Download ANTI-INFLAMMATORY EFFECT OF THE SERRATIOPEPTIDASE

Indian J Physiol Pharmacol 2012; 56(4) : 367–374
ANTI-INFLAMMATORY EFFECT OF THE SERRATIOPEPTIDASE –
RATIONALE OR FASHIONABLE: A STUDY IN RAT PAW
OEDEMA MODEL INDUCED BY THE CARRAGEENAN
KALPESH K. JOSHI AND RAJAN P. NERURKAR
Department of Pharmacology,
TNMC & BYL Nair Charitable hospital,
Mumbai. (Now - Medical Advisor, GlaxoSmithKline Asia Ltd, Mumbai.)
( Received on November 5, 2011 )
Abstract : Introduction – Many drugs, including serratiopeptidases, are
marketed without proven efficacy in clinical trials. It is protein in nature
and claimed to be effective orally.
Methods – 24 albino wistar rats, 6 each in following groups were assigned
– (1) Control group (distilled water, orally) (2) Diclofenac (6.75 mg/kg, IP)
(3) serratiopeptidase (5.4 mg/kg, orally) (4) Combination of serratiopeptidase
(5.4 mg/kg, orally) and diclofenac (2.25 mg/kg, IP). Inflammatory agent,
carrageenan (0.1 ml of 1% w/v) was injected subcutaneously in the ether
anesthetized rat hind paw, half an hour after the administration of drug.
Rat paw volume up to lateral malleolar process was recorded with
plethysmometer at various time periods.
Results – Percentage formation and inhibition of oedema in serratiopeptidase
or combination groups were not significantly different than control group.
Both were significantly less for diclofenac group.
Conclusion – Serratiopeptidase was not effective in this animal model of
oedema/inflammation.
Key words :
enzyme
anti-inflammatory drugs
INTRODUCTION
Inflammation is one of the most common
suffering of human being and antiinflammatory drugs are one of the most
commonly used one. Probably, most
commonly used drugs are Non-Steroidal AntiInflammatory Drugs (NSAIDs). However as
they are effective, they are associated with
side effects as well as cost to the patients.
*Corresponding Author :
analgesic
rational use of drugs
Thus their judicious use is mandatory. There
are many drugs which are marketed without
clear scientific evidence of efficacy. One such
drug is serratiopeptidase.
Serratiopeptidases are obtained from
bacteria, Serratia species, belonging to
enterobactericeae family (1). It is a protein
and endopeptidase in nature with molecular
weight of 60 daltons (2). Generally, proteins
Email : [email protected]
368
Joshi and Nerurkar
Indian J Physiol Pharmacol 2012; 56(4)
are digested to constituent amino acids and
then amino acids are absorbed, not the
parent protein molecule. Still this drug is
claimed to be efficacious when given orally.
This drug is not marketed as drug in
western world where regulatory bodies are
very strict and they issue notices from time
to time to drug manufacturers (3-4). They
are marketed and promoted in countries like
India and other south East Asian countries
(5). This drug does not find its mention in
US pharmacopeia (6) or British National
Pharmacopeia (7) or Indian Pharmacopeia
(8); nor in standard textbook of pharmacology
like – Goodman and Gilman (9).
Thus we wanted to evaluate the antiinflammatory effect of this drug in
carrageenan induced rat paw oedema model.
(10) This is one of the models used for the
evaluation of anti-inflammatory effect in
animals. The study objectives were to
measure the absolute oedema values, to
assess the change in rat paw volume at
various time points as compared baseline
and inhibition of oedema formation as
compared to control group. Adverse effects
or death among the experimental animals
was also noted.
MATERIALS
AND
METHODS
Experiment began after written approval
from Institutional Animal Ethics Committee,
registered under Animal (Scientific
Procedures) Act, 1986. A homogenous group
of adult albino wistar rats (14 males and 10
females) were included in the study. Animals
were procured from Haffkine Institute, parel,
Mumbai. They were housed in animal house.
One week time was allowed for their
acclimatization. They were given water and
food ad libitum. They were kept nil by mouth
for overnight before the day of experiment.
