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					‫69‬

‫‪Original Articles‬‬


        ‫‪EFFECT OF SELECTED NATURAL PRODUCTS, THIOPROLINE AND‬‬
        ‫)‪PEGASYS® ON HEPATIC PLATELET ACTIVATING FACTOR (PAF‬‬
               ‫‪IN CCL4-INDUCED HEPATIC FIBROSIS IN RATS‬‬


                                              ‫*2‪Farid A. Badria1 and Hala A. Attia‬‬



            ‫يه د هذاددلبذب إلددتذي ددمذتويددياذعا د ي ذب ع د ذب كإ د ذب منشددلذ فا د لدذب ع ي د ذ)‪(PAF‬ذفددكذدإ د ذب نددلمباذب ما د ذ‬
            ‫فيفذب كإ ذب ما ل ثذ لب عذدف ري ذب كل ا.ذأخ يل ذعنم ع ذعنذب من ن ذب طإي ي ذعث :ذأحم ضذب إ زويفيد ذ‬
            ‫وب كلداذوب نفياليهزينذ(عا لضلذيادممذأوعندم®ذواد ذعود رذتلدلذب ند ر ذبإلدفينيكيد ذ د فذفيدلوالذبه هد ذ‬
            ‫ب كإ ذب لكذف)،ذعيلبزيد ®ذ(عود رذع د فلذتن ريد ذ و د ذديد باذب إفه ر دي )،ذثيد لو ينذ(ع عد ذواد لكذ فكإد ذع د فلذ‬
            ‫تن ريددذ)،ذ ين ددي ®ذ(عدد فذع دد فلذتن ريدد ذ دد فذفيددلوالذبه هدد ذب كإدد ذب دد لكذف).ذتدداذبخ إدد رذتددذثيلذاددل ذ‬
            ‫ب منم ع ذعفمذعا ىذ‪PAF‬ذفكذدإ ذب نلمباذ ذت لضه ذ ف اماذ لب عذدف ري ذب كل ا.ذو هلبذب غلضذتداذب د ا ب ذ‬
            ‫54ذجلمبذفكذال ذب رب .ذذتاذتواياذب نلمباذي مذ6ذعنم ع ذد ذعنم ع ذت ك اذعدنذ9ذجدلمبا.ذب منم عد ذبوو دمذ‬
            ‫تمددلذع ن هدد ذ لب ددعذدف ريدد ذب كل دد اذفوددل،ذب منم عدد ذذ2ذ–ذ4ذتدداذحونهدد ذ ددىذأوعنددم®،ذعيلبزيدد ®،ذثيدد لو ين،ذ‬
            ‫ين ددي ®ذعفددمذب د ب ك،ذ ينم د ذب منم ع د ذب ا د د ذد ةددلذب منم ع د ذب ض د ط ذ( د واذأ ذع عف د ذ د ثن نذب لوددنذ‬
            ‫مليب).ذذتاذت عيلذب كإ ذفكذد ذب منم ع ذ ثن نذب منم ع ذب ضد ط ذ(ب ذب منم عد ذ1-4)ذعدنذرليداذب لودنذ‬
            ‫ب إلي ةكذ ىذ45%ذرب عذدف ري ذب كل اذب ملب ذفكذزيلذب لرةذ(4.3.4ذع /دنا)ذ3ذعلب ذب إ عي ذ مد ةذ3ذأ د يعذ د ذ‬
            ‫أ إ عذعنذب اماذ لب عذدف ري ذب كل د اذتداذبعطد نذجميدعذب ود ايلذتلدلذبهخ إد رذي عيد ذو مد ةذ3ذأ د يعذعدنذرليداذ‬
            ‫ب لودنذب إلي د ةكذوتداذايد الذتلديدزذعاد ي ذب عد ذب نشددلذ فاد لدذب ع يد ذ(‪)PAF‬ذعدنذرليدداذذ‪HPTLC‬ذ(ب لفيد ذ‬
            ‫ب كلوع ت جلبفكذ طإو ذب لايو ذع ي ذب ك نة).ذذ ينم ذتاذب ا ب ذب مطي فيد ذ ود يلذعاد ي ذيةزيمد ذتدلبة ذأعيند زذ‬
            ‫)‪(ALT, AST‬ذوع دةذاي روداكذ لو ينذ(ب ما ا ع ذد ب دم ذ فيدفذب كإد )،ذوعد دةذعد اذدب ذب ايد ذويةدزياذد تد يزذ‬
            ‫(ب ما ا ع اذدم بدذوب دم ذ جهد دذب ذداد ).ذ ود ذوجد ذأاذعاد ي ذب عد ذب منشدلذ فاد لدذب ع يد ذدد اذأعفدمذ‬
            ‫ع ن ي ذفكذعنم ع ذرب عذدف ري ذب كل اذ(14.2±ذ52.52ذ م لذعك فئ/عنا)ذعن ذعو رة ه ذععذب ض لذب طإي دكذ(94.1ذ‬
            ‫±ذ1...ذ م لذعك فئ/عنا).وعن ذب م ن ذ ك ذعنذأوعندم®ذوعيلبزيد ®ذوثيد لو ينذو ين دي ذفود ذحد ثذبةا د ضذ‬
            ‫ع ن ذفكذعا ي ذب عد ذب منشدلذ اد لدذب ع يد ذ يادإدذ22.4ذ±ذ5..11ذوذ44.1ذ±ذ4.51ذوذذ54.4ذ±ذ.1.31ذوذ94.1ذ±ذ‬
            ‫62.51ذ م لذعك فئ/عناذ،ذعفمذب د ب ك.ذوادل ذب رب د ذيمكدنذأاذتضديفذزيد دةذع و د ذي دمذب عفيد ذب مضد دةذ ف فيدفذ‬
                                                        ‫فملدإ ذب منل ذعنذخ لذيةو صذب ع ذب كإ ذب منشلذ فا لدذب ع ي .ذ‬
                                                                                                                                    ‫ذ‬
                                                                                                                                  ‫ذ‬
                                                                                                                                  ‫ذ‬
            ‫‪This study aimed to estimate hepatic levels of platelet activating factor (PAF) in liver fibrosis‬‬
                                                                                                               ‫ذ‬
            ‫‪induced by CCl4 in rats. A group of selected natural products; boswellic acids, curcumin and‬‬
                                                     ‫®‬
            ‫‪glycyrrhizin (preparation named OMNI ; a drug under clinical trials for treatment of hepatitis C‬‬
                                                                                                               ‫ذ‬
                            ‫®‬
            ‫‪virus), Mirazid (a commercially available schistomicidal drug), Thioproline (a commercially‬‬
            ‫‪available hepatoprotective agent) and Pegasys® (peg interferon alpha-2a; a commercially‬‬
            ‫‪available therapy for treatment of hepatitis C virus) were examined for their effect on hepatic PAF‬‬
            ‫6 ‪after CCl4 intoxication. For this purpose, 54 rats were used in the study. Rats were divided into‬‬
            ‫‪groups each comprised 9 rats. Group 1 was treated only with CCl4, groups 2 to 5 were treated‬‬
            ‫‪with OMNI®, Mirazid®, Thioproline and Pegasys®, respectively, whereas the sixth group was the‬‬
            ‫‪normal control group (with no treatment, except an injection of the vehicle). Liver damage was‬‬
‫1‬
 ‫,‪Department of Pharmacognosy, 2Department of Biochemistry‬‬
‫,‪Faculty of Pharmacy, Mansoura University 35516, Mansoura‬‬
‫.‪Egypt‬‬
‫*‬
 ‫.‪To whom correspondence should be addressed‬‬
‫‪E-mail: halafetoh@yahoo.com‬‬

