THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (SCIENCE) IN CHEMISTRY OF THE UNIVERSITY OF BURDWAN 2013 POLYSACCHARIDES FROM Turbinaria conoides AND Adhatoda vasica : STRUCTURAL FEATURES AND BIOLOGICAL ACTIVITIES Nabanita Chattopadhyay, M. Sc. NATURAL PRODUCTS LABORATORY DEPARTMENT OF CHEMISTRY THE UNIVERSITY OF BURDWAN WEST BENGAL, INDIA
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THESIS SUBMITTED FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY (SCIENCE) IN CHEMISTRY
OF THE UNIVERSITY OF BURDWAN
2013
POLYSACCHARIDES FROM
Turbinaria conoides AND Adhatoda vasica :
STRUCTURAL FEATURES AND BIOLOGICAL
ACTIVITIES
Nabanita Chattopadhyay, M. Sc.
NATURAL PRODUCTS LABORATORY
DEPARTMENT OF CHEMISTRY
THE UNIVERSITY OF BURDWAN
WEST BENGAL, INDIA
Dedicated to my beloved parentsDedicated to my beloved parentsDedicated to my beloved parentsDedicated to my beloved parents
THE UNIVERSITY OF BURDWAN
TO WHOM IT MAY CONCERN This is to certify that the thesis “POLYSACCHARIDES FROM Turbinaria
conoides AND Adhatoda vasica: STRUCTURAL FEATURES AND
BIOLOGICAL ACTIVITIES” is the result of work done by Nabanita
Chattopadhyay, M.Sc., who has registered her name in The University of
Burdwan on 19-03-2007 for the award of Doctor of Philosophy (Science) in
Chemistry under my supervision and Guidance. This work neither in part nor
whole has been submitted for any degree by Nabanita Chattopadhyay or other. It is
also certified that she has successfully completed Ph.D. course work framed by the
Department of Chemistry, following the guidelines of The University of Burdwan,
and that she has delivered one seminar lecture on 02-08-2012 in our department
regarding fulfillment of all the requirements for submitting the thesis for Ph.D.
degree under new regulation, 2009 of this University.
(Naito et al., 2006; Jain, 2006), Parkinson’s disease (Beal, 2003; Chaturvedi et al., 2008),
atherosclerosis (Heinecke, 1997), and AIDS (Sepulveda & Watson, 2002). Natural compounds
offer interesting pharmacological properties for their use as antioxidative agent. In recent years, a
number of polysaccharides containing fractions isolated from various sources as for example,
marine algae (Ruperez et al., 2002; Rocha de Souza et al., 2007; Wang et al., 2008, 2009a),
plants (Aguirre et al., 2009; Yang et al., 2011), and even some enzymatic extracts (Je et al.,
2009) possess antioxidative activity. Some of them, in particular the sulfated polysaccharides
from marine algae such as fucoidan (Zhao et al., 2005; Rocha de Souza et al., 2007; Wang et al.,
2008, 2009b; Wang et al., 2010), sulfated galactan (Barahona et al., 2011), sulfated
polysaccharide fractions containing galactose and xylose residues as constituent sugar, and
rhamnose-rich polysaccharide fractions showed considerable antioxidative properties (Hu et al.,
2010; Yang et al., 2011).
Polysaccharides from higher plants such as arabinogalactan (Fig. 1.4) also showed
antioxidative activity (Chatterjee et al., 2011; Sinha et al., 2011a). The highly branched
11
arabinogalactan isolated from Indian medicinal plant Eugenia jambolana is esterified with
phenolic acid and possess potent dose dependent antioxidative activity (Bandyopadhyay et al.,
2012).
In addition, chemically modified macromolecules such as acetylated and benzoylated ulvans
from Ulva pertusa (Chlorophyta) also possess antioxidative property. Potency of some of these
compounds is higher than the others (Qi et al., 2006).
Structure-activity relationships. The antioxidative property of polysaccharides depends upon a
number of parameters including molecular mass (Qi et al., 2005). In case of sulfated
polysaccharides, potency depends upon their sulfate content (Qi et al., 2005; Hu et al., 2010).
