chapter vii evaluation of anti -inflammatory a...
TRANSCRIPT
Chapter VII
Evaluation of
Anti-inflammatory Activity
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7. EVALUATION OF ANTI-INFLAMMATORY ACTIVITY OF AERIAL
PARTS OF PHYLLANTHUS AMARUS Schum & Thonn.
7.1 Introduction
Inflammation was known as ‘phlogosis’ to the Greeks and as ‘Inflammation’
to the Romans. Nearly 2000 years ago Cornelous Celsius, a Roman doctor first
described the four main signs of inflammation: rubor, turnor, color and dolor. Galen,
in third century A.D, defined inflammation as a reaction of the body against injury.
Subsequently Julius Colintein in 1873 emphasized the role of vessels in the
inflammatory process and E. Metchnikoff in 1892 emphasized mainly on the
migration of leukocytes and on phagocytosis (Metchnikoff et al., 1930). In late 19th
century, the science of immunology had been developed with the pioneering
contributions from Jenner, Pasteur and Landsteiner (Iravani et al., 2002). Thereafter
conclusive evidences have accumulated that body fluids and blood serum have a
protective effect against variety of invasive microorganisms.
Lewis in 1927 proposed that either histamine or H- substances might be
released from the skin in many forms of injury (Lewis, 1927). During 1930’s, Henry
Dale tried to explain the process of inflammation as an auto pharmacological
phenomenon. According to him, most pathophysiological events of inflammation
were mediated by the release of acetyl choline, catecholamine, histamine etc. In the
forties Menkin and Silva (Rocha and Silva, 1994) with co-workers in the fifties,
demonstrated the role of the leukotrienes and kinins, in the process of inflammation.
In the late sixties and seventies there came the prostaglandin phase.
Inflammatory diseases including different types of rheumatic diseases are very
common throughout the world. Although rheumatism is one of the oldest known
diseases of mankind and affects a large population of the world, no substantial
progress has been made in achieving a permanent cure (Salma, 1990; Maleki et al.,
2005). The greatest disadvantages of the presently available synthetic drugs (both
steroidal and non-steroidal drugs) lie in their toxicity and reappearance of symptoms
after discontinuation of treatment. The research on screening and development of
drugs for their activity is therefore, an unending process and there is hope of finding
out anti-inflammatory drugs from indigenous plants. Various plant extracts and their
isolated compounds have been proven to be good anti-inflammatory agents (Iracema
et al, 2005).
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Before embarking on a discussion of this topic, it is necessary to make it clear
that inflammation is not one event, but a series of events occurring in orderly
sequence, though not necessarily dependent on each other for their development. In
fact, no known anti-inflammatory response may act only on one component of the
reaction or even on a single phase of one component.
Inflammation was characterized two thousand years ago by Celsus using the
four Latin words: Rubor (warmth), Calor (reddening), Tumor (pain), and Dolor
(swelling) (Victor et al., 2005). Inflammation is a tissue reaction due to infection,
irritation. It is a part of the host defense mechanism but when it becomes
uncontrollable it is a hopeless condition. There are several tissue factors or
mechanisms that are known to be involved in the inflammatory reactions such as
release of histamine, bradykinnin and prostaglandins.
7.1.1 Plants having anti-inflammatory activity
The research involving screening and development of drugs for their anti-
inflammatory activity is therefore, an unending problem but there is hope of finding
out anti rheumatic drugs from indigenous plants.
The literature survey reveals that the plant species of about 96 genera
belonging to 56 families have exhibited anti-inflammatory activity. Some of the plant
sources used in the traditional system of medicine with pharmacologically or
therapeutically proven anti-inflammatory and anti-rheumatic claims (Gupta et al.,
2005; Perez-Garcia et al., 2005) are mentioned in Table.7.1.