‫7002 ‪Saudi Pharmaceutical Journal, Vol. 15, No.2, April‬‬
SELECTED HEPATOPROTECTIVE AGENT ON HEPATIC PAF                                                                    97

            induced in all groups except normal control group (groups 1 to 5) by i.p. injection of 40% CCl 4 in
            corn oil (0.375 ml/kg) 3 times a week for 3 weeks. One week after CCl 4 intoxication, all tested
            drugs were injected i.p. daily for 3 weeks. Hepatic PAF concentration was estimated by HPTLC
            (high performance thin layer chromatography), while, levels of serum transaminases (ALT, AST),
            hepatic hydroxyproline (as markers of liver fibrosis), serum malondialdehyde and catalase (as
            markers of oxidative stress) were estimated spectrophotometrically. The hepatic PAF levels were
            significantly higher in CCl4 group (24.24±2.01 pmol equiv./mg) as compared to normal control
            (8.81±1.09 pmol equiv./mg) (p<0.001). Treatment with OMNI ®, Mirazid®, Thioproline and
            Pegasys® reduced hepatic PAF significantly to be 11.84±0.22, 14.5±1.00, 13.17±0.54 and
            14.26±1.09 pmol equiv./mg, respectively. This study may add further rational to the anti-fibrotic
            activity of the tested drugs via reduction of hepatic PAF.

            Key words: Platelet activating factor (PAF), OMNI®, Mirazid®, Thioproline, Pegasys® and liver
            fibrosis.



