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Research Article
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Development and Evaluation of Antipyretic and Antinociceptive Activity of Polyherbal Formulation Containing Some Indigenous Medicinal Plants |
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R.K. Sahu,
A. Singh,
R. Gupta,
A. Roy
and
J. Dwivedi
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ABSTRACT
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In the present study, it was envisaged to prepare three polyherbal formulations (F1, F2 and F3) containing leaves of Solanum xanthocarpum and Andrographis paniculata, rhizomes of Curcuma longa and stem of Tinospora cordifolia in varying ratio and evaluating the polyherbal formulations for the antipyretic and antinociceptive activities. The antipyretic activity of methanol extracts (MF1, MF2 and MF3) and aqueous extracts (AF1, AF2 and AF3) of polyherbal formulation were studied in Brewers yeast induced pyrexia in mice. The antinociceptive activity of methanol and aqueous extracts of polyherbal formulation were studied using Eddys hot plate method and tail flick method in mice. The polyherbal formulation of all extracts showed significant reduction in the elevated body temperature of rat which was compared with standard paracetamol. The extract of polyherbal formulation produced significant increase in the reaction time by Eddys hot plate method and tail flick method in mice which was compared with standard morphine sulphate. From these results it may be concluded that AF1 formulation demonstrated maximum significant antipyretic and antinociceptive activities that might be due to combined effect of active constituents present in all plant material.
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Received: November 06, 2011;
Accepted: February 24, 2012;
Published: March 27, 2012
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INTRODUCTION
Herbal Medicine is the oldest form of healthcare known to mankind. In traditional
medicines, various herbal preparations are being used for treating rise in body
temperature (Mounnissamy et al., 2008) and relief
of pain (Gupta et al., 2007). Now polyherbal
formulation are becoming more popular because of simplified treatment regimens,
improved clinical effectiveness, enhanced patient adherence and reduced administrative
costs. Therefore we planned to prepare polyherbal formulation containing leaves
of Solanum xanthocarpum and Andrographis paniculata, rhizomes
of Curcuma longa and stem of Tinospora cordifolia. The leaves
of S. xanthocarpum are used in fever, expectorant in cough and asthma
(Kulcsar, 1976). This plant has been reported beneficial
in treatment of asthma and chronic bronchitis (Gunaselvi
et al., 2010). The leaves and root extracts of A. paniculata
exhibits antimalarial, anti-hepatitic, analgesic and antipyretic properties,
besides its general use as an immunostimulant agent (Lin
et al., 2009; Puri et al., 1993).
The rhizomes of C. longa used as anti-inflammatory, analgesic, antioxidant,
hepatoprotective and antimicrobial properties, in addition to its use in cardiovascular
disease and gastrointestinal disorders (Neha et al.,
2009). The bitter principles present in T. cordifolia show antiperiodic,
antispasmodic, anti-inflammatory and antipyretic properties (Panchabhai
et al., 2008; Vedavathy and Rao, 1991).
This plant is used in Ayurvedic to improve the immune system and body resistance
against infections (Prince et al., 1998). In
the different formulations used by different herbal practitioners, these plants
were the chief ingredients to treat pyrexia, inflammation and related pain.
These plants were reported having flavonoids, triterpenoids, polyphenolic compounds,
glycosides etc. (Bhatt, 2011; Pachaly
and Schneider, 1981; Qudrat-I-Khuda et al., 1964;
Singh et al., 2007). Flavonoids and polyphenolic
compounds are known for their biological efficacy such as antipyretic, anti-hepatotoxic,
analgesic and anti-inflammatory (Hajare et al., 2000;
Singh and Singh, 2010).
However, there were no scientific evaluations records found based on experimental
trial on rats about the antipyretic and antinociceptive efficacy of this medicinal
preparation. So here we planned to make such polyherbal formulation which produce
synergistic effect against antipyretic and analgesic along with increasing the
immune system of body. Therefore, this study was undertaken to develop and evaluate
the antipyretic and analgesic activity of three different polyherbal formulations
containing leaves of S. xanthocarpum and A. paniculata, rhizomes
of C. longa and stem of T. cordifolia in varying ratio.
