Evaluation of protective efficacy of Spirulina platensis against collagen-induced arthritis in rats

RESEARCH ARTICLE

Evaluation of protective efficacy of Spirulina platensis againstcollagen-induced arthritis in rats

Narendra Kumar Æ Surendra Singh ÆNisha Patro Æ Ishan Patro

Received: 2 March 2009 / Accepted: 23 March 2009

� Birkhauser Verlag, Basel/Switzerland 2009

Abstract

Aim To assess the protective efficacy of Spirulina plat-

ensis against collagen-induced arthritis (CIA) in female

Wistar rats based on the changes in paws thickness, serum

albumin, cholesterol, lipid peroxidation, alkaline phos-

phatase and acid phosphatase activities and histology of

paw joints.

Methods Arthritis was induced by intradermal injection

of Collagen and Freund’s adjuvant incomplete suspension

at several sites on the back with a dose of 2 mg kg-1 of

body weight and boosted with 0.1 ml intradermally at the

base of the tail. CIA rats were orally treated with 200 and

400 mg kg-1 per oral of S. platensis from 0 to 45th day.

Results S. platensis at 400 mg kg-1 per oral significantly

elevates serum albumin and decreases the serum choles-

terol, alkaline phosphatase and acid phosphatase activities,

lipid peroxidation, paw thickness as well as normalize the

joint histopathology of CIA rats.

Conclusions S. platensis (400 mg kg-1) significantly

normalizes changes observed in arthritic rats to near nor-

mal conditions, indicates that S. platensis has promising

protective efficacy against CIA rats.

Keywords Spirulina platensis � Collagen � Alkaline

phosphatase � Lipid peroxidation � Pannus

Introduction

Rheumatoid arthritis (RA) is traditionally considered a

chronic, inflammatory autoimmune disorder that causes the

immune system to attack the joints. It is a disabling and

painful inflammatory condition, which can lead to sub-

stantial loss of mobility due to pain and joint destruction.

Most commonly, small joints are affected, but larger joints

can also be involved; the pattern of joint involvement can

differ from patient to patient (Majithia and Geraci 2007).

RA affects women three times more often than men and it

can first develop at any age. The risk of first developing the

disease appears to be greatest for women between 40 and

50 years of age, and for men somewhat later (Alamanos

et al. 2006). There is a significant increase in the lifetime

cost and a decrease in the quality of life (Kobelt et al.

2002). The disease is symmetric, especially as it pro-

gresses, and favors the interphalangeal joints of the hands

and feet, such as the proximal interphalangeal, metacar-

pophalangeal, and metatarsophalangeal joints, as well as

the wrist and ankle (Lee and Weinblatt 2001). Joint

swelling, deformity, pain, stiffness, and weakness are

classic symptoms. Other includes tenderness, synovial

thickening, effusion, erythema, and decreased range of

motion, ankylosis, and subluxation (Lee and Weinblatt

2001). RA is the commonest inflammatory arthropathy

worldwide and affects up to 0.75% of the Indian population

(Malaviya et al. 1993). The collagen-induced arthritis

(CIA) model in the rat is in many aspects similar to RA,

which is perhaps the most commonly used model for

RA today. Intradermal injection in rats with collagen

N. Kumar

School of Studies in Microbiology, Jiwaji University,

Gwalior 474011, Madhya Pradesh, India

S. Singh

Department of Botany, Banaras Hindu University,

Varanasi 221005, Uttar Pradesh, India

N. Patro � I. Patro (&)

School of Studies in Neuroscience, Jiwaji University,

Gwalior 474011, Madhya Pradesh, India

e-mail: [email protected]

Inflammopharmacol

DOI 10.1007/s10787-009-0004-1 Inflammopharmacology

emulsified in IFA leads to a severe, erosive poly-arthritis

developing within 2–3 weeks after immunization followed

by a subsequent chronic relapsing phase (Holmdahl et al.

1994).