                       Introduction                             estimate the level of hepatic PAF after intoxication
                                                                for prolonged time.
    Platelet activating factor (PAF:1-0-alkyl-2-                    The present study aimed to estimate hepatic PAF
acetyl-sn-glycero-3-phosphocholine) is a potent pro-            levels 3 weeks after CCl4-induced liver damage in
inflammatory phospholipid which exerts diverse                  rats. Different agents with different postulated
biological activities including platelet secretion and          mechanisms of action on liver injury were selected;
aggregation, hypotension, bronchoconstriction, incr-            OMNI® (a drug under clinical trials for antiviral
eased vascular permeability and stimulated synthesis            therapy), Mirazid® (Schistomicidal agent), Thiopro-
of mediators such as eicosanoids (1-6). PAF acts as             line (a well known antioxidant agent) and Pegasys®
a mediator of inflammation, allergic reactions and              (long acting interferon). This study, in addition,
shock and as a specific membrane bound adhesion                 aimed to test the influence of these drugs on hepatic
molecule (6-8). The PAF content of the intact liver is          PAF levels after CCl4 intoxication and subsequently
elevated by various types of injury including                   the possible contribution of this effect to their known
ischemia reperfusion (9), endotoxin exposure in vivo            hepatoprotective action.
(10) and obstructive jaundice (11). Plasma and liver                OMNI®, a drug under clinical trials for treatment
tissue PAF levels are increased in experimental                 of hepatitis C virus (HCV), is composed of boswellic
obstructive jaundice and activation of this mediator            acids, curcumin and glycyrrhizin. Boswellic acids
contributes to the ongoing liver injury (12).                   are the effective compounds isolated from the gum-
    PAF has been suspected to play an important                 resin of Boswellia serrata and Boswellia carterii that
role in liver pathophysiology. The cultured Kupffer             have been used for the treatment of rheumatoid
and endothelial cells produce and release PAF in                arthritis and inflammatory bowel diseases (16) and
order to facilitate communication between hepatic               may have cytostatic and apoptotic effects towards a
sinusoidal and parenchymal cells (12). Moreover,                variety of malignant cells (17). Curcumin is a natural
PAF is an important mediator of hepatic injury.                 compound present in the rhizome of plant Curcuma
Inappropriate production of PAF leads to pathologic             longa Linn, it has anti-inflammatory, anti-carcino-
inflammation, and PAF is clearly involved in liver              genic, anti-thrombotic, antispasmodic and potent
damage as PAF receptor blockers reduce hepatic                  antioxidant effects (18). Glycyrrhizin, a major com-
damage after ischemia reperfusion. (13).                        ponent of licorice root, is an anti-inflammatory and
    Villamediana et al. (14) found that, in the                 antioxidant compound isolated from Glycyrrhiza
cirrhotic rats, PAF levels were elevated in systemic            glabra (19). It has inhibitory effect on PAF
blood but made no measurements of the hepatic PAF               production in human neutrophils in a dose dependent
system or altered hepatic responses to PAF. Marathe             manner (20) and it was shown to significantly supp-
et al. (15) reported increased hepatic PAF two hours            ress the production of PAF from the rat peritoneal
after CCl4 administration but did not determine if the          exudate cells (21). The anti-inflammatory and anti-
PAF content of liver was elevated during chronic                allergic effects of glycyrrhizin were suggested to be
injury. Therefore, a thorough study was needed to               due to the suppression of PAF production (21).

Saudi Pharmaceutical Journal, Vol. 15, No. 2, April 2007
98                                                                                        BADRIA & ATTIA

