CCHHAAPPTTEERR--VV

EEFFFFEECCTTSS OOFF GGLLYYCCOOWWIITTHHAANNOOLLIIDDEESS

OONN GGLLYYCCOOPPRROOTTEEIINNSS IINN

SSAALLIIVVAARRYY GGLLAANNDDSS

OOFF

DD--GGAALLAACCTTOOSSEE SSTTRREESSSSEEDD

MMIICCEE

1. INTRODUCTION.

2. MATERIAL AND METHODS.

A. MATERIAL.

a. Animal.

B. METHODS.

a. Estimation of Fucose.

b. Estimation of Sialic Acid.

c. Histochemical Distribution of Glycoproteins.

i. Periodic Acid Schiff (PAS) for neutral Glycoproteins.

ii. Alcian Blue pH 1.0 for sulfated Glycoproteins.

iii. Alcian Blue pH 2.5 for Acidic Glycoproteins.

iv. Alcian Blue pH 1.0 + PAS Double Staining Technique.

v. Alcian Blue pH 2.5 + PAS Double Staining Technique.

vi. PAS-Sodium borohydride Technique for O-acetylated

Sialic Acid

d. Eletrophoretic Separation of Total Proteins and Glycoproteins.

i. Coomassie Brilliant Bule.

ii. Alcian Blue pH 1.0

3. RESULTS.

4. DISCUSSION.

93

1. INTRODUCTION:

Salivary fluid is an exocrine secretion (Ferraris and Munoz, 2006),

consisting of approximately 99% water, containing variety of electrolytes

and proteins. Salivary proteins represented by enzymes, immunoglobulins

and glycoproteins. Up to 26% of the salivary proteins are glycoproteins

(Nieuw Amerongan et al, 1995).

Two types of mucin were present in human saliva viz. oligomeric

mucin glycoproteins (MG 1) and monomeric mucin glycoproteins (MG

2). The submandibular glands exhibit both types of cells i.e. mucous cells

and serous cells and they secrete 30% of the saliva mucin. The

sublingual, labial and palatal glands mainly contain mucous cells secrete

70% saliva mucin. The concentration of mucin secreted by sublingual

gland is higher than secreted by submandibular gland, while parotid gland

is devoid of mucin.

Glycoproteins are the carbohydrate–protein complexes, with

relatively short branched chain with no indication of a specific repeating

unit. The salivary glycoprotein complexes mainly include sulfated

hexoses, fucose and sialic acids. Glycoproteins are classified into two

group viz. neutral and acidic glycoproteins, depending upon number of

positive or negative charges on the molecular entity. Acidity results due

to presence of sulfate, sialic acid or uronic acid moieties in it (Spicer et

al, 1965). Accordingly the acid glycoproteins further classified into

sulphomucins and sialomucins. Odebald and Bostram, (1952) by radio –

autographic technique and Pillai et al, (1998) with histochemical

technique reported that mucus acini of sublingual gland rich in sulfated

glycoproteins. Sillanqukee et al, (1999) obtained histochemical evidence

for the presence of acid mucopolysaccharides in mucous acini with

radiography. Numerous histochemical studies have been made on

glycoproteins elaborated by the secretory cells of salivary glands

94

(Shackleford and Wilborn, 1968; Goldstein and Hayes, 1978; Rothl,

1978; Schalte et al, 1984; Laden et al, 1984). Nisizawa and Pigman,

(1959) reported the presence of sialic acid, hexoses, hexosamine and

fucose in human salivary gland.

Several studies reported age related changes in glycoproteins.

Bruzynski, (1971) reported that, glycoprotein bound hexose protein

nitrogen ratio was significantly decreased in submandibular gland of

aging guinea pig. Glucksmann and Cherry, (1973) identify solid masses

of cells in the acinar components of aged glands, that stain weakly or not

at all for mucous substances in the neoplastic salivary glands. Reduction

in glycoprotein sialic acid in submandibular gland of old rats was

described by Mysliwaski and Zurawska Czupa, (1976); Kuyatt and

Baum, (1981). Rybakova, (1979) reported reduction in basal level of

proteins and mucopolysaccharides in salivary glands of albino rats during

aging. Reduction in N-linked glycosylation was also reported in aged rats

by Kousvelari et al, (1998). Baum, (1989) showed decreased

incorporation 3 H mannose into N-linked glycosylation by aged rats.

Denny et al, (1991 a, b) reported reduction in total mucin levels in mice

during aging but no change in sialic acid content. Vissink et al, (1996) in

human study observed that SIg-A and high and low molecular weight

mucins in mucous saliva were reduced with age. Accili et al, (1999) and

Przybylo, (2004) showed aging changes in glycoproteins with the

development, differentiation and maturation in salivary gland.

Fucose is deoxyhexose sugar. It is required for optimal functions of

the cell to cell communication. The chemical formula of fucose is

C6H12O5 and molecular weight is 164.16 g Mol-1

. Its structural formula is

as follows:

95

Its systemic name is 3S 4R 5R 6S-6 methyloxane 2, 3, 4, 5 tetrol.

Structurally, it is different from other six carbon sugars present in

mammals due to lack of hydroxyl group on the carbon at 6th

position (C –

6) and L-configuration. It is found in N-linked glycans on the

mammalian, insect and plant cell surface. α- 1→3 linked core fucose is a

suspected carbohydrate antigen for Ig-E mediated allergy (Becker and

Lowe, 2003). Fucose is metabolized by enzyme called alpha-fucosidase.

Fucosylated glycans are synthesized by fucosyltransferase.

Thirteen fucosyltransferase genes have been identified in human genome.

All fucosyltransferase utilize a neucleotide–activated form of fucose,

GDP–fucose as a fucose donor in construction of fucosylated

oligosaccharides. GDP–fucose is synthesized by two pathways (Tonetti et

al, 1998) viz denovo path way and salvage pathway. In denovo pathway

GDP–mannose transforms into GDP–fucose. The salvage pathway

synthesizes GDP–fucose from free fucose derived from extracellular or

lysosomal fucose, which is liberated from catabolism of fucosylated

glycanes in the lysosome and then transported in cytosol. GDP–fucose

synthesized by these pathways is then transported into the lumen of Golgi

apparatus, where it becomes available to the catalytic domains of

96

fucosyltransferases (Becker and Lowe, 2003). Free fucose that supplies

the salvage pathway may also derived from lysosomal catabolism of

glycoproteins and glycolipids by one or more fucosidase activities

(Johnson and Alhadeff, 1991; Michalski and Klein, 1999). Fucose

liberated in the lysosomal compartment can be transported, across the

lysosomal membrane into cytosol by a relatively uncharacterized

transport system, which appears to allow efflux of multiple natural sugars

by facilitated diffusion (Jonas et al, 1990). O-fucose reduces on EGF

domain play important role in fate determination during neurogenesis,

angiogenesis and lymphoid development (Artavanis et al, 1999). FUT 1,

FUT 2, are α (1, 2)-fucosyltransferases responsible for synthesis of ‘H’

blood group antigen and related structure (Larsen et al, 1990; Kelly et al,

1995).

Functions of fucose:

i. Fucose concentration is found in synaptic region, macrophases,

kidney, testis, and skin indicating vital need of the sugar in brain

function, kidney function, immunity and reproduction (Becker and

Lowe, 2003). It has been indicated that, it is powerful immune

modulator.

ii. It has significant role in many diseases including cancer and its

spread, showing a promise in the area of inhibiting and reverse

leukemia, breast cancer and suppression of tumor growth. Fucose

and mannose appeared to be most effective of the essential sugar,

involving in slowing the growth of cancer cells.

iii. Important roles for fucosylated glycans have been demonstrated in

a variety of biological settings (Listinsky et al, 1998; Staudacher et

al, 1999). Fucose containing glycanes have important roles in

blood transfusion reactions, selectin mediated leukocyte

97

endothelial adhesion, host–microbe interactions and numerous

ontogenic events.

iv. Fucose deficiency is accompanied by a complex set of phenotypes,

both in humans and mice. Fucosylated glycanes have been

implicated in the pathogenesis of several human diseases (Lee et

al, 1997; Miyake et al, 1992 and Kim and Varki, 1997). Increased

expression of fucosylated glycanes has also been reported on serum

immunoglobins in both juvenile and adult rheumatoid arthritis

patients (Flogel et al, 1998; Gornik et al, 1999). Fucosylation of

mucin also observed to be increased in cystic fibrosis with a

concomitant decrease in sialylation (Scanlan and Glick, 1999).

