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International Journal of Biological Macromolecules 57 (2013) 50– 56

Contents lists available at SciVerse ScienceDirect

International Journal of Biological Macromolecules

jo ur nal home p age: www. elsev ier .com/ locate / i jb iomac

mmuno-modulation effect of sulphated polysaccharide (porphyran)rom Porphyra vietnamensis

aurabh Bhatiaa,∗, Permender Ratheea, Kiran Sharmab, B.B. Chaugulec,abanita Kard, Tanmoy Berad

PDMCOP, Bahadurgah, Haryana, IndiaDepartment of Pharmaceutical Sciences, Jamia Hamdard, New Delhi, IndiaDepartment of Botany, Pune University, Maharashtra, IndiaDepartment of Pharmaceutical Technology, Jadavpur University, Kolkata 700 032, India

a r t i c l e i n f o

rticle history:eceived 2 January 2013eceived in revised form 20 January 2013ccepted 2 March 2013vailable online 13 March 2013

a b s t r a c t

Our investigation explores the immuno-efficiency of sulphated polysaccharides enriched Porphyra viet-namenis. Isolated polysaccharide fraction (17.1–25.8%) was characterized by FTIR and NMR spectroscopywhich showed the presence of typical linear backbone structure called as porphyran. Oral administrationof porphyran (200–500 mg/kg) evoked a significant (P ≤ 0.05) increase in weight of the thymus, spleenand lymphoid organ cellularity. The total leucocyte and lymphocyte count was increased significantly

eywords:orphyra vietnamensislgaeorphyranolysaccharidesmmunomodulatory

(P < 0.005). The increase in the percent neutrophil adhesion to nylon fibres as well as a dose-dependentincrease in antibody titre values was observed. A decreased response to DTH reaction induced by SRBCwas recorded. A potential phagocytic response was seen and significant changes were observed in theformation of formazone crystals. It also prevented myelosuppression in cyclophosphamide drug treatedrats. The results indicated that P. vietnamenis possesses potential immunomodulatory activity and hastherapeutic potential for the prevention of autoimmune diseases.

. Introduction

Defense mechanism of humans is complex and phagocytes arective participants in innate immune response. Now a days, sev-ral infectious agents manifest certain fatal disease which demandshe development of novel therapeutics to augment innate immuneesponses [1]. Seaweeds are ample source of dietary polysaccha-ides and exhibit diverse biological properties [2]. These dietaryolysaccharides are also known to modulate immune functionshich are more structure and molecular-weight dependent [3].lmost all marine algae enriched sulfated polysaccharides arenown to have various effects on innate immune and comple-ent system which may further reduces the pro-inflammatory and

everal allergic reactions. A variety of interests in sulfated polysac-harides are complement activation, nitric oxide synthase and aro-inflammatory cytokine induction, interference with the migra-ion of leukocytes, elevate primary antibody response, binding

o pattern recognition receptors, influence macrophage func-ion and modifying intestinal cecal flora [4,5]. Furthermore they

∗ Corresponding author at: PDMCOP, Bahadurgarh 124 507, Haryana,ndia. Tel.: +91 9991634366.

E-mail address: sbsaurabhbhatia@gmail.com (S. Bhatia).

141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.ijbiomac.2013.03.012

© 2013 Elsevier B.V. All rights reserved.

augment the cytotoxicity of natural killer cells, lymphocytes andmacrophages against tumours [3].

Porphyra (Bangiales, Rhodophyta) is a good taste traditionalmedicinal food which is widely consumed in East Asia [6]. Inour previous work we have reviewed the biological activities ofPorphyra sp. and its cell wall polysaccharide porphyran [7–11]. Por-phyran is one of the active principles in Porphyra having multitudeof biological activities. It suppresses Th-2 type immune systemwithout influencing Th-1 type immune system and also affectsthe IgE and IgG2� [12,13]. However the immunodulation of por-phyran is still unclear. Thus, this study was designed to studythe immunomodulatory activity of isolated porphyran in differentexperimental models of cellular and humoral immunity in animals.