    Mirazid® is a commercially available drug used         ml/kg), groups 2 to 5 were treated with OMNI ®,
as a safe and effective natural product against            Mirazid®, Thioproline and Pegasys®, respectively,
Schistosoma mansoni infection (22). It is a special        one week after CCl4 intoxication, whereas the 6th
formulation of myrrh; the oleo-gum resin obtained          group was the normal control group, which only
from the stems of Commiphora molmol, and                   received an injection of corn oil (0.375 ml/kg). Liver
probably other species of Bursearacae (23). Myrrh          damage was induced in all groups except normal
is useful for treatment of sore throat, bleeding gums,     control group (groups 1 to 5) by i.p. injection of
chronic pharyngitis and amenorrhea (24) and is used        CCl4 3 times a week for 3 weeks. One week after
widely in Somalia for treatment of diarrhea and            CCl4 intoxication, drugs were injected i.p. daily for 3
stomach complains (25). Moreover,            myrrh is      weeks in the following doses; OMNI® (600 mg/kg),
approved by the Food and Drug Administration               Mirazid® (500 mg/kg), Thioproline (100 mg/kg) and
(FDA) for food use (21 CFR 172.510).                       Pegasys® (0.5 μg/rat).
    Thioproline (thiazolidine-4-carboxylic acid, ti-
monacic acid or Hepargen®) is a cyclic sulfur              C) Assessment of liver fibrosis:
containing amino acid and is a condensation product        i- Estimation of liver enzymes (ALT and AST):
of cysteine and formaldehyde (26). Animal studies              At the end of the 3 weeks of study, blood was
confirmed the anti-toxic effects of Thioproline, parti-    withdrawn from the eye ball under ether anesthesia.
cularly on the liver (27). Thioproline has been cli-       The blood was allowed to coagulate then centrifuged
nically used, mainly in the treatment of liver diseases    at 3000 rpm for 10 min. to separate the serum.
and related gastrointestinal disturbances (27).            Serum levels of ALT and AST were measured
          Pegasys® (peg interferon alpha-2a) is a long     immediately according to the method of Reitman
acting interferon used in antiviral therapy in humans,     and Frankel (35).
mainly for viral hepatitis B and C. An anti-fibrotic
effect of interferon has been postulated even in the       ii- Estimation of hepatic hydroxyproline:
absence of antiviral response, suggesting that,                 The content of hepatic hydroxyproline was
interferon directly inhibits fibrogenesis (28-30).         measured to estimate the anti-fibrotic property of the
Moreover, its effect on the reduction of fibrosis has      injected drugs by using the modified method of
been discovered in different experimental rat models       Woessner (36). Briefly, at room temperature, liver
of hepatic fibrosis (31-33).                               tissues of the right lobe were dehydrated by 95%
                                                           alcohol for 5-6 hrs, and defatted by acetone for two
                 Materials and Methods                     days. The obtained defatted tissues were dried at
                                                           110oC and ground into powder. Forty mg of liver
A) Chemicals:                                              tissue powder were hydrolysed in 3 ml of 6 M HCl
   ALT and AST kits were purchased from                    at 110o for 6 hrs. The hydrolysates were filtered and
bioMerieux Co.; Thioproline powder, chloramine-T           diluted to 50 ml with distilled water, and the solution
and perchloric acid were obtained from Aldrich Co.;        was neutralized to pH 6 by 6 M NaOH. Two ml of
p- dimethylamino-benzaldehyde and authentic PAF            the solution and 1 ml of 0.05 M chloramine-T were
(1-O-palmityl-2-acetyl-sn-glycero-3-phosphocholine)        placed in a glass tube, shaken vigorously and left at
were purchased from Sigma Chemical Co.                     room temperature for 20 min., followed by addition
                                                           of 1 ml 3.15 M perchloric acid and 1 ml of 10% p-
B) Animals:                                                dimethylamino-benzaldehyde and incubated at 60oC
    Fifty four Wistar rats, weighing 180-250 gm,           for 20 min. Finally, the reaction mixture was cooled
were used in this study. They were fed with a              in an ice bath for 5 min. and the obtained color was
standard laboratory diet and tap water ad libitum and      measured spectrophotometrically at 550 nm against
housed in individual cages. Houses were kept at            reagent blank. Standard hydroxyproline solutions of
255oC, 555% humidity, and a 12 h light/dark              concentrations 5, 10, 15, 20, 25 and 30 g/ml, were
cycle. Rats were divided into 6 groups each                used for construction of the standard curve.
comprised 9 rats. Based on the treatment protocol
and dosage regimen described previously by the             D) Assessment of oxidative stress:
author (34), animals were treated as follow: Group 1           Lipid peroxidation, a major indicator of oxida-
was treated only with 40% CCl4 in corn oil ( 0.375         tive stress, was assayed by measuring malondi-


Saudi Pharmaceutical Journal, Vol. 15, No. 2, April 2007
SELECTED HEPATOPROTECTIVE AGENT ON HEPATIC PAF                                                                              99

