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The role of apigenin in an experimental model of acutepancreatitis

Pavlos Lampropoulos, BA, MD, MSc, PhD,a,* Maria Lambropoulou, PhD,a,b

Apostolos Papalois, PhD,c Neofitos Basios, MSc,a Maria Manousi, MSc,a

Constantinos Simopoulos, PhD,a,d and Alexandra K. Tsaroucha, PhDa,d

aPostgraduate Program in Hepatobiliary/Pancreatic Surgery, Faculty of Medicine, Democritus University of Thrace, Alexandroupolis, Greeceb Laboratory of Histology, Faculty of Medicine, Democritus University of Thrace, Alexandroupolis, GreececExperimentaleResearch Center, ELPEN Pharmaceuticals, Pikermi, Attica, Greeced2nd Department of Surgery and Laboratory of Experimental Surgery, Faculty of Medicine, Democritus University of Thrace, Alexandroupolis,

Greece

a r t i c l e i n f o

Article history:

Received 10 September 2012

Received in revised form

24 October 2012

Accepted 29 November 2012

Available online 20 December 2012

Keywords:

Apigenin

Experimentally induced pancreatitis

Bilio-pancreatic duct ligation

* Corresponding author. Faculty of Medicine,6957200180; fax: þ30 2102463411.

E-mail address: pav.lampropoulos@gmai0022-4804/$ e see front matter ª 2013 Elsevhttp://dx.doi.org/10.1016/j.jss.2012.11.053

a b s t r a c t

Aim: The aim of the present study is to evaluate pathologic changes in the pancreatic

parenchyma in an experimental model of acute pancreatitis (AP) following bilio-pancreatic

duct ligation. An effort was made to clarify the role of apigenin, a substance that is well-

known for its antioxidant and anti-inflammatory role and its likely beneficial activity to

the pancreatic parenchyma following AP in rats.

Material and method: One hundred twenty-six male Wistar rats 3e4 mo old and weighing

220e350 g were used. At time 0, the following groups were randomly assigned: group sham:

rats were subjected to virtual surgery; group control: rats were subjected to surgery for

induction of AP, by ligation of the bilio-pancreatic duct; group apigenin: rats were subjected

to surgery for induction of AP and enteral feeding with apigenin. Pathologic changes of the

pancreatic parenchymal and myeloperoxidase activity were measured at predetermined

time intervals 6, 12, 24, 48, and 72 h.

Result: From the pathologic reports, by comparing the control group with the apigenin

group, an improvement of pancreatic tissue architecture following apigenin administration

was observed. Inflammatory infiltration, edema, ductal dilation, and necrosis were reduced

following apigenin administration over time (P ¼ 0.049, P ¼ 0.228, P ¼ 0.387, P ¼ 0.046).

Treatment with apigenin significantly reduced the bilio-pancreatic duct ligation and

evoked an increase in pancreatic myeloperoxidase activity (P ¼ 0.030).

Conclusion: Oral apigenin administration in rats, following experimentally induced pancre-

atitis, seems toprotect thepancreatic tissue. Thus, apigenin administration tohumans could

potentially ameliorate the damages to the pancreas.

ª 2013 Elsevier Inc. All rights reserved.

Democritus University of Thrace, Mikras Asias 13, Ilioupolis 16345, Athens, Greece. Tel.: þ30

l.com (P. Lampropoulos).ier Inc. All rights reserved.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) 1 2 9e1 3 7130

1. Introduction 2. Materials and methods

Acute pancreatitis (AP) is a quite common inflammatory

disease which in 10%e20% of all cases leads to severe AP [1],

involving remote organ failure and finally multiple organ

dysfunction syndrome. The precise mechanisms that initiate

an episode of AP are not clearly understood but once initi-

ated; all cases share the same inflammatory and repair

pathways [2]. Acute lung injury is almost always present

in cases of severe AP [3] and manifests itself as acute

respiratory distress syndrome [4], as a result of the system-

ic inflammatory response syndrome. The most important

inflammatory mediators involved in the pathogenesis of AP

are tumor necrosis factor-alpha, interleukin (IL)-1b, IL-6,

platelet activating factor, IL-10, granulocyte macrophage-

colony stimulating factor, C5a, intercellular adhesion

molecule-1, reactive oxygen species, and reactive nitrogen

species [5e7].

