Anti-arthritic activity of agnuside mediated through the down-regulation of inflammatory mediators and cytokines

ORIGINAL RESEARCH PAPER

Anti-arthritic activity of agnuside mediated throughthe down-regulation of inflammatory mediators and cytokines

Anjali Pandey • Sarang Bani • Naresh Kumar Satti •

Bishan Dutt Gupta • Krishan Avtar Suri

Received: 20 October 2010 / Revised: 4 November 2011 / Accepted: 2 December 2011 / Published online: 8 January 2012

� Springer Basel AG 2012

Abstract

Objective and design The purpose of this study was to

elucidate the probable mechanism for the anti-arthritic

activity of agnuside (AGN), a compound isolated from the

leaf extract of Vitex negundo.

Methodology The anti-inflammatory activity of AGN

within a dose range of 1.56–12.50 mg/kg in normal and

adrenalectomized rats was evaluated against different in-

flammagens. An array of pro-inflammatory mediators

(PGE2 and LTB4) and T-cell-mediated cytokines (IL-2,

TNF-a, IFN-c, IL-4, IL-10, IL-17) was assayed using flow

cytometry, in arthritic paw tissue homogenate and

splenocytes of treated animals.

Results Significant anti-arthritic activity was observed

in the polyarthritis test in rats and this was associated with

significant suppression of inflammatory mediators and

T-cell-mediated cytokines (Th1/Th2). The anti-inflammatory

activity in adrenalectomized rats confirmed that the effect of

AGN is not mediated by the pituitary–adrenal axis. AGN also

showed inhibition of vascular permeability and leukocyte

migration in vivo.

Conclusion The study suggests the possible development

of AGN as a therapeutic agent in the treatment of arthritis

by the modulation of the host immune response.

Keywords Vitex negundo � Agnuside �Leukocyte migration � Inflammation �Vascular permeability � Polyarthritis �Cytometric bead array immunoassay � Cytokines

Introduction

Vitex negundo L. (Verbenaceae) is a small tree distributed

in India, Sri Lanka, Malaya, The Philippines and East

Africa. The leaves are astringent, febrifuge, sedative, tonic

and vermifuge [1, 2]. The water extract of fresh mature

leaves is used in Ayurveda medicine as anti-inflammatory,

analgesic and anti-itching agents internally and externally

[3]. This study was undertaken to investigate the role of

agnuside, a compound isolated from the leaf extract of

V. negundo, on various pro-inflammatory cytokines and

immune mediators related to inflammation and arthritis.

Prostaglandin E2 (PGE2) plays a role in exacerbating joint

inflammation and many of the effects of pro-inflammatory

cytokines are associated with PGE2 production. The level of

leukotrine B4 (LTB4) was found to be elevated in the

synovial fluid and membrane from patients with rheumatoid

arthritis (RA) [4, 5]. Studies have shown that LTB4

increases the production of the pro-inflammatory cytokines,

interleukin-1beta (IL-1b) and tumour necrosis factor-alpha

(TNF-a), in a dose-dependent manner in human RA [6]. The

Responsible Editor: Jerauld Skotnicki.

A. Pandey � S. Bani (&)

Department of Pharmacology, Indian Institute

of Integrative Medicine, Jammu Tawi 180001, India

e-mail: [email protected]

A. Pandey


N. K. Satti � B. D. Gupta � K. A. Suri

Natural Product Chemistry Division, Indian Institute

of Integrative Medicine, Jammu Tawi 180 001, India


B. D. Gupta


K. A. Suri


Inflamm. Res. (2012) 61:293–304

DOI 10.1007/s00011-011-0410-x Inflammation Research

123

cumulative evidence suggests that the aetiological cause of

arthritis is related to interferon-gamma (IFN-c) producing

Th1 cells that play a pivotal role in the development of

arthritis in both humans and animal models. Therefore,

recent therapeutic strategies put emphasis on modulating

the response of CD4? T cells and on targeting cytokines.

Neutralization of pro-inflammatory cytokines can effi-

ciently control arthritis, indicating that modulation of the

cytokine balance leading to systemic immune suppression

could be an effective therapeutic strategy.

Materials and methods

Plant material

Vitex negundo was collected on 28 April 2003 (Jammu and

Kashmir state, India) and authenticated by Dr. S. N. Sharma, a

taxonomist at Janki Ammal Herbarium of the Indian Institute

of Integrative Medicine, Jammu, India, where voucher

specimen no. 50311 and Accession no. 19889 is deposited.