For example, sulfated polysaccharides from Undaria pinnitafida had stronger antioxidant
abilities than their de-sulfated derivatives (Wang et al., 2008). Recent study showed that the
position of sulfate group is another important parameter. The increased hydrophobic character of
Fig. 1.4. A model showing the structural features of an arabinogalactan from the Indian medicinal plant Eugenia jambolana (Bandyopadhyay et al., 2012).
12
the macromolecule also leads to higher potency (Qi et al., 2006). Finally, the structure of
polysaccharide also plays an important role in antioxidative property.
1-4.4. Antitumor activity
Polysaccharides also possess antitumor activity (Table 1.1). These macromolecules usually
have low toxicity and few side effects, which is essential for immunotherapy against cancer.
(13%) & minor amounts of other sugars (∼2%) also showed a cough suppressing effect on
laryngopharyngeal type of cough (Nosáľová et al., 2012).
The high molecular mass polysaccharide-polyphenolic conjugate (rhamnogalacturonan
associated with arabinogalactan in Lythrum conjugate) from flowering parts of Lythrum
salicaria that contains 74% of carbohydrates and 17% of phenolics showed antitussive activity
(Sutovska et al., 2012). The polyphenolic–polysaccharide–protein complex (molar mass
11.2 kDa) from flowers of Solidago canadensis L. composed of carbohydrates (43%), protein
(27%), phenolics (12%), uronic acids (10%) and inorganic material (8%) showed antitussive
activity (Sutovska et al., 2013).
Structure-activity relationships. The antitussive properties of polysaccharide from various
sources are different (Fig.1.8), but because of limited data available the relationship between the
structure of polysaccharides and their antitussive activity is not yet clear (Sutovska et al., 2010;
Nosáľová et al., 2011).
19
Mechanism of action. The mechanism behind the antitussive activity of the carbohydrate
polymers from plants is poorly understood, although it has been reported that many antitussive
herbs work by an antispasmodic action or bronchodilator (Ernst, 1998; Pavord & Chung, 2008).
They cause bronchial muscle relaxation in vitro, or decrease airways resistance in vivo. Pavord
(2004) reported that broncho constriction causes or enhances the sensitivity of cough, while
bronchodilation does the opposite. However, the role of other mechanism including bioadhesive
effect of the polysaccharide to the epithelial mucosa (Sutovska et al., 2009a) cannot be ruled out.
Further research should be directed in this area.
1-4.6. Antiviral activity
Many viruses display affinity for cell surface proteoglycans, especially heparan sulfate
proteoglycans, with high biological relevance to virus entry. This raises the possibility of the
application of sulfated polysaccharides in antiviral therapy. In recent years, screening assays of
the antiviral activity of extracts from a number of marine algae and cyano bacteria have led to
the identification of carbohydrate polymers with potent inhibitory effects against several human
and animal viruses, including Herpes simplex virus (Witvrouw & De Clercq, 1997; Balzarini &
Van Damme, 2007; Ghosh et al., 2009a; Rusnati et al., 2009).
0
1
2
3
4
5
6
7
8
9
Num
ber
of c
ough
effo
rts
(NE
)
Control
Codeine
Adhatoda vasica
Glycyrrhiza glabra
Withania somnifera
Rhodella grisea
Fig. 1.8. The influence of the carbohydrate polymer from Adhatoda vasica, Glycyrrhiza glabra, Withania somnifera, and Rhodella grisea,and codeine (positive control) and saline water (negative control) on the citric acid-induced cough efforts (NE) in guinea-pigs recorded at 30, 60, 120 and 300 min time intervals (Chattopadhyay et al., 2011; Saha et al., 2011; Sinha et al., 2011b; Nosál’ova et al., 2011).
N 30 60 120 300 Time interval (min)
20
Table 1.3. Carbohydrate polymers with antiviral activity
Families of
polysaccharide
Source Molecular weight Strain CC50
(µg/ml)
IC 50
( µg/ml)
Reference
Fucan Padina
tetrastromatica
50 kDa HSV-1 & HSV-2 1000 0.30-1.05 Karmakar et al.,
dien-3-one (Sheu et al., 1999). The presence of two new antifungal steroids namely, 3,6,17-
trihydroxy-stigmasta-4,7,24(28)-triene and 14,15,18,20-diepoxy turbinarin have also been
reported (Kumar et al., 2010, 2011). Despite the general interest in phytoconstituents it remains
ironic that research on the chemical and biological aspects of the high molecular weight
bioactive has been neglected. Notably, it is intriguing to observe that polysaccharide from plant
extracts exhibit a large range of pharmacological activities (Inngjerdingen et al., 2006;
Mantovani et al., 2008; Ghosh et al., 2009a; Baek et al., 2010; Thakur et al., 2012).