2. Materials and methods

2.1. Plant material

Porphyra vietnamensis was collected in July 2008 from Ratna-giri coast, Maharashtra, India. Further taxonomic identification was

conducted by Professor B.B. Chaugule at the Department of Botany,Pune University, Pune, Maharashtra, India. A voucher specimen hasbeen deposited in the herbarium at the Laboratory of Ecology underthe voucher specimen number (Bot/571/09).

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S. Bhatia et al. / International Journal o

.2. Structural characterization of porphyran

The polysaccharide fraction was isolated according to theethod of Ishihara et al. [12]. The isolated polysaccharide fractionas initially hydrolyzed by NaBH4–NaOH and then characterized

y Fourier transform infrared (FTIR), and 1H NMR and 13C NMR.TIR spectra were obtained using KBr pellet method in the range00–4000 cm−1 using FTIR spectrophotometer (Jasco, Japan). The

yophilized sample was dissolved in CDCl3 for 13C NMR, and in D2Oor 1H NMR. 1H NMR and 13C NMR spectra were obtained using Var-an 300 MHz Mercury NMR spectrometer from Shimadzu Analyticalechnique Centre, Department of Chemistry, Pune University, Pune14].

.3. Drugs

Accurately weighed quantities of the polysaccharide fractionsere suspended in 2% sodium carboxy methylcellulose (SCMC) torepare suitable forms of the dosages. Cyclophosphamide was useds a standard immunosuppressant. Suspensions of different sam-les of Porphyra were prepared by suspending weighed quantity ofrug in 2% PVP in water.

.4. Experimental animals

Laboratory bred Wistar albino rats (180–200 g) and albino mice20–25 g) of either sex were housed at 25 ± 5 ◦C in a well-ventilatednimal house under 12/12 h light/dark cycle. The mice were pro-ured from National Toxicological Centre, Pune. The experimentalrotocol was approved by Institutional ethical committee (CPC-EA 20/2008). The animals were subjected for quarantine (10 days)rior to experimentation.

.5. Chemicals and their sources

Leishmann’s stain and gluteraldehyde were purchased fromerck (Mumbai, India). Indian ink from HiMedia (Mumbai, India).BC diluting fluid and EDTA from Nice Chemicals (Cochin, India).

BT was purchased from Hi media laboratories Pvt. Ltd., Mumbai.ylon fibres (Local market, Bangalore, India). Cyclophosphamide

CYP) and Dexamethasone (DMS) were used as a standard immuno-uppressant. All the organic solvents and chemicals were ofnalytical grade and used as obtained.

.6. Acute toxicity studies

Lyophilized polysaccharide fraction was dissolved in water anddministered orally to different groups of rats in dosages rangingrom 100 to 1000 mg/kg for the LD50 study using the modi-ed method. There was no lethality in any of the groups after 7ays of treatment. For further studies the dose range was fixedetween 100 and 500 mg/kg b.w. Cyclophosphamide was used at aose of 30 mg/kg in cyclophosphamide-induced myelosuppressionhereas dexamethasone 2 mg/kg, p.o. [15].

.7. Body weight, lymphoid organ weight and cellularity

Animals were divided into four groups (I–IV). Each group com-rised of a minimum of five animals. Group I (Control) receivedormal saline; Group II, Porphyran 100 mg/kg body weight; Group

II, Porphyran 200 mg/kg; and Group IV, Porphyran 300 mg/kg. Thenimals were humanized 24 h after the last dose. Body weight gain

percentage) and relative organ weight (organ weight/100 g of bodyeight) of kidney, liver, spleen and thymus were determined for

ach animal. Single cell suspensions were prepared in RPMI-1640edium from bone marrow (from femur), spleen and thymus for

gical Macromolecules 57 (2013) 50– 56 51

cell count. Cell counting was done by using the Neubauer chamber[16].

2.8. Antigen (immunization)

Fresh blood was collected from sheep sacrificed in the localslaughter house. Sheep red blood cells (SRBCs) were washed threetimes in large volumes of pyrogen free 0.9% normal saline andadjusted to a concentration of 0.5 × 109 cells/ml for immunizationand challenge. The animals were immunized by injecting 1 ml of20% SRBC, i.p. The day of immunization was considered as day 0.On the 5th day, the blood samples were collected from each animalthrough the retro-orbital plexus to carry out the TLC, DLC and NBTtest [17].