MATERIALS AND METHODS Plant material: The leaves of Solanum xanthocarpum and Andrographis paniculata, rhizomes of Curcuma longa and stem of Tinospora cordifolia were collected from the out field of Bhopal city, Madhya Pradesh, India in August 2010. The species were identified by the local people during the time of collection and later on authentication was made by Dr. P. Jayaraman, Botanist, Plant Anatomy Research Centre (PARC), Chennai, India. The plant parts were washed with distilled water to remove dirt and soil and shade dried. The dried materials were powdered and passed through a 10-mesh sieve. The three formulations (F1, F2 and F3) were made by mixing different ratio of plant powdered materials. The composition of different polyherbal formulation was given in Table 1. Preparation of extracts
Preparation of methanol extract: All the three formulations were extracted separately with methanol in a Soxhlet apparatus. The extracts were filtered and concentrated by distilling off the solvents and evaporated to dryness using water bath to get crude methanol extract. Preparation of decoction: Twenty grams of mixture of each formulation with 150 mL of distilled water were macerated at ambient temperature for 24 h. After 24 h the drug macerate was boiled for 45 min and filtered through muslin cloth to get a decoction. The volume of the decoction was adjusted such that 20 g of mixture gave 50 mL of the decoction.
Animals: Swiss albino mice having weight 20-30 g were maintained under
standard conditions of temperature (23±1°C), relative humidity (55±10%)
and 12 h/12 h light/dark cycle and fed with a standard pellet diet with water
ad. libitum. They were housed in standard polypropylene cages with wire
mesh top.
Table 1: |
Composition of polyherbal formulation containing plant material |
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All studies were carried out using six animals in each group. The project proposal
was approved by the Institutional Animal Ethical Committee (1349/ac/10/CPCSEA).
Acute oral toxicity study: Acute oral toxicity was performed by following
OECD guideline-420 fixed dose procedure for methanol and aqueous extract of
polyherbal formulations and it was found that dose increasing up to 2000 mg
kg-1 body wt. shown no toxicity or mortality in experimental rats.
The LD50 of the methanol and aqueous extract as per OECD guidelines-420
is greater than 2000 mg kg-1 (Ecobichon, 1997).
Antipyretic studies: The procedure described by Al-Ghamdi
(2001) was adopted for this study. The body temperature of each albino Wistar
mice was recorded by measuring rectal temperature at predetermined intervals.
Albino Wistar mice were fasted overnight with water ad libitum before
the experiments. Pyrexia was induced by subcutaneously injecting 20% (w/v) brewer's
yeast suspension (10 mL kg-1) into the animal's dorsum region. The
rectal temperature of each mice was again recorded after 24 h of yeast administration.
Mice that did not show a minimum increase of 0.5°C in temperature 24 h after
yeast injection were discarded. Forty eight selected mice were grouped into
eight and immediately treated as follows: group I received normal saline, group
II received 10 mg kg-1 paracetamol, group III to group V received
methanol extract (MF1 to MF3) 100 mg kg-1 i.p.,
while group VI to group VIII received decoction (AF1 to AF3)
20 mL kg-1 body weight orally. Rectal temperature of all the mice
was then recorded by inserting digital thermometer into the rectum of each mice
at 30 min (Woode et al., 2009).
Antinociceptive activity
Hot plate method: The animals were divided into eight groups with six mice
in each group. Group I animals served as control, animals of Group II received
Morphine sulphate at 5 mg kg-1 body weight i.p., while animals of
group III to group V received methanol extract (MF1 to MF3)
100 mg kg-1 i.p., while group VI to group VIII received decoction
(AF1 to AF3) 20 mL kg-1 body weight orally.