The experiment consisted of 24 animals,
6 each in 4 groups :–
(1) Control group given distilled water
orally
(2) Diclofenac sodium IP (6.75 mg/kg,
Intra-peritoneally [IP]).
(3) Serratiopeptidase group (5.4 mg/kg,
orally)
(4) Combination of serratiopeptidase (5.4
mg/kg, orally) and diclofenac sodium
(subtherapeutic dose 2.25 mg/kg, IP).
Sub-therapeutic dose was used to
evaluate the additional synergistic
effect of serratiopeptidase, if any.
Then, 0.1 ml of 1% (w/v) of freshly
prepared carrageenan solution was injected
subcutaneously in the anesthetized rat hind
paw, half an hour after the administration
of drug, in morning time approximately
between 9–10 am. Rats were anesthetized
using ether soaked cotton rolls placed under
bell jar. Then rat paw volume up to lateral
malleolar process was recorded at 0
(baseline – immediately after the injection
of carrageenan), 1, 2, 3, 4, 5, 6 and 24 hours
respectively. After the carrageenan injection
rats were allowed to feed ad libitum. The
volume of rat hind paw was recorded in
millimeters of displacement of mercury in
plethysmometer.
Efficacy parameters
Absolute oedema scores were recorded.
Indian J Physiol Pharmacol 2012; 56(4)
Anti-inflammatory Effect of Enzymes
These recordings were further analyzed by
two formulae – percentage oedema and
percentage inhibition of oedema.
1. Percentage oedema formation within
group (compared to baseline ‘0’ hours) =
{Volume at Nth hour (Vn) – Volume at
base line (Vo)} × 100
Volume at baseline (Vo)
2. Percentage oedema inhibition compared
to control between groups at same time
point =
{Volume of the test group (Vt) – Volume
of control at same hour (Vc)} × 100
Volume of control group (Vc)
369
qualitative data was expressed as proportion
or percentages. The percentage oedema
formation within group was analyzed using
repeated measures ANOVA followed by
Dunnet‘s post test. One way ANOVA with
Dunnet’s post-hoc test was used for the
analysis of percentage edema formation
between groups.The statistical analysis was
done using graphpad instat software version
no. 3.7. The P value less than 0.05 was
considered as statistically significant.
RESULTS
The oedema scores in all four groups are
mentioned in Table I.
Statistical analysis
Significant rise in paw volume was seen
in control, serratiopeptidase and in the
combination group at many time point
interval (Table I). The rise in oedema volume
for diclofenac group was statistically
significant only at 3, 4 and 6 hours.
The quantitative data was expressed as
mean±standard deviation (SD) while
The oedema formation within the groups
rose uniformly from 1 hour to 6 hours and
These were recorded as per above time
points and are mentioned in observation
table.
TABLE I : The mean±SD paw volumes (in mm $ ) for control (distilled water, orally); Diclofenac (6.75 mg/
kg, intra-peritoneally); Serratiopeptidase (5.4 mg/kg, orally) and Combination (serratiopeptidase
(5.4 mg/kg, orally) and diclofenac (2.25 mg/kg, intra-peritoneally)) groups at various time points
(0–6 and 24 hours). PO = orally and IP = intra-peritoneally.
Time
(Hrs)
0
1
2
3
4
5
6
24
P value (by repeated
measures ANOVA)
$
Control group
(mean in mm±SD)
3.08±0.58
3.38±0.69
3.63±0.89
3.80±0.92
3.85±1.06
4.40±0.89**
4.55±0.97**
4.28±99**
0.0004
Diclofenac grroup
(mean in mm±SD)
3.44±0.42
3.76±0.56
3.92±0.76
4.12±0.98**
3.98±0.84*
3.88±0.68
3.96±0.49*
3.74±0.42
0.0435
Serratiopeptidase group
(mean in mm±SD)
Combination group
(mean in mm±SD)
4.07±0.64
4.58±0.55**
4.98±0.59**
5.55±0.80**
5.55±0.77**
5.78±0.85**
6.05±0.79**
4.67±0.66**
4.72±0.41
5.38±0.48**
5.80±0.55**
6.12±0.54**
6.32±0.48**
6.55±0.45**
6.70±0.47**
5.50±0.57**
< 0.0001
< 0.0001
The volume measurements represent the measurements of length mercury column displacement in mm
in plethysmometer. *P value <0.05, **P value <0.01 and ***P value <0.001 by Dunnet’s post-hoc test.