Isolation of agnuside from V. negundo leaves

Extraction

The shade-dried and powdered leaves (1 kg) of V. negundo

were soaked in ethanol (5 L) and kept overnight. The per-

colate was filtered and concentrated under reduced pressure

at below 50�C. The extraction procedure was repeated three

more times using 3 L of ethanol each time. The combined

ethanolic extract was concentrated at 50�C and finally dried

in a vacuum desiccator to give the ethanolic extract (187 g).

Isolation

The ethanolic extract (50 g) of V. negundo was adsorbed

over silica gel (100 g) to make slurry which was packed

over a column of silica gel (1 kg) packed in chloroform.

Elution was done with chloroform followed by a mixture of

chloroform and methanol. Elution with 10% methanol in

chloroform gave agnuside (Fig. 1). The pure compound

was characterized on the basis of 1H-NMR, 13C-NMR mass

spectral data and the final purity of the sample was also

established with the help of HPLC.

HPLC protocol

Agnuside was quantified using the Shimadzu HPLC system

consisting of pump LC-10ATVP, an automatic sampling

unit (Autosampler), SIL-10ADVP, a column oven CTO-

10ASVP, a diode array detector SPD-M10AVP and system

controller SCL-10AVP version 5.40. Shimadzu.Class VP

software version 6.10 was used for data analysis and data

processing. The samples were analysed at 30�C on a Merck

C18 column (5 lm, 250 9 4.0 mm I.D.) by UV detection

at 254 nm. The mobile phase consisted of isocratic con-

ditions, i.e. MeOH:2.0% AcOH in water (30:70) at a flow

rate of 0.8 ml/min.

Agnuside:Linear Y = 0.00011235x ? 0 (r2 = 0.999646).

The stock solution of the pure agnuside was prepared in

HPLC-grade methanol and stored in a refrigerator in dark

at 4�C. From the stock solution, working solutions in the

concentration range of 17.5–35.0 lg/ml were prepared by

dilution with HPLC-grade methanol.

Quantification

The compound exhibited linear response in the concen-

tration range of 17.5–35.0 lg/ml and the calibration curve

was prepared by using a multipoint calibration curve

method. The working solution was injected in different

concentrations and the calibration curve was obtained

(r2 = 0.999646). The calibration curve was determined on

the basis of six levels of concentration.

Chemicals

Cytometric bead array flex, CBA antibodies for IL-2, IFN-

c, IL-4, IL-10, FACS sheath solution (BD Biosciences),

phosphate-buffered saline, Evans blue, carrageenan, dex-

tran, histamine, ibuprofen (IBP), phenylmethylsulfonyl

fluoride, aprotinin, Tween 20 (Sigma-Aldrich, India); gu-

macacia (Hi Media), Mycobacterium tuberculosus (Difco).

Animals

Fourteen-week-old Wistar rats (150–180 g) and 12-week-

old Swiss albino mice (24–30 g) of either sex, bred in the

Institute’s animal house, were used in the study. Animals

Fig. 1 Structure of agnuside

294 A. Pandey et al.

123

were housed under standard conditions (23 ± 2�C,

60–70% relative humidity and 12 h photo-period) and were

maintained on standard rodent pellet diet (Lipton India

Ltd., Bombay) and water ad libitum. In all the experiments,

a control group was maintained (vehicle administered)

whilst the other group received a standard drug for com-

parison and authenticity/credibility of the test. The study

was carried out after approval from the Institutional Ani-

mal Ethics Committee and all the animals used in

experimental work received humane care.

Treatment

IBP was used as a standard drug and was administered at

50 mg/kg per oral dose. 50 mg of IBP was prepared as

suspension in gumacacia (1% w/v) in 10 ml of normal

saline; 1 ml of this was administered per 100 g body

weight to the experimental animals. The treated groups

received freshly prepared agnuside (AGN) as a suspension

in gumacacia (1% w/v) at 1.56 mg/10 ml, 3.12 mg/10 ml,

6.25 mg/10 ml and 12.5 mg/10 ml and administered at

1 ml/100 g body weight to the experimental animals. The

control animals were given the vehicle only. IBP was used

as a standard for authenticity of the experiments. The

oedema was measured using plethysmographic recordings

of paw volume (volume differential meter, Model LE

7500 N, Panlab, Spain) of the experimental rats.

Experimental protocols

Carrageenan-induced oedema in rats

Oedema [7] was induced in left hind paws of the rats by the

sub-plantar injection of 0.1 ml of freshly prepared (1%

w/v) carrageenan (Marine Colloids, USA) suspension in

normal saline 1 h after the administration of the test drug

(AGN). The left hind paw volume was measured before

and 4 h after the carrageenan injection.