Fucosylated glycanes have role in leukocyte recruitment, selectin –

selectin ligand interactions contribute to the development of

arthrosclerosis reperfusion injury following ischemic events,

inflammatory skin diseases and asthma (Varki, 1999). Thus fucose

deficiency in animal causes a large number of phenotypic

consequences, underscores the crucial role of fucosylated glycanes

to many physiological and development processes.

Sialic acid is a terminal sugar component of oligosaccharide chains

of glycoproteins and glycolipids. The chemical formula of sialic acid is

C11H19NO9 and the molecular weight is 309.27. It is water soluble and

named as N-acetyleneuraminic acid. Its structural formula is as follows:

8 6 3 2

9 7

5 4 1

98

In the still growing family of sialic acids, there are more than 40

different derivatives of neuraminic acid, but the interest of the scientific

community is increasing directly towards the O-acetylated species. In the

past two decades, it is evolved that O-acetylated sialic acid plays

fundamental role in the development of organism, in the regulation of the

immunity system, in cancer processes and many other biological as well

as pathological events (Kelm et al, 1997).

Naturally occurring sialic acids, can be O-acetylates at all their

hydroxyl groups that is at position C4 C

7, C

8 and C

9 of these corboxylated

sugars as shown in figure (Schaucer, 1982). In human beings they are

present in body fluids like blood plasma, breast milk, gall bladder

excretions, synovial fluid, sweat, gastric juice, urine and tissue like

salivary glands, stomach, throat, cervix, colon, cartilage, erythrocytes,

leucocytes and platelets (Sillanqukee et al, 1999). High content of sialic

acid is one of the characteristic features of the salivary gland

glycoproteins (Blix et al, 1952; Spicer and Warren, 1960; De Salegui and

Plonska, 1969). Kamerling et al, (1982) reported that, side chain O-

acetylated sialic acid are also expressed on human leukocytes in a range

of 1 to 5% of the total sialic acid content, where they can be used as

marker for specific subsets of T-helper cells known as DW 60. Th-9-O-

acetylated sialic acid is the terminal sugar of the di-sialoganglioside GD3

(Kniep et al, 1995) which is also expressed on activated β-cells (Vater, et

al, 1997). Cytometric flow analysis showed that each major class of

human leukocytes contains a significant fraction of cell expressed 7–O–

acetylate GD3 (Kniep et al, 1995). Large amount of various O-acetylated

sialic acid were also found in bovine submandibular gland (Schauer et al,

1991; Kelm et al, 1997), rat liver (Butor et al, 1993) and healthy human

colon (Corfield et al, 1999).

99

The structure, occurrence and general functions of sialic acids have

been extensively reviewed (Schauer, 1982; Kelm and Schauer, 1997).

Sialic acid is an important structural component of carbohydrate and

glycoconjugates where, it is found to be linked glycosidically to C–3 or

C– 6 of galactose, C– 6 of acetylgalctosamine and to C - 8 of another

sialic acid residue (Tuppy and Gottschalk 1972; Slomiany and Slomiany

1977).

Functions of sialic acid:

i. Sialic acid contribute significantly to the over all negative charge

of cell surface and glycoproteins. The negative charge contributes

to cell to cell repulsion, functioning stability and survival of

glycoproteins in blood circulation and cell to matrix interactions.

Due to the shielding effect sialylated glycanes protect parts of a

glycoprotein from proteolytic attacks.

ii. Membrane sialic acid assists in cell to cell recognition and

interaction. It serves as a component of cell surface receptors for

influenza virus.

iii. Sialic acid plays an essential role of the lubrication and protection

in the digestive tract (Warner et al, 1982)

2. MATERIAL AND METHODS:

A. MATERIAL

a. ANIMALS:

Male mice (Mus musculus) were used for the study. The

breeding pairs were obtained from Hindustan Antibiotics Pune, and were

reared in air-conditioned departmental animal house. They were received

Amrut mice feed (Pranav Agro Industries, Pvt. Ltd, Sangli) and water ad

libitum. The record of their age and body weight was maintained.

Adult male mice of 5 to 6 month age, weighing 50 to 55 ± 2 gm

body weight and old male mice of 16 to 18 month age, weighing 40 to 45

100

± 2 gm body weight are used. Both adult and old male mice were divided

into two main group viz. protective and curative group. Each group

further divided into 4 batches viz control batch, D-galactose stressed

batch, centrophenoxine treated batch and glycowithanolides (WSG)

treated batch (details were described in Chapter II).

B. METHODS:

a. Estimation of fucose: (Dische and Shettles, 1948)

For estimation of fucose the submandibular glands and sublingual

glands were pulled, weighed and homogenized in distilled water,

centrifuged at 5000 rpm for 10 minutes at 10oC. Supernatants were used

for estimation of proteins (Lowry et al, 1951) and fucose. Additions were

made as below.

Sr.No. Standard Blank Sample Control

1. Sample ---- ---- 1.0 ml 1.0 ml

2. Standard

α-D (+) fucose (0.02%)

1.0 ml ---- ---- ----

3. Distilled water ---- 1.0ml ---- ----

4. Cold H2SO4 4.5 ml 4.5ml 4.5 ml 4.5 ml

The tubs were shaken vigorously and place at 20oC for

10 minutes. Then tubes were capped with glass bulbs and

placed in vigorously boiling water bath exactly for 3 minutes.

Then added

5. 3%

cystein hydrochloride

0.1 ml 0.1ml 0.1 ml ----

Mixed well and kept them at room temperature for two hours. The

absorbance was determined at 400 nm and 430 nm adjusting calorimeter

to zero with blank. Calculations were done as described in chapter II.

101

b. Estimation of sialic acid by thiobarbituric acid: (Warren, 1959)

The submandibular and sublingual glands were homogenized in

distilled water, centrifuged at 5000 rpm for 10 minutes at 10oC. Sulphuric

acid was added to the homogenates to make the final concentration of

homogenates 0.1 M. Then homogenates were heated at 80oC for one

hour. Additions were made as below.

Sr.No Standard Blank Sample

1. Sample ---- ---- 0.2 ml

2. N-acetyleneuraminic acid (0.001%) 0.2 ml ---- ----

3. 0.1 N H2 SO4 ---- 0.2 ml ----

4. Periodate solution 0.1 ml 0.1 ml 0.1 ml

Mixed well and allowed to stand at room temperature for 20

minutes and then added.

5. 10% sodium arsenite 1.0 ml 1.0 ml 1.0 ml

Shaken till yellow brown color disappear and then added

6. 0.6% thiobarblturic acid 3.0 ml 3.0 ml 3.0 ml

Mixed the content vigorously by capping and shaking.

It was heated in boiling water for 5 minutes and then added

7. cyclohexanone 4.3 ml 4.3 ml 4.3 ml

Centrifuged for 3 minutes at 1000 rpm and clear top of

cyclohexanone phase was read at 549 nm against blank and µg sialic acid

per mg protein was calculated.

c. Histochemical distribution of glycoproteins:

The glandular tissues were fixed in 2% calcium acetate

formaldehyde (CAF) and 7 µ sections of submandibular and sublingual

glands were subjected to PAS, AB pH 1.0 and AB pH 2.5 staining

technique (detail of procedures was described in Chapter II). These

techniques were used to show the presence of neutral glycoproteins, acid

102

mucosubstances and sulfated mucosubstances. O-acetylated sialic acid

was determined by PAS sodium borohydrate technique.

d. Electrophoretic separation of total proteins and glycoproteins:

Slab gel electrophoresis was done using various samples of

submandibular and sublingual glands as explained in Chapter II and gels

were treated with Coomassie Brilliant Blue for total proteins, and Alcian

blue pH 1.0 for sulfated mucosubstances. (Detailed procedure was

described in Chapter II).

3. RESULTS:

a. Effects of glycowithanolides (WSG) on fucose content (in

µg/mg proteins) in salivary glands of D-galactose stressed mice.

These changes were depicted in Table No. 11 & 12 and graphs No.