Mortality rates of AP are related to necrosis, bacterial

contamination, and pancreatic ascites. Thus, in cases of

pancreatic necrosis, the mortality rate ranges from 7% if the

necrosis is <30% of the pancreatic tissue to 50% if more than

50% of the pancreatic tissue is necrotized. Bacterial contami-

nation triples themortality rate from 10% to 32%, whereas the

presence of pancreatic ascites increases the rate from 9% to

36% [8].

Bearing inmind that inflammatorymediators play a crucial

role in the progression of pancreatitis from mild edema of the

pancreas to multiple organ dysfunction syndrome, there is

an ongoing research to find evidence-based pharmacological

treatment focused on targeted anti-inflammatory drugs [9,10].

Apigenin (4,5,7 trihydroxyflavone) found in fruits, plants, and

vegetables is known for its anti-inflammatory, antioxidant,

anti-allergic, anti-osteoporotic, and even anti-cancerous ac-

tivities [11e14]. To the best of our knowledge, apigenin has

never been studied before as a possible therapeutic agent in

experimental models of AP.

Early events of AP in humans are not possible to be studied

in depth for two main reasons. First, the diagnosis of AP is

usually set late, once damage to the pancreas has been initi-

ated, and second, there is no available biopsy material from

patients with early onset AP. This fact hasmade efforts to find

an ideal experimental model of AP popular. There are

a significant number of such models developed, each with its

own advantages and drawbacks [15e18]. Duct obstruction

induced pancreatitis model can mimic both benign and

malignant disorders of the humans [18]. This model does not

require special surgical dexterity and once ligation occurs

close to the entry of the common bilio-pancreatic duct to the

duodenum, the model resembles gallstone obstruction at the

ampulla of Vater [18e20].

The purpose of the present study is to evaluate the pa-

thologic changes to the pancreatic parenchyma in an ex-

perimental model of AP in rats, following ligation of the

bilio-pancreatic duct close to the duodenum and to examine

the possible beneficial role of apigenin. In order to accomplish

this, myeloperoxidase (MPO) activity of the pancreatic tissue

will be examined, along with all histopathologic parameters

of AP.

2.1. Animals and design of the study

One hundred twenty-six Wistar male rats, 3e4 mo old and

weighing 220e350 g, were used in this study. All rats were

maintained under conventional conditions of controlled

temperature (22e25�C), humidity (55%e58%), and lighting

(12 h light/12 h dark), with free access to tap water and rat

chow diet. The animals were supplied by Pasteur Institute,

Athens, Greece; all experiments took place at the approved

Experimental Research Center of ELPEN Pharmaceuticals,

Athens, Greece, while the pathology examination took place at

the Laboratories of the Medical School of Democritus Univer-

sity of Thrace. The experimental procedures conform to

National Research Council Guide for the Care and Use of

Laboratory Animals and Directive 86/609 of the European

Union, protocol number (K/2284). The experimental animals

were randomly assigned in three groups, namely sham group

(n ¼ 20), control group (induction of pancreatitis) (n ¼ 56),

apigenin group (induction of pancreatitis plus administration

of apigenin) (n ¼ 50). This unbalanced randomization design

does not compromise the statistical power [21,22].

2.2. Experimentally induced AP in rats

Before surgery rats were anesthetized in a specially designed

glass box for about 2 to 3 min with isoflurane, following

a subcutaneous injection of 0.25 cc of butorphenol (Dolorex;

Intervet/Schering/Plough Animal Health, Boxmeer, Holland).

Soon thereafter, endotracheal intubation was performed

under direct laryngoscopy by a trained veterinarian and

research assistants with the use of a 16-G venous catheter

connected toa rodentventilator (HarvardApparatus,Holliston,

MA) at the following settings: tidal volume: 3 mL; rate: 70

breaths/min. Proper intubation was confirmed by observation

of chest expansion and retraction and lung auscultation.

Anesthesiamaintenancewas accomplishedusing amixture of

93% O2, 5% CO2, and 2% isoflurane (Fig. 1A and B).