Histamine-induced oedema in rats

The method of Horakova and Moratova [8] was followed.

Oedema in the left hind paw of the rats was induced by the

sub-plantar injection of 0.1 ml freshly prepared 0.1% w/v

histamine (Sigma Chemical Co., USA) in normal saline 1 h

after oral administration of the test drug. The oedema was

measured before and 1 h after the histamine injection.

Dextran-induced oedema in rats

Acute oedema [9] in the left hind paw of the rats was

induced by the sub-plantar injection of 0.1 ml freshly

prepared 6% w/v dextran (Sigma) in normal saline. The

paw volume was measured before and 1 h after the phlo-

gistic injection. Different doses of the test and reference

drug dissolved in normal saline were given orally (p.o.) 1 h

before the dextran injection.

Adjuvant-induced polyarthritis in rats

Adjuvant arthritis [10] was induced by the sub-plantar

injection of 0.05 ml freshly prepared suspension (5.0

mg/ml) of steam-killed Mycobacterium tuberculosis (Difco,

USA) in liquid paraffin. The volume of the injected

(‘‘primary’’ response) [11], and un-injected (‘‘secondary’’

or immune-mediated response) [12] hind paws were mea-

sured before and on alternate days from day 1 to day 21

after the adjuvant injection. Different doses of the test drug

dissolved in normal saline and the vehicle (normal saline)

were given p.o. 1 h before the injection and then once daily

for 21 days. The erythrocyte sedimentation rate of all the

animals were monitored on day 21 by the Wintrobe method

[13]. The body weight of the animal was also monitored on

day 21. Gastric perforations were examined in the drug-

and standard-treated groups on the day of termination of

the experiment.

Homogenization of tissue

Before homogenization for each assay, frozen paw con-

taining bony tissue was weighed and broken into pieces on

dry ice. Paw tissues were added at 4 ml/g tissue to

extraction buffer containing 1 mM phenylmethylsulfonyl

fluoride, 1 mg/ml aprotinin, and 0.05% Tween 20 in

phosphate-buffered saline. Tissues were homogenized on

ice with a Polytron and the homogenate was centrifuged at

5,000g for 15 min [14]. Supernatants were stored at -80�C

until analysis by cytometric bead array immunoassay.

Cytometric bead array immunoassay

Becton–Dickinson (BD) cytometric bead array (CBA) was

utilized in the arthritis studies. This newly developed

technique uses uniform-size microparticle-based flow

cytometry to measure a panel of five cytokines (IL-2, IFN-

c, TNF-a, IL-4 and IL-10) simultaneously in a single

sample. Supernatants of arthritic tissue homogenate sam-

ples were transported immediately to the laboratory and

stored at -80�C until analysis. The faster, sensitive CBA

flex technology combines the principle of the ‘sandwich’-

based immunoassay with flow cytometry for simultaneous

measurement of multiple cytokines from small sample

volumes. Two-color flow cytometric analysis was per-

formed using a FACS–LSR flow cytometer.

Anti-arthritic activity of agnuside 295

123

Cytokine analysis by CBA

The assay provides the flexibility to select five microbead

populations with distinct fluorescent intensities (FL-3) that

are pre-coated with capture antibodies specific for each

cytokine. Analysis was carried out in two sets; one set was

maintained for standards and the second set was for the

samples (tissue supernatant). 50 ll of standard cytokines (set

one) and 50 ll of tissue supernatant were added to the pre-

mixed microbeads in 12 mm 9 75 mm Falcon tubes (BD).

After the addition of 50 ll of a mixture of PE-conjugated

antibodies against the cytokines, the mixture was incubated

for 3 h in the dark at room temperature. The mixture was

washed and centrifuged at 500g for 5 min and the pellet was

resuspended in 300 ll of wash buffer. The FACS-LSR flow

cytometer (BD Pharmingen) was calibrated with setup beads

and 3,000 events were acquired for each sample. Data were

acquired and analyzed using BD Cytometric Bead Array

software. Forward versus side scatter gating was employed

and data was displayed as two-color dot plots (FL-2 vs.

FL-3) such that the five discrete FL-3 microparticle dye

intensities were distributed along the y-axis.

Quantification of PGE2 and LTB4 in supernatant

from tissue homogenate

Samples on day 14 from different groups of animals were

prepared for the analysis of cytokine mediators as described

above. PGE2 and LTB4 were estimated using commercially

available kits based on sandwich and competitive ELISA

technique according to the manufacturers’ instructions. All

cytokine concentrations were determined by means of col-

orimetric measurement at 450 nm on an ELISA plate reader

by interpolation from a standard curve [14].