Hence the central goal of this study was to investigate the structural features of the different
classes of polysaccharides present in T.conoides. We have also evaluated in vitro the
antioxidative activity of fucoidan, glucan and alginic acid isolated from this brown seaweed.
These polysaccharides may represent a new approach for inhibiting the harm caused by
excessive free radicals.
2-2. CHEMICAL CHARACTERIZATION OF POLYSACCHARIDES
FROM Turbinaria conoides
2-2.1. Preparation of depigmented algal power and sugar compositional analysis
The major goal of this study was to develop antioxidative drug candidate from Turbinaria
conoides. In order to study the chemical structures and antioxidative properties of polymers
present in T. conoides, the dried seaweed was sequentially extracted in a Soxhlet apparatus with
petroleum ether and acetone to leave a depigmented algal powder named as DAP (Fig. 2.1).
29
To understand the type of polysaccharides present in DAP, a knowledge about its sugar
composition is necessary. For the determination of the monosaccharide composition the fractions
obtained were hydrolyzed in 2M TFA, the sugars released were reduced and acetylated as
described in section 4-2.2. The alditol acetates were separated by GC and identified by their (i)
retention time relative to that of inositol hexa-acetate (present as internal standard) and (ii) mass
spectral fragmentation patterns. Fig. 2.2 shows a typical standard chromatogram and how mass
spectroscopy was used in order to verify the identities of alditol acetates.
Fig. 2.2. The GC-MS analysis of the alditol acetates derived from standard sugars.
min
m/z m/z m/z
Gal GlC Man Int. Std. (Inositol)
Xyl Fuc Rha
50 100 150 200 250 3000
25000
50000
75000
100000 43115 170129
9969 85 157145 187 23173 28920121741
303
50 75 100 125 150 175 200 2250e3
50e3
100e3
43
115
14510385
217127 18715873 20017555 218
50 100 150 200 250 300 3500
25000
50000
43
115
139103 187 217157170 25973 28941 361
5.0 7.5 10.0 12.5 15.0
0e3
500e3
1000e3
TIC Ara
Fig. 2.1. Scheme for the preparation of depigmented algal powder from the brown seaweed Turbinaria conoides.
Depigmented algal powder (DAP)
Sequential extraction with petroleum ether and acetone in Soxhlet apparatus
Air dried algal powder
Turbinaria conoides
30
GC analysis of the alditol acetates derived from DAP (Fig. 2.3) showed the presence of fucose
(56%), xylose (11%), galactose (15%), glucose (12%) and small amount of mannose (Table 2.1).
2-2.2. Isolation and sugar composition of polysaccharide fractions
Sugar composition of depigmented algal powder (DAP) revealed that, it contained 35% 2 M
TFA hydrolysable sugars of which about seventh were uronic acids. The main neutral sugar was
fucose and it also contained other sugar including glucose, galactose
5.0 7.5 10.0 12.5 15.0
0.0e6
5.0e6
10.0e6
15.0e6
20.0e6 T IC
Fig. 2.3. GC analysis of alditol acetates derived from DAP of Turbinaria conoides.
Fu
cose
Xyl
ose
Man
nose
Gal
acto
se
Glu
cose
Int.
Std
. (In
osi
tol)
min
SEC
Algal Powder (DAP)
0.1 M HCl, 30-350C
A
AF1
AF2
AF3 Residue
3% Aqueous Na2CO3 Extract B
2% CaCl2
Residue
INS
C
2% CaCl2
Precipitate (C2)
Solution (C1)
AEC
Water, pH~6.5, 800C
C1F1 C1F2
Fig. 2.4. Scheme for the extraction and purification of polysaccharides from the marine brown alga T. conoides.