370
Joshi and Nerurkar
TABLE II :
Time points
Control Group
Diclofenac Group
Serratiopeptidase Group
Combination Group
P value by ONE-Way ANOVA
Indian J Physiol Pharmacol 2012; 56(4)
Percentage formation of oedema within groups compared
to baseline (0 hrs) for all four experimental groups.
1
2
3
4
5
6
24
9.7
9.30
12.70
14.13
0.549
17.8
13.95
22.54
18.68
0.342
23.2
19.77
36.48
22.89
0.0877
24.9
15.70*
36.48
33.92
0.0418
42.7
12.79**
42.21
38.87
0.006
47.6
15.12*
48.77
42.05
0.0183
38.9
8.72*
14.75
16.61
0.0413
*P<0.05; **P<0.01 as compared to the control group value by Dunnet’s post hoc test.
after that was found to decline at 24th hour.
As compared to control group values,
the percentage oedema formation was
significantly less only in diclofenac group at
4, 5, 6 and at 24 hours. Serratiopeptidase
and combination group did not show
significant reduction as compared to control
group values at all time intervals (Table II
and Fig. 1). The percentage inhibition of
oedema compared to the control group was
found more in the diclofenac group than
serratiopeptidase group or combination group
at all time points (Table III and Fig. 2).
TABLE III : Percentage inhibition of oedema compared
between various groups as compared to
control group.
Time
intervals
1
2
3
4
5
6
24
Diclofenac
Seratiopeptidase
Combination
–4.5
–21.9
–15
–37.2
–70.02
–68.3
–77.6
28.6
25.2
55.6
45.5
–2.0
1.9
–62.2
43.6
28.1
26.9
35.6
–9.5
–12.1
–57.6
The negative sign indicates inhibition of oedema,
while positive value indicates increase in oedema.
Fig. 1 : Percentage oedema formation compared to baseline (‘0’ hr value) for all experimental groups.
Indian J Physiol Pharmacol 2012; 56(4)
Fig. 2 :
Anti-inflammatory Effect of Enzymes
371
Percentage inhibition of oedema compared to control group.
The negative sign indicates inhibition of oedema while positive
value indicates increase in oedema.
The negative sign indicates inhibition of
oedema while positive value indicates
increase in oedema.
DISCUSSION
There are many doubts raised about the
efficacy of serratiopeptidases (11). It
is marketed as orally effective antiinflammatory drug despite of its protein
nature. There are many enteric coated
formulations available which claim that they
are not destroyed by gastric acid and get
absorbed by intestines, circulate in blood to
reach the site of inflammation. This further
raise issues like even after enteric coating
how it is protected from intestinal digestive
enzymes – esp. proteases – those of its own
kind. Though there is some evidence that
proteins may be absorbed orally, it is
applicable to only few proteins in ‘extremely
low’ amounts (12-13). This amount may not
sufficient to produce efficacy of a ‘protein
drug.’ In addition to this there is hardly any
evidence present for the oral absorption of
serratiopeptidases. Also, why it is not
recognized as foreign protein by immune
system and not fixed by it remains to be
answered. The other pharmacokinetic data
of the drug is also not well studied – for e.g.
protein binding, metabolism, tissue
penetration; half life and volume of
distribution are not known. Its status of use
in pregnant and lacting females is not known.
Our results demonstrated that there is
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Joshi and Nerurkar
lack of efficacy of serratiopeptidases when
given alone. Also, in combination with subtherapeutic dose of diclofenac no additional
synergistic effects were observed. We have
chosen sub-therapeutic dose of diclofenac in
combination group with the expectation to
observe the anti-inflammatory properties of
serratiopeptidase, if any. However, this study
failed to show anti-inflammatory effects of
serratiopeptidase alone or in combination.