aldehyde (MDA) in serum (37). Serum level of                        nm. The layer was passed through a beam of light,
catalase was determined according to the method of                  and the transmitted energy was measured by
Goth (38).                                                          densitometer (Wilmington, FNC, USA). Authentic
                                                                    PAF (C16); 1-O-palmityl-2-acetyl-sn-glycero-3- pho-
E) Determination of PAF in liver tissues:                           sphocholine (Sigma, GmbH, Germany) was dissol-
    PAF was extracted and purified form liver as                    ved in chloroform to produce a concentration range
described previously (39, 40). PAF was determined                   of 2.5 to 50 fmoles and assayed by the same method.
using high performance thin layer chromatography                        PAF concentrations on HPTLC fractions of
(HPTLC) (41) as described below.                                    unknown samples were calculated directly from PAF
    For lipid extraction, 100 mg of liver were                      C16 standard curve and expressed pmole equivalent
homogenized in 9.5 ml of methanol-chloroform-                       of PAF C16/ mg liver tissue. Total PAF for a given
water mixture (2:1:0.8 v/v). The homogenates were                   sample represented the sum of PAF activity detected
kept at room temperature for one hour, mixed with 5                 in all HPTLC fractions for that sample.
ml chloroform-water (1:1 v/v), and after one hour
the mixture was centrifuged (3000 rpm for 15 min).                  Statistical analysis:
The lower chloroformic layer containing hepatic                         Data were expressed as mean  standard error of
lipids, including PAF, was aspirated into glass tubes               mean (SEM). Statistical analysis was performed
and stored at -20oC.                                                using Instat-3 computer program (Graph pad soft-
    For purification, the chloroformic layer was                    ware Inc, San Diego, CA, USA). The groups were
dried under nitrogen and the obtained residue was                   compared using one way analysis of variance
dissolved in 200 l of chloroform and finally                       (ANOVA) followed by Tukey-Kramer multiple
purified by Sep-Pak columns (Strata, phenomenex,                    comparison test.
USA) using 3 ml of acetone-chloroform (2:3 v/v).
The eluate was dried under reduced pressure at room                                            Results
temperature and then quantitatively transferred with
chloroform into a small vial and evaporated until                       Values of the analyzed parameters and the
dryness under nitrogen.                                             statistical differences in the groups are shown in
    For PAF determination, the residue was                          Table (1) and Figure (1). During the experiment, one
redissolved in 100 l chloroform and spotted on                     rat died from each of the following groups; CCl4,
precoated HPTLC (Merck, Germany) using                              Mirazid® and Pegasys®, while three rats died from
chloroform-methanol-water (65:53:6 v/v/v) as the                    Thioproline group.

developing solvent (41). Spots on a HPTLC were
quantitized spectroscopically by transmission at 254

Table 1: Markers of liver fibrosis and oxidative stress in OMNI ®, Mirazid®, Thioproline and Pegasys® groups
as compared to both control and CCl4 groups 3 weeks after treatment. Values were expressed as meanSEM.
  Group
                                                          AST
                             ALT (IU/ml)                          H. proline (g/ml)   MDA (nmol/ml)         CAT (kU/L)
                                                        (IU/ml)
              Marker
      CCl4 (n=8)              a
                                  151.214.3     a
                                                    219.69.9        a
                                                                         22.051.07      a
                                                                                          17.70.84           219  4.27
                                                                                                                 a

    OMNI®+CCl4 (n=9)              b
                                   76.68.2      b
                                                    164.69.6        b
                                                                         12.90.42      b
                                                                                         10.630.68      b
                                                                                                             289.7  15.2
   Mirazid®+ CCl4 (n=8)           b
                                   70.04.2     a
                                                    205.810.6           b
                                                                          14.50.7       b
                                                                                             11.060.7       295.5  11.7
                                                                                                             b


     Thio+CCl4 (n=6)          b
                               71.258.0            b
                                                   252.618          b
                                                                      13.90.52          b
                                                                                          13.350.9      b
                                                                                                          292.6.4  13.5
   Pegasys®+CCl4 (n=8)        b
                               73.125.6         194.521.7          b
                                                                      13.40.44         b
                                                                                         11.260.38       b
                                                                                                           302.6  11.7
      Control (n=9)           b
                               58.574.7         b
                                                  159.36.4          b
                                                                      12.50.73          b
                                                                                          10.70.26      b
                                                                                                          291.5  10.95
n=number of rats
 a=significance versus control.
 b=significance versus CCl4 group.
Thio=Thioproline, ALT=alanine aminotransferase, AST=aspartate aminotransferase, MDA=malondialdehyde, CAT= catalase, H.
proline=hydroxyproline.