45 to 52.

i. Submandibular gland of adult protective group:

Fucose content in submandibular gland of control mice was 1.32 ±

0.1303 µg. In D-galactose treated it was reduced to 0.458 ± 0.0192 µg

and significantly increased in Dg + cent and Dg + WSG batches to 1.27 ±

0.01581 and 1.29 ± 0.01581 µg respectively. (Table 11, graph 45)

ii. Sublingual gland of adult protective group:

In sublingual gland of control the fucose content was 1.62 ± 0.1303

µg while in D-galactose treatment it was reduced to 0.5 ± 0.01581 µg but

when mice were treated with centrophenoxine and WSG along with D-

galactose, the sublingual gland showed increased amount of fucose to

1.51 ± 0.0380 and 1.558 ± 0.0319 µg respectively. (Table 11, graph 46)

iii. Submandibular gland of adult curative group:

In curative group the fucose content in submandibular gland of

control batch was 1.3 ± 0.0790 µg. In Dg → saline batch fucose content

was reduced to 0.65 ± 0.0790 µg. After 20 days treatment of D-galactose,

when mice received centrophenoxine for further 20 days, the fucose

103

content was increased significantly to 1.158 ± 0.0228 and batch received

glycowithanolides for 20 days after 20 days D-galactose treatment, the

fucose content was increased to 1.25 ± 0.0790 µg. (Table 11, graph 47)

iv. Sublingual gland of adult curative group:

Control batch had 1.6 ± 0.1 µg fucose contents and it was reduced

significantly to 0.5 ± 0.01 µg in Dg → saline batch. After receiving

centrophenoxine and glycowithanolides the fucose content were

increased to 1.12 ± 0.0474 and 1.26 ± 0.01581 µg respectively. The

difference in fucose content of Dg → cent and Dg → WSG batches was

significant. (Table 11, graph 48)

v. Submandibular gland of old protective group:

As compared to adult control, there was significant decrease in

fucose content in submandibular gland of old control and it was 0.39 ±

0.0169 µg and further reduced to 0.216 ± 0.002 µg when old mice

received D-galactose for 20 days. But after receiving centrophenoxine or

glycowithanolides along with D-galactose, significant increase in fucose

content was observed and it was 0.4 ± 0.0689 and 0.42 ± 0.01 µg

respectively. There was no significant difference between fucose content

of Dg + cent and Dg + WSG batches. (Table 12, graph 49)

vi. Sublingual gland of old protective group:

In sublingual gland of control, the fucose content was 0.47 ±

0.0187 µg and it was reduced to 0.27 ± 0.02 µg in Dg-treated batch. In Dg

+ cent and Dg + WSG batches fucose content was significantly increased

to 0.452 ± 0.0130 and 0.47 ± 0.0122 µg respectively. (Table 12, graph 50)

vii. Submandibular gland of old curative group:

In submandibular gland of old control mice, the fucose content was

0.394 ± 0.0270 µg. It was reduced significantly to 0.218 ± 0.0083 µg in

Dg → saline batch. The significant increase in fucose content was

observed in Dg → cent and Dg → WSG batches and it was 0.35 ± 0.0158

104

in and 0.37 ± 0.0158 µg respectively. There was non significant

difference fucose content of Dg → cent and Dg → WSG batches. (Table

12, graph 51)

viii. Sublingual gland of old curative group:

In control batch of curative group, sublingual gland contains 0.464

± 0.0207 µg fucoses. In Dg → saline batch it was reduced to 0.32 ±

0.0158 µg. After receiving centrophenoxine or WSG followed by 20 days

D-galacotse injection, significant increased in fucose content was

observed in both Dg → cent and Dg → WSG batches and it was 0.444 ±

0.0207 and 0.462 ± 0.0130 µg respectively. (Table 12, graph 52)

b. Effects of glycowithanolides (WSG) on sialic acid content (in

µg/mg proteins) in salivary glands of D-galactose stressed mice.

These changes were depicted in Table No. 13 & 14 and graphs No.

53 to 60.

i. Submandibular gland of adult protective group:

In submandibular gland of control batch the sialic acid content was

0.00015 ± 5.4772 µg and it was increased to 0.00024 ± 1.9493 µg in Dg-

treated batch. It was significantly reduced to 0.00011 ± 8.9442 and

0.00011 ± 5.4772 µg in Dg + cent and Dg + WSG batches respectively.

Thus in between Dg + cent and Dg + WSG batches no difference was

observed in sialic content. (Table 13, graph 53)

ii. Sublingual gland of adult protective group:

Sialic acid content in sublingual gland of control batch was

0.00037 ± 8.3666 µg. In Dg-treated batch it was significantly increased to

0.00046 ± 0.00001 µg. There was significant reduction in sialic acid

content in Dg + cent batch to 0.00032 ± 1.6431 µg and in Dg + WSG it

was 0.00022 ± 1.6431 µg. There was significant decrease in sialic acid

content of Dg + WSG batch. (Table 13, graph 54)

105

iii. Submandibular gland of adult curative group:

The control batch showed 0.00018 ± 0.00001 µg sialic acids in its

submandibular gland. After D-galactose treatment, it was increased to

0.000296 ± 5.4772 µg. Significant decrease in sialic acid was observed in

Dg → cent and Dg → WSG batches to 0.000158 ± 8.3666 and 0.000118

± 1.7889 µg respectively. There was significant difference in sialic acid

content in Dg → cent and Dg → WSG batches. (Table 13, graph 55)

iv. Sublingual gland adult curative group:

Sialic acid content in sublingual gland of control batch was

0.000338 ± 4.4721 µg. There was increase in sialic acid content in Dg →

saline batch to 0.000504 ± 5.4772 µg. Decrease in sialic acid was

observed in centrophenoxine and WSG receiving mice to 0.00033 ±

1.5811 and 0.000226 ± 8.9443 µg respectively. In WSG batch significant

decrease in sialic acid content as compared to centrophenoxine receiving

batch. (Table 13, graph 56)

v. Submandibular gland of old protective group:

In control batch the sialic acid content was 0.00026 ± 8.9442 µg

and it was significantly increased to 0.00034 ± 1.6431 µg in D-galactose

treated batch. Reduction in sialic acid content was observed in Dg + cent

and Dg + WSG batches to 0.00021 ± 1.0954 and 0.00019 ± 8.3666 µg

respectively. (Table 14, graph 57)

vi. Sublingual gland of old protective group:

Sialic acid content in sublingual gland of control batch was

0.00049 ± 5.4772 µg. In D-galactose received mice, it was significantly

increased to 0.00054 ± 5.4772 µg. It was decreased to 0.00045 and

0.00042 ± 5.4772 µg in Dg + cent and Dg + WSG batches respectively.

Decrease in WSG batch was significant as compared to centrophenoxine

batch. (Table 14, graph 58)

vii. Submandibular gland old curative group:

106

Submandibular gland of the control batch had 0.000276 ± 5.4772

µg sialic acids. It was increased to 0.000364 ± 1.3416 µg in Dg → saline

batch but significantly decrease to 0.00026 ± 1.5811 and 0.000226 ±

5.4772 µg in Dg → cent and Dg → WSG batches respectively. No

significant difference was observed in sialic acid content between these

two batches. (Table 14, graph 59)

viii. Sublingual gland of old curative group:

Control batch contains 0.000518 ± 8.3666 µg sialic acids in

sublingual glands. There was significant increase in sialic acid content of

Dg → saline batch but decreased significantly to 0.000488 ± 8.3666 and

0.000454 ± 1.1402 µg in Dg → cent and Dg → WSG batches

respectively. There was significant difference in sialic acid content

between these two batches. (Table 14, graph 60)

c. Histochemical changes in glycoproteins.

i. Periodic Acid Schiff reaction (PAS).

PAS stains neutral glycoprotein selectively. It was revealed in

plates 13, 14, 15 and 16.

• Submandibular gland of adult male mice. (Plate 13 Figs. 1 to 8)

In control batch of protective group the secretory acini of

submandibular gland, stained dark magenta in color showing strong PAS

positivity. GCT cells (GC) showed PAS activity more towards the lumen

(Fig. 1).

In submandibular gland of D-galactose stress induced mice the

PAS staining of the acini (AC) was considerably decreased showing a

sporadic number of acini stained only towards their lumen (↑). The GCT

cells (GC) lost their PAS reactivity (Fig. 2).

