In vitro/in vivo effect of Citrus limon (L. Burm. f.) juice on blood
parameters, coagulation and anticoagulation factors in rabbits
Azra Riaz1, Rafeeq Alam Khan1,2*, Talat Mirza3, Tazeen Mustansir3 and Mansoor Ahmed4
1
Department of Pharmacology, Faculty of Pharmacy, University of Karachi, Karachi, Pakistan
Department of Basic Medical Sciences, College of Medicine-Jeddah, King Saud bin Abdul-Aziz University for Health Sciences, KSA
3
Department of Pathology, Dow International Medical College, Dow University of Health Sciences, Karachi, Pakistan
4
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, Karachi, Pakistan
2
Abstract: The genus Citrus of the family Rutaceae includes many species e.g. Citrus indica, Citrus aurantifolia and
Citrus limon, among which Citrus limon L. Burm. f. has been reported to have highest antimicrobial activity. It is used as
antidote against certain venom, due to its platelet inhibitory effect and also reported to have hypocholesterolemic effect.
However its anticoagulant and thrombolytic effect were not been investigated, hence a prospective in-vitro/in-vivo study
was designed to determine the effect of Citrus limon on blood parameters, coagulation and anticoagulation factors. Invitro tests revealed highly significant increase in thrombin time and activated partial thromboplastin time by Citrus
limon, whereas fibrinogen concentration was significantly reduced in comparison to control, however prothrombin time
was not affected significantly. In-vivo testing of Citrus limon was done at three different doses i.e. 0.2ml/kg, 0.4ml/kg
and 0.6ml/kg in healthy rabbits. Significant changes were observed in hematological parameters such as erythrocytes,
hemoglobin and mean corpuscular hemoglobin concentration. Bleeding time and thrombin time was significantly
prolonged and there was increase in protein C and thrombin antithrombin complex levels. These results may be due to
inactivation of thrombin because it significantly decreases fibrinogen concentration and inhibit platelet aggregation.
Citrus limon showed maximal anticoagulant effect at 0.4ml/kg, which suggest that Citrus limon possesses an antithrombin component and could prevent thrombosis playing a cardio protective role.
Keywords: Thrombin time, activated partial thrombin time, Fibrinogen concentration, mean corpuscular hemoglobin
concentration
INTRODUCTION
Defects in coagulation and thrombosis are one of the
important causes of atherosclerosis and cardiovascular
diseases (Little et al., 2002; Wang et al., 2007). Studies
regarding coagulation, anticoagulant agents have been
done for the prevention and treatment of thrombogenic
state (Ahmed et al., 2008; Riaz et al., 2009). Many factors
of blood coagulation like increased level of factor VIII,
von Wille brand factor and platelet activation are
associated with cardiovascular disease as well as hemo
stasis (Abdullah et al., 2010). Few studies indicates the
role of flavonoids and polyphenol compounds in
prevention of cardiovascular diseases either by reversing
endothelial dysfunction (Heiss et al., 2005; McCullough
et al., 2012) or increasing nitric oxide bioavailability or
acting as antioxidant and anti-inflammatory (Kim et al.,
2004; Chun et al., 2008; Grassi et al., 2010).
Fruits and their juices are the major dietary sources of
polyphenol compounds, flavonols, flavanones, and
anthocyanidins. It has been exhibited that Citrus fruit
have highest antioxidant activity (Chun et al., 2008), due
to the presence of abundant flavonoids, vitamin C and
carotenoids (Xu et al., 2008). Common species of the
genus Citrus are Citrus indica, Citrus aurantifolia, and
*Corresponding author: e-mail: rkhan1959@gmail.com
Pak. J. Pharm. Sci., Vol.27, No.4, July 2014, pp.907-915
Citrus limon, in which Citrus limon (L. Burm. f.) is
available in Pakistan and commonly known as Limo. It
has significant economic value for its essential oil and is
reported to be the source of magnesium, potassium,
vitamin C, folic acid, limonoids and flavonoids (Deyhim
et al., 2006). Citrus limon has shown usefulness as
antidote against certain venom, due to its platelet
inhibitory effect (Arias et al., 2005), however it needs
further confirmation. More attention has also been paid on
antioxidant capacity of Citrus limon (Berhow et al., 1995;
Xu et al., 2008), since increase dietary antioxidant
constituents could help to prevent athero-sclerosis
(Hernandez et al., 2009; Gonzalez et al., 2010).
Number of studies has suggested the possibility of Citrus
limon in preventing cardiovascular diseases due to its
hypocholesterolemic activity (Gonzalez et al. Khan et al.,
2010). Citrus limon was thought to produce antithrombotic effect, since hypercholesterolemia and
thrombosis are interrelated (Vazquez et al., 2004; Pfister,
2006; Son et al., 2008). Beneficial effects of Citrus limon
are due to its wide range of bioflavonoids, including rutin,
hesperidin, quercitrin, eriocitrin, narirutin, didymin and
naringin (Nijveldt et al., 2001; Tripoli et al., 2007). Two
more isomers of hesperidin, neohesperidin and
homoeriodictyol rutinoside have also been identified in
Citrus limon (Gonzalez et al., 2010). Other micronutrient
907
In vitro/in vivo effect of Citrus limon juice on blood parameters, coagulation and anticoagulation factors
includes magnesium, potassium, vitamin C, folic acid,
limonoids and xanthoxyletin. Systemic studies on
anticoagulant effects are scare, however there are studies
on Rutin and Hesperidin (Kuntic et al., 2011),
xanthoxyletin (Teng et al., 1992), antifungal activity
(Viuda et al., 2008) and anti-cancer activity of Citrus
limon (Arias et al., 2005).
Present study was designed as a part of the therapeutic
approach to evaluate the anticoagulant and antithrombotic effects of Citrus limon both in-vitro and invivo. The effect on coagulation and anticoagulation
factors was assessed by determining thrombin time (TT),
prothrombin time (PT), activated partial thromboplastin
time (aPTT), fibrinogen concentration (Fb), bleeding time
(BT), platelet function and thrombin antithrobin (TAT)
complex and protein C (PC) levels in rabbit blood.
