Effects of pine needle extracts on plasma cholesterol, fibrinolysis and gastrointestinal motility

Abstract

Pine needle (Pinus densiflora Sieb. et Zucc.) extract has been used to improve cardiovascular disorders, detoxification of nicotine, the infirmities of
age and curing diseases of unidentified symptoms in folk medicine. To determine the facts behind the traditional belief, we
tried to investigate the effects of fresh and self-fermented pine needle extracts of different aging. Fibrinolytic activities
of the extract indicated that activity depends on time and also with aging of the product. It was also found that the extract
can lower the blood plasma cholesterol and triglyceride in cholesterol fed rat. Also, Self-Fermented Pine Needle Extracts
7 years old (SFPE 7) (200 μg/mL) reduce the frequency and amplitude of pacemaker currents in Interstitial Cells of Cajal (ICC)
of murine small intestine by modulating ATP-sensitive potassium channels. Therefore, the investigation indicated that self-fermentation
improves efficacy of the pine needle extracts reducing risk of cardio-vascular related disorders and would be an important
source in nutraceutics.

Full text

Biotechnology and Bioprocess Engineering 2008, 13: 262-268
DOI/10.1007/s12257-008-0039-y
Effects of Pine Needle Extracts on Plasma
Cholesterol, Fibrinolysis and Gastrointestinal
Motility
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Department of Biotechnology and BK21 Research Team for Protein Activity Control, Chosun University, Gwangju 501-759,
Korea
^Äëíê~Åí= Pine needle (máåìë=ÇÉåëáÑäçê~ Sieb. et Zucc.) extract has been used to improve cardiovascular disorders, detoxification of
nicotine, the infirmities of age and curing diseases of unidentified symptoms in folk medicine. To determine the facts behind
the traditional belief, we tried to investigate the effects of fresh and self-fermented pine needle extracts of different aging. Fi-
brinolytic activities of the extract indicated that activity depends on time and also with aging of the product. It was also found
that the extract can lower the blood plasma cholesterol and triglyceride in cholesterol fed rat. Also, Self-Fermented Pine
Needle Extracts 7 years old (SFPE 7) (200 µg/mL) reduce the frequency and amplitude of pacemaker currents in Interstitial
Cells of Cajal (ICC) of murine small intestine by modulating ATP-sensitive potassium channels. Therefore, the investigation
indicated that self-fermentation improves efficacy of the pine needle extracts reducing risk of cardio-vascular related disor-
ders and would be an important source in nutraceutics.
© KSBB
hÉóïçêÇëW=éáåÉ=åÉÉÇäÉ=Éñíê~ÅíëI=ëÉäÑJÑÉêãÉåí~íáçåI=ÑáÄêáåçäóíáÅ=~ÅíáîáíóI=ÅÜçäÉëíÉêçä=~åÇ=íêáÖäóÅÉêáÇÉI=f``= =
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fkqolar`qflk
In many areas, peoples adopt traditional methods in proc-
essing foods to increase its effectiveness or longevity during
storage. These practices follow open storage systems to ob-
tain liquors, pickles, and other products of desired tastes
since unspecified period through critical consideration of
storage conditions like temperature, humidity, and light.
Timing and micro-environmental conditions influence
greatly in obtaining quality products. Microorganisms are
expected to play a key role in changing composition and
quality in targeted food, which is stored.
Pinus densiflora is an evergreen needle-leafed tree indige-
nous to Asia Pacific. Pine products have been used for mil-
lennia for the treatment of multiple ailments. It has been
reported that pine needle extracts improved unidentified
clinical syndrome such as fatigue, depression, anxiety, sleep-
ing disturbance, etc. [1]. Biological activity of pine needle is
the essence for traditional medicine, which uses the pharma-
G`çêêÉëéçåÇáåÖ=~ìíÜçê=
Tel: +82-62-230-6667/7991 Fax: +82-62-224-6678
e-mail: hscheong@chosun.ac.kr
cological efficacy of natural compounds present in pine nee-
dle for treating human diseases. Furthermore, pine needles
are used in preparation of teas, extracts, some alcoholic bev-
erages for tonic, and the health-improving agent [2]. In con-
nection with this, evidence has supported the role that anti-
oxidants, including several compounds, play in the preven-
tion of anti-aging and several chronic diseases such as car-
diovascular disease, cancer, diabetes, and antihypertension
[3-6]. Therefore, pine extract has been processed and used
traditionally to treat multiple disorders.