The submandibular gland of Dg + cent batch showed significant

increase in PAS activity in acinar cells (AC). PAS activity was

107

throughout the cells and GCT (GC) cells showed PAS staining similar to

the control (Fig. 3).

The secretory acini (AC) in submandibular gland of mice receiving

glycowithanolides along with D-galactose showed increase in PAS

positive material in acini (AC) and GCT (GC). PAS reaction was more

towards lumen of GCT (Fig. 4).

Control of curative group showed PAS reaction throughout acini

(AC) as well as in GCT (GC) cells (Fig. 5).

But in Dg → saline treated salivary gland, the PAS reaction in

acinar cells (AC) was almost lost, few acini showed PAS reactivity

towards their lumen. GCT (GC) cells lost all its PAS positive material

(Fig. 6).

Except few acini, no recovery in PAS positive material was

observed in mice, received centrophenoxine after D-galactose treatment

(Fig. 7).

But all acinar cells of Dg → WSG batch showed PAS positive

material nearly equal to that of control, similar PAS reactivity is in GCT

(GC) cells (Fig. 8).

• Sublingual gland of adult male mice. (Plate 14 Figs. 1to8)

Sublingual gland is formed of mucous acini and serous demilune.

Mucous acini (MA) were intensely stained in PAS technique, staining

reaction was dark magenta in color. Demilunar cells (DM) showed weak

PAS activity. Mucous acini (MA) are very rich in PAS positive material

(Fig. 1).

No change was noticed in PAS reaction in D-galactose stressed

mice (Fig. 2). Similarly no increase in PAS positive material was

observed in mucous acini (MA) of centrophenoxine treated batch (Fig. 3).

108

Glycowithanolides treated sublingual gland, mucous acini (MA)

showed PAS positive material, reactions were similar to that control

batch (Fig. 4).

In curative group receiving saline for 40 days, showed PAS

reaction in mucous acini but not in demilune (Fig. 5).

Sublingual glands received saline for 20 days after 20 days

treatment of D-galactose, showed acini stained dark with PAS staining

(Fig. 6).

Demilunar cells showed PAS reaction similar to control in Dg →

cent (Fig. 7) and Dg → WSG (Fig. 8), there was increase in PAS positive

material in mucous acini (MA).

• Submandibular gland of old male mice. (Plate 15 Figs. 1to8)

In control batch acini showed positive PAS material, PAS

positivity was lost in GCT (Fig. 1).

In D-galactose stressed batch PAS positivity in GCT was

completely abolished (Fig. 2) and only few acini showed unlocalised PAS

reaction.

Batch received centrophenoxine along with D-galactose, showed

recovery in PAS positive material in acini (AC) and GCT showed

increased PAS positive material in GC (↑) than control batch (Fig. 3).

Fig. 4 of batch received glycowithanolides along with D-galacotse

showed increased PAS reaction. Reaction was localised in acinar cells

(AC) and it was not localised in GCT cells.

PAS material was localised in acinar cells (AC) of control batch of

curative group (Fig. 5). GCT cells were without PAS reactivity. A batch

received saline after D-galactose treatment showed only few acini

irregularly stained positively with PAS (Fig. 6).

109

Slight recovery in PAS positive material was observed in

centrophenoxine (Fig. 7) and WSG (Fig. 8) treatment after stress. But the

structure of the gland was damaged, some ducts showed PAS reaction

(Fig. 8).

• Sublingual gland of old male mice. (Plate 16 Figs. 1to8)

Some of the cells from mucous acini of both protective and

curative group stained for PAS. Demilunar cells (DM) as well as duct

cells were darkly stained for PAS (Figs. 1&5).

PAS reactivity was increased in some cells of mucous acini (MA)

and demilunar cells in D-galactose treated sublingual glands from both

the groups. Ducts were also intensely stained for PAS (Figs. 2&6).

In centrophenoxine and WSG treated sublingual gland, PAS

reaction was like control in mucus acini (MA) as well as in demilunar

cells (Figs. 3 & 4).

Mucous acini of all batches of curative group treated for

centrophenoxine and ashwagandha, showed PAS reaction in demilunar

cells (DM) and in some of the cells of mucous acini (Figs. 7&8). In duct

cells (↑) PAS reaction was observed in both the cases.

ii. Alcian blue at pH 1.0

Alcian blue at pH 1.0 selectively stains sulfated mucin. These

results were depicted in plates 17, 18, 19, 20.

• Submandibular gland of adult male mice. (Plate 17 Figs. 1 to 8)

The acinar cells of control batch stained blue with Alcian blue,

staining intensity was poor (Fig. 1). Staining reaction was considerably

reduced in some acinar cells of D-galactose stressed batch (Fig. 2). In Dg

+ cent and Dg + WSG batches staining with AB pH 1.0 was increased in

acinar cells (Figs. 3&4). In curative group staining with AB pH 1.0 was

similar to control batch of protective group (Fig. 5). Significant reduction

in staining reactivity with AB pH 1.0 in acinar cells of Dg → saline (Fig.

110

6) and no increase in the AB pH 1.0 reactivity either in centrophenoxine

treated or WSG treated batch (Figs. 7&8).

• Sublingual gland of adult male mice. (Plate 18 Figs. 1 to 8)

Mucous acini (MA) from the control mice, showed strong positive

reaction with AB pH 1.0. Demilunar cells showed negative reaction in

control batch of protective as well as curative group (Figs. 1&5).

Demilunar cells and duct cells of sublingual glands totally negative for

AB pH 1.0 reactivity. Size of D-galactose treated acini was reduced and

some of the cells of mucous acini lost their AB pH 1.0 positivity (Figs.

2&6). Duct and demilunar cells showed no reaction in both groups in

(Figs. 3&4) and (Figs. 7&8).

• Submandibular gland of old male mice. (Plate 19 Figs. 1 to 8)

Acinar cells of submandibular gland of old mice, show AB pH 1.0

reactivity. GCT cells (GC) did not show staining reaction both in

protective and curative group (Figs. 1&5).

In D-galactose treated mice, staining for AB pH 1.0 in acini was

lost (Figs. 2&6) and there was no gain due to centrophenoxine and WSG

(Figs. 3, 7, 4, & 8).

• Sublingual gland of old male mice. (Plate 20 Figs. 1 to 8)

When sublingual gland sections of old mice were studied, in both

the controls acinar cells of mucous acini lost AB pH 1.0 staining reaction,

slight reaction was observed in demilunar cells and duct cells (Figs.

1&5).

In D-galactose treated sublingual glands AB pH 1.0 staining

intensity was decreased in mucous cells, seromucous cells and duct cells

(Figs. 2&6).

Centrophenoxine treated protective group showed AB pH 1.0

reaction in some of the mucous acini and in duct cells (Fig. 3), but in

curative group there was no reaction in ducts (Fig. 7).

111

Intense reaction was observed in mucous acini in WSG treated

protective group (Fig. 4), as well as in curative group (Fig. 8). Demilunar

cells and duct cells lost AB pH 1.0 reaction.

iii. Alcian blue at pH 2.5.

The results were depicted in plates 21, 22, 23, and 24.

Alcian blue at pH 2.5 stains acidic glycoproteins, staining intensity

is always low.

• Submandibular gland of adult male mice. (Plate 21 Figs. 1 to 8)

Acinar cells of submandibular gland of both the controls i.e.

protective and curative group were stained moderately with AB pH 2.5

(Figs. 1&5).

In D-galactose treated submandibular gland lost this reaction (Figs.

2&6) and regained in protective group of both treatments i.e.

centrophenoxine (Fig. 3) and WSG (Fig. 4) but not up to that extent in

curative group (Figs. 7&8)

• Sublingual gland of adult male mice. (Plate 22 Figs. 1 to 8)

In the control of both protective and curative groups the mucous

acini were selectively AB pH 2.5 positive, showed dark blue colored

staining. Few demilunar cells also showed staining reaction (Figs. 1&5).

In D-galactose treatment protective and curative groups, showed

reduction in staining intensity in mucous acini but demilunar cells did not

show staining reaction with AB pH 2.5. (Figs. 2&6).