Table.7.1 Plants having anti-inflammatory activity
Plant species Trade names in India Family
Aconitum napellus Aconite Ranunculaceae
Alpine officinarium Rasna Zingiberaceae
Azadirachta indica Neem Meliaceae
Balanites roxburghii Gari Simarubiaceae
Boerhaavia diffusa Punarnava Nyctaginaceae
Boswellia serrata Salaiguggal Burseraceae
Colchicum autumnale Colchicum Liliaceae
Commiphora mukul Guggul Burseraceae
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Curcuma longa Turmeric Zingiberaceae
Delonix elata Vatanarayana Leguminosae
Glycyrrhiza glabra Liquorice Leguminosae
Hedychium spicatum Kapur kacheri Zingiberaceae
Haliotropium curassavicum Haatisura Boraginaceae
Hemidesmus indicus Margabi Asclepidaceae
Rosa sinensis Jassoon Malvaceae
Indigofera aspalathoides Hakna Fabaceae
Inula racemosa Poshkar Compositae
Iris kashmiriana Padma-pushkara Iridaceae
Lawsonia aspera Henna Lythraceae
Leucas aspera Hulkusha Labiatae
Mammea longifolia (vitexin) Nagkesar Guttiferae
Moringa oleifera Sahinajan Moringaceae
Morus alba Tutri Moraceae
Myrtus communis Baragasha Myrtaceae
Nepeta hindostana (nepitrin) Billilotan Labiatae
Nerium indicum (plumieride) Kaner Apocyanaceae
Nigella sativa Kalonji Ranunculaceae
Nyctanthes arbortristis Seoli Oleaceae
Nymphaea stellata Nilkamala Nymphaceae
Operculina turpethum Nakpatra Convolvulaceae
Ougenia oojeinensis Sandan Fabaceae
Paederia foetida Gandhaki Rubiaceae
Peltophorum pterocarpum Kondachinta Caesalpiniaceae
Phyla nodiflora Jalapeepala Verbenaceae
Piper longum Pippali Piperaceae
Pulchea lanceolata Rasna Compositae
Premna herbacea Bhumjambu Verbenaceae
Prosopsis spicigera Shami Leguminosae
Psoralea corylifolia
(bavachinine) Bakachi Leguminosae
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Pterocarpus santalinus Raktachandhana Leguminosae
Randia dumetorum Maniphal Rubiaceae
Salvadora percica Brihatpilu Salvadoraceae
Teramnus labialis Mashaparni Leguminosae
Tinospora malabarica Sudarsana Menispermaceae
Urena lobata Vanabhenda Malvaceae
Vandal roxburghii Rasna Orchidaceae
Verbena officinalis Karaita Verbenaceae
Vitex negundo Nirgundi Verbenaceae
Withania somnifera Ashwagandha Solanaceae
7.1.2 Experimental models used for evaluating anti-inflammatory activity (Isabel
et al., 2004).
Inflammation involves a series of events that can be elicited by numerous
stimuli, e.g.: infectious agents, ischemia, antigen-antibody interactions and chemical,
thermal or mechanical injury. The response is accompanied by the clinical signs of
erythema, oedema, hyperalgesia and pain. Inflammatory responses occur in three
distinct phases, each apparently mediated by different mechanisms:
1. An acute transient phase, characterized by local vasodilation and
increased capillary permeability.
2. A sub-acute phase, characterized by infiltration of leukocytes and
phagocytic cells.
3. A chronic proliferative phase in which tissue degenerations and fibrosis
occur.
According to these phases, pharmacological methods have been developed for
the screening of anti-inflammatory activity.
In vivo methods for testing acute and sub-acute inflammation are:
1. UV-erythema in guinea pigs (Vayalil et al., 2004).
2. Vascular permeability (Yeshwanten et al.,2009).
3. Oxazolone-induced ear oedema in mice (Ceullar et al., 2001).
4. Croton oil ear oedema in rats and mice (Kim et al., 1999).
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5. Paw oedema in rats (using various phlogistic agents) (Madhukiran and Ganga
Rao, 2012; Winter et al., 1962; Arulmozhi et al., 2005; Muruganandan et al.,
2002; Zeitune et al., 1991).
6. Pedal inflammation induced by chemical agents like histamine, serotonin,
dextran, egg white and kaolin (Arulmozhi et al., 2005; Dewanjee et al.,2009;
Selye and Somogyi, 1967).
