Sur T K et a l / Acta Pharmac ol Sin 20 03 Feb; 2 4 (2): 18 7-192
Chine se Academ y of Sci encesht tp:/ /www.Chi naPh ar.c om
Anti-inflammatory and anti-platelet aggregation activity
SUR Tapas Kumar, BISWAS Tuhin Kanti2, ALI Liaquat3, MUKHERJEE Biswapati4
Department of Pharmacology; 2SN Pradhan Centre for Neurosciences,Dr BC Roy Postgraduate Institute of Basic Medical Sciences 244B, Acharya J C Bose Road, Calcut a 700020, India;3Research Division, BIRDEM, 122 Kazi Nazrul Islam Avenue, Dhaka 1000, BangladeshKEY WORDS placental extracts; inflammation; carrageenin; platelet aggregation; adenosine diphosphate ABSTRACT AIM: To find the anti-inflammatory and anti-platelet aggregatory activity of human placental extract (HPE, Placentrex). METHODS: The HPE was studied for anti-inflammatory effect in Wistar rats on carrageenin, serotonin (5-HT), and prostaglandin E (PGE ) induced edema in acute model and cotton pellet induced granuloma on sub-acute
model. Anti-platelet aggregation was studied against pr otection of adinosine diphosphate (ADP)-induced aggrega- tion of human platelet through in vitro study. RESULTS: HPE showed positive results both in acute and sub-acute models of inflammation. Highly significant (P<0.01) results were obtained against 5-HT induced acute inflamma- tion and cotton pellet induced sub-acute inflammation in comparison with standard (diclofenac sodium) and control (normal saline) drugs. The anti-inflammatory property of HPE in animal model was well supported with clinical
study of platelet aggregation. There was highly significant (P<0.01) inhibition of platelet aggregation with HPE at diff erent doses against ADP. CONCLUSION: Our data suggest that human placental extract may be useful in suppressing inflammation and platelet aggregation. INTRODUCTION
man placental extract has corticotropin releasing factor
The var iety of biological actions of human pla-
(CRF)-like action[1]. Enzyme-linked immunosorbant
cental extract (HPE) is a matter of increasing interest.
assay (E LISA) studies r evealed that human placental
Recent research studies reveal that HPE is the rich re-
cytotrophoblasts which expressed interleukin-8, a
sour ces of var io us bio- active subst ances like
known mediator of inflammation, was suppr essed by
polydeoxyr ibonucleotides ( PDRN), RNA, DNA,
peptides, amino acids, enzymes, tr ace elements, etc[1].
The current study was aimed to find the anti-in-
Therapeutic properties in the treatment of patients with
flammatory activity of HPE in both acute and sub-acute
wounds have been described[2]. It is reported that hu-
experimental models. Platelet aggregation is an impor-
tant pathogenic marker of inf lammation. Hence, onerational approach in the research of anti-inf lammatory
drugs is to search for compounds causing inhibitions
1 Supported by M/s, Albert D avid Ltd, Calcutta, India. 4
of platelet aggregation. Although there are some re-
Correspondence to Dr MU KHERJ EE Biswapati.
ports of placental extract for their anti-platelet aggrega-
tion activity[4,5] but the observations not correlated with
Sur TK et al / Acta Pharmac ol Sin 20 03 Feb; 2 4 (2): 18 7-192
its anti-inflammatory activity. In the current research
trically at 0 h and 3 h after carrageenin injection. The
programme, anti-inflammatory effect of human placental
tr eated drugs wer e administered intramuscular ly 1 h
extract was observed in experimental animal model while
platelet aggregation activity was studied in clinical cases.
5-HT-induced paw edema In this exper iment
0.1 mL of 5-HT (1 g/L) in ster ile saline was injected
MATERIALS AND METHODS
into the sub-planter tissue of the right hind paw of rats. Test drug Human placentas weighing between
The paw volume was measur ed plethysmometr ically
400-600 g collected at the time of full term spontane-
before and after 30 min of the 5-HT injection[8,9] . HPE,
ous deliver y were immediately placed under ice, then
diclofenac sodium, and 0.9 % NaCl were administered
the amniotic membr ane and umbilical cor d wer e
removed, minced into small pieces and washed with
cold normal saline. Aqueous extract with these pieces
landin E (1 mg/kg) was administered into the sub-planter
of placenta was prepared, sterilized, and sealed in am-
region of the right hind paw of rats, in accordance with
pules (2 mL) under inert condition. The extract 1 mL
the method of Willis and Cornelsen[10]. The paw vol-
in the ampule was derived from 0.1 g of placenta. This
ume up to the ankle joints were measured plethysmo-
extract contains protein ( 0.95 g/L), DNA (2.8 mg/L),
metrically before and after 30 min of the prostaglandin
RNA (1.6 mg/L), Na+ ion (27.9 mmol/L), K+ ion (3.07
mmol/L), and Cl– ion (15.1 mmol/L).
