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Der Pharmacia Lettre, 2010: 2 (1) 285-290
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Comparative evaluation of polyherbal formulations for
anti-inflammatory and analgesic activity in rats and mice
Sandhya Desai*, Akbar Ahmad, Mahesh Gite, Bhaskar Gavitre, Yogesh More
K. M. Kundnani College of Pharmacy, Colaba, Mumbai, INDIA
___________________________________________________________________________
Abstract
Polyherbal formulations (MFT09 and MFG09) containing extracts of various plant
constituents viz: Boswellia serrata, Commiphora wightii, Withania somnifera, Curcuma
longa, Tinospora cordifolia, Zingiber officinale, Alpinia galangal, Cyperus rotundus and
Vitex negundo, were evaluated for anti-inflammatory and analgesic activity. Anti-
inflammatory activity were investigated in albino wistar rats using Carageenan induced hind
paw edema model, while the Radiant heat tail flick test was used as a model for evaluating
analgesic activity. Treatment with MFT09 resulted in significant decrease in hind paw
swelling as compared to MFG09. The effect of MFT09 produced significant analgesic
activity against thermal induced pain stimuli in mice at various time intervals post treatment
as indicating by increased latency to flick the tail which suggests that its activity might have
resulted from its central action. Meanwhile, the results of the acute toxicity test, for oral as
well as topical preparation of MFT09 and MFG09 respectively indicate that it is relatively
safe and/or non-toxic to rats. The findings of these experimental animal studies indicate that
MFT09 possesses potential anti-inflammatory and analgesic activity as compared to MFG09.
Keywords: MFT09, MFG09, Carageenan, anti-inflammatory, analgesic activity.
______________________________________________________________________
Introduction
Medicinal herbs as a potential source of therapeutic aids have attained a significant role in
health system all over the world for both humans and animals, not only in the diseased
condition but also as potential material for maintaining good health [1]. Since time
immemorial, medicinal plants, nature’s hidden and to a large extent unexplored treasure, have
been used virtually in all human cultures around the world (over 75 % of the population) as a
source of safe and effective medicines [2].
Inflammation or phlogosis is a pathological response of living tissue to injuries that leads to
the local accumulation of plasmatic fluid and blood cells. It is a body defense reaction in
ISSN 0975-5071
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order to eliminate or limit the spread of injurious agent as well as to remove the consequent
necrosed cells and tissues [3]. Although it is basically a defense mechanism, the complex
events and mediators involved in inflammatory reaction can induce, maintain or aggravate
many diseases [4]. Pain is universally understood as a signal of disease and is the most
common symptom that brings a patient to a physician’s attention, requiring treatment with
analgesic agents [5]. Chronic pain is often accompanied by depression [6]. Selective
serotonin reuptake inhibitors used in the management of depression, by increasing the
serotonin level, inhibit the release of transmitters carrying the pain sensation from the nerve
endings.
Currently available remedy for pain and inflammation mainly include Cortico-steroids and
Non-steroidal anti-inflammatory drugs for the relief of pain and inflammation. All these
therapies are however associated with adverse effects [7]. An investigation on efficacy of
plant based drugs used in the traditional medicine has been paid great attention to because
they are cheap and have fewer side effects [8]. MFT09 and MFG09 contain extracts of
medicinal plants viz Boswellia serrata, Commiphora wightii, Withania somnifera, Curcuma
longa, Tinospora cordifolia, Zingiber officinale, Alpinia galangal, Cyperus rotundus and
Vitex negundo. These constituents are used in folk medicine for the treatment of
inflammation and pain. The present investigation was undertaken to comparatively evaluate
the Polyherbal formulations (MFT09 and MFG09) for possible anti-inflammatory and
analgesic activity.