S. platensis is a cyanobacterium, or more commonly

blue-green alga, appeared on the earth 3,500 million years

ago. Before Columbus, Mexicans (Aztecs) exploited this

microorganism as human food; presently, African tribes

(Kanembu) use it for the same purpose. S. platensis has

been used as food and nutritional supplements for a long

time (Dillon et al. 1995). It is generally regarded as a rich

source of proteins, vitamins, essential amino acids, min-

erals, essential fatty acids such as c-linolenic acid and

sulfolipids (Mendes et al. 2003). It also contains phenolic

acids, tocopherols and beta-carotene that are known to

exhibit antioxidant properties.

Over the last few years, S. platensis has been found to

have many additional pharmacological properties (Belay

et al. 2002; Chamorro et al. 2002; Rathore et al. 2004). S.

platensis also exhibits antiviral (Hernandez-Corona et al.

2002), anti-platelet (Hsiao et al. 2005), anti-cardiotoxic

(Khan et al. 2005), hypocholesterolaemic (Nagaoka et al.

2005), anti-nephrotoxic (Khan et al. 2006), anti-hepato-

toxic (Mohan et al. 2006) and anti-acute allergic rhinitis

(Mao et al. 2005) effects. To the best of our knowledge, the

protective effect of S. platensis has never been reported in

CIA in rats. Keeping in view the nutritive and pharmaco-

logical properties of S. platensis, present investigation was

undertaken to assess the protective effects of S. platensis

against CIA in rats.

Materials and methods

Reagents

All the inorganic and organic chemicals used were of

analytical grade and unless otherwise stated were pur-

chased from the Span Diagnostics Ltd., Surat, India, Merck

India Ltd, Sigma Chemical Co., St. Louis, Missouri (USA)

and BDH, Poole, England.

Mass cultivation and biomass preparation

of S. platensis

S. platensis was axenically grown in Zarrouk’s medium

(Zarrouk 1966). The exponentially growing cells of S.

platensis were harvested by filtration through screen-

printing filter with pore size 305 nm (1,400 pore/cm2) and

the biomass was dried at 50�C. The dried S. platensis

biomass was collected, weighed and used to feed the

experimental animals (rats).

Experimental model

Albino female rats (Wistar strain) of 6–10 weeks of age

were taken as the experimental animals for conducting the

proposed study. The choice of sex of the animals, i.e.,

females, is based on the findings of Holmdahl (1995) that

autoimmune arthritis is mediated by sex hormones and is

associated with a female preponderance for development of

arthritis. In addition, Van den Berg (2004) holds that the

female rats are more susceptible to induced arthritis as

compared to the males. A rat colony was maintained in the

animal house with 12:12 h light:dark schedule and free

access to food and water was given ad libitum. Rats were

subdivided into the following groups: normal rats (n = 6);

Arthritic control rats (n = 6); S. platensis treated arthritic

rats 200 mg kg-1 (n = 6); S. platensis treated arthritic rats

400 mg kg-1 (n = 6). All the experimental protocols were

pre-approved by the animal ethical committee, Jiwaji

University, Gwalior, Madhya Pradesh, India.

Induction of collagen-induced arthritis

Collagen-induced arthritis (CIA) in rats was developed

according to Remmers et al. (2002). Collagen from bovine

tracheal cartilage type II obtained from Sigma chemical

company St. Louis, Missouri, USA (CII) was dissolved in

cold 0.1 N acetic acid (2 mg ml-1) and was emulsified

with an equal volume of freshly opened, cold Freund’s

adjuvant incomplete (IFA) (Sigma, USA). Rats (6–

10 weeks old) were injected intradermally at several sites

on the back with a dose of 2 mg kg-1 of body weight. On

the seventh day after the primary immunization, the rats

were boosted with 0.1 ml (100 lg) of similarly prepared

collagen/IFA emulsion injected intradermally at the base of

the tail.

Treatment of animals with S. platensis biomass

The water suspension of S. platensis (200 and 400 mg kg-1)

was administered orally to arthritic rats with the help of

syringe cannula on a daily basis, whereas sterile water was

given to the control as well as untreated arthritic control rats.

The treatment was started from 0 day up to 45th day.

Measurement of body weight and paw thickness

changes

Severity of arthritis in hind paw was assessed by the

quantification of the changes in paw thickness. Measure-

ments were made with a dial gauge caliper from 0 to 45th

day at an interval of every 5th day. The body weight of rats

N. Kumar et al.

was measured from 0 to 45th day at an interval of every 5th

day.