Expression of intracellular IL-17 in lymphocytes

of arthritic animals

Blood samples from different groups of animals were

processed and lymphocytes were separated. This lympho-

cyte population was evaluated for the intracellular

expression of IL-17 using flow cytometery. Briefly, the

animals were bled retro-orbitally and blood was collected

in EDTA-coated tubes for the estimation of PE-labelled

anti-rat IL-17 monoclonal antibody expression. Analysis

was done by flow cytometry [15].

Intracellular IFN-cdetection by flow cytometry

in splenocytes

Spleens collected from animals in all the test groups under

aseptic conditions, in Hank’s balanced salt solution (HBSS,

Sigma), were minced using a pair of scissors and passed

through a fine steel mesh to obtain a homogeneous cell

suspension. The erythrocytes were lysed with FACS Lysing

solution. After centrifugation (380g at 4�C for 10 min), the

pelleted cells were washed three times in phosphate-buf-

fered saline, and resuspended in complete medium [RPMI

1640 supplemented with 12 mM HEPES (pH 7.1),

0.05 mM 2-mercaptoethanol, 100 IU/ml penicillin, 100

lg/ml streptomycin, and 10% FCS]. Cell numbers were

counted with a haemocytometer by the Trypan blue dye

exclusion technique. Cell viability exceeded 95% [16].

Briefly, splenocytes were seeded into 96-well flat-bottom

microtitre plates (Nunc) at 2 9 106 cells/ml. The expres-

sion of CD4? markers on the cellular surface was evaluated

by monitoring the fluorimetric changes of the corresponding

FITC-conjugated monoclonal antibody with a flow cytom-

eter. After 3 days, splenic lymphocytes were stained with

5 ll of FITC-conjugated anti-CD4, followed by simulta-

neous staining with 5 ll of PE-conjugated anti-rat IFN-cantibody incubated for 30 min at 4�C in the presence of 19

FACS permeabilizing solution. Analysis was carried out on

the flow cytometer (BD, LSR) using Cell Quest Pro soft-

ware [17].

Carrageenan-induced oedema in adrenalectomized rats

The adrenal glands of the animals were removed surgically

[18] and saline was provided to these rats instead of water

for drinking. Two days later the carrageenan acute oedema

test [4] was carried out as described earlier.

Leukocyte migration (in vivo) in rats

Pleurisy was induced in the rats by injecting 0.5 ml of

carrageenan (1% w/v in sterilized normal saline) into the

pleural cavity of the rats following the procedure of Mea-

cock and Kitchen [19]. Different doses of the test and

reference drug were given orally 1 h before and 6 h after

the carrageenan injection. After 24 h of carrageenan

injection, rats were anesthetized, the pleural exudate vol-

ume measured and the total leukocyte count of the exudate

performed using the method of Bauer [20].

Acetic-acid-induced vascular permeability in mice

Using the method of Whittle [21], mice were injected with

a 0.2% solution of Evans blue dye (0.25% w/v in normal

saline) intravenously 30 min after oral administration of

the drug. Fifteen minutes later the mice were injected

intraperitoneally (1 ml/100 g of body weight) with freshly

prepared 0.6% acetic acid (v/v) in normal saline. After

30 min of acetic acid injection, mice in all the groups were

killed, their peritoneal cavities washed with 10 ml of

heparinized sterile normal saline and the wash was


123

centrifuged (3,000g) for 10 min. Absorbance of the

supernatant was measured at 610 nm using a spectropho-

tometer (Uvikon-810, Kontron Instruments, Switzerland).

Acute safety studies

Different oral doses (maximum 2,000 mg/kg) of AGN

were given to groups of three female mice for each dose

(OECD guidelines 423), while one group with the same

number of mice served as control.

Statistical analysis

The data obtained was subjected to statistical analysis

using Student’s t test.

Results

Carrageenan-induced oedema in normal

and adrenalectomized rats

AGN showed a dose-dependent anti-inflammatory effect

against carrageenan-induced oedema in normal and adre-

nalectomized rats. The results from adrenalectomized rats

and from normal animals (adrenals intact) were highly

comparable, thus showing that the mechanism of action of

AGN is independent of stimulation of the pituitary–adre-

nocortical axis (Table 1).