Materials and Methods
Animals
Albino rats of Wistar strain (weighing 100-200 g) and Albino mice of Swiss strain (weighing
25-35 gm) of either sex, obtained from Bharat Serum and Vaccines, Thane, India were
housed under standard conditions of temperature (24+1
0
C), relative humidity (65+10 %), 10-
h light and 14-h dark cycle and fed with standard pellet diet (Chakan Mill Ltd, Pune, India)
with water ad libitum. All the experimental procedures and protocols used in the study were
reviewed by the Institutional Animal Ethics Committee (Approval number of project: 080907
and Registration Number of institute: 25/1999/CPCSEA) and were in accordance with the
guidelines of the CPCSEA, Ministry of Forests and Environment, Government of India. The
animals were deprived of food for 24 hour before experimentation but allowed free access to
water throughout.
Drugs and chemicals
Poly Herbal Formulations MFT09 and MFG09 were a gift sample from Om Pharmaceuticals,
Bangalore. The dry powder of MFT09 was reconstituted using 0.5% w/v Sodium Carboxy
Methyl Cellulose (SCMC) to get 1 mg ml
-1
of MFT09. The suspension was freshly prepared
before use. Carageenan from Sigma Chemical Co, St Louis, MO, USA., Aspirin from Themis
Pharmaceuticals, Mumbai., Piroxicam gel from Cipla Limited, Ahmadabad., and all other
chemicals, reagents used were of analytical grade.
Acute toxicity studies
For oral preparation (MFT09), Albino wistar rats weighing 100-200 g were divided into two
groups of three animals each (three male & three female) the homogenous suspension was
prepared freshly, using 0.5% (w/v) Na-Carboxyl Methyl Cellulose (CMC) using a mortar and
pestle. The animals were administered 2 g/kg dose p.o. of MFT09 suspension (OECD
Guidelines No.423). For topical preparation (MFG09), Albino wistar rats weighing 100-200 g
Sandhya Desai et al Der Pharmacia Lettre 2010: 2 (1) 285-290
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were divided into two groups of five animals each (five male & five female). The animals
were applied 2 g/kg dose topically to 10 percent of the dorsal body surface area of MFG 09
(OECD Guidelines No.402). Then animals were critically observed for clinical symptoms,
behavioural changes and mortality up to 72 h period and then upto a period of 14 days.
The studies were carried out by using nine groups of six animals each for both oral
preparation (MFT09) & topical preparation (MFG09) in anti-inflammatory (Carageenan
induced hind paw edema using albino wistar rats) and analgesic activity (Radiant heat tail
flick test using Albino wistar mice).
Group I received normal saline (0.5% Sodium CMC solution) serve as a control,
For oral preparation:- Test drug (MFT09), Group II received MFT09 (200 mg/kg p.o.),
Group III received MFT09 (400 mg/kg p.o.), Group IV received MFT09 (600 mg/kg p.o.),
Group V received Standard drug (Aspirin 10 mg/kg p.o.),
For topical preparation:- Test drug (MFG09), Group VI received MFG09 (100 mg/kg
topically), Group VII received MFG09 (400 mg/kg topically), Group VIII received MFG09
(800 mg/kg topically) and Group IX received Standard drug (Piroxicam gel 10 mg/kg
topically).
Anti-inflammatory activity (Carageenan-induced paw edema)
Inflammation was induced by a 0.1 ml injection of 1% w/v suspension of carageenan in
saline to the plantar surface of right hind paw [9]. For oral preparation, test and standard
drugs (MFT09 & Aspirin) were administered orally to the respective groups 60 minutes prior
to carageenan injection [10], [11]. For topical preparation, test and standard drugs (MFG09 &
Piroxicam gel) were applied to the plantar surface of the hind paw by gently rubbing 50 times
with the index finger to the respective groups 60 minutes prior to carageenan injection [12].
The change in the inflammatory reaction was measured using Digital plethysmometer on
various time intervals (0, 1,2,3,6 & 24 hr) and compared with control group. The right hind
paw edema inhibition at different doses of Test drug and Standard drug were calculated by
comparing with vehicle treated control rats.