Sample collection and processing

The blood samples were drawn from the retro-orbital

bleeding. The blood was collected in tubes, each blood

sample was centrifuged for 10 min at 5,000 rpm and at

4�C. The serum were collected and stored at -80�C for

further investigation.

Total serum cholesterol and albumin

Serum cholesterol level was estimated by CHOD-PAP

method using kit (Merck India Ltd.). Total serum albumin

was assayed according to Doumas et al. (1971) by using kit

(Merck India Ltd.). Albumin forms blue-green complex

with bromocresol green at slightly acidic pH, which is

measured spectrophotometrically at 540 nm.

Serum alkaline phosphatase activity

Serum alkaline phosphatase activity was assayed according

to Kind and King’s (1954) using commercially accessible

kits (Span Diagnostics Ltd., Surat, India).

Serum alkaline phosphatase converts phenyl phosphate

to inorganic phosphate and phenol at pH 10.0. Phenol, so

formed, reacts in alkaline medium with 4-aminoantipyrine

in the presence of the oxidizing agent potassium ferricya-

nide and forms an orange-red colored complex, which is

measured spectrophotometrically at 510 nm. The color

intensity is proportional to the enzyme activity, which is

expressed as Kings and Armstrong Unit (KAU).

Serum acid phosphatase assay

Serum acid phosphatase activity was assayed according to

King and Jegatheesan (1959) using commercially accessi-

ble kits (Span Diagnostics Ltd., Surat, India). Acid

phosphatase from serum converts phenyl phosphate to

inorganic phosphate and phenol at pH 4.9. Phenol, so

formed, reacts in alkaline medium with aminoantipyrine in

the presence of oxidizing agent potassium ferricyanide and

forms an orange red colored complex, which is measured

spectrophotometrically at 510 nm. The color intensity is

proportional to the enzyme activity, which is expressed as

Kings and Armstrong Unit (KAU).

Serum lipid peroxidation

Serum lipid peroxidation was measured following the

method of Okhawa et al. (1979). In this method, the

released malondialdehyde (MDA) serves as an index of

lipid peroxidation. To 0.2 ml of serum sample, 0.2 ml of

8.1% sodium dodecyl sulfate, 1.5 ml of 20% acetic acid

(pH 3.5) and 1.5 ml of 0.8% thiobarbituric acid (TBA)

aqueous solution were added. Distilled water (0.6 ml) was

added to make up the final volume 4 ml.

The content was vortexed, kept in water bath at 100�C

for 60 min and cooled in ice bath or in tap water for 5 min.

To the content, 1 ml water and 5 ml n-butanol/pyridine

mixture (15:1 v/v) were added and the content was shaken

vigorously. The content was centrifuged at 4,000 rpm for

10 min at 4�C and the absorbance of organic upper layer

was recorded at 532 nm against a reagent blank. The

concentration of thiobarbituric acid reactive substance was

expressed as nmol MDA ml-1 of serum using 1,1,3,3-tet-

raethoxypropane (TEP) as the standard.

Histopathological examination

Paw joints were collected at the end of experiment (45th

day) and after that tissue samples of the joints

(10 mm 9 5 mm thick pieces) were fixed in 10% (v/v)

neutral formalin. The formalin fixed tissues were cut into

thin pieces (2–3 mm thick) and decalcified in 10% EDTA

for 21 days. The decalcified tissue pieces were washed in

running tap water for overnight and washed twice with

distilled water for 30 min. The decalcified tissues were

dehydrated in ascending grades of alcohol and embedded

in paraffin blocks, 7l thick sections were cut with Leica

RM 2135 rotary microtome and mounted on slides and

stained with haematoxylin and eosin (Humason 1972) to

study the histopathological changes associated with CIA

and S. platensis treatment. The stained slides were visual-

ized using a Leica DM 6000 (Germany) equipped with

digital camera and the images were captured using a Leica

Application Suite software.