Histamine-induced oedema in rats

Oral administration of AGN inhibited histamine-induced

oedema and the effect was 10.08–36.97% depending upon

the dose (Table 2). There was no significant difference in the

magnitude of the suppressive effect between AGN and IBP.

Dextran-induced oedema in rats

AGN, when administered orally in graded doses, caused

inhibition of dextran-induced oedema (12.35–34.05%).

The effect was dose-dependent (Table 2).

Adjuvant-induced polyarthritis in rats

AGN caused significant inhibition of the oedema in the

injected limb and simultaneously inhibited the late second-

ary swelling in the un-injected limb. The secondary changes

in the forepaws and tail were also significantly inhibited by

AGN (data not included). The effect of AGN on the 21-day

course of adjuvant arthritis in two limbs is shown in Figs. 2

and 3. The anti-arthritic effect of AGN appeared to be

approximately equal to IBP in the injected limb. The eryth-

rocyte sedimentation rate (ESR) of all the adjuvant-induced

rats was monitored on day 21 and our study showed that

AGN prevented the increase of ESR in arthritis rats in a dose-

dependent manner (Table 3). At high dose levels of 6.25 and

12.5 mg/kg p.o., the ESR levels were decreased by 26.79

and 29.67% relative to controls, respectively. IBP at the

50 mg/kg p.o. dose showed 12.30% inhibition of ESR. The

weight loss in arthritic animals was inhibited by AGN,

reflecting its protective effect (Table 4). At the termination

of the experiment on Day 21, the stomachs of rats from all the

groups were observed. The group treated with IBP showed

gastric perforations, whereas none of the groups treated with

AGN showed gastric perforations (Fig. 4).

Cytometric bead array immunoassay

All five cytokines were detectable in the supernatant of

arthritic paw tissue homogenate from both control and

treated arthritic groups of rats. This microparticle-based flow

cytometric immunoassay has been proven in comparison

Table 1 Effects of oral administration of AGN on anti-inflammatory response to carrageenan induced oedema in normal and adrenalectomized

rats

Treatment Dose (mg/kg) Normal rats Adrenalectomized rats

Oedema (ml)a (mean ± SE) Inhibition Oedema (ml)a (mean ± SE) Inhibition

Control – 0.95 ± 0.01 – 0.92 ± 0.04 –

AGN 1.56 0.79 ± 0.04 16.84%; 0.81 ± 0.08 11.95%;

AGN 3.12 0.68 ± 0.07 28.42%; 0.73 ± 0.07 20.65%;

AGN 6.25 0.59 ± 0.03* 37.89%; 0.63 ± 0.04* 31.52%;

AGN 12.50 0.48 ± 0.02** 49.47%; 0.51 ± 0.07** 44.56%;

IBP 50.00 0.47 ± 0.06** 50.52%; 0.44 ± 0.06** 52.17%;

n = 6 per group

%; represents the percentage inhibition of oedema by the drug treatment

* P \ 0.01; ** P \ 0.001; Student’s t testa Values represent the increase in paw volume (mean ± SE) of six animals in each group


123

studies to have comparable analytical sensitivity to con-

ventional ELISAs. Moreover, it is possible to measure all

the five cytokines in a single sample. The expression of

IL-2, IFN-c, TNF-a, IL-4 and IL-10 was significantly lower

in the blood from AGN-treated animals, with maximum

inhibition at higher dose levels (6.25 and 12.50 mg/kg p.o.)

(Fig. 5).

Down-regulation of pro-inflammatory mediators PGE2

and LTB4 by AGN

AGN at graded doses of 6.25 and 12.5 mg/kg significantly

decreased LTB4 levels in a dose-dependent manner. In

contrast, PGE2 inhibition was not significant, even at

higher dose levels. IBP, as a standard drug, maximally

suppressed PGE2 when compared to the groups treated

with the test drug (Fig. 6).

AGN-administered arthritic rats have lower levels

of intracellular IL-17 in lymphocytes from peripheral

blood

The expression of intracellular IL-17 in blood from rats

treated with AGN doses of 6.12 and 12.5 mg/kg were 12.17

and 11.04%, respectively (Fig. 7). This finding was in sharp

contrast to arthritic rats in non-AGN-fed control groups,

where higher amounts of IL-17 (19.71%) were detected.

Oral administration of AGN thus appears to inhibit the

production of IL-17 in the lymphocytes of these animals.