MATERIALS AND METHODS
Citrus limon L. was purchased from local market,
identified by center of plant conservation, University of
Karachi and voucher specimen no C.L 11-11 was
deposited in Department of Pharmacognosy, University of
Karachi. Fruit were cut and squeezed by hand to yield
fresh juice which was filtered immediately before use.
In-vitro Study
Blood Samples
In-vitro study was carried out on blood samples drawn
from marginal vein of nine white healthy rabbits. 5 ml
sample from each rabbit was collected in coagulation
tubes containing 3.8% tri sodium citrate solution (9:1
v/v).
Design of experiment
Thrombin time (TT), Prothrombin time (PT), activated
partial thromboplastin time (aPTT) and Fibrinogen
concentration (Fb) were estimated using standard kits of
Merck (Germany) by coagulation analyzer, Humaclot
Duo (Kung-chi et al., 2007). Determination of TT, PT,
aPTT and Fb was done by taking equal volume of plasma
with fresh Citrus limon juice or water for injection or
heparin sodium, 25,000IU (Huons Co. Ltd) to measure
test, control and standard reading respectively.
Measurement of TT, PT, aPTT and Fb
Sample collection and animal handling was in accordance
with the NCCLS approved guideline H21-A3 (Wayne,
1998). Plasma from each sample was immediately
separated at 1500 rpm for 15 minutes by centrifugation in
14 K Humax centrifuge machine. The separated plasma
samples were stored at -20ºC for determination. The
principle used in Humaclot duo coagulometer (Human,
Germany) was turbidimetric clot detection to assess
coagulation endpoint. While TT was measured by mixing
100ul Citrus limon juice or water for injection or heparin
908
and 100ul plasma incubated with 100ul thrombin reagent.
PT was measured by mixing 50ul Citrus limon juice or
water for injection or heparin and 50ul plasma incubated
with 200ul pre-warmed thromboplastin reagent. aPTT was
measured by mixing 50ul Citrus limon juice or water for
injection or heparin and 50ul plasma incubated for 1-2
min at 37˚C, followed by addition of 100ul aPTT-EL, then
incubated with 100ul CaCl2.
Incubation time before the addition of respective reagent
for all these tests was 3 min at 37˚C. The timer was
started with addition of reagent and time was recorded
required for clot formation. Fb concentration was
measured as described by McNerlan et al. (1997) using
Clauss, (1957) method.
In-vivo Study
Animal Selection
Sixty healthy white rabbits of either sex were selected for
in- vivo study. All animals had mean body weight of 1300
± 50 grams. Body weights of the animals were measured
weekly during 60 day study. Rabbits were housed
individually in steel rod bottom cages, under controlled
condition of temperature 23±2°C, humidity 50-60%. Diet
and water was provided ad libitum.
Design of experiment
Animals were divided into six groups with ten rabbits in
each group. Three groups were given Citrus limon juice,
once daily in three doses i.e. 0.2ml/kg, low Citrus limon
dose (LCLD), 0.4ml/kg, moderate Citrus limon dose
(MCLD) and 0.6ml/kg, high Citrus limon dose (HCLD).
Fourth group was given saline in same dose equivalent to
their body weights and considered as control group. Fifth
and sixth groups were given aspirin and warfarin as
standard drugs. Aspirin was suspended in normal saline
and administered in the dose of 150 mg/kg once daily for
6 days a week (Merchant et al., 2004). Warfarin was
suspended in distilled water and dose was scheduled for 6
days only, 5mg/kg for first 3 days and 10mg/kg next three
days (Warfarin dosing guideline, 2009). All drugs were
given by gastric intubation for 60 days. Blood samples
were collected from ear vein in the EDTA containing
tubes, trisodium citrate (3.8%) tubes in the ratio of 9:1
and gel tubes at 30 and 60 day at the end of dosing
period.
Measurement of TT, PT, aPTT and Fb
Blood samples collected were centrifuged in Humax 14 K
(Human, Germany) at 2000 xg for 10 min to separate
plasma. Tests were performed on Humaclot duo (Human,
Germany) coagulometer using the principle of
turbidimetric clot detection to assess coagulation endpoint
by measuring change in optical density in plasma
samples. To measure TT 200ul plasma was incubated with
100ul thrombin reagent, while PT was measured by
incubating 100ul plasma with 200ul pre-warmed
Pak. J. Pharm. Sci., Vol.27, No.4, July 2014, pp.907-915
Azra Riaz et al
thromboplastin reagent. While aPTT was measured by
incubating 100ul plasma for 1-2 min at 37˚C, with 100ul
aPTT-EL reagent, followed by adding 100ul CaCl2.
Incubation time was for 3 min at 37˚C; Fb concentration
was measured as described by McNerlan et al., (1997)
using Clauss, 1957 method. All parameters were
determined using standard kits by Human, Germany.
Measurement of bleeding time (BT)
Bleeding time was measured by cutting the ear tip as
describe by (Johnstone et al., 1990; Garcia et al., 2001; Li
et al., 2005). First the ear was shaved then small incision
of 5mm long and 1mm deep was made to the central ear
artery using a template bleeding device. The incision sites
were carefully blotted at 30 sec intervals with filter paper
until bleeding has ceased.
