chapter vii evaluation of anti -inflammatory a...

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

85

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).

86

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

87

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

89

(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).

90

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


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


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


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


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.


prophylactic treatment with the ethyl acetate extract of P.amarus (PAEA)

aerial parts and Indomethacin.

Treatment


the maximal paw oedema

during 6 hours



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***


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



***

***

*** ***

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


7.4.3.1 Aim

To evaluate the anti-inflammatory activity of methanolic extract of P.amarus aerial

parts (PAME).



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).


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:


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


prophylactic treatment with the methanolic extract of P.amarus aerial parts

(PAME) and Indomethacin.


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


the maximal paw

oedema during 6 hours



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



***

***

******

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


7.4.4.1 Aim

To evaluate the anti-inflammatory activity of Phyllanthin (PAPH) and

Hypophyllanthin (PAHP) on carrageenan induced rat paw oedema.



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


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


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.


Treatment


the maximal paw

oedema during 6 hours



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


PAHP 1.3x10-5 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



***

*** ******

Drug vehicle

Indomethacin




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.

chapter vii evaluation of anti -inflammatory a...

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