Pine extract contains several different organic compounds
including carbohydrates, proteins, lipids, terpenoids, alka-
loids, and several others. Pine leaves have essential oils
(0.3~1.3%) including α-pinene, β-pinene, camphene, phel-
landrene, limonene, borneol (6.8%), and bornyl acetate
(3.8%) [7] that are helpful in reducing cardiovascular dis-
eases and possess anticancer properties [8]. Especially pine
needle and bark are abundant in terpenoids. The essential oil
of pine needles has found in wide commercial use [9]. Fla-
vonoides and other plant phenolics such as phenolic acids,
stilbenes, and tannins are important for normal growth and
defense against infection and injury [10].
Fibrinolytic enzymes dissolve the blood clots, which are

Biotechnol. Bioprocess Eng. OSP=
formed by the conversion of fibrinogen into fibrin via the
proteolytic action of thrombin. When clots are not lysed,
they accumulate in blood vessels and cause thrombosis
leading to myocardial infarction and other cardiovascular
diseases. Intravenous administration of urokinase and
streptokinase, which are capable of degrading fibrin, has
been widely used for this thrombosis therapy. However,
these enzymes have a low specificity for fibrin and very
expensive [11-15]. Therefore, it has been reported that
there are some proteases of pine needle showing fibri-
nolytic activity.
Hypercholesterolemia, resulting from cholesterol meta-
bolic changes, is a major cause of cardiovascular disturbance,
such as atherosclerosis and coronary heart disease [16,17].
Epidemiologic data showed that a high consumption of
vegetables and fruits is consistently associated with a low
risk of cancer and cardiovascular disease [18,19].
Pacemaker activities are ubiquitously existed in organs
systems such as circulatory, vascular, digestive systems, etc.
which are conducted by group of cells called pacemaker
cells. The pacemaker cells have ability of modulating organ
movements and the movements are easily modified from
foods and drugs.
Alimentary canal is the main place for digestion of food
materials taken. There is a myonteric movement of bowls
that helps the downward movement of food in gut where the
continuous contracting and relaxing cells are existed [20,21].
Such cells are called the Interstitial Cells of Cajal (ICC),
small spindle-shaped or stellate cells having numerous mito-
chondria capable to modulate the gastrointestinal movement
through the alteration of the spontaneous inward currents
generated through influences of external agents [21-24]. The
current generated is called pacemaker current that enables
the tissues producing continuous rhythm of contraction and
relaxation in the smooth muscle tissues of bowl. Therefore,
these cells play key role as basic regulators of gastrointesti-
nal motility, many hormones, neurotransmitter, and various
substances can modulate GI tract motility by influencing
ICC. Abnormalities in these currents also cause gastrointes-
tinal irregularities, which is also implicated in the use of cer-
tain drugs.
The study also aimed to assess whether the extracts show
multiples response with fibrinolysis, cholesterol, triglyc-
erides, and intestinal motility viewing the points that the
impairment of these components are vowed relate with
cardio-vascular or circulatory disorders. There is always
lacking information in role of self-fermentation in functional
efficacy of the pine needle extracts. Therefore, present study
would play an important role in fulfilling the existing gap.