Effect of AGN on intracellular IFN-cexpression by flow

cytometry

We cultured the splenocytes with fluorochromes to assess

intracellular cytokine contents. Predictably, we noted a

Table 2 Effects of oral administration of AGN on anti-inflammatory response to histamine and dextran induced oedema in rats

Treatment Dose (mg/kg) Histamine Dextran

Oedema (ml)a (mean ± SE) Inhibition (%) Oedema (ml)a (mean ± SE) Inhibition (%)

Control – 1.07 ± 0.04 – 1.19 ± 0.04 –

AGN 1.56 0.92 ± 0.06 14.01%; 1.07 ± 0.01 10.08%;

AGN 3.12 0.86 ± 0.03 19.62%; 0.96 ± 0.02 19.32%;

AGN 6.25 0.79 ± 0.01* 26.16%; 0.83 ± 0.08** 30.25%;

AGN 12.50 0.69 ± 0.08** 35.51%; 0.75 ± 0.07** 36.97%;

IBP 50.00 0.74 ± 0.07** 30.84%; 0.69 ± 0.02** 42.01%;

n = 6 per group

%; represents the percentage inhibition of oedema by the drug treatment

* P \ 0.01; ** P \ 0.001; Student’s t testa Data shows oedema in ml (mean ± SE) in paw volume of six animals in each group

Fig. 2 Effect of AGN on

Mycobacterium tuberculosus-induced polyarthritis (injected

day 0) over 21 days. The data

(plotted as mean ± SE change

in the paw volume in ml) show

that AGN (1.56, 3.12, 6.25 and

12.50 mg/kg p.o.) markedly

inhibited the primary reaction

(injected paw)


123

higher expression of of IFN-c (28.30%) in the arthritic

control group compared to 11.84% expression in the

naı̈ve control group. To clarify the characteristics of IFN-

c-related cytokine-producing lymphocyte subsets, we

examined IFN-c-producing cells among CD4? T cells.

We noted a lower level of intracellular IFN-c in AGN-

treated splenocytes at graded doses. Maximum suppres-

sion was observed with the 12.50 mg/kg p.o. dose

(Fig. 8).

Leukocyte migration and exudate volume (in vivo)

in rats

AGN showed a dose-dependent inhibition of both exudate

volume, where inhibition was 8.99–32.64%, and the total

leukocytes count, where inhibition was 16.98–32.69%,

against control depending on the dose given. IBP at the dose

of 50 mg/kg showed 22.10 and 28.88% inhibition of exudate

volume and total leukocyte count, respectively (Table 3).

Fig. 3 Effect of AGN on

Mycobacterium tuberculosus-induced polyarthritis (un-

injected day 10 onward) over

21 days. The data (plotted as

mean ± SE change in the paw

volume in ml) show that AGN

(1.56, 3.12, 6.25 and 12.50 mg/

kg p.o.) markedly inhibited the

secondary (delayed) response to

adjuvant reaction (un-injected

paw) thereby showing

immunosuppressive activity

Table 3 Effects of AGN on (in vivo) leukocyte migration, vascular permeability and erythrocyte sedimentation rate (ESR)

Treatment Dose

(mg/kg)

TLC mm 9 10-3

(mean ± SE)aExudate volume

(mean ± SE)aDye concentration lg/25 g

mouse (mean ± SE)bESR (mm) (day 21,

polyarthritis) (mean ± SE)c

Naı̈ve control – – – 0.20 ± 0.02 6.04 ± 0.40

Control – 54.29 ± 1.33 3.89 ± 0.34 0.59 ± 0.08 14.22 ± 0.56

AGN 1.56 45.07 ± 1.28 (16.98%;) 3.54 ± 0.20 (8.99%;) 0.51 ± 0.09 (13.55%;) 14.05 ± 0.79 (1.19%;)

AGN 3.12 42.19 ± 2.04 (22.28%;) 3.25 ± 0.23 (16.45%;) 0.46 ± 0.03 (22.03%;) 11.86 ± 1.02* (16.59%;)

AGN 6.25 39.42 ± 1.86* (27.36%;) 3.01 ± 0.34* (22.62%;) 0.37 ± 0.08** (37.28%;) 10.41 ± 0.67** (26.79%;)

AGN 12.50 36.54 ± 2.07** (32.69%;) 2.62 ± 0.16** (32.64%;) 0.33 ± 0.07** (44.06%;) 10.00 ± 0.68** (29.67%;)