7. Pleurisy tests.
8. Granuloma pouch technique (Srinivas et al., 2000).
9. Hyaluronidase inhibition (Sumantran et al., 2007).
Chronic inflammation can be studied using the methods for testing granuloma
formation such as:
1. Cotton wool granuloma (Mengi and Bakshi, 2009)
2. Glass rod granuloma (Yeshwanten et al., 2009)
3. PVC sponge granuloma
Further, methods for testing immunological factors have been developed such as:
1. Adjuvant arthritis in rats (Lily et al., 2005; Fan et al., 2005).
2. Experimental allergic encephalomyelitis (Lassmann et al., 1988).
3. Schultz-Dale-reaction (Chand et al., 1979).
4. Passive cutaneous anaphylaxis (Ovary and Taranta, 1963).
5. Arthus type immediate hypersensitivity (Munoz, 1967).
6. Delayed type hypersensitivity (Henningsen et al., 1984).
These are the various models available for testing anti-inflammatory activity.
The models with reasonable accuracy, minimum time and less sample consumption
are described below.
7.1.2.1 Acute models for inflammation
a) Carrageenan induced rat paw oedema model (Winter et al., 1962).
Among the many methods used for screening of anti-inflammatory drugs, one
of the most commonly used techniques is based upon the ability of such agents to
inhibit the oedema produced in the hind paw of the rat after injection of phlogistic
agent. Various measuring systems used for the assessment of induced oedema in the
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paw include: volume, paw thickness, paw weight and painfulness to monitor the
development of the induced oedema in the paw.
Acute hind paw oedema is induced either in mice or in rats by injecting
0.05mL to 0.1mL of 1%w/v carrageenan which reaches a peak oedema level at 2-3hrs
after carrageenan injection. Although oedema can be induced by many other
phlogistic agents like dextran, formaldehyde, 5- hydroxytryptamine, histamine,
bradykinin, prostaglandin E1 etc for routine screening, carrageenan induced oedema
model is employed (Arulmozhi et al., 2005; Muruganandan et al., 2002; Zeitune et
al., 1991).
b) U.V. radiation induced erythema model (Vayalil et al., 2004).
Exposure to U.V radiation also induces acute erythema which is used as
model for testing anti-inflammatory activity.
7.1.2.2 Chronic models for inflammation
a) Cotton pellet test (Srinivas et al., 2000; Mengi and Bakshi , 2009)
Chronic inflammation is induced by the implantation of the sterile cotton
pellets (50±1 mg) on the back or axilla of the rats aseptically. The peak effect is
reached within 7 days.
b) Granuloma pouch test (Moura et al., 2005).
Pouch on the back of the rat is produced by injecting 20mL of air and 1.0mL
of 1%croton oil in olive oil or 0.5 mL of turpentine oil in the subcutaneous tissues in
between the shoulder girdles. The effect is seen after 7 days.
c) Formaldehyde induced arthritis (Eun-mi and Jae-kwan, 2003).
Arthritis is induced by injecting 0.1 mL of 2% formaldehyde solution into the
sub-planter region of one of the hind paws of rat on the first and third day of the 10
days experiment.
d) Adjuvant induced arthritis (Lily et al., 2005 and Fan et al., 2005).
Chronic arthritis in rats is induced by injection of 0.5 mg of killed
Mycobacterium tuberculosis (Freund’s adjuvant) suspended in 0.1 mL of liquid
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paraffin into one of the hind paws. The effect is observed for 40 days after irritant
injection.
7.2 Evaluation of Anti-inflammatory Activity
The complexity of inflammatory process and the diversities of the drugs that
have been found effective in modifying the process have resulted in the development
of numerous methods for detecting anti-inflammatory substances. In the present
investigation, the anti-inflammatory activity of hexane (PAHE), ethylacetate (PAEA)
and methanolic (PAME) extracts of Phyllanthus amarus aerial parts and the isolated
lignan molecules from hexane extract i.e., phyllanthin (PAPH) and hypophyllanthin
(PAHP) were also tested for the anti-inflammatory nature against the carrageenan
induced paw oedema model (Winter et al., 1962).
7.3. Materials and Methods
7.3.1 Materials
1. Hexane (PAHE), ethylacetate (PAEA) and methanol (PAME) extracts of
aerial parts of Phyllanthus amarus Schum & Thonn.
2. Phyllanthin (PAPH) and hypophyllanthin (PAHP) isolated from hexane
extract of P.amarus aerial parts.