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Fresh pine needles were selected and harvested from Ko-
rean red pine trees (Pinus densiflora Sieb. et Zucc.) in Gok-
seong, Jeollanam-Do, Korea.
mêÉé~ê~íáçå=çÑ=mb=~åÇ=pcmbë= =
Harvested pine needles were cleaned with tap water,
dipped with a charcoal in water to final wash, and dried,
ground for 1 min to homogenize. The preparation was al-
lowed to settle for 3 h at 4°C and the supernatant was recov-
ered. This supernatant was used to sample and stored at 4°C
for assays. PE was stored for years favor self-fermentation in
the stored extracts. The effects of the extract were examined
for fresh pine needle extract (PE) as well as after 3 and 7
years of self-fermentation designing as Self-Fermented Pine
Needle Extracts 3 years old (SFPE 3) and Self-Fermented
Pine Needle Extracts 7 years old (SFPE 7).
cáÄêáåçäóëáë=^ëë~ó=
Fibrinolytic activity of self-fermented pine needle extract
was measured on fibrin plate. The fibrin agarose plate was
made to a 5 mm thickness. To prepare fibrin assay plates, 5
mL of 1% (w/v) fibrinogen solution in distilled water was
mixed with 10 mL of 1.2% agarose solution and 20 µL of
thrombin solution (0.1 U/µL). The solution was then poured
into a Petri dish and allowed to stand for 1 h at room tem-
perature to form a fibrin clot. Then 20 µL of self-fermented
pine needle extract was carefully dropped onto the plate. The
plate was incubated for 1 h at 37°C. And the diameter of the
lytic circle was measured. In the fibrin plate method, a clear
region is observed in which fibrin is hydrolyzed, and its di-
ameter is directly proportional to the potency of the fibri-
nolytic activity.
`ÜçäÉëíÉêçä=~åÇ=qêáÖäóÅÉêáÇÉ=
Male Sprague-Dawley rats (200 ± 20 g) approximately
12-weeks-old were used in all experiments. Animals were
housed four per cage and maintained under control envi-
ronmental conditions (22 ± 2°C, 12 h light/dark cycle).
Food (Mouse E.P. from Suprfeed Co. Ltd.) and tap water
were supplied for animals. All efforts were made to mini-
mize animal suffering and to reduce the number of animals
used. For study, 15% cholesterol and 1% sodium cholate
were mixed with 84% corn oil to get cholesterol mixture.
Rats were grouped into control and the test groups where
each group contained 3 individuals and the experiment was
repeated for three times. Rats were separated into Choles-
terol non-administered (−C), Cholesterol administered (+C),
PE, and SFPEs as below. The animas were adjusted for two
weeks before administration of cholesterol and/or pine
needle extracts. Except the control, all rats were adminis-
tered cholesterol (0.5 mL) continuously at once a day for 4
weeks (for first 2 weeks cholesterol only and remaining 2
weeks cholesterol along with pine needle extracts). The
control group was fed DW (0.5 mL/day) during feeding
periods. For one group of rats they were continuously ad-
ministered cholesterol (0.5 mL) and SFPE 7 (0.5 mL) once
a day throughout 4 weeks. Analysis was done using auto-
matic biochemical analyzer (Hitachi 7600, Hitachi Tokyo,
Japan).

OSQ=
Week
Group 1 2 3 4
−C Distilled water (0.5 mL/day)
+C Cholesterol (0.5 mL/day)
PE Cholesterol
(0.5 mL/day)
Cholesterol + PE
(0.5 mL/day)
SFPE 3 Cholesterol
(0.5 mL/day)
Cholesterol + SFPE 3
(0.5 mL/day)
SFPE 7 Cholesterol
(0.5 mL/day)
Cholesterol + SFPE 7
(0.5 mL/day)
SFPE 7-2 Cholesterol + SFPE 7 (0.5 mL/day)
bäÉÅíêçéÜóëáçäçÖó=
j~íÉêá~äë=
Glibenclamide and pinacidil were purchased from RBI
(USA). For stock solutions, all chemicals were dissolved in
distilled water or dimethylsulfoxide, and stored at −20°C
until analyzed.