Platelet Function Assays
Blood samples were drawn into tubes containing trisodium citrate 3.8% with 9:1 v/v ratio and processed
within 2 hours. Platelet rich plasma (PRP) was obtained
as a supernatant fluid after centrifuging blood at 100 g for
10-15 minutes in centrifuge machine (Humax 14 K,
Human, Germany). The remaining blood was further
centrifuged at 1600-2000 g for 10-15 minutes to prepare
platelet-poor plasma (PPP). Each PRP sample was
standardized (approx. 250 000/mm3) with autologous PPP
as needed (Son et al., 2008). Platelet reactivity was traced
for 10 minutes at 37°C as previously described by Jeong
et al., (2010). The absorbance of the untreated PRP mixed
with the aggregation reagent represent 0% aggregation
and the absorbance of PPP control represents 100%
aggregation. Platelet aggregation was induced by addition
of Adenosine diphosphate (20µM), Collagen (10µg/ml),
Epinephrine (300 µM), Ristocetin (1500 µg/ml) and
Arachidonic acid (500µg/ml). Platelet aggregation assay
was performed with turbidimetric monitoring device,
Helena Agg RAM aggregometer (Helena Laboratories
Corp, Beaumont, TX, USA), according to manufacturer’s
instructions. Briefly pipette 450µl PRP into cuvettes
incubate at 37˚C, insert PPP cuvette into appropriate
channel and set to 100% aggregation, add 50µl of
aggregating reagent. Resulting aggregation, measured as a
change of light transmission, and was expressed as
percentage of the PPP transmission value.
Protein C and Thrombin-antithrombin complex
The activity level of PC and TAT complex in plasma was
measured by commercial Protein C and Thrombinantithrombin (TAT) complex Elisa kit (Cusabio Biotech
Co. LTD). Standard curve was prepared for these two
parameters taking absorbance of standard plasma at
450nm. The activity level of TAT complex and Protein C
in the samples was expressed as percentage related to the
activity level of standard plasma. The entire tests were
performed under NCCL guideline (Wayne, 1998).
Pak. J. Pharm. Sci., Vol.27, No.4, July 2014, pp.907-915
Hematological Examination
Huma Count (Human, Germany) fully automated
hematology analyzer was used to examine red blood cell
count (RBC), white blood cell count (WBC), platelet
count (PLT), hemoglobin (Hb), Hematocrit (Ht), mean
corpuscular volume (MCV), mean corpuscular
hemoglobin (MCH) and mean corpuscular hemoglobin
concentration (MCHC).
Liver Function Tests (LFTs)
Blood collected in gel tubes, was allowed to clot at room
temperature. Serum was separated by centrifugation at
2500 rpm for 10 minutes and used for estimation of liver
function by measuring SGPT (serum glutamic-pyruvic
transaminase), γGT (Gamma glutamyltransferase) and
total bilirubin concentration (Fischbach et al., 1992) by
using standard kits of Human, Germany.
Histopathological Examinations
Microscopic changes were observed through random
selection of liver samples from each test and control
animals. Tissues were preserved in 10% formalin
followed by dehydration in ascending grades of alcohol.
Clearing by xylene and embedding in paraffin wax.
Paraffin sections (5µm thickness) were stained with
hematoxylin and eosin (H & E) for histological
examination (Diab et al., 2012).
STATISTICAL ANALYSIS
Data entry and analysis was performed using Superior
Performance Statistical Software (SPSS) version 20. Data
was presented as mean ± SD with 95% confidence
interval. ANOVA followed by post hoc was performed for
comparisons of values with control. Values of p<0.05
were considered significant and p<0.005 as highly
significant.
RESULTS
Table 1 shows in vitro comparison between water for
injection, Citrus limon and heparin sodium. There was
highly significant increase in TT and aPTT in animals of
Citrus limon group as compare to control (water for
injection), aPTT values were very near to the value of
heparin sodium. Whereas Fb concentration was
significantly reduced by Citrus limon as compare to
control, however PT was not affected significantly.
Table 2 shows the effect of Citrus limon juice on BT, TT,
PT, aPTT and Fb. There was highly significant increase in
BT and TT in animals at moderate dose and significant
decrease in Fb level as compared to control at 30 and 60
days. However at 60 days only BT was increased
significantly in animals at low dose, whereas other
changes were insignificant both at LCLD and HCLD.
909
In vitro/in vivo effect of Citrus limon juice on blood parameters, coagulation and anticoagulation factors
Table 1: In vitro comparison of Citrus limon, heparin and control on coagulation parameters
Parameters
Thrombin time (Sec)
Prothrombin time (Sec)
Activated partial thromboplastin time (Sec)
Fibrinogen concentration (mg/dl)
Groups
Citrus limon
422.2±49.4**
5.3±0.1
509.7±35.5**
11.26±2.05**
Water for injection
5.8±0.2
8.4±0.2
193.0±29.8
102.03±18.18
Heparin sodium
600.0±0.0**
600±0.01**
569.9±20.7**
7.21±2.51**
n=10, Values are means ± S.E.M., **p<0.005 highly significant as compared to control
Table 2: In vivo effect of Citrus limon juice and warfarin on coagulation parameters
Parameters (Sec)
BT
TT
PT
Aptt
Fb(mg/dl)
Control
99.60
±5.35
9.36±
0.92
5.2±
0.12
8.25±
0.62
439.47
±43.70
30 DAY
LCLD MCLD
103.8
152.9±
±5.25
8.77**
9.2±
12.4±
0.13
0.59**
5.12±
6.34±
0.02
0.73
12.52±
12.1±
2.08
1.55
412.17 333.11±
±28.71 29.55*
HCLD
107.7
±5.54
8.8±
0.08
5.84±
0.04
8.67±
0.11
380.23±
32.62
Control
101.66
±5.8
9.41±
1.02
5.32±
0.12
8.33±
0.61
437.50±
43.14
LCLD
128.10
±10.3*
9.21±
0.41
5.22±
0.02
12.51
±1.82
407.08
±28.65
60 DAY
MCLD
HCLD
168.50±
109.8±
8.32**
5.28
12.86±
9.17±
0.54**
0.09
6.73±
6.57±
0.89
0.14
12.22±
9.13±
1.30
0.07
336.33± 339.58±
26.48*
24.35*
Warfarin
133.9±
12.7*
16.58±
1.74**
13.59±
1.73**
15.73±
1.77**
315.40±
25.54*
n=10, Values are means ± S.E.M
*P ≤0.05 significantly different as compared to control, **P ≤0.005 highly significant as compared to control
LCLD: Low Citrus limon dose 0.2ml/kg/day; MCLD: Moderate Citrus limon dose 0.4ml/kg/day; HCLD: High Citrus limon dose
0.6ml/kg/day.