The animals were placed on Eddys hot plate kept at a temperature of 55±0.5°C.
A cut off period of 15 sec was observed to avoid damage to the paw. Reaction
time was recorded when animals licked their fore or hind paws, or jumped prior
to and 0, 30, 60 and 90 min after administration of the samples (Zakaria
et al., 2006; Franzotti et al., 2001).
Hot tail flick method: The animals were divided into eight groups of
six animals each. Group I served as control, Group II served as standard and
were injected Morphine sulphate (5 mg kg-1) intraperitoneally, group
III to group V received methanol extract (MF1 to MF2)
100 mg kg-1 i.p., while group VI to group VIII received decoction
(AF1 to AF3) 20 mL kg-1 b.wt. orally After
treatment of mice, the tip of tail was dipped up to 5 cm into hot water maintained
at 50±0.7°C and tail flick responses were recorded. The same experiment
was repeated after 30, 60, 90 and 120 min. Cut off time of 10 sec was maintained
to avoid damage to the tail for all groups (Sanmugapriya
and Venkataraman, 2010).
Statistical analysis: The results are expressed as Mean±SEM of
six independent experiments. Statistical significance between group was evaluated
by one-way Analysis of Variance (ANOVA) followed by Dunnetts test. A p<0.05
value was considered as statistically significant.
RESULTS
Antipyretic activity: Antipyretic activity of methanolic and aqueous
extracts of polyherbal formulation was determined in pyrexia induced model.
It was evident from the Table 2, after 1 h administration
of extracts, the methanol extracts (MF1 and MF3) and aqueous
extracts (AF1, AF2 and AF3) of polyherbal formulation
produced significant (p<0.05) antipyretic activity, while methanol extracts
(MF2) showed significant (p<0.05) antipyretic activity at 1.5
h. The aqueous extract AF1 of polyherbal formulation showed maximum
decrease in body temperature (35.24±0.17), exhibited highest antipyretic
activity. Results were comparable with the standard drug, paracetamol, at 10
mg mL-1 concentration. The aqueous extract of polyherbal formulation
showed more potent activity than methanol extract.
Hot plate method: The result of hot plate test indicated that methanol extracts (MF1 to MF3) and aqueous extracts (AF1 to AF3) produced significant (p<0.05) increase in reaction time at 0.5, 1 and 1.5 h as comparable to the reference drug morphine sulphate (5 mg kg-1; i.p.,) which is showed in Table 3. The methanol extracts (MF1 to MF3) and aqueous extracts (AF1 to AF3) showed dose dependent increase in the reaction time at 1.5 h. The aqueous extract AF1 of polyherbal formulation showed maximum increase in reaction time (12.96±0.47 sec), exhibited highest antinociceptive activity.
Hot tail flick method: The tail withdrawal reflex time following administration
of the methanol extracts (MF1 to MF3) and aqueous extracts
(AF1 to AF3) of polyherbal formulation was found to increase
from 0.5 to 1.5 h. The results obtained from hot tail flick experiments are
shown in Table 4, in this model, administration of methanol
and aqueous extract of polyherbal formulation showed significant (p<0.05)
protection against the pain induction. The methanol extracts (MF1
to MF3) and aqueous extracts (AF1 to AF3) showed
dose dependent increase in the reaction time at 1.5 h.
Table 2: |
Antipyretic effect of methanol and aqueous extracts of polyherbal
formulation |
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Values are expressed as Mean±SEM, n = 6 in each group,
*p<0.05 compared to control group, MF: Methanolic extract, AF: Aqueous
extract |
Table 3: |
Analgesic effect of methanol and aqueous extracts of polyherbal
formulation by hot plate method |
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Values are expressed as Mean±SEM, n = 6 in each group,
*p<0.05 compared to control group, MF: Methanolic extract, AF: Aqueous
extract |
Table 4: |
Analgesic effect of methanol and aqueous extracts of polyherbal
formulation by tail flick method |
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Values are expressed as Mean±SEM, n = 6 in each group,
*p<0.05 compared to control group, MF: Methanolic extract, AF: Aqueous
extract |
The aqueous extract AF1 of polyherbal formulation showed maximum
increase in reaction time (9.05±0.25 sec), exhibited highest antinociceptive
activity. The formulation AF1 showed maximum activity as compared
to other formulations.