Following formula was used:
% inhibition of paw edema = (Vt-Vo)
control
- (Vt-Vo)
treated
X 100
(Vt-Vo)
control
Where,
Vt is the rat paw volume at time‘t’, Vo is the initial rat paw volume (before Carageenan injection), (Vt-Vo)
control
is edema produced in control group and (Vt-Vo)
treated
is edema produced in treatment groups.
Analgesic activity (Radiant heat tail flick test)
A radiant heat tail flick analgesiometer was used to measure response latencies. For oral
preparation, test and standard drugs (MFT09 & Aspirin) were administered orally to the
respective groups 30 minutes before taking response [13]. For topical preparation, test and
standard drugs (MFG09 & Piroxicam gel) were applied topically to the tail of respective
groups 30 minutes before taking response [14]. Basal reaction time of all the albino wistar
mice to radiant heat was recorded by placing the tail (1.5 cm measured from the root of the
tail) on the radiant heat source. The cut-off reaction is fixed at 15 sec to avoid tissue damage.
The tail withdrawal (flicking action) from the heat source i.e, reaction time was recorded at 0
min, 30 min, 1 hr, 2 hr & 4 hr.
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Statistical analysis
All the values are expressed as mean ± S.E.M. The results were analyzed statistically by
Analysis of Variance (ANOVA) followed by Dunnett’s test. P values <0.05 were considered
significant.
Results and Discussion
In the preliminary acute toxicity study, MFT09 & MFG09 seems to be safe up to 2 g/kg
because even at this high dose no toxic or deleterious effects were observed immediately or
during 3 days and upto 14 days of observation period.
Anti-inflammatory activity
The anti-inflammatory effects of the MFT09 & MFG09 are shown in Table-1. The MFT09
(200,400 and 600 mg/kg, p.o.) showed very significant results and caused an inhibition in the
carageenan-induced hind paw edema in rats compare to MFG09 (100, 400 and 800 mg/kg,
topically). The MFT09 showed dose dependent inhibition of paw edema in the first and
second phase as compared to MFG09.
Table 1: Effect of MFT09 & MFG09 in Carageenan induced hind paw edema model
MFT09-Test drug, N = 6 in each group, Values are mean ± S.E.M, one way ANOVA followed by Dunnet’s test,
* P< 0.05 vs Arthritic control, ** P< 0.01 vs arthritic control. Values in the bracket indicate percent inhibition.
Treatment and dose
(mg/kg) p.o.
Paw volume (ml)
0hr 1hr 2hr 3hr 6hr 24hr
Group I
Control
0.84
±0.007
1.07
±0.03
1.30
±0.01
1.49
±0.03
1.36
±0.04
0.92
±0.01
Group II
MFT09 (200)
0.86
±0.008
1.04
±0.01
(21.73)
1.26
±0.009
(13.04)
1.28
±0.031**
(35.4)
1.16
±0.028**
(42.3)
0.91
±0.02
(37.5)
Group III
MFT09 (400)
0.84
±0.012
1.14
±0.028
(-30.4)
1.22
±0.023
(16.3)
1.22
±0.021**
(41.5)
1.07
±0.028**
(55.7)
0.90
±0.014
(25)
Group IV
MFT09 (600)
0.87
±0.01
1.04
±0.017
(21.73)
1.18
±0.02**
(32.6)
1.26
±0.02**
(40)
1.03
±0.015**
(69.2 )
0.91
±0.023
(50)
Group V
Aspirin (10)
0.87
±0.015
1.12
±0.018
(-8.69)
1.20
±0.029∗
(28.2)
1.12
±0.02**
(61.5)
0.99
±0.01**
(76.9)
0.87
±0.019
(75)
Group VI
MFG09 (100)
0.88
±0.009
1.31
±0.01
(-0.15)
1.36
±0.02
(0.43)
1.37
±0.01
(1.7)
1.32
±0.01
(1)
1.0
±0.02
(0.49)
Group VII
MFG09 (400)
0.89
±0.016
1.3
±0.017
(0.76)
1.35
±0.025
(1.16)
1.35
±0.03
(3.1)
1.3
±0.01
(3)
0.96
±0.02
(4.4)
Group VIII
MFG09 (800)
0.87
±0.01
1.30
±0.01
(-0.53)
1.33
±0.01
(0.07)
1.28
±0.02
(6.5)
1.21
±0.03
(5.5)
0.93
±0.02
( 5.2)
Group IX
Piroxicam gel (10)
0.86
±0.01
1.30
±0.009
(0.38)
1.33
±0.01
(2.5)
1.28
±0.02*
(8.3)
1.21
±0.03**
(9.3)
0.93
±0.02
(9.1)
Sandhya Desai et al Der Pharmacia Lettre 2010: 2 (1) 285-290
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Analgesic activity
In Radiant heat tail flick test, Treatment with MFT09 (200,400 and 600 mg/kg, p.o.)