IBP 50.00 38.61 ± 1.87* (28.88%;) 3.03 ± 0.39* (22.10%;) 0.36 ± 0.06** (38.98%;) 12.47 ± 0.65 (12.30%;)

n = 6 per group

* P \ 0.01; ** P \ 0.001; Student’s t testa Data shows increase in total leukocytes count (TLC) and exudate volume (mean ± SE) in carrageenan-induced pleurisy in rats (in vivo) and,

within parentheses, the percentage reduction of total leukocyte count and exudate volume by AGN treatmentb Data shows increase in concentration of dye due to excessive vascular permeability (mean ± SE) in mice, and, within parentheses, the

percentage reduction of dye concentration by AGN treatmentc Data shows increase of ESR (mean ± SE) in polyarthritis rats, and, within parentheses, the percentage inhibition of erythrocyte sedimentation

rate (ESR) by AGN treatment


123

Acetic-acid-induced vascular permeability in mice

AGN produced a dose-related reduction in vascular per-

meability in mice and the effect was 13.55–44.06%

depending on the dose. IBP at 50 mg/kg showed 38.98%

inhibition (Table 3).

Acute safety studies

There was no mortality or any behavioural change when

AGN was administered up to a single maximum dose of

2,000 mg/kg p.o. over a period of 14 days. No adverse

safety effects were observed at doses as high as 2,000

mg/kg; however, these studies need to be interpreted with

caution since blood levels of AGN were not measured.

More extensive studies are required to confirm the safety

findings. No adverse effects were observed at any of the

efficacious doses, indicating that levels of AGN in the

animals were high enough to be anti-inflammatory without

any noticable side effects.

Discussion

The therapy of arthritis usually employs NSAIDs, steroids

or disease-modifying drugs. The long-term use of these,

however, may not limit the disease progression leading to

joint deformity. In addition, all of these drugs have side

effects and the search for a novel anti-arthritic drug

continues.

An oral dose of AGN as high as 2,000 mg/kg did not

cause any sign of mortality or any observable negative

Table 4 Effect of AGN on body weight in adjuvant-induced arthritic

rats

Groups Dose

(mg/kg)

Initial body

weight (g)

day 0

(mean ± SE)

Final body

weight (g)

day 21

(mean ± SE)

Normal control – 144.71 ± 1.34 151.82 ± 1.07

Arthritis control – 141.66 ± 1.56 137.81 ± 1.79

AGN 1.56 142.21 ± 1.34 141.00 ± 1.32

AGN 3.12 142.79 ± 1.88 142.53 ± 1.33

AGN 6.25 144.11 ± 1.33 146.10 ± 1.55

AGN 12.50 145.22 ± 1.11 149.62 ± 1.75

IBP 50 140.24 ± 1.00 146.11 ± 1.43

No significant reduction was seen in body weight in groups treated

with AGN

n = 6 per group

Fig. 4 Gastric perforations in experimental animals: animals treated

with IBP had gastric perforations, whereas AGN treated animals did

not have any perforations

Fig. 5 CBA analysis for the expression of IL-2, IFN-c, TNF-a (Th1 cytokines) and IL-4, IL-10 (Th2 cytokines) in supernatant from arthritic paw

tissue homogenate from a group of rats treated with different concentrations of AGN


123

symptoms in the general behavior of mice over a period of

2 weeks, indicating its apparent safety. Repeated once-

daily dosing up to 100 mg/kg p.o. in a poly-arthritis test did

not cause any deviation from normal general behavior

when compared to the control group, or induce mortality in

rats up to 30 days.

AGN demonstrated significant anti-inflammatory activ-

ity against the acute inflammation induced by carrageenan,

histamine and dextran in rats (Tables 1, 2) and did not

appear to act through the activation of adrenal–pituitary

axis since there was significant inhibition of oedema

induced by carrageenan in adrenalectomized rats (Table 1).

Thus the test compound seems to be acting by inhibiting

inflammation induced by different phlogistic agents [22,

23]. The Mycobacterium tuberculosis-induced adjuvant

arthritis model is considered to be the closest to human RA

[24] and AGN showed significant anti-arthritic activity at a

dose range of 1.56–12.5 mg/kg p.o. in this model. AGN

prevented the increase in erythrocyte sedimentation rate

(ESR) in this model. The increase in ESR is a common

feature in rheumatoid arthritis [25]. The appearance of

secondary lesions in arthritic rats is the manifestation of

cell-mediated immunity and their enhancement suggest

immunostimulant activity [11, 26] and suppression sug-

gests immunosuppressive activity. In adjuvant arthritis the

secondary reaction is a manifestation of cell-mediated

immunity and AGN suppressed this response, suggesting

immunosuppressive activity.