Acute pancreatitis was induced as described previously

[18], by ligation of the common bilio-pancreatic duct close to

the duodenum. Briefly, soon after anesthesia, a 3 cm midline

incision was performed under sterile conditions and entry to

the abdominal cavity was accomplished. The bilio-pancreatic

duct was identified and ligated close to the duodenum with

a4-0 silk suture (Fig. 1C). Before closureof theabdominal cavity

with vicryl 2-0, 1 cc of natural saline and 1 cc D5W were

instilled. All animals regained consciousness as soon as they

were extubated. For the sham operated animal group, the

experiments were terminated without the ligation step. Soon

after the midline incision, the intraperitoneal pancreas was

identified, manually mobilized, and no further action was

taken before closure of the abdominal cavity. For the apigenin

animal group, after termination of the surgical ligation proce-

dure, a prepared 4 cc apigenin solution was administered

orally, as described later (Fig. 1D). Analgesia (2 cc/kg butor-

phenol, Dolorex) for all animals was given subcutaneously at

a predetermined time every 4 h, subsequently according to the

animal’s need based on the clinical picture.

Fig. 1 e Photographs taken during experiment: animal after anesthesia with endotracheal intubation (A), animal following

termination of the experiment, showing midline incision (B), black arrow shows bilio-pancreatic duct of the animal (C),

administration of apigenin orally post-surgery (D). (Color version of figure is available online.)

Table 1 e Modified immunochemistry scoring system bySidhu et al. of different markers of severity on AP [23].

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) 1 2 9e1 3 7 131

Eight rats died before scheduled euthanasia, one during the

surgical experiment due to rupture of the trachea from the

control group and seven post-surgery, one from the apigenin

group and six from the control group. These animals were not

included in the statistical analysis. Animals were sacrificed

according to the protocol: control group (at 6 h 12 rats, 12 h 12

rats, 24 h 10 rats, 48 h 12 rats, and 72 h 12 rats, post-surgery);

apigenin group (at 6 h 10 rats, 12 h 10 rats, 24 h 10 rats, 48 h 10

rats, and 72 h 10 rats, post-surgery). The rats were sacrificed

at a predetermined time as detailed above with ketamine

(Narcetan; Vetoquinol, Buckingham, UK) 0.3e0.6 cc and xyla-

zine (Rompun; Bayer, Uxbridge, UK) 0.1e0.3 cc up to triple

dosage.

Edema 0dAbsent or rare

1dEdema in interlobular space

2dEdema in intralobular space

3dMarked and diffuse edema, greater

than 2

Inflammatory

infiltration

0dAbsent

1dMild inflammatory cell infiltration

2dModerate inflammatory cell

infiltration

3dSevere inflammatory cell infiltration

Duct dilatation 0dAbsent

2.3. Apigenin solution preparation

Apigenin was purchased from Sigma-Aldrich, Taufkirchen,

Germany in vials with >99% purity by thin layer chromatog-

raphy, as yellow powderwith tan cast. Apigeninwas dissolved

as follows: 660 mg apigenin was dissolved in 500 cc corn oil,

2 cc dimethyl sulfoxide, and 10 cc Tween 80. The final con-

centration was 5 mg Apigenin in 4 cc solution.

1dMinor peripheral dilatation

2dAs 1 and/or central dilatation

3dAs 2 and/or marked central dilatation

Hemorrhage 0dAbsent

1dMild

2dModerate

3dSevere

Acinar necrosis 0dAbsent

1dSmall foci or small multiple foci

2dLarger confluent foci

3dExtensive and diffuse

2.4. Histopathology and histopathologic evaluation

Histopathologic examination was performed in hematoxylin-

eosin stained slides and involved the evaluation of the

following parameters for each case: hemorrhage, ducts dila-

tation, edema, acinar necrosis, and inflammation. Alterations

were quantified according to a scoring system that ranged

fromabsence to severe lesions (0: none, 1:mild, 2:moderate, 3:

severe). Histological scoring was accomplished according to

Sidhu’s scoring system with modification (Table 1) [23]. The

scores of each parameter for each slide were added to obtain

the histopathologic score [24].

2.5. Immunohistochemistry

All animals were sacrificed, exsanguinated, and the pan-

creatic tissue was harvested. The immunohistochemistry

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) 1 2 9e1 3 7132

procedure has been described elsewhere [25]. Briefly, 4-mm

thick sections were mounted on glass slides, dewaxed, and

rehydraded. The kit, En Vision HRP mouse/rabbit detection

system (K5007; DAKO, Carpinteria, CA) for the streptavidin

biotin technique was used. To inhibit endogenous peroxidase,

the specimens were incubated with 3% H2O2 (200 cc H2O and

6 cc H2O2) for 15 min in a dark room. Before the primary

antibody was applied, the sections were immersed in 10 mM

citrate buffer (pH 6.0), rinsed in Tris-buffered saline, and

subsequently heated in a microwave oven (650e800 W) for

three cycles of 5 min. The slides were washed with Tris-buff-

ered saline before application of the primary antibody in order

to reduce nonspecific binding of antisera. Control slides were

used as common negative controls for all antibody staining.