3. Carrageenan – E.Merck Co., Mumbai, India.
4. Saline.
5. Sodium. Carboxy Methyl Cellulose (Sodium.CMC) - E.Merck Co., Mumbai,
India.
6. Indomethacin- Micro Labs, Bangalore, India.
7. Distilled water.
8. Zeitlin’s Apparatus.
All the materials used for this experiment were of analytical grade and were
purchased from Lotus Enterprises, Visakhapatnam.
7.3.2 Animals
Wistar albino rats of either sex weighing between 180-200g were obtained
from Mahaveer enterprises, Hyderabad, Andhra Pradesh. The animals were housed
under standard environmental conditions, one week before the start and also during
the experiment as per the rules and regulations of the institutional ethics committee
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(Regd No. 516/PO/c/01/CPCSEA). They were fed with standard laboratory diet.
Water was allowed ad libitum during the experiment.
7.3.3. Preparation of carrageenan suspension
Suspension of carrageenan sodium salt 1% was prepared by sprinkling 100mg
of carrageenan powder in 10 mL of saline (0.9% Nacl) and set aside to soak for 1
hour, and then the suspension was mixed thoroughly using magnetic stirrer.
7.3.4 Preparation of sodium CMC suspension
Stock suspension of sodium CMC was prepared by triturating the powder
sodium CMC (1g) finely in 2.5 mL of water, 1:10 dilution of this stock solution made
in distilled water was used for suspending the test and standard drugs.
7.3.5 Experimental Protocol
Animals were divided into XV groups (each contains 6 rats). The rats were
given doses orally with extracts at different dose levels 18 hrs and 2 hrs prior to the
induction of carrageenan subcutaneously (SC) into the sub-plantar tissue of the hind
paw of each rat, 0.1 mL of 1% carrageenan suspension.
The drug effects were estimated by comparing the maximal paw oedema
response during 6 h in the drug as extract treated group with that of vehicle treated
group as control. Group I normal rats treated with Drug vehicle (1% Sodium CMC)
and served as normal control and Group II rats were treated with Indomethacin at a
dose of 1.3x10-5
moles/kg b.w. Remaining groups were treated with the selected plant
extracts at different dose levels all the doses were administered orally according to the
body weight of the animals.
7.3.6 Statistical analysis
Data of Paw thickness was analyzed by using One-Way ANOVA followed by
post hoc Dunnett’s test using Graph pad Prism-5 software. The results are expressed
as Mean ±S.E.M (mean standard error). p < 0.05 was considered to be significant (*p
< 0.05; **p < 0.01; ***p < 0.001).
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7.3.7 Evaluation of Model
To evaluate this model, the percentage increase in paw thickness was plotted
against the time (Hour) and the maximal paw oedema response induced during the 6 h
was determined. The results showed the ability of the model in detecting that the time
course changes in the paw size was associated with carrageenan induced rat paw
oedema. The paw oedema was constantly increased during 4 h and reached peak of
oedema at 4th
hour. At the 5th
and 6th
hour, the oedema was gradually reduced. The
percentage increase in paw oedema thickness was calculated by using the following
formula.
% Increase in paw thickness = Yt – Yo x 100
Yo
Yt = Paw thickness at time (1, 2, 3, 4, 5 and 6 th
) after injection
Yo= Paw thickness at 0 hr (before injection)
Data was expressed in terms of mean values + S.E.M.
The % increase in paw oedema response during 6 hr was determined. The
percentage inhibition of paw oedema thickness is calculated using the formula.
Percentage Inhibition = 100 x (1-Y1/Yo)
Y1= Average increase in paw thickness in groups treated with test
extracts/compounds.
Yo= Average increase in paw thickness in control.
MeanS.E.M (n=6)
0 1 2 3 4 5 60
10
20
30
40
50
60
70
80
90
100Drug Vehicle
Time(hrs)
%In
cre
ase i
n p
aw
oed
em
a
Fig.7.1. Progression of the carrageenan-induced rat paw oedema over 6 h as
monitored with Zeitlin’s apparatus
91
Fig.7.2. Zeitlin’s apparatus was used to measure the paw thickness Zeitlin’s
constant loaded lever (Paw thickness measuring device) (Battu, et al., 1999; Madhukiran
and Ganga Rao, 2012).