Table 3 shows the effect of Citrus limon on hematological
parameters. There was significant increase at MCLD in
RBC, hemoglobin concentrations, MCHC and highly
significant decrease in red cell distribution width (RDw)
at 30 and 60 day, while animals received high dose of
Citrus limon showed significant increase in RBC both at
30 and 60 day. Whereas at low dose there was no
significant affect on any hematological parameters.
However hematocrit, MCV, WBC and platelets count
were not affected by any dose of Citrus limon.
Table 4 shows the influence of three doses of Citrus limon
on platelet aggregation. There was significant inhibition
in platelet aggregation at moderate dose induced by
adenosine diphosphate (ADP), collagen (Col),
epinephrine (Epi) and arachidonic acid (AA) both at 30
and 60 days. However significant inhibition in platelet
aggregation induced by ristocetin (Risto) was only
observed at high dose both at 30 and 60 days, whereas
reduction in platelet aggregation was not affected at low
dose.
Fig. 1 shows the effect of Citrus limon on Protein C (PC)
at different doses. There was significant increase in PC
level at moderate dose both after 30 and 60 days. Whereas
PC level was not changed significantly at low and high
dose.
910
Fig. 2 shows the effect of Citrus limon on thrombin
antithrombin (TAT) complex. There was significant
increase in TAT complex at moderate dose of Citrus
limon, after 30 days and highly significant increase after
60 days. While significant increase was observed at high
dose both after 30 and 60 days. However there was no
significant change in TAT complex level at low dose of
Citrus limon.
Liver function as determined by measuring SGPT, γGT
and total bilirubin was not changed significantly as
compared to control (data not shown). No histological
changes were also observed in hepatic tissues of treated
groups at any dose (data not shown).
DISCUSSION
Fruits and their juices have been increasingly studied due
to their health promoting effects. Several studies have
shown effectiveness of vegetables, fruits and their juices
or extracts for the treatment and/ or prevention of chronic
diseases. However little work has been done to observe
the effect of Citrus limon on coagulation and
hematological parameters.
In present study no significant effect of Citrus limon on
PT was observed both in-vivo and in-vitro. Hence it
Pak. J. Pharm. Sci., Vol.27, No.4, July 2014, pp.907-915
Azra Riaz et al
shows that Citrus limon has no effect on extrinsic
coagulation factors, since prolong PT is due to deficiency
of extrinsic coagulation factors, I, II, V, VII, and X (Rao
et al., 2000).
Similarly there was no significant change in aPTT in-vivo.
It means that Citrus limon has no effect on intrinsic
coagulation factors in vivo. Since prolong aPTT is due to
deficiency of intrinsic coagulation factors I, II, V, VIII,
IX, X, XI and XII (Khanin et al., 1998; Mann et al., 2003;
Kanahara et al., 2008), but there was significant
prolongation in aPTT in-vitro, Kuntic et al., (2011)
showed that Citrus flavonoids-Rutin and Hesperidin could
prolong aPTT in vitro. However, in-vitro results alone not
sufficient to draw definite conclusion about the usefulness
of flavonoids in the diet. Vitamin C has role in prevention
of blood protein as reported by Gonzalez et al., (2010).
Hence it could be suggested that Citrus limon may play a
crucial role in preventing the change in PT, APTT or
coagulation factors due to essential content, vitamin C.
Table 3: Effect of Citrus limon and aspirin on Hematological parameters of rabbits
Groups
Days
30
Control
60
30
Aspirin
60
30
LCLD
60
30
MCLD
60
30
HCLD
60
RBC
(x10³/cm)
3.62±
0.34
3.84±
0.34
3.44±
0.363
3.64±
0.370
4.34±
0.26
4.69±
0.43
4.81±
0.321*
4.94±
0.30*
4.67±
0.31*
4.81±
0.288*
WBC
(x10³/cm)
3.22±
0.42
3.28±
0.43
4.38±
0.395*
4.43±
0.511*
3.77±
0.28
3.84±
0.28
4.14±
0.36
4.38±
0.412
4.17±
0.308
4.27±
0.285
Hb
(g/dl)
9.72±
0.37
9.77±
0.36
5.31±
0.18*
5.24±
0.50*
9.78±
0.22
9.90±
0.21
10.98±
0.55*
10.95±
0.55*
10.02±
0.20
10.31±
0.37
PARAMETERS
Ht
MCV
RDW
(%)
(%)
(%)
27.96±
61.00
16.04
3.70
±0.63
±0.22
28.23
62.30
16.18
±3.70
±0.70
±0.22
20.27±
63.60
14.27±
0.56*
±0.73* 0.05**
20.56±
65.10
14.22±
0.51*
±0.83* 0.08**
26.56
60.40
15.75±
±2.85
±0.68
0.19
25.63
61.70
15.91±
±2.78
±0.77
0.22
30.23
61.40
14.16±
±2.67
±0.56
0.06**
32.37
62.30
14.33±
±2.37
±0.44
0.06**
31.63
61.50
15.85±
±7.96
±1.00
0.21
33.94
61.90
15.81±
±2.64
±0.75
0.20
MCH
(Pg/cell)
19.55±
0.64
20.55±
0.64
21.71±
0.79*
22.90±
0.67*
20.10
±0.79
20.61
±0.66
19.73
±0.62
20.53
±0.53
19.17
±0.68
20.17
±0.68
MCHC
(%)
29.82
±0.65
30.88
±0.69
29.14
±0.68
30.47
±0.75
28.88
±0.43
29.77
±0.50
31.72
±0.70*
33.29
±0.59*
30.67
±0.64
31.07
±0.56
PLT
(x10³/cm)
271.40
±27.57
269.90
±26.88
281.60
±32.37
324.60
±15.27
296.50
±33.37
283.10
±36.78
287.10
±37.29
298.80
±29.90
294.10
±27.68
285.60
±15.27
n=10, Values are means ± S.E.M.