Statistical analysis

The values were presented as mean ± SE of six rats per

group. Results were analyzed by one-way analysis of var-

iance (ANOVA) followed by Tukey test (all pair wise

multiple comparison procedure) (Sigma Stat 3.5, Systat

Software Inc. USA). A value of *P \ 0.05 was considered

significant to arthritic control versus normal and treated

groups.

Results

Effect of S. platensis on arthritic symptoms

The sign of arthritis appeared from 20–22 days after

immunization and reached its maximum level at 45th day.

Protection of arthritis by Spirulina platensis

The macroscopic sign of severe arthritis at 45th day

included swelling, redness deformity and ankylosis in hind

paw and ankle joints. The symptoms of arthritic control

rats showed significant difference as compared to the

hind paw of normal rats. Such symptoms were, however,

found to be very less in the forelimbs. Whereas S. platensis

(200 mg kg-1) treated arthritic rats showed redness and

swelling only with moderate arthritis, the arthritic rats

treated with S. platensis (400 mg kg-1), however, showed

almost no sign of arthritis and appeared essentially similar

to normal rats (Fig. 1).

Effect of S. platensis on paw thickness changes

A significant increment in hind paw thickness from

4.17 ± 0.05 to 5.35 ± 0.20 mm was observed in arthritic

control rats from 0 to 45th day during development

of arthritis. Arthritic control rats, however, showed a

significant difference in paw thickness from normal rats

from 4.27 ± 0.06 to 4.38 ± 0.04 mm on 30th–45th day,

respectively. Whereas, S. platensis (400 mg kg-1) treatment

resulted in significant decline of paw thickness (4.60 ±

0.17 mm) in arthritic treated rats at 45th day, the arthritic rats

treated with S. platensis (200 mg kg-1), however, did not

show significant decline in paw thickness during develop-

ment of arthritis as compared to their arthritic control

counterpart (Fig. 2).

Effect of S. platensis on body weight

In the first 3 weeks, the absolute increment in the body

weight was found to be almost similar in all the groups of

rats, and no significant differences were observed between

them. However, after 3 weeks, a loss in body weight was

observed in the arthritic control rats as compared to their

normal and S. platensis treated counterparts. The body

weight of arthritic control rats was declined significantly at

35th, 40th and 45th day as compared to its normal coun-

terpart. S. platensis (400 mg kg-1) treated arthritic rats

showed significant increment in their body weight as

compared to their arthritic control counterparts. Arthritic

rats treated with S. platensis (200 mg kg-1) also showed

Fig. 1 Hind paws and ankle

joint of representative rat groups

at 45th days. a Normal rat,

b arthritic control rat showing

redness, swelling, deformity and

ankylosis with severe arthritis

(symptoms maximum in the

group), c S. platensis treated

(200 mg kg-1) rat showing

redness and swelling only with

moderate arthritis (symptoms

maximum in the group),

d S. platensis treated

(400 mg kg-1) rat showing

almost no sign of arthritis and

appeared essentially normal

(symptoms minimum in the

group). Pictures are

representative of six distinct rats

per group (colour figure online)

N. Kumar et al.

increment in their body weight as compared to arthritic

control rats, however, the difference was found to be sta-

tistically non-significant (Fig. 3).

Biochemical studies

Effect of S. platensis on total serum cholesterol

The total cholesterol levels in serum of arthritic control rats

were found to be significantly elevated from 72.30 ± 1.83

to 89.64 ± 2.23 mg dl-1 from 0 to 45th day, respectively,

during the development of arthritis. It also showed signif-

icant difference form normal rats at 45th day (71.60 ±

2.57 mg dl-1). S. platensis (400 mg kg-1) treatment resul-

ted in a significant decline of serum cholesterol level

(74.53 ± 2.14 mg dl-1) in arthritic treated rats at 45th day

as compared to the serum cholesterol level of arthritic control

rats. S. platensis (200 mg kg-1) treatment also resulted in a

decline in serum cholesterol level (82.66 ± 2.54 mg dl-1)

in arthritic treated rats at 45th day as compared to arthritic

control rats, but no significant difference was recorded in

these groups (Fig. 4).