31
and xylose (Table 2.1). The DAP was fractionated by the sequential extraction procedure as
shown in Fig. 2.4, was based on the different solubility, molecular mass and charge distribution
of polysaccharides from brown seaweeds. Glucans are soluble in warm waters and therefore
extracted at 80°C. Fucoidans were extracted with diluted hydrochloric acid, whereas alginates
were extracted with sodium carbonate. Alginates form insoluble precipitates with bivalent
calcium and at acidic pH, and are soluble in solution between pH 6 and 9. Procedures described
in the materials and methods (Section 4-1.1.).
GC and GC-MS analysis of the alditol acetates derived from A (Fig. 2.5) revealed the
presence of fucose as the major monosaccharide together with smaller amounts of xylose and
galactose (Table 2.1). No amino sugars were detected during GC-MS analysis of the derived
alditol acetates.
50 100 150 200 250 3000
25000
50000
75000
100000 43115 170129
9969 85 157145 187 23173 289201 21741
303
5.0 7.5 10.0 12.50.0e6
1.0e6
2.0e6
3.0e6
4.0e6 TIC
Gal
acto
se
Int.
Std
. (I
nos
itol)
Fu
cose
Xyl
ose
m/z
min
Fig. 2.5. GC-MS analysis of the alditol acetate derived from the fraction A of T. conoides.
32
Table 2.1. Yield and chemical composition (mol %) of fractions obtained from T. conoides
*percent weight of fraction dry weight.†mol percent of anhydro sugar - = not determined. nd = not detected. Tr = trace. TS = Total sugar
2-2.3. Purification of the fucoidans by chromatography
Anion exchange chromatography
on a DEAE Sepharose column
separated fraction A of T.
conoides into three sub-fractions
(AF1, AF2, and AF3) (Fig. 2.4).
AF1, which accounted for 11% of
the total sugars recovered from
the anion exchanger, was the
minor component of A. It
consisted mostly of fucose
together with smaller amount of
galactose, xylose and glucose (Table 2.1). The second fraction AF2, eluted at the beginning of
the salt gradient, is the second largest fraction. AF3 was the major fraction, amounting to 51% of
the total polymers recovered from the column. Fucose, accounted for more than 54% of the total
sugars of AF3 together with smaller amount of galactose and xylose units (Table 2.1). Thin layer
chromatographic analysis of the monosaccharides present in the hydrolysate indicates the
presence of an uronic acid with an Rf value
similar to that of glucuronic acid. GC analysis of
the TMS derivatives of the derived methyl
glycosides confirmed this result. The presence of
glucuronic acid has already been reported in
fucoidan from brown seaweeds (Adhikari et al.,
2006; Mandal et al., 2007).Therefore, AF3 is
essentially a fucoidan that might contain a high
number of sulfate groups, as indicated by its late
elution. Indeed, the high charge density of this
polysaccharide was confirmed by its sulfate
content (4%, w/w). The FT-IR spectrum
Fractions DAP A AF3 AF3D B C1 C1F2
Yield 100 8.8 nd nd 22.6 6.2 nd
TS* 35 36 37 56 46 41 58
Sulfate* nd 3 4 Tr 1 2 Tr
Protein* nd 1.3 nd nd 2.4 2.1 nd
Fucose† 56 57 54 52 Tr 59 6
Xylose† 11 15 18 19 Tr 12 5
Mannose† 7 Tr Tr - Tr Tr Tr
Galactose† 15 23 28 29 Tr 18 -
Glucose† 12 5 - - - 11 89
Fig. 2.6. FT-IR spectrum of fucoidan (AF3) obtained from T. conoides.
Sulfate band 1252 cm-1
33
0
0.5
1
1.5
2
2.5
3
00.0
80.1
50.23 0.3
0.380.45
0.53 0.6 0.68
0.75
0.83 0.9
0.98
Fig. 2.7. Elution profile of the fucoidan (AF3) of T. conoides on Sephacryl S-200 column with 500 mM sodium acetate buffer (pH 5.0) at 20 mL/h. Elution of polysaccharide was expressed as a function of the partition coefficient Kav [Kav = (Ve - V0)/(Vt - V0) with Vt and V0 being the total and void volume of the column determined as the elution volume of glucose and blue dextran, respectively, and Ve is the elution volume of the sample].