DISCUSSION The aqueous extract of polyherbal formulation showed more potent antipyretic and antinociceptive activities than methanol extract of polyherbal formulation. The aqueous extract of first polyherbal formulation (AF1) showed maximum significant antipyretic and antinociceptive activities compared with all other methanol and aqueous extract.
Antipyretic are the agents, which reduce the elevated body temperature. Regulation
of body temperature requires a delicate balance between production and loss
of heat and the hypothalamus regulates the set point at which body temperature
is maintained (Akindele and Adeyemi, 2007). In fever
this set point elevates and a drug like paracetamol does not influence body
temperature when it is elevated by the factors such as exercise or increase
in ambient temperature. Yeast induced fever is called pathogenic fever. Its
etiology includes production of prostaglandins, which set the thermoregulatory
center at a lower temperature (Goodman and Gilman, 1996).
The present results show that polyherbal formulation possesses a significant
antipyretic effect in yeast-provoked elevation of body temperature in mice and
its effect is comparable to that of paracetamol (standard drug). So inhibition
of prostaglandin synthesis could be the possible mechanism of antipyretic action
as that of paracetamol. Also, there are several mediators or multiprocesses
underlining the pathogenesis of fever. Inhibition of any of these mediators
may bring about antipyresis (Danquah et al., 2011).
The hot-plate and tail-flick tests are useful in elucidating centrally mediated
antinociceptive responses, which focuses mainly on changes above the spinal
cord level. The tail flick and hot plate methods are further distinguished by
their tendency to respond to the pain stimuli conducting through neuronal pathways
as the tail flick mediates a spinal reflex to nociceptive stimuli, while the
hot plate involves higher brain functions and is a supraspinally organized response
morphine and related opioid analgesic drugs exert their antinociceptive effects
by interacting with different opioid receptors both at spinal and supraspinal
sites. From the result it has been observed that narcotic analgesic drug, morphine
(5 mg kg-1, i.p.) exhibited significant antinociceptive effects in
the hot plate (supra spinal) as well as in the tail flick (spinal) tests. The
significant increase in pain threshold produced by the methanol and aqueous
extracts of polyherbal formulation in these models suggests involvement of central
pain pathways (Kumar and Rajani, 2011; Okokon
et al., 2008). The antinociceptive effect produced by the extract
may be via central mechanisms involving these receptor systems or via peripheral
mechanisms involved in the inhibition of prostaglandins, leucotrienes and other
endogenous substances that are key players in pain (Srinivasan
et al., 2003). The ability of methanol and aqueous extracts of polyherbal
formulation inhibits prostaglandin synthesis and other mediators and it produces
antinociceptive activity. However, the mechanism behind the central antinociceptive
action of the extracts in the hot plate and tail flick tests is not completely
understood and may need further investigation.
CONCLUSIONS
In conclusion, present findings demonstrated that the methanol and aqueous
extracts of polyherbal formulation has the favorable antipyretic and antinociceptive
activities, which are involved in possible inhibition of the central synthesis
of prostaglandins. However, further studies are necessary to fully elucidate
the mechanism of action of the polyherbal formulation. AF1 formulation
demonstrated maximum significant antipyretic and antinociceptive activities
that might be due to combined effect of active constituents present in all plant
material. Further investigations are in progress in our laboratory for fully
development of this aqueous polyherbal formulation, so that it can be prepare
easily in home when required.
ACKNOWLEDGMENT The authors acknowledge to Director of Oriental College of Pharmacy, Bhopal (M.P.), India, for providing all the facilities and successfully completion of work.
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