Sections then were briefly counterstained with Mayer’s

hematoxylin, mounted, and examined under a Nikon Eclipse

50i microscope (Nikon Instruments Inc, NY).

Scoring was assigned according to the proportion of cells

with cytoplasmic staining. Sections with greater than 10%

stained cells were considered as being positive (0: negative; 1:

low; 2: moderate; and 3: high expression). The positive expres-

sion of MPO was determined by counting the number of stain-

ed cells. The average labeling index of MPO was assessed

according to the proportion of positive cells, after scanning the

entire section of the specimen. The results of MPO expression

were graded as negative (0) for�10% of stained cells, low (1) for

>10% and �30% of cells stained, moderate (2) for >30% and

�70% cells stained, and high (3) for <70% cells stained.

2.6. Determination of pancreatic MPO activity

The antibody MPO (rabbit polyclonal), DAKO (A 0398 DAKO,

Carpinteria, CA), was used at 1:400 dilution. In brief, pancre-

atic tissue was homogenized in 50 mmol/L potassium phos-

phate buffer (pH 6) with 0.5% hexadecyl-trimethyl ammonium

bromide [26]. The homogenates were then centrifuged for

10 min at 40,000 at 4�C. The resultant supernatant reacted

with o-dianisodine dihydrochloride and H2O2 and the absor-

bance was determined spectrophotometrically at 450 nm (and

540 nm as control wavelength). MPO activity serves as

a marker for neutrophil infiltration [27].

Fig. 2 e Representative results of H and E staining in rat

pancreatic tissue: sham group, 3200 at 24 h (A), control

group3100 at 24 h (B), and apigenin group 3200 at 24 h (C).

(Color version of figure is available online.)

3. Statistical analysis

Weutilized SPSS v. 15, (SPSS Inc., Chicago, IL) for the statistical

analysis of the data. Out of 126 rats initially entered in the

experiment, eight died before the scheduled time and were

therefore not considered in further analysis. Owing to the

ordinal nature of inflammatory markers used (number of

crosses indicated intensity of findings) and the lack of

normality in distribution, we used nonparametric statistical

analysis for qualitative and ordinal data (Fisher’s exact test,

KruskaleWallis test and ManneWhitney test for independent

samples). Through Fisher’s exact test we examined the qual-

itative differentiation of markers because of the segregation

into three separate treatment groups (sham, apigenin,

control), while through the KruskaleWallis test we examined

this segregation also taking into account the ordinal (semi-

quantitative) nature of the data.

Finally, the ManneWhitney test was further used for

comparison between groups, using two groups at a time (all

possible combinations), to investigate a possible differentia-

tion and the direction of this trend. The above approach was

performed for the overall sample (all times of death, 118 rats)

and then repeated for each scheduled time of death (at 6, 12,

24, 48, and 72 h postoperatively), separately, to highlight

differences depending on time elapsed from initial surgical

Table 2e Scoring of hemorrhage (SD[ 0, 0.53 ± 0.54, 0.93 ± 0.45), edema (SD[ 0, 0.57 ± 0.5*, 0.45 ± 0.5*), duct dilatation (0.5± 0.51*, 0.88 ± 0.56, 0.77 ± 0.42*), acinar necrosis (SD[ 0, 0.28 ± 0.46*, 0.12 ± 0.33*), inflammatory infiltration (0.5 ± 0.51*, 1.06± 0.69, 0.79 ± 0.54), andMPO activity (0.65 ± 0.81, 1.77 ± 0.74, 1.45 ± 0.54) in test subjects, in the three separate groups (Sham,Control, Apigenin, Mean ± SD).

