPLC chromatogram of isolated tannins (Figure 2B) was characterised by four peaks
(numbers 3 to 6) with the retention times same
as those in chromatogram 2A. UV spectra of the
separated compounds attributed to peaks 2–6
showed maxima at 300, 292, 301, and 302 nm
(Figures 3: 2, 3, 6), respectively. Compounds 3, 5,

Figure 3. UV spectra of compounds separated by SE-HPLC; the numbers are related to the peaks from Figure 2

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_Vol. 27, 2009, No. 5: 386–391_ _Czech J. Food Sci._

and 6 are most likely hydrolysable tannins, whereas
compound 4 was clearly a condensed tannin. This
conclusion is in accordance with our previous
results showing the presence of both condensed
and hydrolysable tannins in the bearberry-leaf
extract (Pegg _et al_ . 2005).

The presence of the peaks coming from tannin
constituents in chromatogram 2A indicates that the
myosin-tannins complex is not stable. Based on the
conditions of the SE-HPLC (i.e., the basic pH of the
mobile phase, 8.3), a majority of tannins are liberated from the complex. An interesting observation
is that compound 5, which is the main constituent
in the tannins fraction, shows a weak activity in the
formation of a complex with myosin.

The application of SE-HPLC reported in this study
is a new method used for the detection of complexes
of phenolic compounds with proteins. Before us, Bartolomé _et_ _al_ . (2000) used gel filtration on Sephadex
G-50 to investigate the interaction of procyanidins
with bovine serum albumin (BSA). The main methods
used by scientists are RP-HPLC and capillary electrophoresis. Hagerman _et al_ . (1998), for example, employed a C18 (ODS) column to study the mechanism
of protein precipitation with pentagalloyl glucose and
procyanidin. To estimate the binding constants between the protein and phenolic ligand, capillary electrophoresis was applied. The presence of complexes
of rapeseed proteins and phenolic constituents was
monitored by capillary electrophoresis and RP-HPLC
methods (Amarowicz _et al_ . 2003; Kosińska _et al_ .
2006). Preferential binding of sorghum tannins with
γ -kafirin was investigated using RP-HPLC and free
zone capillary electrophoresis (FZCE) (Taylor _et_
_al_ . 2007).

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Received for publication December 20, 2008

Accepted after corrections July 28, 2009

_Corresponding author:_

Doc. Dr. Ryszard Amarowicz, Division of Food Science, Institute of Animal Reproduction and Food Research,
Polish Academy of Sciences, ul. Tuwima 10, 10-747 Olsztyn, Poland
tel.: + 895 234 627, fax: + 895 240 124, e-mail: amaro@pan.olsztyn.pl

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