NSAID ingestion is associated with erosions, petechiae,

type C gastritis, ulceration, interference with ulcer healing,

ulcer complications and injury to the small and large

intestine [27]. The number of lesions present on the gastric

mucosa is indicative of the ulcer severity [28]. The AGN-

treated group did not develop gastric perforations, whereas

continuous administration of IBP enhanced the severity of

ulcer formation. Despite decreasing the inflammation, IBP

induced gastric ulceration in the treated animals, which

was not observed in AGN-treated experimental animals.

Inflammatory mediators are responsible for the devel-

opment of clinical symptoms of arthritis. They cause

vasodilation, increase permeability of blood vessels and

induce migration of leukocytes to the site of inflammation.

AGN at graded doses of 1.56, 3.12, 6.25 and 12.5 mg/kg

significantly decreased LTB4 levels in a dose-dependent

manner, showing significant inhibition at the higher dose of

12.5 mg/kg p.o. PGE2 was, however, not significantly

Fig. 6 Effect of AGN on the

levels of a PGE2 b LTB4

expressed in supernatant from

arthritic paw tissue homogenate


123

inhibited. IBP as a standard drug suppressed PGE2 signif-

icantly when compared to the groups treated with the test

drug. The non-significant inhibition of PGE2 by AGN at

different doses may be one of the important features of its

non-ulcerogenic potential, as PGE2 has a protective effect

on gastric mucosa.

Cytokines are locally acting protein mediators that are

involved in almost all biological processes including cell

Fig. 7 Flow-cytometric data representing the effect of AGN on expression of intracellular IL-17 in Mycobacterium tuberculosus-induced

inflammatory arthritis in rats

Fig. 8 Effect of AGN on intracellular IFN-c expression by flow cytometry in splenocytes from arthritic rats


123

growth and activation, inflammation, immunity and dif-

ferentiation. Analysis of the expression of cytokines at the

mRNA levels in patients with arthritis has revealed that

many pro-inflammatory cytokines are abundant in synovial

tissues [29–31]. A preferential activation of type 1 cells

suggests that Th1 cytokines are involved in the pathogen-

esis [32]. Up-regulation of IL-17 is considered to be

involved in the pathological process as inhibitors of IL-17

provide effective anti-inflammatory therapy and cause

significant improvements in the signs and symptoms of

RA. These results are supported by our data which clearly

show that rats given AGN had considerably reduced

amounts of intracellular IL-17 expressed by the lympho-

cytes in the peripheral blood.

Since the balance of Th1/Th2 cytokines is thought to

influence autoimmune diseases like arthritis, studying this

balance advances understanding of the main mechanisms

involved in these diseases [29–31]. Consequently, we tested

the ability of AGN to control Th1/Th2 cytokine balance in

arthritic Wistar rats. IL-2 produced by CD4? T-helper cells

is a central regulator of the immune response that stimulates

the synthesis of IFN-c in T cells as well as induces the

secretion of pro-inflammatory cytokines such as TNF-a by

activated macrophages, neutrophils and other cell types.

The inhibition of IL-2 by AGN is possibly responsible for

reducing IFN-c expression by T cells and TNF-a by mac-

rophages and neutrophils. Since IFN-c and TNF-a are Th1-

type (pro-inflammatory) cytokines, their inhibition shows a

strong correlation with the anti-arthritic activity of the test

compound. AGN also inhibited Th2 cytokine (IL-4 and

IL-10) expression, as well as CD4? T-cell-mediated IL-2

expression, indicating that AGN causes non-specific inhi-

bition of both pro- and anti-inflammatory cytokines. The

cell-mediated immune response plays an important role

during the development of arthritis [33] and inhibition of

this response by AGN, particularly IFN-cproduced by

CD4? T cells, correlates strongly with anti-arthritic activity.

AGN inhibited migrating leukocytes and reduced the

exudate volume in the pleural cavity in rats (Table 3),

indicating inhibition of vascular permeability, which is

known to cause oedema in this model. Histamine,

5-hydroxytryptamine and bradykinin all increase vascular

permeability [34, 35] and act in conjunction with prosta-

glandins to cause oedema [36]. The process involves the

maintenance of a pro-inflammatory response which is

inhibited effectively by AGN. The effect of AGN may be

explained by inhibition of cell migration into the site of

inflammation via a reduction in chemotactic factors as well

as inhibition of primary inflammatory mediators.

Such observations strongly suggest that agnuside may

be of potential interest in the development of new therapies

for the management of certain inflammatory processes,

including arthritis.

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Documents

Anti-arthritic activity of agnuside mediated through the down-regulation of inflammatory mediators and cytokines