Diclofenac alone in therapeutic doses was
effective in reducing oedema. The results
obtained were different in another study with
formalin injection model for induction of
acute as well as chronic oedema. In this
study serratiopeptidase group was found to
be effective equally as compared to diclofenac
(0.5 mg/kg) (14). In another similar
animal experiment, it was found that
serratiopeptidases were effective when
injected into joint during eradication of
staphylococcal infection. However it was
given locally, not orally. In another
experiment for evaluation of distribution of
cefotiam in lung and trachea of rabbits
combined with serratiopeptidases (injected),
it was found that cefotiam concentration was
not significantly increased in respiratory
track (15).
The
evidence
of
efficacy
of
serratiopeptidase is based on some
animal experiments, personal letters or
uncontrolled trials with poor quality.
Whatever studies conducted in randomized
double blind manner are generally of poor
quality with either randomization failure or
other methodological flaws in it (16).
Thus firm evidence about efficacy of
serratiopeptidase is lacking. Also, ignorance
of safety data of such agents may be
Indian J Physiol Pharmacol 2012; 56(4)
dangerous. US FDA regularly serves notices
to marketing of the drugs which are not
approved. It even serves notices to online
marketing websites (17). It is requirement
of US FDA to do at least one placebo
controlled clinical study to get approval for
the marketing. However in India such norms
are rarely followed. Its lack of mention
of this drug in standard textbooks
of pharmacology shows that it is not
unanimously accepted by medical community.
Serratiopeptidase,
alone
or
in
combination with NSAIDs is very costly and
prescribed very commons by physicians and
more commonly by surgeons. This adds
significantly to cost of treatment. It is
claimed to have no side effects (18). When
asked about the drug, many physicians were
also unaware about the active ingredient
in various formulations, its mechanism
of action, rationale of combination
with NSAIDs and cost of treatment.
Pharmaceutical companies make aggressive
marketing of such drugs and doctors are
pursued to prescribe it. Sadly so, there lies
hardly any scientific rational for it.
Below is the comparison of cost of various
formulations of serratiopeptidase either
alone or with diclofenac combination as
compared to diclofenac alone. One can clearly
observe the combination or serratiopeptidase
monotherapies are costlier. [19] The average
cost of plain serratiopeptidase is more than
43 Indian rupees (INR) whereas diclofenac
alone costs Rs 8.5. The combination of
diclofenac and serratiopeptidase ranges from
31 to 71. This similarly applies to aceclofenac
and ibuprofen based combinations. Even
combinations with tramadol are also
available.
Indian J Physiol Pharmacol 2012; 56(4)
Anti-inflammatory Effect of Enzymes
373
Rational use of medicine is important.
Often drugs are prescribed ‘fashionably’,
but not rationally. There are extensive
marketing campaigns by pharmaceutical
companies for these drugs. In countries like
India, where affordability of medicines is still
very low, one should be very cautious about
prescribing such drugs. The readers should
also acknowledge the limitations of this
study like use of only one animal model or
only one inflammatory agent and small
number of rats per group. Thus, we
encourage further experimental and clinical
research in this area.
must be further research on these aspects
of a drug, apart from simply efficacy. Also,
there should be emphasis on the rational
use of drugs in the medical curricula.
About 40 years before, Prof. Cochrane, a
pioneer in clinical epidemiology, coined a
concept of evidence ladder stepped by efficacy
(can it work?), effectiveness (does it work?)
and cost-effectiveness (is it worth using?).
In the era of evidence based medicine, lack
of firm efficacy data, while effectiveness and
cost-effectiveness is far away, poses an
objection on the use of such drugs. There
ACKNOWLEDGEMENTS
Conclusion
From this animal experiment, it could
be concluded that serratiopeptidase is not
effective as an anti-inflammatory agent.
Further it adds to cost and may add to side
effects as well. Thus it is recommended to
conduct human studies and find out its
efficacy in humans.
We would like to thank TNMC research
society for funding the project. Authors
would like to thank anonymous reviewer for
his patience and time while reviewing this
article. His efforts have improvised the
article and will add significant value to
scientific discussion.
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