mêÉé~ê~íáçå=çÑ=`Éääë=~åÇ=qáëëìÉë=
Balb/C mice (8~13 days old) of both sexes were anesthe-
tized with ether and sacrificed by cervical dislocation. The
small intestines from 1 cm below the pyloric ring to the
cecum were removed and washed with Krebs-Ringer bi-
carbonate solution, the tissues were pinned to the base of a
Sylgard dish, and the mucosae were removed by sharp
dissection. Small strips of intestinal muscle (containing
both circular and longitudinal muscles) were equilibrated in
Ca2+-free Hanks solution (containing in mM: KCl 5.36,
NaCl 125, NaOH 0.34, Na2HCO3 0.44, glucose 10, sucrose
2.9, and HEPES 11) for 30 min. The cells were then dis-
persed with an enzyme solution containing collagenase
(Worthington Bio-chemical Co., USA) 1.3 mg/mL, bovine
serum albumin (Sigma) 2 mg/mL, trypsin inhibitor (Sigma,
USA) 2 mg/mL, and ATP 0.27 mg/mL. Thereafter they
were plated onto sterile glass cover slips coated with mur-
ine collagen (2.5 µL/mL, Falcon/BD) in 35 mm culture
dishes, and cultured at 37°C in a 95% O2-5%CO2 incubator
in SMGM (smooth muscle growth medium, Clonetics
Crop., USA) supplemented with 2% antibiotics/antimy-
cotics (Gibco, USA) and murine stem cell factor (SCF, 5
ng/mL, Sigma).
m~íÅÜ=`ä~ãé=bñéÉêáãÉåíë=
The whole-cell configuration patch-clamp technique as
used to record the membrane currents (voltage clamp) and
potentials of the cultured ICC (current clamp), and Axopatch
1-D (Axon Instruments, USA). Command pulses were ap-
plied using an IBM-compatible personal computer and
pClamp software (version 7.2; Axon Instruments). Data
were filtered at 5 kHz and displayed on an oscilloscope, a
computer monitor, and a pen recorder (Gould 2200, Gould,
USA). The cells were bathed in a solution containing (in
mM): KCl 5, NaCl 135, CaCl2 2, glucose 10, MgCl2 1.2, and
HEPES 10, adjusted to pH 7.4 with Tris. The pipette solution
contained (in mM): KCl 140, MgCl2 5, K2ATP 2.7, Na2GTP
0.1, disodium creatine phosphate 2.5, HEPES 5, and EGTA
0.1, adjusted to pH 7.2 with Tris. All experiments were per-
formed at 30°C.
obpriqp=^ka=afp`rppflk=
Different parts of the red pine, such as the needles, cones,
cortices, and pollen, have been used as folk medicine or as
food. Particularly, pine needles are used in folk medicine to
treat liver disease, gastrointestinal diseases, nervous system
disease, circulatory diseases, and skin problems [25,26].
Pine needle extracts have been found to lower blood lipid
levels, and to have antioxidative, antitumor, antimutagenic,
and antibiotic effects [27-31]. In identifying the effects of
pine needle extracts fresh or during and/or after self-
fermentation we have tried to analyze in following aspects
relating to vascular stimulation. Suspicion of hypercholes-
terolemia is relating to atherosclerosis that ultimately links
with vascular related disease. Increasing concerns of people
nowadays is linked with reducing cholesterol level which is
hard to achieve. Our perspective was to assess whether the
pine products lower the cholesterol level in rat blood and
other blood related systems.
oçäÉ=çÑ=mb=~åÇ=pcmbë=áå=cáÄêáåçäóëáë=
Fibrinolysis is one of the important aspects in medicine
linking to the blood clotting and its removal procedures. We
tried to assess whether the pine needle extracts show fibri-
nolytic activities in fibrin plates experiments. Also we were
intended in searching whether the fermentation has role in
altering fibrinolytic properties. Twenty µL of each sample
was carefully placed on fibrin plate. The plate was incubated
for 30 min, 1 and 2 h at 37°C and the diameter of the lytic
circle was measured. After 30 min incubation, lytic circle
formed by PE, SFPE 3, and SFPE 7 on fibrin plate were 11,
12, and 14 mm, respectively. At after 1 h, the circles were 13,
14, and 18 mm, respectively. In 2 h of incubation, lytic circle
of PE, SFPE 3, and SFPE 7 were 17, 18, and 23 mm, respec-
tively (Fig. 1). Studies on extracts from bark or French pine
was able to show the fibrinolytic behavior [32,33]. It seems
that fermentation facilitates fibrinolytic activity in pine ex-
tract. And SFPEs might be an important ingredient and be
potential source as a functional food (health related food) for
thrombosis prevention.