Effect of S. platensis on total serum albumin

A highly significant decline in total serum albumin levels

from 3.76 ± 0.15 (0 day) to 2.74 ± 0.12 g dl-1 (45th day)

was recorded in arthritic control rats as compared to the

total serum albumin levels from 3.77 ± 0.30 (0 day) to

4.08 ± 0.34 g dl-1 (45th day) in normal rats during

development of arthritis. Arthritic control rats treated with

S. platensis (200 mg kg-1), however, did not show any

significant change in serum albumin levels from

3.64 ± 0.10 (0 day) to 3.26 ± 0.11 g dl-1 (45th day) as

compared to the total serum albumin levels in arthritic

control rats. However, arthritic control rats treated with S.

platensis (400 mg kg-1) showed significant increase in

serum albumin level (4.28 ± 0.18 g dl-1) at 45th day as

compared to arthritic control rats (Fig. 5).

Effect of S. platensis on serum alkaline phosphatase

activity

A highly significant increment in alkaline phosphatase

activity from 10.02 ± 0.63 (0 day) to 20.04 ± 0.98 KA

unit (45th day) was observed in arthritic control rats during

the development of arthritis with respect to the normal rats

Fig. 2 Changes in paw thickness before (0 day) and after collagen

immunization in normal, arthritic control and S. platensis (200 and

400 mg kg-1) treated rats. Data are expressed as mean ± standard

error of six rats per group. *P \ 0.05: arthritic control versus normal

and treated groups

Fig. 3 Changes in the body weight before (0 day) and after collagen

immunization in normal, arthritic control and S. platensis (200 and

400 mg kg-1) treated group. Data are expressed as mean ± standard

error of six rats per group. *P \ 0.05, #P \ 0.001: arthritic control

versus normal and treated groups

Fig. 4 Changes in total serum cholesterol level before (0 day) and

after collagen immunization in normal, arthritic control and S. plat-ensis (200 and 400 mg kg-1) treated rats. Data are expressed as

mean ± standard error of six rats per group. *P \ 0.05: arthritic

control versus normal and treated groups

Protection of arthritis by Spirulina platensis

having the alkaline phosphatase activity of 10.04 ± 0.37

and 10.86 ± 0.47 KA unit on 0 and 45th day, respectively.

However, S. platensis (400 mg kg-1) treatment showed a

significant decline of serum alkaline phosphatase activity

in arthritic treated rats at 30th (14.86 ± 0.64 KA unit) and

45th day (13.63 ± 0.68 KA unit) as compared to their

arthritic control counterparts. Arthritic control rats treated

with S. platensis (200 mg kg-1) also showed significant

decline in their serum alkaline phosphatase activity at 30th

(15.64 ± 0.89) and 45th day (15.63 ± 0.78 KA unit) as

compared to their arthritic control counterparts (Fig. 6).

Effect of S. platensis on serum acid phosphatase assay

A highly significant increase in acid phosphatase activity

from 4.02 ± 0.38 (0 day) to 6.47 ± 0.35 KA unit (45th

day) was recorded in arthritic control rats during the

development of arthritis with respect to the normal rats

having acid phosphatase activity of 4.08 ± 0.32 and

4.20 ± 0.39 KA unit at 0 and 45th day, respectively.

S. platensis (400 mg kg-1) treatment resulted in a signifi-

cant decline in serum acid phosphatase activity in arthritic

treated rats at 30th (4.7 ± 0.26) and 45th day (5.12 ± 0.18

KA unit) as compared to its arthritic control. In contrast,

S. platensis (200 mg kg-1) treated arthritic control rats did

not show significant decline in serum acid phosphatase

activity at 30th (5.79 ± 0.31) and 45th day (6.09 ± 0.32

KA unit) as compared to their arthritic control counterpart

(Fig. 7).