Saudi Pharmaceutical Journal, Vol. 15, No. 2, April 2007
100                                                                                                                    BADRIA & ATTIA

                        30                                                             levels in comparison with CCl4 group and insigni-
                                  a                                                    ficant differences were observed as compared to
                                                                                       control group. On the other hand, serum AST
 PAF (pmol equiv./mg)




                        20                                                             decreased significantly (p<0.01) only in OMNI ®-
                                                 ab
                                                                                       treated group as compared to CCl4 group, whereas,
                                                                 ab
                                          b
                                                           b                           Mirazid®, Thioproline and Pegasys® did not signi-
                        10
                                                                         b             ficantly affect AST as compared to CCl4 group. No
                                                                                       significant differences were found among drug-
                                                                                       treated groups in ALT and hydroxyproline levels.
                        0
                                        OMNI® Mirazid® Thio   Pegasys®Control
                                 CCl4
                                        + CCl4 + CCl4  + CCl4 + CCl4
                                                                                       b) Oxidative stress markers:
                                                                                           Serum MDA levels were significantly higher
Figure 1. Hepatic platelet activating factor (PAF)                                     (p<0.001) in CCl4 group than in control. OMNI®,
levels in OMNI®, Mirazid®, Thioproline and Pega-                                       Mirazid®, Thioproline and Pegasys® significantly
sys® groups as compared to both control and CCl4                                       reduced (p<0.001) serum MDA levels as compared
groups 3 weeks after treatment. Values were                                            to CCl4 with no significant differences in compa-
expressed as meanSEM.                                                                 rison with control. On the other hand, serum catalase
a = Significance versus control group.                                                 levels decreased significantly (p<0.05) in CCl4 group
b = Significance versus CCl4 group.                                                    as compared to control. All tested drugs significantly
                                                                                       increased catalase levels as compared to CCl4 group
                                                                                       (p<0.01). No significant differences were observed
Table 2: Correlation between levels of PAF and                                         in levels of MDA and catalase among tested drug
levels of both MDA and catalase in CCl4, OMNI®,                                        groups.
Mirazid®, Thioproline and Pegasys® groups.

                                                                                       C) Hepatic PAF levels:
                                         Correlation between Correlation between           A highly significant increase in hepatic PAF
                                              PAF and MDA        PAF and Catalase      levels was observed in CCl4 group as compared to
                                                                                       control (24.242.01 Vs 8.811.09 pmol equiv./mg,
       CCl4 group (n=8)                    Significant, p<0.01   Significant, p<0.05   p<0.001). Highly significant reductions (11.84±
                                                 r =0.87              r = -0.81        0.22, 14.5±1.00, 13.17±0.54 and 14.26±1.09 pmol
                             ®
                    OMNI +CCl4             Significant, p<0.05   Significant, p<0.01   equiv./mg, p<0.001) were detected by OMNI ®,
                         (n=9)                   r =0.79              r = -0.76
                                                                                       Mirazid®, Thioproline and Pegasys®, respectively, as
                             ®
                                                                                       compared to CCl4 group. OMNI® and Thioproline
                Mirazid +CCl4                 Not significant      Not significant     groups showed no significant differences in hepatic
                         (n=8)                   r =0.67              r = -0.50        PAF as compared to control, while PAF levels in
     Thioproline+CCl4                      Significant, p<0.05     Not significant     Mirazid® and Pegasys® groups were still signify-
                         (n=6)                   r =0.82              r = -0.55        cantly higher (p<0.01) than those of control. No
                             ®                                                         significant differences were shown among tested
               Pegasys +CCl4                  Not significant      Not significant
                                                                                       drug groups.
                         (n=8)                   r =0.52              r = -0.57            As shown in Table (2), positive correlation was
                                                                                       found between levels of PAF and MDA in CCl4 and
                                                                                       tested drug groups. The correlation was significant
a) Liver fibrosis markers:                                                             in CCl4, OMNI® and Thioproline groups, while
    Three weeks after CCl4 administration; levels of                                   insignificant correlation was seen in Mirazid® and
serum transaminases (ALT, AST) and hepatic                                             Pegasys® groups. On the other hand, negative
hydroxyproline were significantly higher (p<0.001)                                     correlation was observed between levels of PAF and
in group 1 (rats received CCl4 only) than those of                                     catalase, significantly in CCl4 and OMNI® groups
control. Groups 2 to 5 (OMNI®, Mirazid®, Thio-                                         and insignificantly in Mirazid®, Thioproline and
proline and Pegasys®) showed significant reductions                                    Pegasys® groups.
(p<0.001) in serum ALT and hepatic hydroxyproline


Saudi Pharmaceutical Journal, Vol. 15, No. 2, April 2007
SELECTED HEPATOPROTECTIVE AGENT ON HEPATIC PAF                                                               101