*P≤0.05 significantly different as compared to control. **P≤0.005 highly significant as compared to control
Table 4: Effect of Citrus limon juice and aspirin on inhibition of Platelet aggregation
Groups
Control
Aspirin
LCLD
MCLD
HCLD
Days
30
60
30
60
30
60
30
60
30
60
ADP (20µM)
42.20±2.13
42.30±4.12
29.90±0.85*
24.84±0.70**
32.01±0.75
31.28±0.69
31.43±0.85*
26.09±0.77*
50.50±7.62
47.66±8.09
% Inhibition of Platelet Aggregation
Col (10µg/ml)
Epi (300 µM)
Risto (1500 µg/ml)
18.15±0.92
13.88±0.98
17.48±2.29
18.21±1.14
13.41±0.73
21.82±2.61
10.43±0.77*
12.85±1.54
9.98±0.93*
10.72±0.93*
13.0±2.62
12.76±1.76*
22.53±1.95
11.77±1.30
13.15±2.40
22.53±1.95
11.77±1.30
21.67±2.79
10.34±0.75*
9.37±0.48*
12.73±2.35
10.25±0.72*
10.77±0.80*
21.40±2.58
22.78±2.03
11.98±1.08
10.67±0.97*
21.76±1.94
13.0±2.62
13.89±2.86*
AA (500µg/ml)
69.81±8.91
75.77±6.66
44.57±8.95*
46.14±8.88*
65.87±5.72
61.68±8.51
43.99±8.73*
48.19±7.29*
65.30±5.62
49.70±5.87*
n=10, Values are means ± S.E.M.
*P≤0.05 significantly different as compared to control. **P≤0.005 highly significant as compared to control
Pak. J. Pharm. Sci., Vol.27, No.4, July 2014, pp.907-915
911
In vitro/in vivo effect of Citrus limon juice on blood parameters, coagulation and anticoagulation factors
In present study Citrus limon have shown identical results
both in-vitro and in-vivo i.e. prolonged TT, BT and
decrease Fb, which might be due to impaired activity of
thrombin, since thrombin plays an important role in
platelet aggregation and conversion of fibrinogen to
fibrin, during coagulation (Lane et al., 2005; Wolberg,
2007; Doormaal et al., 2008). These results suggest that
Citrus limon have anticoagulant effects due to inhibition
of thrombin, rather than altered activity of coagulation
factors.
Fig. 1: Effect of Citrus limon on plasma activity of
Protein C
n=10; Values are means ± S.E.M. Columns with (A) are
significantly different, P≤0.05
Results of present study showed significant inhibition of
platelet aggregation by Citrus limon at MCLD as well as
HCLD in presence of ristocetin and arachidonic acid a
response identical to aspirin (Walter et al., 1969). This
might be due to flavonoids, vitamin C and coumarin
compound-xanthoxyletin which are reported to inhibit the
activity of cyclooxygenase and lipoxygenase pathways
(Nijveldt et al., 2001) or modify membrane fluidity of
platelets (Furusawa et al., 2003). There are several studies
which show that these compounds have antiatherosclerotic and anti-platelet aggregation effects (Teng
et al., 1992; Heiss et al., 2005; Kim et al., 2008; Violi et
al., 2010; Bojic et al., 2011). Hence it may be proposed
that inhibition of platelet aggregation by Citrus limon is
due to these essential components.
Fig. 2: Effect of Citrus limon on plasma activity of
Thrombin antithrombin (TAT) complex
n=10; Values are means ± S.E.M; Columns with (A) are
significantly different, P≤0.05; Columns with (B) are highly
significant, P≤0.005.
Results of present study also reveals marked decrease in
Fb, hence it may be concluded that inhibition of platelet
aggregation might be due to this effect of Citrus limon,
since platelets need fibrinogen to activate aggregation of
platelets (Denninger et al., 1987; Peterson et al., 1989).
More over there are several studies which show that
platelet hyperaggregability and hyper-fibrinogenaemia are
associated risk factors for coronary heart disease (DeMaat et al., Silva et al., 1996; Bach et al., Miesbach et al.,
Seppanen-Laakso et al., 2010).
912
Present study shows increase in PC and TAT complex
which suggest that anticoagulant and antiplatelet action of
Citrus limon may be caused by impaired activity of
thrombin. Since PC and TAT complex are important
measures of inhibited thrombin (Arid, 2004; Chandler et
al., 2003; Lipe et al., 2011). Several studies have shown
that the level of PC and/or TAT complex, were decreased
in thrombotic disease patients and cardio-vascular events
(Moriau et al., 1995; Little et al., 2002). Hence Citrus
limon by elevating PC and TAT complex, may provide
beneficial effects in patients of thrombotic diseases.
Result of the present study showed significant changes in
hematological parameters by Citrus limon. There was
significant increase in RBC and Hb as compare to control.
While aspirin showed significant increase in WBC and
significant decrease in Hb, however there was no change
in RBC which is in consistency to the result of Merchant
et al., (2004). According to this report aspirin causes
chronic blood loss due to alteration of iron uptake. It
could be suggested that beneficial hematological effects
of Citrus limon are due to vitamin C, flavonoids, iron and
pyridoxine as their essential components. Hence several
studies showed their role in iron absorption due to
antioxidant action or nitric oxide synthesis their
consumption reduces the risk of death from CVD
(Gonzalez et al., 2010; Grassi et al., 2010; McCullough et
al., 2012).
Results of present study showed no change in PLT, Hct
and WBC by Citrus limon. Since normal platelet count is
essential for normal blood coagulation that might not
leads to bleeding problem in normal healthy subjects
(Harrison et al., 2007). No significant change in Hct is
beneficial effect with respect to platelet aggregation.
Since increase Hct enhance platelet adherence and
aggregation (Aarts et al., 1983). WBC’s were not
significantly changed because present study is on healthy
rabbits and Citrus limon might affect WBC in disease
conditions. Since Immuno-modulating and antibacterial
effects of Citrus limon and vitamin C has been reported in
several studies (Prabuseenivasan et al., 2006; Sun et al.,
2009).