Slight recovery was observed in staining with AB pH 2.5 in

sublingual gland of both antioxidant treatments. (Figs. 3, 4, 7, & 8)

• Submandibular gland of old male mice. (Plate 23 Figs. 1 to 8)

Submandibular gland of mice is formed of main three secretory

components viz. acini, granular convoluted tubules and ducts. Acini are

PAS as well as AB pH 2.5 positive. Acini are moderately stained with

AB pH 2.5.

112

In old this AB pH 2.5 positivity was lost, some acini stained blue

in control (Figs. 1&5). Few acini showed poor staining for AB pH 2.5 in

D-galactose treated batches (Figs. 2&6). Same intensity was observed in

protective group (Fig. 2) and almost lost in curative group (Fig. 6).

In antioxidant treatment number and size of acini was increased,

slight recovery was observed in AB pH 2.5 positive material in both

protective and curative groups (Figs. 3, 4, 7, & 8)

• Sublingual gland of old male mice. (Plate 24 Figs. 1 to 8)

Sublingual gland of mice is mainly formed of mucous acini, serous

demilune and ductal elements. In old large area of the gland is covered by

ducts. Mucous acini were darkly stained with AB pH 2.5, demilunes and

ducts were AB pH 2.5 negative. (Figs. 1&5)

In D-galactose treated mice there was loss of AB pH 2.5 staining

intensity in mucous acini of sublingual gland. (Figs. 2&6)

In centrophenoxine and WSG treatment considerable recovery was

observed and attained dark blue staining in mucous acini of both

protective and curative groups. (Figs. 3, 4, 7 & 8)

iv. AB pH 1.0 + PAS double staining.

This staining is used to detect presence of both sulfated and neutral

glycoproteins. These results were shown in plates 25, 26, 27 and 28.

• Submandibular gland of adult male mice. (Plate 25 Figs. 1 to 8)

In combined staining technique (AB pH 1.0 + PAS) acinar cells

were stained with AB pH 1.0 and GCT cells were PAS positive in both

control groups. It was purple in acinar and GCT cells of submandibular

glands of protective and curative group (Figs. 1&5).

In D-galactose treated mice the acinar cells of submandibular gland

showed no reaction with AB pH 1.0 but few acini were PAS positive.

GCT cells also did not showed presence of AB pH 1.0 positivity but

showed PAS positive material with poor staining (Figs. 2&6).

113

In centrophenoxine treated submandibular gland acinar cells were

AB pH 1.0 and PAS positive. GCT did not showed AB pH 1.0 staining

but stained faint with PAS (Figs. 3&7)

Mice received glycowithanolides along with D-galactose showed

AB pH 1.0 and PAS positive material and GCT cells showed poor PAS

reactivity in protective group (Fig. 4), while in curative group acinar cells

showed less AB pH 1.0 but more PAS staining. GCT cells showed

positive reaction with PAS only (Fig. 8).

• Sublingual gland of adult male mice. (Plate 26 Fig. 1 to 8)

In case of sublingual gland all mucous cells were intensely purple

but demilunar cells and duct cells were less purple or most of them were

pink in color (Figs. 1&5), staining intensity was very high so no

alterations could be noticed in all the batches of protective and curative

groups (Figs. 2, 3, 4, 6, 7 & 8)

• Submandibular gland of old male mice. (Plate 27 Figs. 1 to 8)

In combined AB pH 1.0 + PAS technique few acini were AB pH

1.0 positive and few showed very poor staining with PAS (Figs. 1&5).

Both staining intensities were reduced in D-galactose treatment (Figs.

2&6). In centrophenoxine treated submandibular gland PAS positive

reaction and AB pH 1.0 reactions were observed in GCT and acinar cells

respectively (Figs. 3&7). Reactions were not localized in (Figs. 4&8).

• Sublingual gland of old male mice. (Plate 28 Figs. 1 to 8)

In double staining with AB pH 1.0 + PAS technique sublingual

gland of both controls showed AB pH 1.0 staining and absence of PAS

reaction in mucous acini. Demilunes and duct were AB pH 1.0 negative

but PAS positive (Figs. 1&5).

In D-galactose stressed batch both AB pH 1.0 and PAS staining

was reduced in all components of sublingual gland (Figs. 2&6).

114

In centrophenoxine treatment mucous acini were strongly AB pH

1.0 positive and demilunes were negative (Figs. 3&7)

In WSG treatment mucous acini were strongly PAS positive and

poorly AB pH 1.0 in both groups (Figs. 4&8).

v. AB pH 2.5 + PAS double staining.

This staining is used to detect presence of acidic as well as neutral

glycoproteins. These observations were depicted in plates 29, 30, 31 and

32.

• Submandibular gland of adult male mice. (Plate 29 Figs. 1 to 8)

The control of both protective and curative groups submandibular

gland showed presence of both acidic and neutral glycoproteins indicated

by purple staining in acinar cells as well as in GCT (Figs. 1&5).

D-galactose stressed batch showed, decreased staining intensity

with AB pH 2.5 and PAS in acinar cells as well as in GCT cells (Figs.

2&6).

There was recovery in acidic glycoprotein and neutral

glycoproteins content in acinar cells as well as in GCT of

centrophenoxine (Figs. 3&7) and WSG (Figs. 4&8) treatments. The

recovery was observed up to level of control batch.

• Sublingual gland of adult male mice. (Plate 30 Figs. 1 to 8)

The mucous acini of sublingual glands were intensely purple but

demilunar and duct cells were poorly stained or more of them were pink

in color. Staining intensity was very high so no alterations could be

noticed in (Figs. 1 to 8).

• Submandibular gland of old male mice. (Plate 31 Figs. 1 to 8)

In combined (AB pH 2.5 + PAS) technique the staining reaction

was very poor, only few acini showed AB pH 2.5 and PAS positive

materials (Figs. 1&5). Staining intensities were reduced in D-galactose

treatment (Figs. 2&6).

115

In both protective and curative groups antioxidant treatments

showed increased staining activity with (AB pH 2.5 + PAS) in acinar

cells and PAS positivity in GCT cells (Figs. 3&7, 4&8)

• Sublingual gland of old male mice. (Plate 32 Figs. 1 to 8)

Mucous acini were AB pH 2.5 positive whereas, demilunes and

duct cells were PAS positive in all the batches. Staining intensity was

high, so no alterations could be noticed in (Figs. 1 to 8).

vi. PAS – Sodium borohydride technique.

This staining technique is used to demonstrate O-acetylated sialic

acid. Changes in staining intensity are depicted in the plates 33, 34, 35,

and 36.

• Submandibular gland of adult male mice. (Plate 33 Figs. 1 to 8)

Control of protective and curative groups showed light staining

reaction in acinar cells and GCT cells of submandibular gland with PAS –

borohydride staining (Figs. 1&5).

In D-galactose stressed submandibular gland of protective and

curative groups, there was destruction of glandular architecture but

remaining acinar cells and GCT cells showed dark staining reaction with

PAS – Sodium borohydride, indicating increased sialomucins (Figs.

2&6).

Centrophenoxine and WSG treated submandibular gland showed,

remarkable decrease in staining intensity for O-acetylated sialic acid.

Staining reaction was completely abolished in GCT during stress and

after stress (Figs. 3&7, 4&8.).

• Sublingual gland of adult male mice. (Plate 34 Figs. 1 to 8)

The control batch showed weak staining reaction with PAS-sodium

borohydride in membrane of mucous acini and in demilune (Fig. 1). The

control of curative group showed slight increase in staining reaction (Fig.

5)

116

The remarkable increase in staining intensity was observed in

membrane of mucous acini, represented by dark magenta pink color,

demilune also stained dark (Fig. 2&6).

In protective and curative groups received centrophenoxine showed

decreased staining intensity in mucous acini but demilune did not shown

any staining reaction (Figs. 3&7)

There was remarkable decrease in sialomucins in protective and

curative groups received glycowithanolides. The intensity of staining in

acini was lighter than control batch (Figs. 4&8).

• Submandibular gland of old male mice. (Plate 35 Figs. 1 to 8)

The both controls showed remarkable increase in O-acetylated

sialic acid content in acinar cells of submandibular gland, indicated by

dark magenta pink color. GCT cells also showed slight staining (Figs.

1&5).

Old male mice received D-galactose, showed still pronounced

increase in staining intensity with PAS–Sodium borohydride. Intense

staining was observed in acinar cells as well as in GCT cells (Figs. 2&6).