2.9. TLC and DLC

For TLC the blood was collected from mice through retro-orbitalplexus and diluted with Turk’s fluid in WBC pipette, in which redcells were lysed without affecting the leucocyte. Leucocyte countwas done using a Neubauer’s chamber. Furthermore for DLC, smearof each blood samples was made on a clean glass slide and stainedwith Leishman’s stain. Among the hundred leukocytes counted, dif-ferentiation of leukocytes was observed based on the cell size andthe presence of granules with respect to their size, shape and colourof the nucleus present in the different cells. The observation wasmade under an oil immersion [18].

2.10. NBT reduction

The blood samples were incubated at 37 ◦C for 30 min in a cleanglass slide and smear was fixed then washed gently with cold salinesolution to remove the debris. To this, 0.4 ml of NBT medium (0.2 mlof 0.34% sucrose solution, 0.2 ml of 0.28% NBT and 0.2 ml of inactiv-ated foetal calf serum) was added and incubated at 37 ◦C for 30 min.After incubation slides were washed with cold saline and stainedwith safranin [18].

2.11. Haemagglutinating antibody (HA) titre

Followed by porphyran treatment for 45 days, animals in allgroups including control were immunized by i.p. administrationof 2 ml of 0.5 × 109 SRBC/ml/mouse. The day of immunization wasconsidered as day 0. On day 1, blood samples were collected fromall test animals by puncturing the retro-orbital plexus in ependrofftube. The serum was separated at a spin of 2500 rpm for 10 min andused to estimate the haemagglutinating antibody (HA) titre againstthe antigen SRBC at 0.025 × 109 cells [19].

2.12. Delayed-type hypersensitivity (DTH) response

The rats were challenged by injection of 0.5 × 109 cells SRBCs inright hind foot pad. Foot thickness was measured after +24 h, +48 hand +72 h of this challenge. The differences obtained for pre- andpost challenge foot thicknesses were taken for the measurement ofDTH and were expressed in mm. The porphyran was administeredorally on day 0 and continued till day 7 of challenge [20].

2.13. Neutrophil adhesion test

This test was performed following the method of Wilkinson,

simultaneously along with haematological parameters [21]. Afterthe determination of neutrophil count and other haematologicalparameters blood samples were incubated with 80 mg/ml of nylonfibre for 15 min. at 37 ◦C. The incubated blood samples were again

5 f Biological Macromolecules 57 (2013) 50– 56

atc

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w

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3.73

3.02

4.06

3.853.40

3.30

1.18

3.58

3.58

4.21;3.96

O

O

OH

OH O

S O

O

-O

precursor units (3-�-d-Gal-(1-4)-linked-�-l-Gal-6-sulfate(G-L6S))were G01, 103.4; G02, 69.4; G03, 81.2; G04, 69.2; G05, 75.7;G06, 61.5; L1, 101.3; L2, 69.3; L3, 71.3; L4, 78.7; L5, 70.9; L6,67.7 ppm). The characteristic signals at 73.3, 71.3, and 59.4 ppm

2 S. Bhatia et al. / International Journal o

nalyzed for TLC and DLC. The product of TLC and DLC indicates neu-rophil index of blood samples. Percent neutrophil adhesion wasalculated as below:

Neutrophil adhesion

= neutrophil count of control − neutrophil count of testneutrophil count of control

.14. Carbon clearance test [22]

Determination of phagocytic index by carbon clearance test isn indicative of phagocytic activity of the immune system (non-pecific immunity). All the animals received the porphyran for

days. On the 8th day, 0.2 ml off camel ink was administeredntravenously through tail vein in all groups, 30 min after the oraldministration of the porphyran. Blood samples were collectedrior to challenge and at time interval of 0, 10 and 20 min afterhallenge from retro-orbital plexus 25 �l blood samples lysed by

ml of 25% glacial acetic acid. The optical density of lysed samplesas analyzed spectrophotometrically at 675 nm till transparency

quivalent to standard sample (collected before challenge) wasbserved. Optical density was plotted against time on a logarith-ic scale and the line was regressed. The phagocytic index was

etermined from the given data applying following formula:

hagocytic index : PI = K sampleK standard

here, K = slope of regressed line.