Effect of S. platensis on serum lipid peroxidation

A highly significant elevation in serum lipid peroxidation

(LPO) level from 2.86 ± 0.20 (0 day) to 5.92 ± 0.25

MDA nmol ml-1 (45th day) was recorded in arthritic

control rats during the development of arthritis with respect

to the normal rats having lipid peroxidation level of

2.87 ± 0.21 and 2.91 ± 0.23 MDA nmol ml-1 at 0 and

45th day, respectively. S. platensis (400 mg kg-1) treat-

ment resulted in a significant decline in serum LPO levels

of 3.61 ± 0.47 and 3.25 ± 0.29 MDA nmol ml-1 in

arthritic treated rats at 30th and a 45th day, respectively, as

compared to the LPO level of their arthritic control coun-

terpart. In contrast, arthritic control rats treated with

Fig. 5 Changes in total serum albumin level before (0 day) and after

collagen immunization in normal, arthritic control and S. platensis(200 and 400 mg kg-1) treated rats. Data are expressed as

mean ± standard error of six rats per group. *P \ 0.05: arthritic

control vs. normal and treated groups

Fig. 6 Changes in serum alkaline phosphatase (ALP) activity before

(0 day) and after collagen immunization in normal, arthritic control

and S. platensis (200 and 400 mg kg-1) treated rats. Data are

expressed as mean ± standard error of six rats per group. *P \ 0.05,#P \ 0.001: arthritic control versus normal and treated groups

Fig. 7 Changes in serum acid phosphatase (ACP) activity before

(0 day) and after collagen immunization in normal, arthritic control

and S. platensis (200 and 400 mg kg-1) treated rats. Data are

expressed as mean ± standard error of six rats per group. *P \ 0.05,#P \ 0.001: arthritic control versus normal and treated groups

N. Kumar et al.

S. platensis (200 mg kg-1), however, did not show sig-

nificant decline in serum LPO level at 30th (5.14 ± 0.19)

and 45th day (5.29 ± 0.21 MDA nmol ml-1) as compared

to the LPO level of their arthritic control counterparts

(Fig. 8).

Histopathological analysis of paw joints

Histology of joints of arthritic control rats showed vigorous

proliferation of synovial cells, resulting in pannus forma-

tion and infiltration of mononuclear cells and neutrophils to

the subsynovial region. Pannus destroyed the cartilage and

bone. In contrast, normal rats showed normal histology

without any synovial infiltration, pannus formation, carti-

lage and bone destruction. S. platensis (200 mg kg-1)

treated arthritic rats showed minimal to moderate synovial

cell infiltration with less destruction of cartilage, however,

they did not show any bone destruction. Whereas S. plat-

ensis (400 mg kg-1) treated arthritic rats showed almost no

sign of synovial cell infiltration, pannus formation, syno-

vitis, cartilage and bone destruction (Fig. 9).

Discussion

The primary drugs used in the treatment of RA are nonste-

roidal anti-inflammatory (NSAIDS) and disease-modifying

anti-rheumatic drugs (DMARDs). In most cases, these drugs

have been proved to be of only limited value. They often

suppress the symptoms, but accelerate factors that promote

the disease. However, patients frequently become unable to

continue long-term treatment with these agents due to tox-

icity and/or loss of benefit. A number of additional, non-

pharmacologic treatments like therapeutic fasting, dietary

supplementation of essential fatty acids and exercise for RA

have also been tried. On the basis of these insights, new

therapies have been developed, and clinical trials have

shown the efficacy of aggressive treatment of patients with

active disease. Despite increased use of these combination

therapies, new treatments for active RA are clearly needed.

Fig. 8 Changes in serum lipid peroxidation (LPO) before (0 day) and

after collagen immunization in normal, arthritic control and S.platensis (200 and 400 mg kg-1) treated rats. Data are expressed as

mean ± standard error of six rats per group. *P \ 0.05, #P \ 0.001:

arthritic control versus normal and treated groups

Fig. 9 Histological analysis of

joint morphology.

Photomicrographs (2509) of rat

phalangial joint after

haematoxylin and eosin staining.

a Normal rat with normal

synovial joint containing intact

cartilage, bone and synovium,

b Representative arthritic control

rat with ulceration and damaging

of cartilage, pronounced

synovitis, cell infiltration and

destruction bone compartment,

c S. platensis (200 mg kg-1)

treated with reduced synovitis,

cell infiltration and cartilage

ulceration, d S. platensis(400 mg kg-1) treated with

almost no sign of synovitis or

adherent cell to the cartilage and

appear essentially normal.

b bone, c cartilage, s synovium.