Submandibular gland of old mice received centrophenoxine during

and after D-galactose stress showed considerable decrease in O-

acetylated sialic acid in acinar cells (Figs. 3&7).

In WSG received mice submandibular gland, the staining intensity

in acinar cells as well as in GCT cells was significantly reduced than

naturally old and D-galactose stressed old male mice (Figs. 4&8). Even

the intensity of staining was weak than centrophenoxine treatment

indicating significant reduction in O-acetylated sialic acid.

• Sublingual gland of old male mice. (Plate 36 Figs. 1 to 8)

In protective as well as in curative groups sublingual gland of old

mice showed intense staining, with PAS-sodium borohydride in

demilunar cells and in membrane of mucous acini (Figs. 1&5).

117

In D-galactose treated old male mice the numbers of secretory

units were reduced and remaining secretory units showed intense dark

staining in demilunar cells and mucous acini membrane (Figs. 2&6). The

intensity of stain was more in excretory duct.

Mice treated with centrophenoxine and WSG showed significant

decrease in staining intensity (Figs. 3&7, 4&8).

d. Electrophoretic separation of total proteins and glycoproteins.

These were revealed in electrophoresis plate 1 to 8.

i. Total proteins:

• Submandibular gland of adult male mice: (Electrophoresis Plate-1)

Proteins from submandibular gland samples (20 µl) were separated

using polyacrylamide gel, stained with Coomassie Brilliant Blue. From

both the control of protective and curative groups, proteins were

separated in to 9 bands viz. I to IX (Fig. 1C & Fig. 2C)

From D-galactose treated gland samples (20 µl) proteins were

separated in to 7 bands viz. I to VII in protective group and 6 bands viz. I,

II, IV, V, VII and VIII in curative group. Proteins from band number VIII

and IX and band number III, VI and IX were lost from protective and

curative groups respectively.

Protective group treated with centrophenoxine (Fig.1 Dg +cent)

and WSG (Fig.1 Dg +WSG) all the bands were separated. There was no

loss of proteins. Curative group treated with centrophenoxine (Fig.2

Dg→cent) and WSG (Fig..2 Dg→WSG) proteins were separated in to 7

bands. Band number VI and IX were lost.

• Sublingual gland of adult male mice: (Electrophoresis Plate - 2)

Proteins from sublingual gland of both protective and curative

controls were separated in to 8 (Fig.1C) and 7 bands (Fig.2C)

respectively. Proteins from band number II were not separated in curative

group (Fig.2C).

118

Proteins from D-galactose treated sublingual gland of both

protective and curative groups were separated in to 3 bands (Fig.1 Dg,

Fig.2 Dg → saline). Proteins from band number I, II, IV and VI were lost

from protective group and proteins from band number II, IV, V, VI &

VIII were lost from curative group.

Centrophenoxine protective group revealed 7 bands, proteins from

V band was lost (Fig.1 Dg + cent). Proteins from centrophenoxine

curative group separated in 6 bands. Proteins from II &IV bands were lost

(Fig.2 Dg → cent). Where as in case of WSG protective group revealed

all 8 bands (Fig. 1 Dg + WSG). But proteins from II band were lost in

curative group (Fig.2 Dg → WSG).

• Submandibular gland of old male mice: (Electrophoresis Plate–3)

In control, proteins were separated in 7 bands (Fig. 1C & Fig.2C).

Proteins were lost in D-galactose treated groups, in protective groups

there were only 3 bands viz. II, III, & VII (Fig. 1Dg) and from curative

group there were 4 bands viz. II III VI &VII (Fig.2Dg→saline).

Both the protective and curative groups treated with

centrophenoxine (Fig. 1 Dg + cent and Fig. 2 Dg → cent) and WSG (Fig.

1 Dg + WSG and Fig. 2 Dg → WSG) all proteins were separated in to 7

bands.

• Sublingual gland of old male mice: (Electrophoresis Plate – 4)

In old control proteins were separated in to 7 and 6 bands in

protective (Fig. 1 C) and curative (Fig. 2 C) groups respectively. In Dg-

treated proteins from both the groups protective (Fig. 1 Dg) and curative

(Fig. 2 Dg → saline) lost, only two bands (II and VII) were revealed in

the both the cases. Proteins separated in controls were protected in

centrophenoxine and WSG treatment. (Fig. 1 Dg + cent and Fig. 1 Dg +

WSG) but in curative group some proteins from Dg → cent and Dg →

WSG were lost from the band number III and VI

119

ii. Glycoproteins:

• Submandibular gland of adult male mice: (Electrophoresis Plate–5)

In control glycoproteins were separated into 6 and 5 bands in Fig.1

C and Fig.2 C respectively. In D-galactose treated batches only two bands

viz. II and III (Fig. 1 Dg) of protective group and a band viz. I (Fig. 2 Dg

→ saline) of curative group were revealed.

There was no loss of proteins either with the centrophenoxine (Fig.

1 Dg + cent) or with WSG (Fig. 1 Dg + WSG) in the protective group.

But in case of curative groups proteins from band number III and V were

lost (Fig. 2 Dg → cent and Fig. 2 Dg → WSG)

• Sublingual gland of adult male mice: (Electrophoresis Plate–6)

Glycoproteins were separated into 7 bands in control of protective

group (Fig. 1 C) and curative group (Fig. 2 C). In D-galactose stress

sublingual gland, all proteins were lost except VII band (Fig. 1 Dg and

Fig. 2 Dg → saline). But mice receiving centrophenoxine and WSG along

with D-galactose protein synthesis were protected and they were

separated in all bands from I to VII (Fig. 1 Dg + cent and Fig. 1 Dg +

WSG). Curative group received centrophenoxine or WSG after the

galactose treatment, also able to regain glycoprotein synthesis in both the

groups (Fig. 2 Dg → cent and Fig. 2 Dg → WSG).

• Submandibular gland of old male mice: (Electrophoresis Plate–7)

In old submandibular gland, little glycoproteins were synthesized,

electrophoretically in control only 3 bands viz. I, II and VII in protective

group (Fig.1 C) and 2 bands viz. I and VII in curative group (Fig.2 C).

They were lost in D-galactose treatment (Fig. 1 Dg and Fig. 2 Dg →

saline); only glycoproteins from band VII were remained. In D-galactose

stress with centrophenoxine or with WSG three glycoproteins were

separated (Fig. 1 Dg + cent and Fig. 1 Dg + WSG); intensity of the bands

was very less. Similar regain was also in curative group, where proteins

120

were separated into 2 bands viz. I and VII (Fig. 2 Dg → cent and Fig. 2

Dg → WSG)

• Sublingual gland of old male mice: (Electrophoresis Plate–8)

Like submandibular gland there was loss of glycoproteins in old

sublingual gland but exhibit 2 less intense bands viz. I and VII (Fig.1 C

and Fig.2 C). One of them is lost in protective and curative groups in D-

galactose treatment (Fig. 1 Dg and Fig. 2 Dg → saline) but both of them

recovered in protective (Fig. 1 Dg + cent and Fig. 1 Dg + WSG) and

curative (Fig. 2 Dg → cent and Fig. 2 Dg → WSG) groups treated with

antioxidants.

DISCUSSION:

Salivary glands secret saliva continuously in the mouth composed

of water, proteins and low molecular mass substances. About 26% of the

salivary proteins are glycoproteins (Nieuw Amerongen et al, 1995).

Among three major salivary glands submandibular and sublingual have

major contribution in the secretion of glycoproteins. Concentration of

mucin, secreted by sublingual gland is higher than that of submandibular

gland. Glycoproteins are believed to play essential role of lubricant as

well as protection of mouth cavity and digestive tract (Warner et al,

1982). If saliva is not secreted in the mouth, it becomes dry and faces

several problems like xerostomia (Yeh et al, 1998; Ship et al, 1990; Ship

et al, 2002). Salivary glands have been hypothesized to account for loss

of acinar cells in normal aging (Scott et al, 1986; Baum et al, 1992).

Physiologically, stressful conditions like diseases, surgery, radiotherapy,

and pharmacotherapy might be strain reserve capacity; hindering their

ability to compensate for increased metabolic demand and results

ultimately into functional compromise (Evers et al, 1994).

In present investigation, mice were stressed giving D-galactose.