1. Place, where the paws use to be kept to measure the thickness.
2. Constant loaded lever.
3. Graduated scale numbered between1-10 and divided by 0.5cm equal to 20
divisions.
4. Thread to pull down the lever with right leg in order to facilitate to keep the
paw in between pointer 1a and basement 1b.
7.4. Results
Hexane (PAHE), ethyl acetate (PAEA) and methanol (PAME) extracts were
tested at doses of 125mg/kg, 250mg/kg and 500mg/kg b.w., p.o. The percentage
protection produced by the standard and extracts of Phyllanthus amarus were
calculated based on the time course changes in the paw size was associated with
carrageenan induced rat paw oedema. The paw oedema was constantly increased
during 4 h and reached peak of oedema at 4th
hour. At the 5th
and 6th
hour, the oedema
was gradually reduced. The percentage protection of extracts was calculated on
inhibition of paw oedema at 4th
hour.
92
7.4.1. Effect of Hexane extract of Phyllanthus amarus aerial parts (PAHE) on
carrageenan induced rat paw oedema
7.4.1.1. Aim
To evaluate the anti-inflammatory activity of hexane extract of Phyllanthus
amarus aerial parts (PAHE).
7.4.1.2. Materials and Methods
Wistar albino rats of either sex (150-200 gm, n=6), Carrageenan and the
Zeitlin’s constant lever (Fig.7.2) for measuring paw thickness, were used.
The PAHE was administered orally at doses of 125mg/kg, 250mg/kg and
500mg/kg b.w. in sodium carboxy methyl cellulose suspension 18 hrs and 2 hrs prior
to the induction of oedema by carrageenan injection and monitored the oedema
progression. The maximal paw oedema response and the area under the time-course
(AUC) as total oedema response are shown in Fig.7.3 (A & B).
The extracts were administered orally in the following order
Group I Received vehicle orally (Sodium. CMC)
Group II Received Indomethacin (1.3x10-5
moles/kg b.w).
Group-III Received PAHE orally at a dose of 125 mg/kg b.w.
Group-IV Received PAHE orally at a dose of 250 mg/kg b.w.
Group-V Received PAHE orally at a dose of 500 mg/kg b.w.
7.4.1.3. Results
Indomethacin at dose of 1.3x10-5
moles/kg b.w. and PAHE at doses of 125
mg/kg, 250 mg/kg and 500mg/kg doses significantly inhibited the maximal paw
oedema response by 62.01±0.22, 22.92±0.47, 31.59±0.27and 38.29±0.2 respectively
and total paw oedema (AUC) by 69.09±0.15, 23.38±0.59, 35.35±0.36 and 38.62±0.62
during the 6 h of the carrageenan-induced rat paw acute inflammation, when
compared to the control group treated with drug vehicle. The results were given in
Table 7.2.
93
Table.7.2. Percentage inhibition of Carrageenan induced paw oedema in rats by
prophylactic treatment with the hexane extract of P.amarus (PAHE) aerial
parts and Indomethacin.
Treatment
Percentage inhibition of
the maximal paw oedema
during 6 hours
Percentage inhibition of
total AUC paw oedema
during 6 hours
Group I 0.0 0.0
Group II 62.01±0.22*** 69.09±0.15***
Group III 22.92±0.47*** 23.38±0.59***
Group IV 31.59±0.27*** 35.35±0.36***
Group V 38.29±0.2*** 38.62±0.62***
Results are expressed as Mean±SEM (n=6); Significance: ***P<0.001.
A)
0 1 2 3 4 5 60
20
40
60
80
100Drug vehicle
Indomethacin
PAHE 125 mg/kg
PAHE 250 mg/kg
PAHE 500 mg/kg
Time(hrs)
% In
cre
ase in
rat
paw
oed
em
a
Mean±SEM (n=6)
94
B)
0
10
20
30
40
50
60
70
80
90
100
Drug vehicle
Indomethacin
PAHE 125 mg/kg
PAHE 250 mg/kg
PAHE 500 mg/kg
P<0.001; Results were analysed by using One-Way ANOVA followed Dunnett's post-hoc test.