Hemorrhage score Edema score

0 1 2 0 1

n (%) n (%) n (%) n (%) n (%)

Sham 20 (100.00) 0 (0) 0 (0) 20 (100.00) 0 (0)

Apigenin 7 (14.29) 39 (79.69) 3 (6.12) 27 (55.10) 22 (44.90)

Control 24 (48.98) 24 (48.98) 1 (2.04) 21 (42.86) 28 (57.14)

Total 51 (43.22) 63 (53.39) 4 (3.39) 68 (57.63) 50 (42.37)

Duct dilatation score Acinar necrosis score

0 1 2 0 1

n (%) n (%) n (%) n (%) n (%)

Sham 10 (50.00) 10 (50.00) 0 (0) 20 (100.00) 0 (0)

Apigenin 11 (22.45) 38 (77.55) 0 (0) 43 (87.76) 6 (12.24)

Control 11 (22.45) 33 (67.35) 5 (10.20) 35 (71.43) 14 (28.57)

Total 32 (27.12) 81 (68.64) 5 (10.20) 98 (83.05) 20 (16.95)

Inflammatory infiltration score

0 1 2 3

n (%) n (%) n (%) n (%)

Sham 10 (50.00) 10 (50.00) 0 (0) 0 (0)

Apigenin 13 (26.53) 33 (67.35) 3 (6.12) 0 (0)

Control 9 (18.37) 29 (59.18) 10 (20.41) 1 (2.04)

Total 32 (27.12) 72 (61.02) 13 (11.02) 1 (2.04)

MPO Score

0 1 2 3

n (%) n (%) n (%) n (%)

Sham 11 (55.00) 5 (25.00) 4 (20.00) 0 (0)

Apigenin 0 (0) 28 (57.14 20 (40.82) 1 (2.04)

Control 0 (0) 20 (40.82) 20 (40.82) 9 (18.37)

TOTAL 11 (9.32) 53 (44.92) 44 (37.29) 10 (8.47)

* Variable contains two attributes.

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) 1 2 9e1 3 7 133

intervention. Because of the almost perfect correlation

between one of the groups and specific findings, logistic

regression, in any form, was not possible to perform.

4. Results

4.1. Histopathologic changes in our experimental modelof AP

Histopathologic examination of the sham group showed

normal architecture of the pancreatic parenchyma, with no

edema, hemorrhage, inflammatory infiltration, duct dilata-

tion, or necrosis (Fig. 2A). On the contrary, rats in the Control

group (bilio-pancreatic duct ligation induced AP) exhibited

major architectural disturbance, with severe edema, hemor-

rhage, inflammatory infiltration, duct dilatation, and necrosis

(Fig. 2B). Rats in the apigenin group (bilio-pancreatic duct

ligation induced AP þ apigenin) showed milder morphologic

changes in the pancreatic parenchyma overall compared with

the control group (Fig. 2C).

4.2. Apigenin does not improve hemorrhage in rats withexperimental AP

At all times of death, the control group shows less hemor-

rhage compared with the apigenin group (P < 0.001). At

separate times of death, apigenin vs. control group compari-

sons significantly favored the control group at 12 (P value

0.016) and 48 (P value 0.006) h postoperatively. At 72 h,

however, apigenin the group shows less hemorrhage than the

control group but it does not reach statistical significance

(P ¼ 0.292) (Table 2, Fig. 3A).

4.3. Apigenin improves duct dilation in rats withexperimental AP, at 24 h onwards postsurgery

At all times of death, the apigenin group had improved

duct dilatation compared with the control group, with no

Fig. 3 e Immunochemical manifestations of experimentally induced AP in the three studied group (sham,

control, apigenin). The time course over 72 h is shown for (A) hemorrhage, (B) duct dilatation, (C) edema, (D) acinar

necrosis, (E) inflammatory infiltration, and (F) MPO activity. ManneWhitney test of apigenin versus control group

with statistical significance (P < 0.05) at 6 h for MPO activity (P [ 0.002), at 12 h for hemorrhage (0.016) and MPO

activity (P [ 0.049), at 48 h for hemorrhage (0.006) and MPO activity (P [ 0.001), at 72 h for duct dilatation

(P [ 0.008), edema (P [ 0.038), acinar necrosis (P < 0.001), inflammatory infiltration (P < 0.001), and MPO activity

(P < 0.001). (Color version of figure is available online.)

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) 1 2 9e1 3 7134

statistical significance (P ¼ 0.387). Even though at 6 and 12 h,

the apigenin group showed more duct dilatations than the

control group, we observed an opposite effect at 24 h in

favor of the apigenin group at 24 h (P ¼ 0.385), reaching

statistical significance at 72 h post-surgery (P ¼ 0.008)

(Table 2, Fig. 3B).