Pictures are representative of six

distinct rats per group. All the

pictures are of same

magnification (colour figure

online)

Protection of arthritis by Spirulina platensis

Recently, Spirulina is gaining more attention as a nu-

traceutical and source of potential pharmaceuticals. In the

present study, we have evaluated the protective efficacy of

dietary S. platensis against CIA in rats. During the entire

study, we have examined the physiological, biochemical,

histological parameters to evaluate the effect of S. platensis

on CIA in rats. S. platensis suppressed the development of

arthritis in rats in preventive manner and inhibited effec-

tively the development of macroscopic as well as

microscopic or histological symptoms in arthritic treated

rats as compared to their arthritic control counterparts.

The macroscopic sign of severe arthritis included

swelling, redness, deformity and ankylosis in hind paw and

ankle joints. The symptoms in hind paw of S. platensis

(400 mg kg-1) treated arthritic rats showed significant

difference with almost no sign of arthritis and appeared

essentially similar to normal. A significant increment in

hind paw thickness was also observed in arthritic control

rats during the development of arthritis. S. platensis

(400 mg kg-1) treatment, however, resulted in significant

decline of paw thickness in arthritic treated rats as com-

pared to arthritic control rats.

In RA, the systemic complaints include fatigue, fever

and weight loss. The weight loss occurs progressively as

the disease progresses (Lee and Weinblatt 2001). In the

present study, a significant loss in body weight was

observed in the CIA rats as compared to their normal

counterparts. However, S. platensis treated arthritic rat at

higher doses (400 mg kg-1) showed significant increase in

their body weight as compared to their arthritic control

counterparts. The gain of body weight in arthritic rats

during S. platensis treatment may be due to enrichment of

their diet with S. platensis having rich protein content.

Similar results on the increment of body weight in rats

following Spirulina fusiformis treatment have also been

reported where S. fusiformis treatment significantly

increases the body weight of animal model of adjuvant-

induced arthritis (Rasool et al. 2006). Normal rats fed with

Spirulina also gain body weight as compared to the rats fed

with normal diet (Nagaoka et al. 2005).

The typical decrease of serum albumin and increase of

globulin in RA are well known (Gutman 1948). Clinical

activity was found to have a significant positive correlation

with total protein ratio and a negative correlation with

serum albumin (Stidworthy et al. 1957). The serum total

cholesterol and serum triglycerides levels were recorded

elevated in RA patients (Mishra et al. 2007). The serum

cholesterol concentration was found to be directly pro-

portional to the risk of active RA (Heliovaara et al. 1996).

The increased level of serum alkaline phosphatase is a

common feature in RA and osteoarthritis (Nanke et al.

2002). The activity of acid phosphatase enzyme in serum

and bone were found to be significantly higher in male and

female CIA rats as compared to normal male and female

rats (Ganesan et al. 2008). The elevated lipid peroxidation

has been reported in patients with RA (Taysi et al. 2002;

Walwadkar et al. 2006). The elevation of serum lipid

peroxidation (Agha and Gad 1995) and plasma lipid per-

oxidation levels were recorded (Geetha et al. 1998)

significantly higher in adjuvant induced arthritic rats.

Riss et al. (2007) have reported that orally administered

phycocyanin or whole Spirulina ‘‘powerfully prevents the

development of atherosclerosis’’ in cholesterol-fed ham-

sters. C-phycocyanin, a novel protein from Spirulina,

clearly demonstrated a higher serum cholesterol lowering

effect in rats than casein (Nagaoka et al. 2005). The levels

of cholesterol in serum and liver were significantly reduced

in Spirulina treated experimental diabetic male and female

mice (Hernandez et al. 2001). The serum albumin levels

increased significantly after Spirulina feeding in exercised

rat as compared to normal exercised rats (Rogatto et al.

2004).