                         Discussion                        hepatoprotective      drugs     (OMNI®,      Mirazid®,
                                                                                      ®
                                                           Thioproline and Pegasys ) on these levels was also
    Metabolism of CCl4 by hepatocytes and/or               studied.
endogenous macrophage like Kupffer cells (42),                 The results showed a highly significant increase
resulted in severe hepatic necrosis and fibrosis as        in hepatic PAF levels in CCl4 group as compared to
indicated by elevated liver enzymes (ALT and AST)          control (Fig. 1). This is in agreement with the results
and hepatic concentration of hydroxyproline. In this       obtained by Yang et al. (60). It has been found that,
established model, liver injury is derived from a          Kupffer cells isolated from cirrhotic rats release
mixture of free radicals and reactive oxygen species       significantly more PAF than those obtained from
(43, 44), lipid peroxidation (45, 46) and activated        control rats (61). Hepatic endothelial cells and
Kupffer cells (47), each of which has an underlying        systemic macrophage recruited to injured liver may
role in resulting liver damage. The administration of      also contribute to elevated hepatic PAF levels.
OMNI®, Mirazid®, Thioproline and Pegasys® to               Marathe et al. (15) found that, the livers of rats
CCl4-intoxicated rats for 3 weeks limited the              metabolizing CCl4 contain inflammatory phospho-
metabolism of CCl4 and improved the biochemical            lipids, mainly PAF. They also found that, oxidants,
parameters. These results were obvious from their          which stimulate cellular PAF generation in vitro,
effect on lowering liver enzyme (ALT), hepatic             such as H2O2, (62) lead to the accumulation of
hydroxyproline and serum MDA in addition to                significant amounts of hepatic PAF. In addition,
increasing serum catalase (Table 1).                       CCl4 metabolism by cytochrome P-450 generates the
    The antioxidant activity of OMNI® is derived           reactive trichloromethyl (CCl3) radical and this event
mainly from curcumin which is a very potent                generates superoxide and H2O2 (45). H2O2 stimulates
antioxidant (18, 48-50) and glycyrrhizin which             the synthesis of PAF (62,63) and PAF is synthesized
antioxidant activity (51-53) and its role in reducing      by stimulated macrophages and Kupffer cells (61).
liver fibrosis (54) has been established. Thioproline      All these findings coincide with our results in CCl4
(thiazolidine-4-carboxylic acid) is a well known           group that revealed a strong positive correlation
intracellular sulfhydryl antioxidant and free radical      between PAF and MDA as well as a significant
scavenger (27, 55). Diet supplementation with this         negative correlation between PAF and catalase
antioxidant slowed the aging process, prolonged life       (Table 2).
span and stimulated the immune system in old mice              It is also postulated that, the elevated hepatic
(55) and reversed age-related behavioral dysfunction       PAF in this model is related to reduced PAF
in prematurely-aging mice (56).                            metabolism by the liver due to low secretion of PAF-
    Many studies suggest an antioxidant and anti-          actylhydrolase, an enzyme predominantly responsi-
fibrotic activity of interferon alpha (IFN-) (29,32,      ble for hydrolysis of PAF to lysoPAF (64).
57-59). Lu et al.(58) suggested that, IFN- may                All tested drugs (OMNI®, Mirazid®, Thioproline
enhance biological defense activities against oxida-       and Pegasys®) exerted a significant reduction of
tive stress and function as a potent fibro-suppressant     hepatic PAF after 3 weeks of treatment (Fig. 1).
by protecting hepatocytes and hepatic stellate cells       These results may add further explanation to the
from lipid peroxidation. In the study of Serejo et al.     anti-fibrotic activity of these drugs. However, the
(29), IFN- promoted a long term inhibition of             levels of hepatic PAF were still higher than those of
oxidative stress with concomitant improvement of           control (significantly in Mirazid® and Pegasys®
activity of fibrosis. Madro et al. (32) found positive     groups and insignificantly in OMNI® and Thio-
effects of high doses of IFN- in improving liver          proline groups). This may suggest the need for either
function and reducing liver fibrosis induced by CCl4       higher doses or prolonged time of treatment.
in rats.                                                       The effect of the tested drugs on hepatic PAF
    Platelet activating factor (PAF) is a potent pro-      could be attributed to their inhibitory effect on PAF
inflammatory and hypotensive phospholipid (1-6).           production, through their antioxidant activity
Inappropriate or excessive PAF has the potential to        revealed by lowering MDA levels and increasing
modify liver function, and PAF is an established           catalase (Table 1). This explanation could be confir-
hepatotoxic agent (9, 11). Our study aimed to              med by the positive correlation observed in the
estimate hepatic levels of PAF in CCl4-induced liver       tested drug groups between levels of PAF and MDA
fibrosis in rats. In addition, the effect of different     as well as the negative correlation found between