4.4. Apigenin improves edema in rats with experimentalAP at 72 h post-surgery

At all times of death, the control group showed much more

edema formation of the pancreatic acinar cells, interlobular

and intralobular space, compared with the apigenin group

(P ¼ 0.228). At different time intervals, the apigenin group had

improved edema compared with the control group (at 12 h

P ¼ 0.167, at 24 h P ¼ 0.081), with opposite results at 48 h

(P ¼ 0.167). At 72 h, however, apigenin administration showed

its beneficial effect over the control group (P ¼ 0.038) (Table 2,

Fig. 3C).

4.5. Apigenin improves necrosis in rats withexperimental AP at 72 h post-surgery

At all times of death, the control group showed much more

necrotic elements, compared with the apigenin group

(P ¼ 0.046). At 72 h post-surgery, the apigenin group improved

necrosis compared with the control group, reaching statistical

significance (P < 0.001) (Table 2, Fig. 3D).

4.6. Apigenin improves inflammatory infiltration in ratswith experimental AP at 72 h post-surgery

Between the two groups of control and apigenin treatment,

the apigenin group versus the control group significantly

favored apigenin for inflammatory infiltration (P value 0.049).

Results at different time intervals up to 48 h post-surgery do

not reach statistical significance; at 72 h, however, apigenin

shows its anti- inflammatory effect (P< 0.001) (Table 2, Fig 3E).

4.7. Apigenin reduces MPO activity in rats withexperimental AP overall

In the control group, pancreatic MPO activity, was signifi-

cantly elevated when compared with the sham group

(P < 0.001). Treatment with apigenin significantly reduced the

bilio-pancreatic duct ligation evoked increase in pancreatic

MPO activity (P ¼ 0.030) (Fig. 4). At separate times of death,

apigenin versus control comparisons presented significant

differences for MPO activity, favoring the control group at 6

(P ¼ 0.002) and 12 (P ¼ 0.049) h, whereas the result was

reversed and presented in favor of the apigenin group at 48

(P < 0.001) and 72 (P value < 0.001) h postoperatively (Table 2,

Fig. 3F).

Fig. 4 e Representative results of MPO immunostaining in

rat pancreatic tissue: sham group, 3200 at 72 h (A), control

group3200 at 72 h (B), and apigenin group 3200 at 72 h (C).

(Color version of figure is available online.)

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) 1 2 9e1 3 7 135

5. Discussion

Apigenin is an active member of the flavones family, constit-

uent of polyphenolic compounds [28], which are abundant in

nature, mostly in aromatic plants (i.e., chamomile, mint,

parsley), fruits, vegetables, honey, andwheat [29,30]. Apigenin

has been described to have anti-inflammatory, antioxidant,

anti-tumorous, anti-allergic, and anti-osteoporotic effects

[11e14]. Anti-inflammatory effects of apigenin are accom-

plishedbyvariousmechanisms.Onepossiblemolecular action

is the inhibition of the cytokine-induced adhesion protein

expression in endothelial cells [31]. Furthermore, apigenin is

suggested to be able to augment apoptosis of recurrently acti-

vated human T cells [31], by interfering in both extrinsic and

intrinsic pathways. However, apigenin has no action on

primarily activated CD4þ T cells [32]. Another study [33]

demonstrated that chamomile extract inhibits the release of

PGE2 from lipopolysaccharide-activated RAW 264.7 macro-

phages in vitro. This is accomplished by dose-dependent inhi-

bition of cyclooxygenase (COX)-2 enzyme activity. In addition,

chamomile extract may reduce COX-2 messenger RNA and

protein expression, but has no effect on the activity or

expression of COX-1. Furthermore, apigenin reduces inflam-

mation and leukocyte infiltration in an experimental model of

inflammation induced via the simultaneous injection of car-

aginan and prostaglandin E1 [34].

The present result are in accordance with these findings,

as it has been shown that apigenin improves all pathologic

parameters of inflammation to the pancreatic tissue and

reducesMPO activity, amarker of inflammation and leukocyte

invasion. However, this effect is time-dependent as benefits

from apigenin administration are seen at 72 h post-apigenin

administration in our experimental model. It seems that per

osadministrationofapigeninrequires sometimeinorder toact

through activation of anti-inflammatory signaling pathways

or inhibition of inflammatory molecular markers (i.e., IL-4

production) [35]. In order to understand this time-dependent

action of apigenin, it is crucial to analyze its intestinal absorp-

tion, first-passmetabolism, and bioavailability. Recently, it has

been shown that apigenin is quickly absorbed into the circu-

lation, following oral administration [35]. In this study, the

pharmacokinetic parameters of apigenin in comparison with

other polyphenolic compounds were analyzed K (h�1) ¼ 0.16�0.02, T1/2Ke (h) ¼ 2.11 � 0.03, Tpeak (h) ¼ 0.50 � 0.01, Cmax (ng/

mL) ¼ 42 � 2, AUC (ng�h/mL) ¼ 659 � 25 [36].