.15. Cyclophosphamide-induced myelosuppression

Ziauddin et al. method was employed for cyclophosphamide-nduced myelosuppression. Albino rats were divided into sixroups designated as: control (I) and treatment (II–VI) groups,ach group containing six rats. The control group received salineolution. Group II was administered with only cyclophosphamidet the dose of 30 mg/kg, i.p. while groups III, IV, V, and VI ratseceived cyclophosphamide with varied concentrations of Por-hyran (200–500 mg/kg, p.o.) for 10 days. On day 11, blood samplesere collected from the retro-orbital plexus of individual animals

nd analyzed for haematological and serological parameters [23].

.16. Statistical analysis

All values are expressed as mean ± SEM. The data obtained werenalyzed statistically using Instat 2 (graph pad software). Statisti-al significance was calculated using one-way ANOVA followed byunnet comparison test. P values < 0.05 were considered signifi-ant.

. Results

.1. FTIR analysis

P. vietnamensis yields 17.6–25.7% of sulphated polysaccharide.TIR spectral analysis demonstrated the absorption spectrum oforphyran with functional peaks at 3418 (broad peak, due to

H stretching and O H group is in polymeric association); 1421due to C O H bending which may be overlapping with CH2 andH3 bending peaks); 1225 (due to C O stretching); 1154 and

072 (due to ether group), 930 (due to out of plane O H def).he two strong bands at 930 and 819 cm−1 may be attributed tohe asymmetric and symmetric epoxide ring deformation bandsespectively. The peaks at 1225 cm−1 and 819 cm−1 reflects the

Fig. 1. 1H NMR estimation of polysaccharide fraction extracted from Porphyra viet-namensis.

presence of sulphate group which is absent in the alkali modi-fication. Whereas peak obtained at 930 cm−1 may be due to thepresence of 3,6-anhydrogalactose which is responsible for impart-ing gelling property to porphyran [14].

3.2. NMR spectroscopy

1H NMR spectrum revealed the presence of aliphatic protons(anomeric resonances at 5.17–5.29 ppm) originated from 4-linked-�-l-galactopyranose units (Fig. 1). A signal at 5.29 ppm may beof anomeric proton of the porphyran precursor 4-O-linked �-l-galactose-6-sulfate units (Fig. 2). The other signal at 5.17 ppm maybe of anomeric proton of the 3,6-anhydro-�-l-galactose units. Theratio of the intensity of the two signals (5.29–5.17) was 1.15:1. Themolar ratio of sulfate to l-galactose is approximately 1.3:1, whichcomply with the data obtained from NMR spectrum reported ear-lier. A strong signal at 3.30 ppm may be due attributed to the methylgroup of 6-O-methyl-d-galactose. The degree of substitution bymethyl groups, estimated by the 1H NMR spectrum, was 0.62 forthe d-galactose residue.

The interpretation of the signals obtained by 13C NMR forthe polymer was accomplished by comparison with previouslyreported spectra of model compounds, porphyran, and otherrelated polysaccharides like agarose. Two groups of signals corre-sponding to the repeating units of the porphyran and the porphyranbiological precursor, respectively, were detected. The signals of 3-�-d-Gal-(1-4)-linked 3,6-anhydro-�-l-Gal (G-A) units, that is, theporphyran repeating units are (G1, 102.3; G2, 70.0; G3, 82.3; G4,69.3; G5, 75.7; G6, 61.7; A1, 102.5; A2, 70.3; A3, 80.2; A4, 77.1; A5,75.5; A6, 61.6 ppm). The signals obtained for porphyran biological

Fig. 2. 1H NMR spectrum of polysaccharide fraction from Porphyra.

S. Bhatia et al. / International Journal of Biological Macromolecules 57 (2013) 50– 56 53

O

O

OH

OH O

S O

O

-O

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83.375.7

72.2

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19.965.4

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3c

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Fig. 4. Nitro blue tetrazolium reduction test in porphyran treated animals. The NBTreduction was significantly altered for porphyran as compared to control. Values

TE

V

TE

V(

ig. 3. 13C NMR estimation of polysaccharide fraction extracted from Porphyra sp.ppm relation to TMS).

ndicate methylation at the C-6 position of �-d-galactose unit.-O-methyl-3, 6-anhydro-�-l-galactose showed a weak signal at8.2 ppm (Fig. 3).