Sub-acute inflammation Sub- acute inflamma-
Acute toxicity (LD )
tion was produced by cotton pellet induced granuloma
to evaluate the therapeutic dose as well as for screening
in rats[11]. Sterile cotton (15±1) mg was implanted sub-
of the CNS toxicity. HPE was administered f or this
cutaneously bilaterally in axilla under ether anesthesia.
purpose at 0.4, 0.5, 0.6, 0.7, 0.8 , and 0.9 mL per 20 g
The treated drugs were administered for consecutive 6
of mice in intra peritoneal route. The studies were car-
d in the same dose as mentioned earlier. The animals
wer e sacrificed on d 7. The gr anulation tissues with
Animals Male Wistar rats weighing 150-180 g
cotton pellet were dried at 60 °C overnight and then the
were used for present research programme. They were
dr y weight was taken. The weight dif ferences were
housed in groups under 12:12 h regime (lights on from
considered as the weight of granulation f ormation.
7:00 h to 19:00 h) at (23±2) °C prior to the experiments. Platelet aggregation study
They were supplied pellet diet and water ad libitum.
Selection of subject Total 15 volunteers of ei-
Drugs Carrageenin (Sigma), serotonin hydrochlo-
ther sex were selected from the medical out patients’
ride (Sigma), prostaglandin E (Sigma), and adenosine
department of Bangladesh Institute of Research and Re-
diphosphate (Sigma) were used in this study.
habilitation on Diabetes, Endocrine & Metabolic Disor-
Anti-inflammatory activity
ders (BIRDE M), Dhaka, Bangladesh. A careful drug
Acute inflammation Acute paw edema was in-
history was taken from the subjects. Patients not re-
duced in groups of ten rats, each using three diff erent
ceiving for last two weeks the dr ugs like aspir in,
experimental models. The rats were deprived of food
sulf inpyr azone, chlor pr omazine, amitr iptyline,
for 24 h before the induction of inflammation, but wa-
furosemide, penicillin and its derivatives, dextran, which
ter was allowed ad libitum. The HPE was adminis-
interfere with the platelet aggregation activity, were se-
ter ed at dose of 300 mg/kg intr amuscular ly[ 6] .
lected for the present research programme.
Diclofenac sodium (10 mg/kg, im) and 0.9 % NaCl (5
Collection of blood Specimens of blood samples
mL/kg, im) were used as reference drugs. After each
were collected using 3.2 % sodium citrate at the ratio 1:
treatment paw volume of the animals were measured
9 with the blood in plastic container with minimum
trauma or stasis at the venipuncture site. Testing was
per formed 30 min after venipuncture at the r oom
flammation was induced by carrageenin according to
the model of Winter et al[7]. For this purpose 0.1 mL of
Preparation of plasma Samples of blood and
1 % suspension of carr ageenin in normal saline was
anticoagulants were gently inverted up and down, avoid-
injected into the sub-planter tissues of right hind paw in
ing shaking. Platelet rich plasma (PRP) was pr epared
rats. The paw volume was measur ed plethysmome-
by centrifuging at 100×g under 4 °C for 15 min. PRP
Sur T K et a l / Acta Pharmac ol Sin 20 03 Feb; 2 4 (2): 18 7-192
thus prepared wer e collected by caref ul pipetting in a
Acute inflammation From the study it was ob-
sterile polypr opylene tube and closed. Platelet poor
served that there was significant (P<0.01) inhibition of
plasma (PPP) was prepared by centrifuging at approxi-
paw edema in the animals treated with HPE both on
mately 2400×g for 20 min. PPP was collected in a
carr ageenin (54. 3 %) and PGE (39. 7 %) induced
inflammation. These results are almost same as in the
Study methods of platelet aggr egation The
case of diclofenac sodium ( 57.1 % and 44.4 %
aggr egation was measured on a dual channel Chr ono-
respectively) treated group. However, the rate of inhi-
L og Optical Platelet Aggr egometer (Chrono- Log
bition (P<0. 01) of edema in 5-HT induced acute in-
Cor poration, Havertown PA 19083-4691) at constant
flammation was even better (49.0 %) than diclof enac
stirring of 1200 rpm (1000 rpm in 50 Hz Units) and
sodium (39.4 %) treated group (Tab 1).
electr onically controlled temperature (37±2) °C. The
Sub-acute inflammation In cotton pellet induced
light tr ansmission was set at 0 % with PRP and at
sub-acute inf lammation model, there was highly sig-
100 % with PPP[10]. Aggregation was induced with
nificant (P<0.01) decrease of the weight of granuloma
aggregating agent ADP at a concentration of 1 mmol/L.
tissue (39.5 % ) in HPE tr eated gr oup. However, the
HPE was added at different doses of 2.5, 5, 10, and 20
rate of inhibition of granuloma tissue weight in diclofenac
µL/mL, 5 min before addition of ADP.
sodium treated group (52.1 %) was found to be better
Statistical analysis T he results of animal ex-
periments were analysed by unpaired Student’s t test.