In the present study, 4cc of apigenin solution were

administered as a single dosage, containing 5 mg apigenin,

dosage (11.11e22.73 mg/Kg). This dosage proved to be capable

of satisfying our hypothesis that apigenin is a potent anti-

inflammatory agent. This dosage selection was based on

previous studies investigating daily dietary exposure of api-

genin to humans [37]. There have been some questions raised

regarding the safety of apigenin. It is suggested that apigenin

given in 100 or 200mg/kg dosage induces oxidative stress liver

damage in Swiss mice [38]. Similar studies show that apigenin

induces cytotoxicity [39], and produces phenoxyl radicals and

reactive oxygen species [40e42]. However, as shown in our

experiment, apigenin is not cytotoxic but, on the contrary, is

a strong shield against inflammatory processes.

In our results, hemorrhage was more pronounced in the

apigenin group than the control group. This result shows that

apigenin is incapable of reducing hemorrhage in our experi-

mental model of AP. Many investigators have studied the

hemorrhagic effect of apigenin through different mechanisms.

Wright et al. concluded that apigenin, together with other struc-

turally distinct flavonoids, interfere with collagen-stimulated

platelet function by blockage of Fyn kinase activity and the

j o u r n a l o f s u r g i c a l r e s e a r c h 1 8 3 ( 2 0 1 3 ) 1 2 9e1 3 7136

tyrosine phosphorylation of Syk and PLCg2, after internalization

of flavonoids in a megakaryocytic cell line [43]. In a similar

fashion, it has been shown that apigenin does not interfere with

thrombin receptors but rather apigenin impairs platelet

aggregation through signaling pathways that inhibit kinase

activation [44]. At last, it has been shown that apigenin, syner-

gistically with aspirin, suppresses the arachidonic acid pathway

[45]. All these important findings may explain our results

regarding increased hemorrhage of the pancreatic tissue in the

apigenin group.

Apigenin may interfere with drug metabolism and this

may increase toxicity from co-administered drugs, or amelio-

rate side effects of others. Apigenin as a member of the

flavones is a potent inhibitor of CYP2C9-mediated diclofenac

40-hydroxylation through competitive or noncompetitive

inhibition, depending on the site of binding of the enzyme [46].

Thus, apigenin may disrupt physiological pharmacokinetics

of drugs such as warfarin, tolbutamide, and phenytoin. In

another study that investigated the mutagenic and anti-

genotoxic effects of different doses of apigenin, alone or in

combination with cyclophosphamide and doxorubicin, in vitro

and in vivo, apigenin was reported to decrease doxorubicin-

induced, but not cyclophosphamide-induced, mutagenicity

in vitro [47]. Furthermore, apigenin is suggested to reduce the

chance of developing secondary tumors after chemotherapy in

patients with cancer, since treatment with apigenin results in

a significant, dose-dependent decrease in the genotoxic effect,

induced by mitomycin C and cyclophosphamide [48].

In conclusion, we have proven that per os administration

of apigenin in an experimental model of pancreatitis in rats

ameliorates all pathologic parameters of inflammation and

reduces MPO activity. This observation is an attractive

and promising research result in the fight against inflam-

matory diseases, including pancreatitis. However, more

studies are warranted to apply this observation to human

beings. Questions to be answered in the future are to

investigate the ultimate dosage of the natural compound

and whether it should be used as a therapeutic or as

a protective agent against pancreatitis. Finally, caution is

needed in case this natural compound is used in combina-

tion with other drugs.

Acknowledgments

The authors acknowledge Argyro Zacharioudaki, DVM, Elef-

theria Karampela, MSc, Maria Karamperi, and Kalliopi Tsarea

for their important contribution in the completion of this

study. This work was supported, mainly, by a research

scholarship of the Experimental Research Center of the ELPEN

Pharmaceuticals.

The authors declare that they have no conflict of interest.

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