Saudi Pharmaceutical Journal, Vol. 15, No. 2, April 2007
102                                                                                                              BADRIA & ATTIA

PAF and catalase (Table 2). Higher catalase levels                     7.    Braquet P, Paubert-Braquet M, Bourgain RH, Bussolino F
                                                                             and Hosford D. PAF/cytokine autogenerated feedback
achieved by these drugs leads to H2O2 scavenging
                                                                             networks in microvascular immune injury: consequences in
limiting its role in PAF production (62,63).                                 shock, ischemia and graft rejection (published erratum
    Most likely, the drugs in this study may affect                          appears in J Lipid Mediat 1989; 1:361). J Lipid Mediat 1989;
the activity of cholinephosphotransferase (CPT) or                           1:75–112.
                                                                       8.    Lorant DE, Patel KD, McIntyre TM, McEver RP, Prescott
PAF-acetylhydrolase; the enzymes which are res-
                                                                             SM and Zimmerman GA. Coexpression of GMP-140 and
ponsible for synthesis and degradation of PAF,                               PAF by endothelium stimulated by histamine or thrombin: a
respectively. It has been found that, CPT was                                juxtacrine system for adhesion and activation of neutrophils. J
reduced significantly in renal cell carcinoma patients                       Cell Biol 1991; 115:223–234.
                                                                       9.    Zhou W, McCollum MO, Levine BA and Olson MS.
who had received IFN- compared with those who
                                                                             Inflammation and platelet activating factor production
had not (65). These findings suggest that IFN- may                          during hepatic ischemia/reperfusion. Hepatol 1992; 16: 1236-
modulate the production of PAF. Further studies are                          1240.
recommended to study the modulatory effect of                          10.   Olson MS, Kitten AM, Eakes AT, Howard KM, Miller JE
these drugs on the enzymes that regulate PAF levels.                         and Mustafa SB. The role of lipid and peptide mediators in
                                                                             hepatic cell communication. In: Shimazu T, ed. Liver
                                                                             Innervation London: John Libbey& Co. Ltd 1996, pp. 75–85.
                          Conclusion                                   11.   Zhou W, Chao W, Levine BA and Olson MS. Role of platelet
                                                                             activating factor in hepatic responses after bile duct ligation
                                                                             in rats. Am J Physiol 1992; 263:G587–G592.
    Hepatic PAF level showed an increase in CCl4-                      12.   Coker A, Coker I, Huseyinov A, Sokmen S and Karademir, S.
induced liver fibrosis in rats. The treatment of                             Is mannitol effective against platelet activating factor (PAF)-
intoxicated rats with OMNI®, Mirazid®, Thioproline                           induced liver damage in obstructive jaundice? Hepatogastro-
and Pegasys® daily for 3 weeks modulated the toxic                           enterology 2001; 48(40): 1134-1137.
                                                                       13.   Yamakawa Y, Takano M, Patel M, Tien N, Takada T and
effect of CCl4 via either antioxidant activity and/or                        Bulkley GB. Interaction of platelet activating factor, reactive
lowering PAF contents of liver tissues. This study                           oxygen species generated by xanthine oxidase and leukocytes
may add further explanation to the anti-fibrotic                             in the generation of hepatic injury after shock/resuscitation.
activity of the tested compounds via reducing                                Ann Surg 2000; 231:387-398.
                                                                       14.   Villamediana LM, Sanz E, Fernandez-Gallardo S, Caramelo
hepatic PAF levels.                                                          C, Sanchez Crespo M, Braquet P and Lopez-Novoa JM.
                                                                             Effects of the platelet activating factor antagonist BN 52021
                      Acknowledgment                                         on the hemodynamics of rats with experimental cirrhosis of
                                                                             the liver. Life Sci 1986; 39:201–205.
                                                                       15.   Marathe GK, Harrison KA, Roberts LJ, Morrow JD, Murphy
   We thank Mr. Ahmad Abbas; the technician of                               RC, Tjoelker LW, Prescott SM, Zimmerman GA and
Pharmacognosy Department, Faculty of Pharmacy,                               McIntyre TM. Identification of platelet activating factor as
Mansoura University for technical assistance, care of                        the inflammatory lipid mediator in CCl4-metabolizing rat
animal house and his effort throughout the whole                             liver. J Lipid Res 2001; 42:587–596.
                                                                       16.   Syrovets T, Buchele B, Krauss C, Laumonnier Y and Simmet
work.                                                                        T.      Acetyl-boswellic acids inhibit lipopolysaccharide-
                                                                             mediated TNF-alpha induction in monocytes by direct
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