oçäÉ=çÑ=mb=~åÇ=pcmb=áå=qçí~ä=mä~ëã~= `ÜçäÉëíÉêçä=~åÇ=
qêáÖäóÅÉêáÇÉ=iÉîÉä=
To determine whether the pine needle extracts involves in
alteration of total cholesterol and triglyceride levels in blood
plasma of rats, we checked the effects of PE and SFPEs. As
the experimental animals were categorized into different test
groups we administered them cholesterol and pine needle
extracts. The average total plasma cholesterol level in cho-
lesterol fed rats was 50.5 ± 0.7 mg/dL. The level was found

Biotechnol. Bioprocess Eng. OSR=
cáÖK=NK Fibrinolytic activity of self-fermented pine needle extracts.
Fibrinolytic activities of SFPEs increase with aging and it
is the effect is time dependent.
34.5 ± 2.12, 35 ± 3.5, and 23 ± 2.8 mg/dL, respectively, in
PE, SFPE 3, and SFPE 7. However, the level was remained
29 ± 1.4 mg/dL in rats which were administered cholesterol
(0.5 mL) and SFPE 7 (0.5 mL) throughout for 4 weeks at
once a day (Fig. 2A). It was revealed that the percentage
decrease in total plasma cholesterol level in cholesterol and
pine needle extracts fed rats was 23.3, 22.2, and 48.9%, by
PE, SFPE 3, and SFPE 7, respectively. However, the level
was 35.6%, in 4 weeks continuous SFPE 7 and cholesterol
administered rats (Fig. 2B).
In cholesterol fed control, the level of plasma triglyceride
was found as 24 ± 4.3 mg/dL. In PE, SFPE 3, and SFPE 7
(0.5 mL) administered rats for final two weeks were re-
corded as 13.5 ± 4.9, 15 ± 1.5, and 14.5 ± 7.8 mg/dL, respec-
tively. However the level was sharply reduced to 6 ± 3.1 mg/
dL in 4 week continuous administration of SFPE 7 with cho-
lesterol (Fig. 3A). The plasma triglyceride level in choles-
terol administered rats was effectively reduced by continu-
ous feeding of SFPE 7 (0.5 mL) for all 4 weeks (Fig. 3B).
Hypercholesterolemia, resulting from cholesterol metabolic
changes, is a major cause of cardiovascular disturbance, such
as atherosclerosis and coronary heart disease [16,17]. Epi-
demiologic data showed that a high consumption of vegeta-
bles and fruits is consistently associated with a low risk of
cancer and cardiovascular disease [18,19]. Present study
indicated that PE and SFPE are useful in lowering hypercho-
lesteromic condition and which might improve blood circu-
lation and could be a good source of functional food devel-
opment.
oçäÉ=çÑ= pcmb=áå=bäÉÅíêçéÜóëáçäçÖáÅ~ä=^ÅíáîáíáÉë=áå=f``=
çÑ=jìêáåÉ=pã~ää=fåíÉëíáåÉ=
ICCs cultured from the murine small intestine are c-kit-
positive positive cells that have distinct morphology contain-
ing spindle shaped structures and form a network within
smooth muscles. Recording from cultured ICC under current
clamp mode (I = 0) showed spontaneous pacemaker poten-
tials. The resting membrane potential was −53 ± 3 mV and
amplitude 23 ± 5 mV. In voltage clamp mode at a holding
potential −70 mV, ICC generated spontaneous inward cur-
rents called ‘pacemaker currents’. The average frequency of
the currents was 14 ± 2 cycles/min and the amplitude aver-
aged −436 ± 62 pA. SFPE 7 has been tested for the analysis
^=
_=
cáÖK=OK Assessment of total cholesterol in blood plasma of cho-
lesterol fed rats. The level of cholesterol was found low-
ered after administration of PE and SFPEs. (A) Shows to-
tal plasma cholesterol in cholesterol administered rats.