In a previous study, S. fusiformis treatment significantly

normalize the serum alkaline phosphatase activity in mice

with mercury induced hepatic toxicity (Kumar et al. 2005)

and significantly decreases the level of alkaline phospha-

tase activity and increase in acid phosphatase activity in

plasma, liver and spleen in adjuvant induced arthritic mice

(Rasool et al. 2006). In previous studies, a significant

decline in serum lipid peroxidation level was also recorded

in Spirulina treated rats as compared to control rats (Lu

et al. 2006; Hernandez et al. 2001). The plasma lipid per-

oxidation levels also decline significantly in Spirulina

treated rats as compared to control rats (Thaakur and Jyothi

2007; Mohan et al. 2006).

Histology of joint of arthritic control rats showed vigorous

proliferation of synovial cells, resulting in pannus formation

and infiltration of mononuclear cells and neutrophils to

the subsynovial region. S. platensis (400 mg kg-1) treated

arthritic rats showed almost no sign of synovial cell infil-

tration, pannus formation, synovitis, cartilage and bone

destruction.

The significant decline in symptoms of arthritis, paw

thickness, histological symptoms histological score and

normalization of biochemical parameters in S. platensis

treated arthritic rats may be due to the anti-inflammatory

and anti-oxidant effect of C-phycocyanin, a biliprotein

found in the S. platensis (Romay et al. 1998a). These

effects are presumed to be mediated by the scavenging

action of phycocyanin against reactive oxygen species

(ROS) such as OH, RO and RO2, anti-lipoperoxidative

activity, and inhibitory effects on prostaglandin and leu-

kotriene biosynthesis (Romay et al. 1999, 2000). The

inhibitory effect of Spirulina on mast cell mediated-type

allergic reaction in rats has also been reported (Kim et al.

1998). Phycocyanin was first tested in an experimental

N. Kumar et al.

model of inflammation, in which the inflammatory

response was induced by peroxide in the mouse paw.

Phycocyanin at doses of 100 and 200 mg kg-1 per oral

significantly inhibited the paw edema in a dose dependent

manner (Romay et al. 1998a). Phycocyanin was also

effective in the carrageenan-induced rat paw edema, which

is considered to be a suitable model for in vivo assessment

of COX inhibitors and also of novel antioxidants. The anti-

inflammatory activity of phycocyanin was greater in the

arachidonic acid induced ear edema (Romay et al. 1998b).

The inhibitory effect of phycocyanin from Spirulina was

also studied in zymosan induced arthritis in mice, an

experimental model of RA in which complement is acti-

vated via alternative pathway and the secretion of

lysosomal enzymes into the knee joint synovial fluid is

induced. This activity is correlated with the histomorpho-

logical changes observed in the joint, such as vasculitis,

synovitis and pannus formation (Remirez et al. 1999). In

our opinion, the mechanism by which Spirulina exerts its

anti-chemoattractant action on synovial cell infiltration or

pannus formation in joints may be due to its inhibitory

effects on the biosynthesis of the neutrophil chemotactic

mediator LTB4. This view is also supported by previous

findings that phycocyanin from Spirulina reduced LTB4

levels in the arachidonic acid induced mouse ear inflam-

mation test (Romay et al. 1999). Our finding are also

supported by Remirez et al. (2002) where Spirulina treat-

ment revealed a marked decrease of histology score, the

inflammatory reaction was substantially reduced and there

was no destruction of joint architecture or pannus forma-

tion and a reduction in bone erosion was observed in

zymosan induced arthritis in mice (Remirez et al. 2002).

Conclusion

Spirulina platensis showed the protective role against

arthritis by modulation of various biochemical and histo-

logical parameters. The present study concluded that dietary

S. platensis is able to suppress the physiological, histological

and biochemical changes produced during CIA in rats. This

suppressing ability of dietary S. platensis could be due to its

antioxidant constituents such as phycocyanion, carotenoids,

vitamin B1, B2, C and E and other micronutrients. However,

further investigations on its effectiveness and mechanisms

of action are warranted before recommending S. platensis as

a food supplement for the treatment of RA.

Acknowledgments Financial assistance provided by Ministry of

Environment and Forest, New Delhi, India for Narendra Kumar is

highly acknowledged. We acknowledge use of equipment facilities

developed through the DBT-HRD and Bioinformatics Infrastructure

grant to the School of Studies in Neuroscience.

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