Kav
Su
gar
Co
nce
ntr
atio
n (µg
/mL)
(Fig. 2.6) of AF3 showed an absorption band at 1252 cm-1 related to a >S=O stretching vibration
of the sulfate group. An additional sulfate absorption band at 848 cm-1 (C–O–S, secondary axial
sulfate) indicated that the sulfate group is located at position 4 of the fucopyranosyl residue
(Patankar et al., 1993).
Solvolytic desulfation of the purified (AF3) fucoidan (as pyridinium salt) produces a
desulfated derivative (AF3D). Preliminary experiments showed a higher recovery with this
method compared to methanol-HCl and auto-desulfation methods. In the IR spectrum of
desulfated derivative (AF3D) absorbances at 1252 cm−1 and 848 cm−1 become weak,
demonstrating that they were associated with sulfate groups. This purified sulfated fucan had
negative specific rotation [α]D -109o (c 0.2, H2O), revealing that fucose in AF3 belongs to the L-
series, like other sulfated fucans from brown seaweeds (Kariya et al., 2004; Adhikari et al.,
2006; Mandal et al., 2007). AF3 was subjected to further chemical analysis.
2-2.4. Molecular mass
AF3 was subjected to
further chemical analysis. First,
the apparent molecular mass
was determined by size
exclusion chromatography. The
elution profile of this
macromolecule on Sephacryl
S-200 suggests that this
polymer is homogeneous (Fig.
2.7). Based on calibration with
standard dextrans, the apparent
molecular mass of AF3 would
be 50 kDa.
2-2.5. Linkage analysis
Table 2.2 shows that the desulfated fucoidan yielded partially methylated alditol acetates
(PMAA) corresponding to (1→2)-Xylp (xylopyranose), (1→3)-linked Fucp, and (1→3)- and
(1→2,3)-linked Galp residues, consistent with the presence of fucoidan. The identity of each
34
PMAA was confirmed by their relative retention time and mass spectral fragmentation pattern.
Fig. 2.8 shows EI-MS fragmentation pattern of selected PMAA. The presence of large quantities
of T-Xylp (terminal xylopyranose) and T-Fucp residues suggest that this polymer is highly
branched with 29 terminals for every 71 residues in the main chain. Interestingly, 33–34% of the
total fucose residues are terminal, 27–28% are (1→3)-linked, 21–22% are branched points
(Table 2.2). Small amount of (1→4)- and (1→2)-linked Fucp residues were also present. So far,
fucose residues in algal fucoidans are either (1→3)- or (1→3)- and (1→4)-linked (Cumashi et
al., 2007; Pomin & Mourao, 2008). Linkage analysis of the native fucan sulfate AF3 yielded a
variety of monomethylated, dimethylated and trimethylated products (Table 2.2) indicating that
the structure of this polymer is highly complex. The amount of sulfate groups in the native
Methylation
products†
Deduced linkage Peak area* AF3 AF3D
2,3,4-Xyl Xylp(1→ 11 12
3,4-Xyl →2)Xylp(1→ 7 9
2,3,4-Fuc Fucp(1→ 8 17
2,4-Fuc →3)Fucp(1→ 3 14
2,3-Fuc →4)Fucp(1→ 3 3
3,4-Fuc →2)Fucp(1→ 5 6
2-Fuc →3,4)Fucp(1→ 17 5
3-Fuc
Fuc
→2,4)Fucp(1→
→2,3,4)Fucp(1→
2
16
6
-
3,4,6-Gal →2)Galp(1→ 6 7
2,4,6-Gal
4,6-Gal
2-Gal
→3)Galp(1→
→2,3)Galp(1→
→3,4,6)Galp(1→
10
6
6
10
6
5
polysaccharide as calculated from methylation was not in good agreement with the
experimentally determined sulfate. But, methylation of sulfated polysaccharides does not always
yield reliable proportions of methylated alditol acetates (Pereira et al., 1999; Adhikari et al.,
2006). 2-O-methyl fucitol, and unmethylated fucitol were amongst the abundant products of
methylation analysis of the native polymer. The increase in the proportions of 2,4-di-O-methyl
*Percentage of total area of the identified peaks. †2,3,4-Fuc denotes 1,5-di-O- acetyl-2,3,4- tri-O-methylfucitol, etc. - = not detected.