(B) Shows the percentage change in total plasma choles-
terol in cholesterol rats. +C, Cholesterol administered; PE,
fresh pine needle extract; SFPE, self-fermented pine
needle extracts.
of the effects on alteration of pacemaker activities in the ICC.
In whole cell patch clamp technique at 30°C, ICC generate
spontaneous pacemaker potential under current clamp mode (I
= 0) and inward currents (pacemaker currents) under voltage
clamp mode at a holding potential of −70 mV [21]. When
SFPE 7 (200 µg/mL) treated in ICC, under currents clamp
mode decreased both the frequency and amplitude of pace-
maker currents, and increased the resting currents in outward
direction. Also, SFPE 7 inhibit the pacemaker currents in a
dose-dependent manner [34]. Glibenclamide, a blocker of
potassium channel, reversed the effect developed by SFPE 7
indicating the SFPE 7 cause the opening of the potassium
channels during modulation of pacemaker current (Figs.
4A~4C). In identifying whether SFPE affects ATP-sensitive
potassium channels, we had tested the effects of pinadicil, an
ATP-sensitive potassium channel opener and glibenclamide,
an ATP-sensitive potassium channel blocker on pacemaker
current. The result demonstrated that the (Fig. 4A) pinacidil
(10 µM) decreased the frequency and amplitude of pace-
maker currents and increased the resting membrane potential

OSS=
^=
_=
cáÖK=PK Level of triglyceride blood plasma of cholesterol fed rats.
Blood plasma triglyceride decreases with administration
of 0.5 mL PE and SFPEs. (A) Shows plasma triglyceride
in cholesterol administered rats. (B) Shows the percent-
age change in plasma triglycerides in cholesterol rats. +C,
Cholesterol administered; PE, fresh pine needle extract;
SFPE, self-fermented pine needle extract.
in outward direction which was reversed by glibenclamide
(10 µM). The agonistical effect was observed in the test of
SFPE 7 (200 µg/mL) with pinacidil which effect was also
reversed by the application of glibenclamide. Further more,
pretreatment of glibenclamide and co-treatment with SFPE 7
showed no alteration in pacemaker currents in ICC of murine
small intestine indicated the role of SFPE in opening ATP-
sensitive potassium channels (Figs. 4B and 4C).
`lk`irpflk=
Assessment of pine needle is the essence for traditional
medicine using pharmacological efficacy of natural com-
pounds present in pine needle for treating possible human
diseases. Fibrinolytic activities of the extract indicated that
activities depend on time and also with aging of the pine
needle products. It was also found that the extract can lower
the blood plasma cholesterol and triglyceride in cholesterol
fed rat. SFPE 7 (200 µg/mL) inhibited the pacemaker current
of ICC in murine small intestine. Therefore, the self-
fermented pine needle products would be additive in lower-
ing blood plasma cholesterol and also can reduce obesity and
^=
_=
`=
cáÖK=QK Effects of self-fermented pine needle extracts on pace-
maker currents recorded in cultured ICC from murine
small intestine. Figure shows the effect of SFPE 7 on
pacemaker currents pre and co-treating cells with gliben-
clamide. (A) Shows the effect of pinacidil, an ATP-
sensitive potassium channel opener was reversed by ap-
plication of glibenclamide. (B) SFPE7 (200 µg/mL) re-
duced the frequency and amplitude of pacemaker cur-
rents deviating resting current towards outward direction.
(C) Pre- and co-treatment of glibenclamide stopped the
effect of SFPE indicating the involvement of SFPE in
modulating ATP-sensitive potassium channel. SFPE:
self-fermented pine needle extract.
helpful in removing blood clots. The study unveils many
potential aspects in the self-fermentation improve nutraceuti-
cal pine needle extracts product processing that further needs
follow up studies in assessing them detail.
^ÅâåçïäÉÇÖÉãÉåíë This Research was supported by the
RCPM (R11-2000-083-00000-0) in Chosun University and
“Program for the Training of Graduate Students in Regional
Innovation”, which was conducted by the Ministry of Com-
merce, Industry and Energy of the Government of Korea.
Received March 3, 2008; accepted April 3, 2008
obcbobk`bp=
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