produced significant analgesic action against thermal induced pain stimuli in albino wistar
mice at various time points post treatment by increasing latency to flick the tail as compared
to MFG09 (100, 400 and 800 mg/kg, topically) (TABLE-2).
Table 2: Effect of MFT09 & MFG09 on reaction time in Radiant heat tail flick method
Treatment and
dose
(mg/kg)
Pre-drug (mean
±SEM) reaction
time (in second)
Reaction time in sec (mean ± SEM)
30 min 1 hour 2 hour 4 hour
Group I
Control
4.438± 0.18 4.215±0.09 4.14±0.16 4.043±0.08 3.973±0.058
Group II
MFT09 (200)
4.373± 0.18
4.918±0.27
∗
5.933±0.26
∗
∗
6.97±0.06
∗
∗
6.905±0.24
∗
∗
Group III
MFT09 (400)
3.757±0.20
5.31±0.16
∗
∗
6.79±0.29
∗
∗
7.68±0.21
∗
∗
7.888±0.35
∗
∗
Group IV
MFT09 (600)
4.323±0.19
6.192±0.18
∗
∗
7.378±0.14
∗
∗
8.95±0.18
∗
∗
8.847±0.21
∗
∗
Group V
Aspirin (10)
3.918±0.17
6.94±0.08
∗
∗
7.562±0.15
∗
∗
8.98±0.073
∗
∗
8.735±0.09
∗
∗
Group VI
MFG09 (100)
4.01±0.206 3.74± 0.232 3.642±0.199 3.60±0.245 3.738±0.190
Group VII
MFG09 (400)
4.02±0.221 3.795± 0.210 3.942±0.128 4.18±0.172 3.995±0.179
Group VIII
MFG09 (800)
3.71±0.238 3.267± 0.156 3.037±0.289 3.59±0.258 3.555±0.258
Group IX
Piroxicam gel(10)
3.66±0.248 3.313± 0.264 3.647±0.206 4.91±0.282* 4.603±0.151*
N = 6; Each data suggest Mean ± SEM, One-way ANOVA followed by Dunnett’s test is applied for statistical
analysis, Drug treated groups were compared with control group. ∗∗ Significant at p < 0.01, ∗ Significant at p <
0.05
The most widely used primary test for screening of anti-inflammatory agents is carageenan
induced edema in rat hind paw [15]. The development of the paw edema in the rats after the
injection of carageenan has been described as a biphasic event, the first phase is due to
release of histamine and serotonin (5-HT) (1 hr), first plateau phase is maintained by kinin
like substance (2hr) and second accelerating phase of swelling is attributed to Prostaglandin
release (3hr) [16]. Histamine and 5-HT are mainly responsible for vasodilatation and
increased vascular permeability. Kinins, once released, are able to activate B
1
and/or B
2
receptors, releasing other inflammatory mediators, such as Prostaglandins (PGs),
Leukotrienes (LTs), Histamine, Nitric oxide (NO), platelet activating factor (PAF) and