.3. Effect of plant extract on body weight and lymphoidellularity

Body weight plays an important role in determination of naturef drug. Immunomodulatory drug show increase in body weighthile immunosuppressant show decrease in body weight in com-arison to control. Porphyran showed a significant increase in bodyeight at 100 mg/kg and 200 mg/kg. Body weight gain differencesere recorded in various groups of animals (Table 1).

At doses of 100 and 200 mg/kg, a significant increase in rela-ive organ weight of thymus was observed but there was no effectt a dose of 300 mg/kg in its weight. Lymphoid organ cellularityata indicate the significant increase in the cellularity at 100 and00 mg/kg as mentioned in Table 1.

.4. TLC and DLC

Analysis of haematological parameters suggested that por-hyran sample have dose dependent stimulatory effect on blood

ells like RBC, WBC, Hb count especially on platelet count andence exerts prominent effect on TLC (cellular immunity) (Table 2).urthermore it was observed that the lymopcyte, monocyte andosinophil were increased whereas basophil populations were

able 1ffect of porphyran on the relative organ weights and cellularity of lymphoid organs of m

Group Relative organ weight (means ± SEM) in grams

Spleen Thymus Liver Kid

CL 0.61 ± 0.02 0.21 ± 0.01 5.31 ± 0.19 1.26P100 0.78 ± 0.03 0.31 ± 0.01* 6.87 ± 0.13 2.29P200 0.86 ± 0.08 0.36 ± 0.01* 7.71 ± 0.22** 2.35P300 0.91 ± 0.02 0.34 ± 0.01 7.75 ± 0.07** 2.36

alues are expressed as means ± SEM, n = 6. *p < 0.01; **p < 0.05 considered significant as

able 2ffect of porphyran on blood cells and differential leucocyte count of mice.

GRP Total count (TLC)

RBC WBC Hb PTs NP

CL 6.33 ± 0.0117 11.88 ± 0.39 14.08 ± 0.223 592 ± 0.281 45P100 4.97 ± 0.287 11.01 ± 212 13.58 ± 0.223 536 ± 0.224 38P200 6.56 ± 0.301 12.36 ± 370 15.67 ± 0.310 672 ± 0.371 44P300 2.89 ± 0.248 8.01 ± 270 9.71 ± 0.165 376 ± 0.212 33

alues are expressed as means ± SEM; one-way analysis of variance, ANOVA followed by

CL) animals.

are expressed as mean ± SEM of six observations (one-way ANOVA, followed byTukey–Kramer multiple comparisons).

decreased and the population of neutrophills was not significantlychanged in comparison with control (Table 2). The enhancementof various haematological parameters has been correlated with theimmune stimulating nature of the drug.

3.5. NBT reduction test

The SRBC immunized mice in the NBT reduction test was signifi-cantly altered for the porphyran at two different doses compared tothe control group. The enhancement of NBT reduction was observedmore significantly at higher doses, 300 mg/kg (P < 0.01) than that oftheir lower dose 100 mg/kg when compared to control (Fig. 4).

3.6. Haemagglutinating antibody (HA) titre

The HA titre was used to assess humoral immune response. Thehumoral antibody titre value was found to be 122.23 + 12.7. Admin-istration of porphyran produced a dose-dependent increase in theHA titre after 1 h incubation with SRBCs. Administration of higher

dose, i.e. 300, 400 and 500 mg/kg, respectively produced significantincrease in HA titre as evident from as evident from haemaggluti-nation after incubation of serum with SRBCs (Table 3).

ice.

Cellularity (means ± SEM ×106)

ney Spleen Thymus Bone Marrow

± 0.05 277.23 ± 12.84 68.21 ± 3.11 14.34 ± 1.11 ± 0.07 375.33 ± 22.73 103.1 ± 10.06* 24.13 ± 1.81* ± 0.06 420.62 ± 51.73 111.10 ± 6.1* 31.32 ± 2.1** ± 0.08 415.51 ± 23.61 107.20 ± 8.22 29.22 ± 1.16

compared to control (CL).