Galactose stress brings about formation of advanced glycation end

121

products (AGEs), which gives rise to free radicals and free radical effects

(Song et al, 1999) which takes place in normal aging. During the stress of

D-galactose and aging, if mice were treated with centrophenoxine–a

synthetic antioxidant and glycowithanolides–a natural antioxidant

(glycowithanolides extracted from W. somnifera leaves powder with

chloroform, then spray dried. The spray dried residue contains

withanolides, flavonoids. Bhattacharya et al, 1997 tested the spray dried

residue by HPTLC and showed that the residue is rich in

glycowithanolides) showed recovery in fucose content, which was

reduced during aging and stress. Gain was highly significant in case of

glycowithanolides receiving mice than centrophenoxine.

Salivary gland glycoproteins contain hexoses like fucose and sialic

acid. D-galactose receiving stressed mice and naturally aged mice showed

reduction in fucose content per mg proteins.

Fucose is hexose sugar present in wide variety of organisms.

Fucose frequently exists as terminal modification of glycation structure. It

is common component of many N-O linked glycan and glycolipids

produced by mammalian cells. Important role of fucosylated glycan has

been demonstrated in a variety of biological studies (Listinsky et al,

1998; Staudacher et al, 1999). Fucosylated glycan synthesis is catalyzed

by fucosyl transferase. Thirteen fucosyl transferase genes have been

identified in the human genome (Kelly et al, 1995; Larsen et al, 1990). In

contest to its role as a terminal modification of oligosaccharides, fucose

may also have direct linkage to hydroxyl groups of serine and thionin

residues of polypeptide chain. This glycosylation event is known as an O-

fucosylation (Harris and Spellman, 1993). They act as specific sequences

in EGF like modules of cell surface and secretory proteins (Harris and

Spellman, 1993; Moloney and Haltiwanger, 1999), though O-fucose

residue does not appear to affect tertiary protein structure, de-fucosylation

122

of the EGF domain abolished its mitogenic activity (Rabhani et al, 1992).

GDP-fucose, is a fucose donor in construction of fucosylated

oligosaccharides. GDP-fucose is synthesized in de-novo (Tonetti et al,

1998) or by salvage pathway. Salivary glands undergo changes with age.

The gland parenchyma is atrophic in aged individuals (Scott, 1977a) and

shows morphological alterations (Gresik et al, 1980). Changes in acinar

cell organelles (Sashima, 1986) and reduction in proportion of volume of

acini (Scott et al, 1986) and decreased saliva production are common

(Seagrave et al, 1996). Free radical reactions are continuously going on

throughout the cells and tissues causing random damage to DNA, RNA,

protein and enzymes. There is a progressive decline in the rate of protein

synthesis with age in the salivary gland (Baum et al, 1983) and decreased

level of secretory granules. Reduction in fucose, glycoproteins in stressed

salivary glands may be due to reduction in protein synthesis and altered

lysosomes may unable to supply fucose for fucosylation (Becker et al,

2003).

There are alterations in sialic acid of salivary gland in old and

during stress in adult. Alterations are prevented by supplying antioxidants

like centrophenoxine and glycowithanolides. Sialic acid comprises N or

O acid derivative of carbon sugar neuraminic acid, sialic acids are

terminal sugar component of the oligosaccharide chain of the

glycoproteins and glycolipids. They are present in tissues like

erythrocyte, platelets, salivary gland, throat, stomach, colon, cartilage

(Sillanqukee et al, 1999). In tissues, major sialic acid is NeuSAc, the

structure occurrence and general functions of sialic acids have been

extensively reviewed by Schauer, (1982) and Kelm et al, (1997) sialic

acid contributes significantly to the overall negative charge on cell

surface. The negative charge contributes to cell to cell repulsion (anti-

adhesion effect), functioning stability and survival of glycoprotein in

123

blood circulation and cell to cell matrix interaction. Due to the shielding

effect sialylated glycan protect part of glycoprotein from proteolytic

attacks. Sialic acid content per mg salivary glands proteins of stressed

mice were increased remarkably from both submandibular and sublingual

gland.

Salivary glycoproteins were studied using PAS technique, AB pH

2.5, AB pH 1.0 and PAS-sodium borohydride for the presence of neutral

mucins, acidic mucins, sulfated and O-acetylated sialomucins

respectively. In both the glands, there was decrease in these glycoproteins

in D-galactose stressed mice and naturally aged mice. The alterations in

these glycoproteins can be reversed by giving antioxidant during the

stress or to some extent after the stress. Study indicates the presence of

neutral mucins, acidic mucins and acidic and sulfated mucins in the

submandibular and sublingual gland. At histochemical level

polysaccharides containing vicinal hydroxyl have been reported by Spicer

et al, (1965). Spicer and Warren, (1960) showed presence of acid

mucopolysuccharides which have been characterized as sialomucins and

sulphomucins in mucous acini. Sublingual gland is rich in sulfated

glycoproteins. In the submandibular gland acini exhibited, PAS and AB

pH 2.5 activity and less of AB pH 1.0, indicating less quantity of sulfated

mucins in the mucous acini of submandibular gland.

Histochemically, it has been observed that PAS positive

glycoprotein and AB pH 2.5 and AB pH 1.0 positive glycoproteins were

present in the granular convoluted tubules and acini of the submandibular

gland respectively. In D-galactose stress and aging salivary glands, these

glycoproteins were reduced.

In aging salivary glands, there is reduction in the volume of acini

with concomitant increase in the ductal volume (Kim and Allen, 1993).

Ducts of salivary glands do not stain or are stained poorly by any of these

124

techniques described above, shown absence of glycoproteins in various

ducts.

However, the salivary acinar cells of aged animals synthesize

secretary proteins at an elevated rate, just as effectively as those from

their younger counter parts in response to external stimuli (Kim and

Allen, 1993). In the present investigation, glycoproteins of stressed and

aged mice salivary glands, studied biochemically and histochemically

showed decrease in various glycoproteins secreted by various parts of

salivary glands. Their secretion when studied in the presence of

antioxidants like glycowithanolides and centrophenoxine, D-galactose

stressed and in old animals there was increase in salivary glycoproteins

but not efficiently as those from their unstressed condition and younger

counter parts. Even though there is decrease in glycoproteins in stressed

and old conditions on the other hand O-linked sialo-glycoproteins are

increased. Increase could be prevented in the presence of antioxidants or

giving treatment of antioxidants after stress to adult or in old mice. There

is close relationship between O-sialoglycoproteins and pathological

conditions (Marquina et al, 1996) and carcinoma (Mello, 1996)

The salivary gland contains mixture of mucous, serous and

seromucous cell types. One of the main functions of salivary gland is

secretion of glycoproteins. The mucins are high molecular weight

glycoproteins (Gindzienski et al, 1987; Paszkiewiez et al, 1995). Various

bands were decreased or lost during stress and in old mice. Proteins when

separated from these glands, they were separated in 9 and 8 bands

respectively in submandibular and sublingual glands. Numbers of bands

were reduced in stressed and old conditions.

When these animals were treated with centrophenoxine and

glycowithanolides, all bands were again revealed in protective group and

almost all in curative group. But in case of old, there was complete

125

recovery at least in submandibular gland. In sublingual glands there was

separation of proteins in equal number of bands as an adult but intensity

was very low. In old age D-galactose stressed mice there is increased free

radical formation beyond the cell’s natural free radical scavenging

capacity. This capacity is increased by supplying antioxidants.

Centrophenoxine is powerful chemical antioxidant (Zs-Nagy, 1980;

1989). Centrophenoxine possess OH¯ radical scavenging capacity (Zs-

Nagy, 1989) which can help to protect cellular damage. W. somnifera is a

powerful natural antioxidant (Bhattacharya et al, 1997; Naidu et al, 2006;

Kumar et al, 2005). It increases activity of cell’s antioxidant enzymes i.e.

superoxidedismutase, (SOD), catalase (CAT) and glutathion peroxides

(GPx) (Gupta et al, 2003; Naidu et al, 2006). The antioxidant potential of

W. somnifera inhibits ROS induced lipid peroxidation, which might

prevent damage of lysosomes, Golgi, ER and other cell organelles.

Kumar and Kulkarni, (2006) reported that W. somnifera decreases lipid

peroxidation in mice brain by rapping hydroxyl radicals, Gupta et al,

(2003) in Wister rats, Palanyandi et al, (2006) in Swiss albino mice.