All groups were compared with drug vehicle group.
***
***
***
***
To
tal
ed
ema(A
UC
) as
%
of
mea
n c
on
tro
l re
spo
nse
Fig.7.3. Effect of hexane extract of P.amarus aerial parts (PAHE) and
Indomethacin (1.3x10-5
moles/kg b.w) A) The maximal and B) The total paw
oedema in carrageenan induced rats.
7.4.2 Effect of ethylacetate extract of P.amarus aerial parts (PAEA) on
carrageenan induced rat paw oedema
7.4.2.1. Aim
To evaluate the anti-inflammatory activity of ethyl acetate (PAEA) extract of
P.amarus aerial parts.
7.4.2.2 Materials and Methods
Wistar albino rats of either sex (150-200 gm, n=6), Carrageenan and the
Zeitlin’s Isotonic Lever (Fig.7.2) for measuring paw thickness, were used.
The PAEA was orally administered at different doses in sodium carboxy
methyl cellulose suspension at 18 h and 2 h prior to the induction of oedema by
carrageenan injection and monitored the oedema progression. The maximal paw
oedema response and the area under the time-course (AUC) as total oedema response
are shown in Fig. 7.4(A & B).
95
The extracts were administered orally in the following order
Group-VI Received PAEA at a dose of 125 mg/kg b.w., p.o.
Group-VII Received PAEA at a dose of 250 mg/kg b.w., p.o.
Group-VIII Received PAEA at a dose of 500 mg/kg b.w., p.o.
7.4.2.3 Results
Indomethacin at a dose of 1.3x10-5
moles/kg b.w. and ethyl acetate extracts of
P.amarus aerial parts (PAEA) significantly inhibited the maximal paw oedema
response by 62.01±0.22, 19.22±0.53, 35.37±0.49 and 39.63±0.48 and total oedema
response (AUC) was inhibited by 69.09±0.15, 20.87±0.37, 38.67±0.41 and
42.33±0.64 respectively during the 6 h of the carrageenan-induced rat paw acute
inflammation, when compared to the control group treated with drug vehicle. The
results are given in Table 7.3.
Table.7.3. Percentage inhibition of Carrageenan induced paw oedema in rats by
prophylactic treatment with the ethyl acetate extract of P.amarus (PAEA)
aerial parts and Indomethacin.
Treatment
Percentage inhibition of
the maximal paw oedema
during 6 hours
Percentage inhibition of
total AUC paw oedema
during 6 hours
Group I 0.0 0.0
Group II 62.01±0.22*** 69.09±0.15***
Group VI 19.22±0.53*** 20.87±0.37***
Group VII 35.37±0.49*** 38.67±0.41***
Group VIII 39.63±0.48*** 42.33±0.64***
Results are expressed as Mean±SEM (n=6); Significance: ***P<0.001.
96
A)
0 1 2 3 4 5 60
20
40
60
80
100Drug Vehicle
Indomethacin
PAEA 125mg/kg
PAEA 250mg/kg
PAEA 500mg/kg
Time(hrs)
% In
cre
ase in
rat
paw
oed
em
aMean±SEM (n=6)
B)
0
10
20
30
40
50
60
70
80
90
100
Drug vehicle
Indomethacin
PAEA 125 mg/kg
PAEA 250 mg/kg
PAEA 500 mg/kg
P<0.001; Results were analysed by using One-Way ANOVA followed Dunnett's post-hoc test.
All groups were compared with drug vehicle group.
***
***
*** ***
To
tal
edem
a(A
UC
) as
%
of
mea
n c
on
tro
l re
spo
nse
Fig.7.4.Effect of ethyl acetate extract of P.amarus aerial parts (PAEA) and
indomethacin (1.3x10-5
moles/kg b.w) on A) The maximal and B) The total paw
oedema in carrageenan induced rats.
97
7.4.3 Effect of methanolic extract of P.amarus aerial parts (PAME) on
carrageenan induced rat paw oedema
7.4.3.1 Aim
To evaluate the anti-inflammatory activity of methanolic extract of P.amarus aerial
parts (PAME).