Differential count

s EPs BPs MNc LMp

.5 ± 0.71 0.78 ± 0.74 1.21 ± 0.32 1.62 ± 0.38 49.73 ± 1.82

.2 ± 1.28 0.88 ± 0.82 0.58 ± 0.223 1.09 ± 0.41 52.3 ± 2.20

.56 ± 1.301 1.24 ± 121 0.68 ± 310 1.60 ± 0.43 57.23 ± 0.56

.74 ± 0.248 1.87 ± 170 0.73 ± 0.15 0.89 ± 0.27 57.92 ± 0.42

Tukey–Kramer multiple comparisons test (n = 6), values are compared with control

54 S. Bhatia et al. / International Journal of Biolo

Tab

le

3Ef

fect

of

eth

anol

ic

extr

act

of

P.

viet

nam

ensi

s

on

HA

titr

e,

DTH

resp

onse

(usi

ng

SRB

Cs

as

an

anti

gen

)

AN

D

neu

trop

hil

adh

esio

n

in

rats

.

HA

titr

e

and

DTH

resp

onse

usi

ng

SRB

Cs

Neu

trop

hil

adh

esio

n

GR

DT-

1

DT-

2

DT-

3

HA

-T

PI

TLC

NA

NI

%N

A

UTB

TB

UTB

TB

UTB

TBC

L

100.

29

±

0.09

0.28

±

0.9

0.28

±

0.1

26.2

2

±

0.31

2.31

±

0.49

7.6

±

0.03

7.1

±

0.30

57

±

0.13

53

±

0.02

433

±

0.01

377

±

0.30

12.9

±

0.75

CP

30–

–

–

–

–

5.8

±

0.10

5.3

±

0.10

49

±

0.03

47. ±

0.02

285

±

0.02

253

±

0.01

11.4

±

0.01

P

200

0.38

±

0.04

0.35

±

0.04

0.35

±

0.01

43.1

6

±

0.07

3.64

±

0.22

8.2

±

0.04

7.4

±

0.01

a61

±

0.01

a56

±

0.02

503

± 0.

03

417

±

0.03

16.9

±

0.02

P

300

0.42

±

0.17

a0.

36

±

0.01

a0.

35

±

0.12

a71

.20

±

6.0a

4.85

±

0.37

8.9

±

0.12

8.0

±

0.01

a67

±

0.01

a62

±

0.01

599

± 0.

01

496

±

0.01

17.1

±

0.01

P

400

0.56

±

0.02

a0.

43

±

0.02

a0.

41

±

0.01

a10

2.03

±

9.7a

6.01

±

0.25

9.0

±

0.03

8.1

±

0.01

a68

±

0.01

a63

±

0.01

603

± 0.

01

510

±

0.01

17.2

±

0.01

P

500

0.61

±

0.40

a0.

49

±

0.40

a0.

47

±

0.12

a12

2.23

±

12.7

a7.

12

±

0.31

9.2

±

0.02

8.1

±

0.04

a69

±

0.04

a64

±

0.05

633

±

0.04

522

±

0.01

17.6

±

0.01

aV

alu

es

are

mea

n

±

S.D

.,

n

=

6,

P

<

0.05

sign

ifica

nt;

DT-

1

resp

onse

(mm

)

24

h;

DT-

2

resp

onse

(mm

)

48

h;

DT-

3

resp

onse

(mm

)

72

h;

con

trol

(10m

l/kg

veh

icle

con

trol

):

CL.

gical Macromolecules 57 (2013) 50– 56

3.7. Delayed-type hypersensitivity (DTH) reactions

The cell-mediated immune response of porphyran was assessedby DTH reaction, i.e. foot pad reaction. As shown in Table 3, theporphyran produced a significant, dose-related increase in DTHreactivity in rats. Increase in DTH reaction in rats in response to celldependent antigen revealed the stimulatory effect of porphyran onT cells.

3.8. Carbon clearance test

Determination of phagocytic index by carbon clearance testis an indicative of phagocytic activity of the immune system.The enhancement of phagocytic index implies the faster removalof foreign particle from the body suggesting faster elicitation ofimmune response. The dose dependent enhancement in phago-cytic activity is an indicative of immunostimulation (Table 3). Theporphyran (7.12 ± 0.31) most strongly enhance rate of carbon clear-ance (non-specific immunity) as compare to control (3.01 ± 0.49).Thus phagocytic index (K) increased significantly (P < 0.05) in a dosedependent manner for porphyran treated group.