Contrary to this expectation, all observed beneficial
effects in this study at MCLD seem to be depressed at
Pak. J. Pharm. Sci., Vol.27, No.4, July 2014, pp.907-915
Azra Riaz et al
HCLD group, which might be due to high contents of
vitamin C. Since high dose of vitamin C cause no change
as compare to low dose (Antunes et al., 1998). Hence
dosage adjustment is necessary to maintain drug
concentrations within their therapeutic windows (Tripoli
et al., 2007). It is suggested that different duration and
dosage of Citrus limon juice may play important role in
all their observed effects.
In recent years a dual role of thrombin has been revealed.
It is not only involved in blood coagulation, but also
associated with inflammatory response, cell-mediated
immunity and cell death (Di-Cera, 2008; Jenkins et al.,
2006; Krupiczojc et al., 2008). Anti-inflammatory
response of the flavonoids of Citrus limon is already
being documented (Chun et al., 2008). Hence it may be
proposed that Citrus limon may retard progression of
atherosclerosis and prevent cardiovascular diseases due to
its anti-inflammatory affect. Since coagulation and
inflammation has been reported as biological mediators of
cardiovascular disease (Arid, 2004; Rallidis et al., 2004;
Hamer et al., 2008).
On the basis of present data it may be concluded that the
Citrus limon have maximum anticoagulant and antiplatelet effects in rabbits at moderate dose i.e 0.4ml/kg,
which opens the door for further investigation on different
doses of Citrus limon, since it is a food rich in flavonoids
and vitamin C which may play vital role in the reduction
of CVD risks.
REFERENCES
Aarts PA, Bolhuis PA, Sakariassen KS, Heethaar RM and
Sixma JJ (1983). Red blood cell size is important for
adherence of blood platelets to artery subendothelium.
Blood, 62: 214-217.
Abdullah WZ, Moufak SK, Yusof Z, Mohamad MS and
Kamarul IM (2010). Shortened activated partial
thromboplastin time, a hemostatic marker for
hypercoagulable state during acute coronary event.
Translation. Res., 155: 315-319.
Ahmed A, Khan RA and Mesaik MA (2008). Antiimflamatory effect of natural honey on bovine
thrombin induced oxidative burst in phagocytes.
Phytother. Res., 22: 1-8.
Antunes LMG and Takahashi CS (1998). Effects of high
doses of vitamin C and E against doxorubicin-induced
chromosomal damage in Wistar rat bone marrow cells.
Mutation Res., 419: 137-143.
Arias BA and Ramon LL (2005). Pharmacological
properties of citrus and their ancient and their medical
use in the mediteraanean region. J. Ethnopharmacol.,
97: 89-95.
Arid CW (2004). Natural anticoagulant inhibitors:
activated Protein C. Best Practice & Res. Clinical
Haematol., 17(1): 161-182.
Pak. J. Pharm. Sci., Vol.27, No.4, July 2014, pp.907-915
Bach J, Haubelt H and Hellstern P (2010). Sources of
variation in factor VIII, von Willebrand factor and
fibrinogen measurements: Implications for detecting
deficiencies and increased plasma levels. Thrombosis
Res., 126: 188-195.
Berhow MA and Smolensky D (1995). Development and
substrate specificity of hesperetin-7-O-glucosyltransferase activity in Citrus limon tissues using highperformance liquid chromatographic analysis. Plant
Sci., 112: 139-147.
Bojic M, Debeljak Z, Tomicic M, Medić-Saric M and
Tomic S (2011). Evaluation of antiaggregatory activity
of flavonoid aglycone series. Nutrition, 10(73): 2-8.
Chandler LW and Valen T (2003). Estimating the rate of
thrombin and fibrin generation in vivo during
cardiopulmonary bypass. Blood, 101: 4355-4362.
Chun OKY, Chung S-J, Claycombe KJ and Song WO
(2008). Serum C-reactive protein concentrations are
inversely associated with dietary flavonoid intake in
U.S. Adults. Nutrition, 138: 753-760.
Clauss A (1957), Rapid physiological coagulation method
in determination of fibrinogen. Acta. Hematologica.,
17(4): 237-246.
De-Maat MPM, Pietersma A, Kofflard M, Sluiter W and
Kluft C (1196). Association of plasma fibrinogen levels
with coronary artery disease, smoking and
inflammatory markers. Atherosclerosis, 121: 185-191.
Denninger MH, Jandrot-Perrus M, Elion J, Bertrand O,
Homandberg GA, Mosesson MW and Gullin MC
(1987). ADP-induced platelet aggregation depends on
the confirmation or availability of the terminal gamma
chain sequence of fibrinogen. Study of the reactivity of
fibrinogen Pairs 1. Blood, 70: 558-563.
Deyhim F, Garica K, Lopez E, Gonzalez J, Ino S, Garcia
M and Patil BS (2006). Citrus juice modulates bone
strength in male senescent rat model of osteoporosis.
Nutrition, 22: 559-563.
Di-Cera E (2008). Thrombin. Review. Mol. Aspect of
Med., 29: 203-254.
Diab KAE, ELmakawy A, Abd-Elmoneim OM and Sharaf
HA (2012). Assessment of genotoxicity and
histopathological changes induced by polyethylene
glycol (PEG6000) in male mice. J. Cytology & Histol.,
3(153): 1-7.
Doormaal FFv, Buller HR and Middeldorp S. (2008).
Development in anticoagulant therapy. Critical Review
I. Oncol. Hematol., 66: 145-154.
Fischbach F and Zawta B (1992). Age dependent
reference limit of several enzyme in plasma at different
measuring temperatures. Klin. Lab., 38: 555-561.
Furusawa M, Tsuchiya H, Nagayama M, Tanaka T,
Nakaya K-ichi and Iinuma M (2003). Anti-platelet and
membrane-rigidifying flavonoids in brownish scale of
onion. J. of health sci., 49(6): 478-480.