7.4.3.2 Materials and Methods
Wistar albino rats of either sex (150-200 gm, n=6), Carrageenan and the
Zeitlin’s Isotonic Lever (Fig.7.2) for measuring paw thickness, were used.
The PAME at different doses were administered p.o in sodium Carboxy
methyl cellulose suspension 18 h and 2 h prior to the induction of oedema by
carrageenan injection and monitored the oedema progression. The maximal paw
oedema response and the area under the time-course (AUC) as total oedema response
are shown in Fig.7.5 (A & B).
The extracts were administered orally in the following order
Group-IX Received PAME at a dose of 125 mg/kg b.w., p.o.
Group-X Received PAME at a dose of 250 mg/kg b.w., p.o.
Group-XI Received PAME at a dose of 500 mg/kg b.w., p.o.
7.4.3.3 Results:
Indomethacin (1.3x10-5
moles/kg b.w.) and methanolic extract of P.amarus
aerial parts (PAME) significantly inhibited the maximal paw oedema response by
62.01±0.22, 28.17±0.22, 38.25±0.45 and 42.7±0.25and the total paw oedema (AUC)
by 69.09±0.15, 29.90±0.19, 40.52±0.14 and 44.72±0.32 respectively during the 6 h of
the carrageenan-induced rat paw acute inflammation, when compared to the control
group. The results were given in Table 7.4.
98
Table.7.4. Percentage inhibition of Carrageenan induced paw oedema in rats by
prophylactic treatment with the methanolic extract of P.amarus aerial parts
(PAME) and Indomethacin.
Results are expressed as Mean±SEM (n=6); Significance: ***P<0.001.
A)
0 2 4 60
20
40
60
80
100Drug vehicle
Indomethacin
PAME 125 mg/kg
PAME 250 mg/kg
PAME 500 mg/kg
Time(hrs)
% In
cre
ase in
rat
paw
oed
em
a
Mean±SEM (n=6)
Treatment
Percentage inhibition of
the maximal paw
oedema during 6 hours
Percentage inhibition of
total AUC paw oedema
during 6 hours
Group I 0.0 0.0
Group II 62.01±0.22*** 69.09±0.15***
Group IX 28.17±0.22*** 29.90±0.19***
Group X 38.25±0.45*** 40.52±0.14***
Group XI 42.7±0.25*** 44.72±0.32***
99
B)
0
10
20
30
40
50
60
70
80
90
100
Drug vehicle
Indomethacin
PAME 125 mg/kg
PAME 250 mg/kg
PAME 500 mg/kg
P<0.001; Results were analysed by using One-Way ANOVA followed Dunnett's post-hoc test.
All groups were compared with drug vehicle group.
***
***
******
To
tal
edem
a(A
UC
) as
%
of
mea
n c
on
tro
l re
spo
nse
Fig.7.5. Effect of methanolic extract of P.amarus aerial parts (PAME) and
standard drug Indomethacin (1.3x10-5
moles/kg b.w) on A) The maximal and B)
The total paw oedema in carrageenan induced rats
7.4.4 Effect of Phyllanthin (PAPH) and Hypophyllanthin (PAHP) on
carrageenan induced rat paw oedema
7.4.4.1 Aim
To evaluate the anti-inflammatory activity of Phyllanthin (PAPH) and
Hypophyllanthin (PAHP) on carrageenan induced rat paw oedema.
7.4.4.2 Materials and Methods
Wistar albino rats of either sex (180-200 gm, n=6), Carrageenan and the
Zeitlin’s Isotonic lever (Fig.7.2) for measuring paw thickness, were used.
The isolated lignans (PAPH & PAHP) were administered orally at two doses
(1.3x10-5
moles/kg & 2.6 x10-5
moles/kg) in sodium carboxy methyl cellulose
suspension 18 h and 2 h prior to the induction of oedema by carrageenan injection and
monitored the oedema progression. The maximal paw oedema response and the area
under the time-course (AUC) as total oedema response are shown in Fig. 7.6(A & B).
100
The extracts were administered orally in the following order
Group XII Received PAPH at a dose of 1.3x10-5
moles/kg b.w., p.o.
Group XIII Received PAPH at a dose of 2.6x10-5
moles/kg b.w., p.o.