Thus, due to decreased lipid peroxidation lysosomes, Golgi bodies and

endoplasmic reticulum remain intact to carry out cellular functions.

Sialic acid content was increased in old and D-galactose stressed

mice. This might be due to accumulation of glycoproteins in the acini.

During old age, there occurs an alteration in β-adrenergic simple

transduction path ways which results into inhibition of protein secretion

(Baum, 1987; Rajakumar and Scarpace, 1991).

Glycoproteins concentration in old age, increase in most of the old

age related diseases like xerostomia, Sjogren’s disease or increase in

intracellular density in acinar cytoplasm. There is increase in intracellular

ionic strength causes considerable condensation of the intracellular

colloid, leading to gradual increase in intracellular dry mass during aging

126

(Zs Nagy et al, 1987). Accumulation of acid and neutral glycoproteins in

acinar cells of submandibular glands of rat during aging was reported by

Emmanouil–Nikoloussi et al, (1992). Increased accumulation of

glycoproteins content may also be due to increase in lipid peroxidation.

Excessive production of lipid peroxides makes the membrane to loose the

fluidity and integrity (Reicher, 1987; Machlin and Bendich, 1987).

Gokmen et al, (2000) have suggested that either the shedding or secreting

of sialic acid from the cell or cell membrane surface may be partly

responsible for increased sialic acid concentration. Increased sialylation

of serum proteins may increases sialic acid (Flynn et al, 1999) or

reduction in desialylation of plasma glycoproteins (Morell et al, 1971)

increases sialic acid.

Most of the studies have shown an elevation in serum sialic acid

concentration in coronary heart diseases and positive correlation between

the raised serum sialic acid and the severity of the coronary lesions was

observed (Nigam et al, 2006).

In present study in D-galactose treated adult and old mice as well

as in naturally aged mice, the sialic acid content was increased. This

might be due to increased lipid peroxidation. Increased lipid peroxidation

in old age was reported by (Tappel, 1980; Donato and sohal, 1981;

Schroeder, 1984; Harman, 1992; Rattan, 1995; Lee et al, 1997). Increased

lipid peroxidation due to D-galactose was reported by Ashokan and Pillai,

(1999) in brain; Song et al, (1999); in brain; Vora et al, (2009) in

mitochondrial fraction. The treatment of D-galactose accelerates

formation of Advanced Glycation End Products (AGEs) which further

stimulates production of free radicals, exerting oxidative stress in the

brain cells (Song et al, 1999; Deshmukh et al, 2006). As a result the

normal functioning of salivary gland may gets disturbed and there by

increasing the sialic acid. Increase in sialic acid in D-galactose stressed

127

mice was also reported by Kalamade et al, (2007) in prostate gland,

Sonavane, (2007) in salivary glands and Tomake, (1998) in naturally old

male mice.

The treatment of centrophenoxine and glycowithanolides (WSG) in

protective and curative groups of both adults as well as old male mice

showed improvement in glycoproteins distribution and their level in

salivary gland of stressed adults up to the level as before but quite

significant in old. This shows both protective and curative role of WSG

and centrophenoxine against D-galactose and natural aging induced lipid

peroxidation. The antioxidant potential of WSG (Bhattacharya et al,

1997) and centrophenoxine (Zs – Nagy, 1989) prevent cellular damage,

due to ROS (Palaniyandi et al, 2006) and ultimately reduces lipid

peroxidation (Kumar and Kalonia, 2007) by increasing activity of cell’s

antioxidant enzymes. Thus, due to prevention of lipid peroxidation,

accumulation of sialic acid in acini might be prevented and there by

decreases sialic acid content of salivary glands.

Decrease in sialic acid content by treatment of antioxidants like

Pertroselinum crispum was observed by Sonavane, (2007) in salivary

glands of D-galactose stressed and old male mice, Bacopa moniera by

Kalmade et al, (2007) in prostate gland of D-galactose stressed male

mice. Scanlin and Glick, (1999) reported that increased fucosylation in

cystic fibrosis, with concomitant decrease in sialylation. In present study

fucose content in D-galactose stressed and old male mice was decreased,

increased in centrophenoxine and WSG treatment. Opposite is true in

case of sialic acid.

The histochemical studies by PAS, AB pH 1.0 and AB pH 2.5

staining and electrophoretic study by Coomassie blue and AB pH 1.0,

staining showed alterations in total proteins, neutral glycoproteins,

128

sulfated and acidic glycoproteins in submandibular and sublingual gland

of adult and old male mice.

In the electrophoretic separation 6 bands of glycoproteins were

observed in submandibular glands whereas, in sublingual glands 7 bands

of glycoproteins resulted and are altered in stressed condition but all these

bands reappeared on treatment with centrophenoxine and WSG.

There was reduction in glycoproteins content of submandibular and

sublingual glands of D-galactose stressed adult and old mice as well as in

naturally aged mice. Decrease in glycoproteins with age was reported by

several researchers, Bruzynski, (1971) showed significant decrease in

glycoproteins bound hexose protein–nitrogen ratio in submandibular

gland of aging guinea pig. Rybakava (1979) observed reduction in basal

level of proteins and mucopolysaccharides in salivary glands of albino

rats during aging. Kuyatt and Baum, (1981) reported that reduction in

proteins, sialic acid and neutral sugar in submandibular gland of old rat.

Denny et al, (1991 a, b) investigated reduction in total mucin levels in

mice during aging. Vissink et al, (1996) in human study observed

reduction in SIg-A, high and low molecular weight mucins in mucous

saliva with age, Sonavane, (2007); Mankapure, (2007) reported decrease

in glycoproteins in salivary glands of D-galactose stressed mice. Decrease

in glycoproteins might be due to decrease in synthesis of glycoproteins.

The decrease in glycoprotein synthesis by acinar cells might be due to

changes in the chromatin material. Chromatin material in many aged

acinar cells exists in relatively condensed form, when compared with

young acinar cells. Diffused chromatin material is primarily responsible

for the transcription of RNA while condensed chromatin is relatively

inactive in the transcription mechanism in old (Litta et al, 1964;

Granboulan and Granboulan, 1965). Pyhtila and Sherman, (1968)

reported that an age related increase in the thermal stability of

129

nucleoprotein from beef thymus and correlative decrease in effectiveness

of this nucleoprotein to serve as template for RNA polymerase. So, there

must be altered transcription of RNA in D-galactose stressed and

naturally aged submandibular and sublingual glands, which results into

decreased synthesis of glycoprotein.

Decrease in glycoproteins may also be due to the loss of

glycosylation at post translational level. Reduction in N-linked

glycosylation was described by Baum et al, (1992) and Kousvelari et al,

(1988).

Reduction in glycoproteins in D-galactose stressed and naturally

aged mice, can also be correlated with structural damage of salivary

glands, which results in salivary gland hypofunction. It has been reported

that, the aging process either natural or induced may cause free radicals

mediated oxidative stress which damages salivary gland’s secretory units

and macromolecules (Rattan, 1989; Sohal et al, 1993; Lee et al, 1997;

Sitte et al, 2000; Bajra and Herrero, 2000; Kujoth et al, 2005). Age

related damage to macromolecules, decrease protein synthesis and also

decrease total protein glycosylation profile in salivary glands (Tomake

and Pillai, 2000; Przybylo et al, 2004) of aged mice.

In mice, which received centrophenoxine and glycowithanolides,

glycoproteins were increased. This might be due to antioxidant activity of

these antioxidants. Firstly, the antioxidant property of WSG

(Bhattacharya et al, 1997; Gupta et al, 2003; Kumar et al, 2005) and of

centrophenoxine (Zs – Nagy and Nagy, 1980) brings increase in cell’s

antioxidant enzyme like SOD, CAT, GPx (Gupta et al, 2003). Secondly,

by preventing free radicals mediated cellular damage, reduce lipid

peroxidation, protect macromolecules and bring increased protein

synthesis and glycosylation of proteins.

130

When antioxidant potentials of glycowithanolides and

centrophenoxine are compared, glycowithanolides is more effective than

centrophenoxine. Secondly, glycowithanolides is natural antioxidant. Its

positive effects have been reported in many other ailments with no

associated toxicity.