3.9. Neutrophil adhesion test

This test is performed to determine the adhesion of neutrophilto nylon fibre. The different fractions from Porphyra sp. increasethe process of margination of cells in blood vessels. The neutrophiladhesion was significantly (P < 0.05) increased. The parameter hasbeen correlated to the ability of neutrophils to stimulate non-specific immune response as indicated by the peripheral movementof neutrophils i.e. greater adhesion (Table 3). Porphyran showedmaximum (17.60 ± 0.21) neutrophil adhesion as compared to con-trol group in a dose dependent manner (12.90 ± 0.75).

3.10. Cyclophosphamide-induced myelosuppression

Cyclophosphamide at the dose of 30 mg/kg, i.p. caused a sig-nificant reduction in the haemoglobin, RBCs, WBCs and plateletscount. Combined treatment of cyclophosphamide and Porphyran(200–500 mg/kg, p.o.) resulted in a restoration of bone marrowactivity as compared with cyclophosphamide treatment alone. Sig-nificant reduction in white blood cell count was observed in animalstreated with cyclophosphamide alone (Group II) as compared to thecontrol group (Group I). Porphyran increased the levels of WBC ina dose-dependent manner as compared to the group treated withcyclophosphamide and ethanolic extract together. It was observedthat porphyran at the doses of 300, 400 and 500 mg/kg, respectivelyrestored the levels of WBC back to normal (Fig. 5).

4. Discussion

Seaweed polysaccharides are extensively used in food as wellas medicinal industries. Their molecular weight and sulfate con-centration plays an important role in influencing its biologicalproperty. Some sulfated polysaccharides acts proinflammatory asimmunosuppressor and some are immunostimulator. Immuno-stimulation was greatly influenced by various parameters such asstructure and molecular weight of sulfated polysaccharides, sul-fated sites heterogeneity, especially the degree of disaccharidesulfation, position of the sulfate moiety and organization of sul-fated domains. Structural modification of sulfated polysaccharidesgreatly affects sulfated polysaccharide-protein interaction which

may be useful in treating inflammatory related diseases [4].

Isolated porphyran from P. vietnamensis yield ranged from 17.6to 25.7% and was characterized by FTIR, 1H NMR and 13C NMR.Porphyran which has ability to stimulate T-cell formation and

S. Bhatia et al. / International Journal of Biological Macromolecules 57 (2013) 50– 56 55

Fig. 5. Effects of porphyran on blood cells of rats treated with cyclophosphamide for 10 days. Group I: control (no treatment), Group II: cyclophosphamide-treated group;Group III: treatment with Porphyran (200 mg/kg); Group IV: cyclophosphamide (30 mg/kg, i.p.) and Porphyran (200 mg/kg) treated group; Group V: treatment with Porphyran(300 mg/kg); Group VI: cyclophosphamide and Porphyran (300 mg/kg) treated group; Group VII: treatment with Porphyran (400 mg/kg); Group VIII: cyclophosphamide andPorphyran (400 mg/kg) treated group; Group IX: treatment with Porphyran (500 mg/kg); Group X: cyclophosphamide and Porphyran (500 mg/kg) treated group. Values arem

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ean ± S.D., n = 6; P < 0.05 significant.

otentiate the induction of different types of antitumor effectorells such as cytotoxic T-cells, NK cells and macrophages [24]. Por-hyran at doses of 100 and 200 mg/kg increased the bone marrowell counts indicating its stimulatory effect on haematopoietic stemells of bone marrow. It appears that 100 and 200 mg/kg are theptimum doses in mice. An increase in dose might have inducedown-regulation of immune functions. Moreover increase in thy-us weight was accompanied by increase in its cell counts. Thisay be partly due to stimulatory effect of porphyran on the lym-

hocytes and bone marrow haematopoietic cells, which ultimatelyome in the thymus.