Anti-platelet aggregation activity Results of
Paired t test method was applied for the analysis of the
platelet aggregation were expressed as a percent of ag-
gregation at a given time interval (5 min) from reagentaddition. Cent per cent aggregation was defined as the
differences between the 0 % baseline (PRP) and 100 %
Acute toxicity (LD )
baseline (PPP). Highly significant responses (P<0.01)
of anti-platelet aggr egation were observed with all the
observed that the HPE is safe upto 45 mL/kg body weight
doses of HPE . T her e wer e 83. 9 % , 76 . 6 % ,
Anti-inflammatory activity
59.2 %, and 60.2 % aggregation of platelet with HPE atthe doses of 2.5, 5, 10, and 20 µL/mL of plasma (PRP)
Tab 1. Effect of human placental extract (HPE) and diclofenac sodium on carrageenin induced rat paw edema. Mean±SD. n=10. cP< 0.01 vs control.
Experimental model Paw volume Paw volume
Tab 2. Effect of human placental extract (HPE) and diclofenac sodium on sub-acute inflammatory model in rat. Mean±SD. n=10. cP< 0.01 vs control.
granuloma granuloma Inhibition/% granuloma Inhibition/% tissue/mg tissue/mg tis sue/mg
Sur TK et al / Acta Pharmac ol Sin 20 03 Feb; 2 4 (2): 18 7-192
Tab 3. Effects of human placental extract (HPE) against
observed after HPE treatment (Tab 1) on carrageenin-
ADP-induced platelet aggregation in human PRP.
induced paw edema in r ats may be mediated through
Mean±SD. n=10. cP< 0.01 vs control.
either any of these mediators alone or in combination.
Hence, HPE was fur ther studied against paw edema
Treatment groups and dose Platelet aggregation
induced by individual agent like 5- HT or PGE .
5-HT is present in mast cells and is consider ably more
potent than histamine in increasing vascular permeabil-
ity in r ats[16]. As ther e was considerable r eduction
(49.0 %) of inflammation in 5-HT pre-treated r ats by
application of HPE, it might be conjectured that the in-
hibitory action was 5- HT mediated. Pr ostaglandins
(PGs) biosynthetic pathway or the cyclooxygenase
(COX) activity of the enzyme is the site of action of
non-steroidal anti-inflammatory drugs or NSAIDS[17,18]. Diclof enac sodium is a widely used potent NSAI DS
with pronounced analgesic and anti-pyretic activity. It
is used mainly for long term symptomatic treatment ofrheumatoid arthr itis, osteoarthritis, and ankylosing
spondylitis[19,20]. Therefore, diclofenac sodium was se-
lected in this study as positive control (Tab 1, 2). Henceit may be assumed that HPE exhibits their anti-inflam-
matory responses either through inhibition/inactivation
of chemical mediators or by dir ectly modulating PGproduction by suppression of COX. It has been recog-
nized recently that mammalian cells explain two forms
of COX activity. COX-1 is the major form present inplatelets, while COX-2 is only found in elevated levels
in inflammatory exudates[20,21]. The action of HPE on
Fig 1. Effect of different doses of HPE for anti-platelet ag-
PGE induced edema (Tab 1) explains that it may modu-
gregation activity.
late PGs production by inhibition of COX.
The sub- acute anti-inflammatory activity of HPE
was studied by investigation of its inhibitory ef fect on
respectively with respect to 100 % PPP control (Tab 3,
the granuloma formation (Tab 2). Cotton pellet granu-
loma is a model of non-immunological type of inflam-
DISCUSSION
mation mediated by the activation of the chemical me-
Inflammation covers a series of reparative and
diators of inflammation, mostly kinins[23]. T he action
protective responses in tissue injury, whether caused
of kinin involves the activation of two membr ane
by infection, auto-immune stimuli or mechanical injury.
receptors, B and B . B -receptor plays an important
Several classes of compounds such as plasma proteins,
role in inflammatory pr ocesses[24,25]. In this present re-
vasoactive amines, tissue digestive enzymes, biologi-
search programme highly signif icant (P <0. 01) result
cally derived oxidant and eicosanoids are all associated
was obtained with HPE in cotton pellet induced sub-
with inflammatory response[13,14]. Most of all investiga-
acute inflammation model indicating that it may act by
tors have reported that inhibition of carrageenin-induced
the way of inhibiting the B -receptor.