Garcia MA, Gonzalez L, Lemini C and Rubio PC (2001).
Standardization of rat blood clotting tests with reagents
913
In vitro/in vivo effect of Citrus limon juice on blood parameters, coagulation and anticoagulation factors
used for humans. Proc. West. Pharmacol. Soc., 44:
153-155.
Gonzalez ME, Domínguez PR, Moreno DA and García
VC (2010). Natural bioactive compounds of Citrus
limon for food and health. J. of Pharm. and Biomed.
Anal., 51: 327-345.
Grassi D, Desideri G and Ferri C (2010). Flavonoids:
Antioxidants against atherosclerosis. Nutrients, 2: 889902
Hamer M and Emmanual S (2008). The accumulative
effects of modifiable risk factors on inflammation and
Haemostasis. Brain, Behavior and Immunity, 22: 10411043.
Harrison P, Frelinger AL, Furman I M and Michelson DA
(2007). Measuring antiplatelet drug effects in the
laboratory. Thromb. Res., 120: 323-336.
Heiss C, Kleinbongard P, Dejam A, Perre S, Schroeter H,
Sies H and Kelm M (2005). Acute consumption of
flavanol-rich Cocoa and the Reversal of Endothelial
Dysfunction in Smokers. J. of American Col. of
Cardiol., 46(7): 1276-1283.
Hernandez MV, Montes MMA and Elizalde GMP (2009).
Study of the thermal degradation of citrus seeds.
Biomass. Bioenergy., 33: 1295-1299.
Jenkins RG, Su X, Su G, Scotton CJ, Camerer E and
Laurent GJ (2006). Ligation of protease-activated
receptor 1 enhances alpha, beta 6 integr independent
TGF-beta activation and promotes acute lung injury. J.
Clin Invest, 116: 1606-1614.
Jeong YH, Hwang JY, Kim IS, Park Y, Hwang SJ, Lee
SW, Kwak CH and Park SW (2010). Adding cilostazol
to dual antiplatelet therapy achieves greater platelet
Inhibition than high maintenance dose clopidogrel in
patients with acute myocardial infarction results of the
adjunctive cilostazol versus high maintenance dose
clopidogrel in patients with AMI (ACCEL-AMI)
Study. Circ Cardiovasc Interv., 3: 17-26.
Johnstone MT, Andrews T, Ware JA, Rudd MA, George
D, Weinstein M and Loscalzo J (1990). Bleeding time
prolongation with streptokinase and its reduction with
1-desamino-8-D-arginine vasopressin. Circulation, 82:
2142-2151.
Kanahara M, Kai H, Okamura T, Wada T, Suda K,
Imaizumi T and Sagawa K (2008). Usefulness of highconcentration calcium chloride solution for correction
of activated partial thromboplastin time (APTT) in
patients with high-hematocrit value. Thrombos. Res.,
121: 781-785.
Khan Y, Khan AR, Afroz S and Siddiq A (2010).
Evaluation of hypolipidemic effect of Citrus lemon. J.
Basic. Applied Sci., 6: 39-43.
Khanin MA, Rakov DV and Kogan AE (1998).
Mathematical model for the blood coagulation
prothrombin time test. Thrombosis res., 89: 227-232.
Kim HP, Son KH, Chang HW and Kang SS (2004). Antiinflammatory plant flavonoids and cellular action
mechanisms. J. of Pharmacol. Sci., 96: 229-245.
914
Kim MJ and Yun-CHS (2008). Antiplatelet effects of
flovonoids and flovonoid-glycosides from Sophora
japonica. Arch. Pharm. Res., 31(7): 886-890.
Krupiczojc MA, Scotton CJ and Chambers RC (2008).
Coagulation signalling following tissue injury: Focus
on the role of factor Xa. The International J. of
Biochem. & Cell Biol., 40: 1228-1237.
Kung-chi C, Mei-chi Y and Wan-Ju C (2007). Effect of
diallyl trisulfide-rich garlic oil on blood coagulation
and plasma activity of anticoagulation factors in rats.
Food. Chemical Toxicol., 45: 502-507.
Kuntic V, Filipovic I and Vujic Z (2011). Effects of rutin
and hesperidin and their Al (III) and cu (II) complexes
on in vitro plasma coagulation assays. Molecules, 16:
1378-1388.
Lane AD, Philippou H and Huntington AJ (2005).
Directing thrombin. Blood, 106: 2605-2612.
Li H, Cone J, Fong M, Kambayashi J, Yoshitake M and
Liu Y (2005). Anti-platelet and Anti-thrombotic
activity of clostazol is potentiated by dipyridamole in
rabbits and dissociated from bleeding time
pronlongation. Basic pharmacol. Cardiovas. Drugs and
Therap., 19: 41-48.
Lipe B and Ornstein LD (2011). Deficiencies of natural
anticoagulants, protein C, protein S and ntithrombin.
Circulation, 124: 365-368.
Little WJ, Miller SC, Henry GR, McIntosh AB, Fla N and
Ky L (2002). Antithrombotic agents: Implication in
dentistry. Oral surg oral med oral pathol oral radiol
endod., 93: 544-551.
Mann KG, Butenas S and Brummel K (2003). The
dynamics of thrombin formation. Atherioscler.
Thombos. Vasc. Biol., 23: 17-25.
McCullough ML, Peterson JJ, Patel R, Jacques PF, Shah
R and Dwyer JT (2012). Flavonoid intake and
cardiovascular disease mortality in a prospective cohort
of US adults. Am. J. Clinical. Nutrit., 95(2): 454-464.
McNerlan SE, Crawford VLS and Stout RW (1997).
Measurement of fibrinogen in frozen plasma.
Thrombos. Res., 88: 481-484.
Merchant MA and Modi DN (2004). Acute and chronic
effects of aspirin on hematological parameters and
hepatic ferritin expression in mice. Indian J.