Further it was observed that porphyran caused significantncrease in TLC, lymphocyte and to some extent monocyte popula-ion indicating the presence of immunological effects of porphyran.eutrophils are capable of a wide range of responses such ashemotaxis, phagocytosis, exocytosis and both intracellular andxtracellular killing [25]. The neutrophil adhesion to nylon fibresescribes the margination of polymorphonuclear lymphocyte inhe blood vessels and the number of macrophages reaching theite of inflammation. This might be due to the activation of the b2ntegrins, present on the surface of the neutrophils through whichhey adhere firmly to the nylon fibres [18]. Both low and high dosef porphyran (100 and 500 mg/kg, p.o.) show a substantial rise inhe neutrophil adhesion to nylon fibres which may potentially helpn increasing immunity of body against microbial infections [26].ence, it was inferred that porphyran causes stimulation of neu-

rophils towards the site of inflammation.Phagocytosis is the process in which phagocytes, ingests and

emoves microorganisms, malignant cells, inorganic particles andellular debris [27]. The carbon clearance test was done to eval-ate the effect of drugs on the reticuloendothelial system. Theeticuloendothelial system (RES) is a diffuse system consisting ofhagocytic cells. Cells of the RES play a vital role in the clearancef particles from the bloodstream. When colloidal carbon particlesn the form of ink are injected directly into the systemic circula-ion, the rate of clearance of carbon from the blood by macrophages governed by an exponential equation [28]. Porphyran showedemarkable augmentation in the phagocytic index by exhibiting aose-related increase in clearance rate of carbon by the cells of theeticuloendothelium system, it is speculated that it might be due toncrease in the activity of the reticuloendothelial system by priorreatment of animals with porphyran (Table 3).

IgG and IgM antibodies are involved in the complement activa-ion, opsonization, neutralization of toxins, etc. [27]. The successiveral treatment of porphyran for 45 days showed significantesponse in antibody production against SRBC compared to the

control group. The maximum HA titre was noted in the animalstreated with porphyran at 500 mg/Kg b.wt. (Table 3). The enhance-ment of antibody responsiveness to SRBC in mice, in this study,indicated the enhanced responsiveness of macrophages and Blymphocyte subsets involved in the antibody synthesis. There-fore augmentation of the humoral immune response to SRBCsby porphyran, as evidenced by increase in the antibody titre inmice (Table 3) indicated the enhanced responsiveness of T and Blymphocyte subsets, involved in the antibody synthesis [26]. Thehigh values of haemagglutinating antibody titre obtained in thecase of porphyran indicated that immunostimulation was achievedthrough humoral immunity.

In NBT reduction test, while NBT is phagosomed by the cells,intracellular dye is converted into an insoluble blue crystalline form(formazone crystals) [29]. The population of the positive cells withthe formazone granules was counted. Upon treatment with por-phyran, the test effectively showed the dose-dependent activity inthe reduction of tetrazolium dye.

Cell-mediated immunity involves effectors mechanisms car-ried out by T lymphocytes and their products (lymphokines) [27].In immuno-inflammatory DTH reaction, macrophages and Th1cells plays a major role. This reaction requires a specific anti-genic substance which will release cytokines by activation withT-lymphocytes [30]. Here SRBC was used as the antigenic substancewhich elicits the hypersensitivity reaction in mice. Therefore,increase in DTH reaction in rats in response to T cell dependentantigen revealed the stimulatory effect of porphyran on T cells(Table 3).

Moreover porphyran administration significantly amelioratedthe total WBCs count, RBCs count, haemoglobin and plateletscount, and also restored the myelosuppressive effects induced bycyclophosphamide (Fig. 5).

5. Conclusion

Current research explores the immunomodulatory effect ofagarose like sulphated polysaccharide present in new Porphyrasp. which has stimulated both cellular and humoral immuneresponses. Its linear backbone structure constituting 3,6 anhydro-galactose was confirmed by FTIR and NMR spectroscopy. Theeffectiveness of fractions treated animals in overcoming the sideeffects of drug-induced myellosuppression provides sufficient evi-

dences for balancing and adaptogenic efficacy of the P. vietnamensis.Thus, from the results obtained, it can be concluded that P. vietna-mensis has therapeutic potential and could be served as an effectiveimmunomodulatory candidate.

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cknowledgement

Authors are grateful to Dr. Tanmoy Bera, Jadhavpur University,olkata for giving proper direction to the manuscript.

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