inflammation in rats is one of the most suitable test pro-
Platelets are essential for nor mal haemostasis.
cedure to screen anti-inflammatory agents[9]. The de-
Activation of the clotting cascade by trauma results in
velopment of car rageenin-induced edema is bi-phasic,
platelet activation, which is followed by aggregation.
the first phase is attributed to the release of histamine,
The major COX metabolite in platelets is thromboxane
5-HT, and kinins, while, the second phase is r elated to
A (TXA ), which is capable of initiating aggr egation.
the release of prostaglandins[7,15]. The inhibitory action
NSAIDS inhibit TXA production and thus inflamma-
Sur T K et a l / Acta Pharmac ol Sin 20 03 Feb; 2 4 (2): 18 7-192
tion[26,27]. The aggregation of human platelets induced
rats . Acta Pharmacol Sin2001; 22: 1113-6.
by ADP was used[12] to study the anti-platelet effect of
Winter CA , Ris ley EA, Nuss CW. Carrageenin-induced
HPE (Tab 3). T here was 84 %±13 % aggregation of
oedema in hind paw of the rats as an assay for anti-inflamma-tory drugs. Proc Soc Exp Biol Med 1962; 111: 544-7.
platelet against ADP with a dose of 2. 5 µL/mL HPE
Kalbhen D A, Smalla HD. Pharmakologis che s tudies zur
which gradually decreased (77 %±16 % f or 5 µL/mL)
antiphlogistischen wirkung von pentos anpolysulfat in
with the increase in dose, r eaching minimum (59 %±
kombination mit metamizol. Drug Res 1977; 27: 1050-7.
33 %) with a dose of 10 µL/mL. There was no appre-
Vogel HG, Vogel HW. In: Drugs dis covery and evaluation.
ciable change on f urther incr ease of the dose (60 %±
14 % for 20 µL/mL). ADP is contained within the
10 Willis A L, Cornelsen M. Repeated injection of prostaglan-
din E in rat paws induces chronic swelling and a marked
platelet in storage organelles and released from the platelet
decrease in pain threshold. Prostaglandins1973; 3: 353-7.
during formation of the primary haemostatic plug and
11 Penn GB, As hford A. The inflammatory respons e to im-
thereby induce further platelet aggregation[28,29]. There
plantation of cotton pellets in the rat. J Pharm Pharmacol
ar e so many activation pathways leading to platelet
aggregation. PGs and 5-HT are considered as the ma-
12 O’Brien J R. Platelet aggregation: some res ults from a new
jor chemical mediators of platelet aggregation[30,31]. The
method of study. J Clin Pathol 1962; 15: 452-5.
13 Larsen GL, Henson PM . Mediators of inflammation. Ann
clinical study of platelet aggregation reflects that HPE
can either inhibit PGs synthesis pathway or 5-HT release.
14 Brooks PM, Day RO. Nonsteroidal anti-inflammatory drugs:
These data indicate that the anti-inflammatory ef-
differences and similarities. N Engl J Med 1991; 324: 1716-
fect of HPE might be mediated through the suppression
of chemical mediators. Further experiments are needed
15 Clarke G D , Macphers on IS , P etrone G , S pangler RS .
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Antinoceptive effects of non-s teroidal anti-inflammatorydrugs in a rat model of unilateral hind paw inflammation.Eur
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16 Fuller RW. Pharmacology of central serotonin neurons. Ann
ACKNOWLEDGEMENTS Authors are thankful to M/
s, Albert David Limited, Calcutta for the financial sup-
17 Vane J, Booting R. N ew insights into the mode of action of
port of the resear ch progr amme. Sincere thanks are
anti-inflammatory drugs. Inflamm Res1995; 44: 1-10.
also due to the Head of the Department of Pharmacology,
18 Willoughby DA. Effects of prostaglandins PGF2α and PGE1
on vascular permeability. J Pathol Bacteriol 1998; 96: 381-7.
University College of Medicine, Calcutta and to the tech-
19 Todd PA, S orkin EM. D iclofenac sodium, a re-appraisal of
nical staffs of the department of pathology and research
its pharmacodynamic and pharmacokinetic properties, and
division BI RDEM, Dhaka, Bangladesh and Director,
therapeutic efficiency. Drugs 1988; 35: 244-85.
Central Drug Laboratory, Calcutta for various help.
20 Tonussi CR, Ferreira SH. Mechanism of diclofenac analgesia:
direct blockade of inflammatory s ens itization. Eur J
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