Pharmacol., 36: 226-230.
Miesbach W, Schenk J, Alesci S and Lindhoff-Last E
(2010). Comparison of the fibrinogen clauss assay and
the fibrinogen PT derived method in patients with
dysfibrinogenemia. Thrombos. Res., 126: 428-433.
Moriau M, Lavenne-Pardonge E, Crasborn L and
Frenckell Rv (1995). The treatment of severe of
recurrent deep venous thrombosis. Beneficial effects of
the co-administration of antiplatelet agents with or
without rheological effects and anticoagulants.
Thrombosis res., 78: 469-482.
Nijveldt R, Nood Ev, Hoorn DECv, Boelens PG, Norren
Kv and Leeuwen PAMv (2001). Flavonoids: A review
Pak. J. Pharm. Sci., Vol.27, No.4, July 2014, pp.907-915
Azra Riaz et al
of probable mechanisms of action and potential
applications. Am. J. Clin. Nutr., 74: 418-25.
Peterson AJ, Visentin PG, Newman JP and Aster SR
(1989). A recombinant soluble form of the Integrin
?IIbB3 (GPIIb-IIIa) assumes an active, ligand-binding
conformation and is recognized by GPIIb-IIIa - specific
monoclonal, allo auto and drug-dependent platelet
antibodies. Blood, 92: 2053-2063.
Pfister SL (2006). Aortic thromboxane receptor deficiency
alters vascular reactivity in cholesterol-fed rabbits.
Atheroscler., 189: 358-363.
Prabuseenivasan S, Jayakumar M and Ignacimuthu S
(2006). In vitro antibacterial activity of some plant
essential oils. BMC Complement and Alternat. Med.,
6(39): 1-8.
Rallidis LS, Paschos G, Maria LP, Georgios KL,
Demosthenes BP, Georgios A and Zampelas A (2004).
The effect of diet enriched with α-linolenic acid on
soluble cellular adhesion molecules in dyslipidaemic
patients. Atheroscler., 174: 127-132.
Rao LV, Okorodudu AO, Petersen JR and Elghetany MT
(2000). Stability of prothrombin time and activated
partial thromboplastin time tests under different storage
conditions. Clinica Chimica. Acta., 300: 13-21.
Riaz A, Khan RA and Ahmed SP (2009). Assessment of
anticoagulant effect of evening primrose oil. Pak. J.
Pharm. Sci., 22: 355-359
Seppanen-Laakso T, Laakso I, Lehtimak T, Rontu R,
Moilanen E, Solakivi T, Seppo L, Vanhanen H,
Kiviranta K and Hiltunen R (2010). Elevated plasma
fibrinogen caused by inadequate α-linolenic acid intake
can be reduced by replacing fat with canola-type
rapeseedoil. Prostagland. Leukotr and Essent. Fat.
Acids., 83: 45-54.
Silva MP, Ferhndez-RJM and Luque CMD (1996). Light
scattering-based determination of fibrinogen in human
plasma using an automated continuous system.
Analytica Chimica Acta., 327: 101-106.
Son DJ, Lee HW, Shin HW, Lee JJ, Yoo HS, Kim TJ, Yun
YP and Hong JT (2008). Enhanced release of
sphingosine-1-phosphate from hypercholeserolemic
platelets: Role in development of hypercholesterolemic
Atherosclerosis. Prostagland. Leukotr and Essent. Fat.
Acids, 78: 383-390.
Sun P, Li D, Dong B, Qiao S, Ma X and Chen X (2009).
Vitamin C: An immunomodulator that attenuates
Pak. J. Pharm. Sci., Vol.27, No.4, July 2014, pp.907-915
anaphylactic
reactions
to
soybean
glycinin
hypersensitivity in a swine model. Food chemistry,
113: 914-918.
Teng CM, Li HL, Wu TS, Huang SC and Huang TF
(1992). Antiplatelet actions of some coumarin
compounds isolated from plant sources. Thromb. Res.,
66(5): 549-57.
Tripoli E, Guardia ML, Giammanco S, Di MD and
Giammanco M (2007). Citrus flavonoids: Molecular
structure, biological activity and nutritional properties:
A review. Food Chemistry, 104: 466-479.
Vazquez PRL, Martinez DE, Perona SJ and Ruiz GV
(2004). Effect of different dietary oils on inflammatory
mediator generation and fatty acid composition in rat
neutrophils. Metabol., 53: 59-65.
Violi F, Pignatelli P and Basili S (2010). Nutrition,
supplements and vitamins in platelet function and
bleeding. Circulation, 121: 1033-1044.
Viuda MM, Ruiz NY, Fernandez LJ and Perez AJ (2008).
Antifungal activity of lemon (Citrus lemon L.),
mandarin (Citrus reticulata L.), grapefruit (Citrus
Paradisi L.) and orange (Citurs sinensis L.) essintial
oils. Food Control, 19: 1130-1138.
Walter BEJ, Owen JR and Charles A (1969). Aspirin,
Platelets, and Bleeding. Circulation, 40: 757-760.
Wang X-M YH, Steinbacher TE, Monticello TM,
Schumacher WA (2007). Quantification of platelet
composition in experimental venous thrombosis by
real-time polymerase chain reaction. Thrombosis Res.,
119: 593-600
Warfarin dosing guideline, 2009, DISCLAIMER:
guidelines were prepared by the Department of
Surgical Education, Orlando Regional Medical Center.
www.Surgical Critical Care.net
Wayne PA (1998). Collection, transport and processing of
blood specimens for coagulation testing and general
performance of coagulation assays. Approved guideline
H21-A2, 3rd Edition, National Committee for Clinical
Laboratory Standards, 1998. (NCCLS Document H21A3).
Wolberg SA (2007). Thrombin generation and fibrin clot
structure. Blood, 21: 131-142.
Xu G, Liu D, Chen J, Ye X, Ma Y and Shi J (2008). Juice
component and antioxidant capacity of citrus varieties
cultivated in China. Food chemistry, 106: 545-441.
915