Group XIV Received PAHP at a dose of 1.3x10-5
moles/kg b.w., p.o.
Group XV Received PAHP at a dose of 2.6x10-5
moles/kg b.w., p.o.
7.4.4.3 Results
Indomethacin (1.3x10-5
moles/kg b.w.) and isolated lignans (PAPH & PAHP)
significantly inhibited the maximal paw oedema response by 62.01±0.22, 47.78±0.72,
50.11±0.64, 41.98±0.49 and, 46.62±0.86 and the total paw oedema (AUC) was
inhibited by 69.09±0.15, 51.12±0.94, 54.25±0.47, 47.62±0.56 and 51.29±0.45
respectively during the 6 h of the carrageenan-induced rat paw acute inflammation,
when compared to the control group. The results were given in Table.7.5.
The isolated lignans of P.amarus i.e., PAPH and PAHP when orally
administered at doses of 1.3x10-5
moles/kg and at 2.6x10-5
moles/kg b.w exhibited
highly significant reduction (P<0.001) in paw thickness when compared to control
group treated with drug vehicle at all evaluated intervals of time. The results were
depicted in Fig.7.6 (A & B).
Table 7.5. Percentage inhibition of Carrageenan induced paw oedema in rats by
prophylactic treatment with the phyllanthin (PAPH) and hypophyllanthin
(PAHP) and Indomethacin.
Results are expressed as Mean±SEM (n=6); Significance: ***P<0.001.
Treatment
Percentage inhibition of
the maximal paw
oedema during 6 hours
Percentage inhibition of
total AUC paw oedema
during 6 hours
Group I 0.0 0.0
Group II 62.01±0.22*** 69.09±0.15***
Group XII 47.78±0.72*** 51.12±0.94***
Group XIII 50.11±0.64*** 54.25±0.47***
Group XIV 41.98±0.49*** 47.62±0.56***
Group XV 46.62±0.86*** 51.29±0.45***
101
A)
0 1 2 3 4 5 60
20
40
60
80
100Drug vehicle
Indomethacin
PAPH 1.3x10-5 moles/kg
PAPH 2.6x10-5 moles/kg
PAHP 1.3x10-5 moles/kg
PAHP 2.6x10-5 moles/kg
Time(hrs)
% In
cre
ase in
rat
paw
oed
em
a
Mean±SEM (n=6)
B)
0
10
20
30
40
50
60
70
80
90
100
P<0.001; Results were analysed by using One-Way ANOVA followed Dunnett's post-hoc test.
All groups were compared with drug vehicle group.
***
*** ******
Drug vehicle
Indomethacin
PAPH 1.3x10-5 moles/kg
PAPH 2.6x10-5 moles/kg
PAHP 1.3x10-5 moles/kg
PAHP 2.6x10-5 moles/kg***
To
tal
ed
em
a(A
UC
) as %
of
mean
co
ntr
ol
resp
on
se
Fig.7.6. Effect of phyllanthin (PAPH) and hypophyllanthin (PAHP) along with
standard drug indomethacin on A) The maximal and B) Total paw oedema in
carrageenan induced rats
102
7.5. Discussion
The results suggested that the standard drug indomethacin and the tested plant
extracts and isolated lignans significantly inhibited paw oedema. Among the three
tested extracts, methanol extract (PAME) showed significant inhibitory effect, ethyl
acetate (PAEA) and hexane (PAHE) extracts showed moderate percentage of
inhibition. Among the isolated compounds phyllanthin (PAPH) showed better
inhibitory action than hypophyllanthin (PAHP).
In the preliminary phytochemical analysis of P.amarus posses different
compounds like lignans, steroid, alkaloids, glycosides, flavanoids, terpenoids,
phenolics etc. From the previous reports natural products like lignans (Dixit et al.,
2011), sterols and terpenoids (Toshihiro Akihis and Ken Yasukawa 2001), glycosides
(Chennpracha et al., 2010; Maoxing Li et al., 2010), phenolic compounds (Sergent et
al., 2010; Elizabeth et al., 2005), alkaloids (Pearce et al., 2007; Kathryn et al., 2012)
possess anti-inflammatory activity. The present anti-inflammatory activity of
P.amarus may be due to the presence of these bioactive compounds.