Table 2.2. Partially methylated alditol acetates derived from the fucoidan (AF3) of T. conoides and its desulfated derivative (AF3D)
35
fucitol after desulfation, as a consequence of decreased proportions of 2-O-methyl fucitol
residues, suggests that sulfate groups, when present, are located at position 4 of the (1→3)-linked
fucosyl residues. This result also confirms the prediction made by IR analysis regarding the
position of sulfate group.
CHDOAc
CHOMe
CHOAc
CHOMe
CHOAc
CH2OMe
118
161
129
234
102
CHOAc
CHDOAc
CHOMe
CH3
CHOMe
CHOMe
118
131 162 175 115
m/z 50 75 100 125 150 1750
5000
10000
15000
20000
25000
118
10289
13172
59
175162
CHDOAc
CHOMe
CHOMe
CHOMe
CH2OAc
118161
117162
102
101
(2)
m/z
Fig. 2.8. EI-MS fragmentation pattern of (1) 1,3,5-tri -O-acetyl-2,4-di-O-methyl fucitol; (2) 1,5-di-O-acetyl -2,3,4-tri-O-methyl fucitol; (3) 1,3,5 tri-O-acetyl-2,4,6-tri-O-methyl galactitol; (4) 1,5 di-O-acetyl -2,3,4 tri-O-methyl xylitol of T. conoides.
50 75 100 125 150 175 200 225 250 2750e3
50e3
100e3
150e3
43 118
129
101
16174 8759
234174143 203 217175 277
m/z
(1)
CHDOAc
CHOMe
CHOAc
CHOMe
CHOAc
CH3
247118
131234
174
m/z
(3)
50 75 100 125 1500
25000
50000
43101 117
59 8871 162131
(4)
m/z
36
HOD
H-6
H-1
α H
-1β
Chemical shift,
Fig. 2.9. 1H NMR spectrum at 500 MHz of the fucoidan (AF3) of T. conoides. The spectrum was recorded at 80°C for sample in D2O solution. H-1α, H-1β and H-6 refer to anomeric signals of α-linked fucose, β-linked sugars and methyl protons of fucose residues, respectively. The signal for the residual water was designated as HOD.
6
2-2.6. NMR spectroscopy
We employed 1H NMR spectroscopy to determine the anomeric configuration and the
sulfation pattern of the fucoidan AF3. The native
polysaccharide has a very complex 1H NMR
spectrum (Fig. 2.9). A number of separate spin
systems ranging from 5.02 to 5.43 ppm
attributable to anomeric protons of α-fucose
residues were distinguishable in the spectrum of
the pure fucoidan (Mourao et al., 1996; Kariya et
al., 2004; Mandal et al., 2007). The appearance
of large number of anomeric signals suggest that
the environment of the fucose residues are
different and this may arise from varied
sulfation pattern, different glycosidic
linkage positions and/or diverse sequence
of monosaccharide residues. This spectrum
also included resonances characteristic of
fucoidan, such as signals from ring protons (H-2 to H-5) between 3.34 and 4.34 ppm, and intense
signals from the methyl protons H-6 at ~1.02–1.28 ppm. This later envelope of signals confirms
the presence of different types of fucosyl residues. The signals between 4.45 and 4.90 ppm were
attributed to anomeric protons of β-linked sugars. The high proportion of xylose and galactose
residues must be responsible for these signals in the spectrum, but was not possible to assign any
particular signals to these residues. Therefore, it is apparent that the NMR spectrum of this
polysaccharide is complex as observed for fucoidan from other marine brown algae (Mourao et
al., 1996; Pereira et al., 1999; Adhikari et al., 2006; Mandal et al., 2007).
ppm
37
p
G M G
HOD
Chemical shift, ppm Fig. 2.11. 1H NMR spectrum at 500 MHz of the alginic acid containing fraction (B)
isolated from the brown seaweed T. conoides. G1 and M1 are the anomeric signals of
guluronic acid and mannuronic acid, respectively, and G5 is the signals of the H-5 proton of
guluronic acid residue. The spectrum was recorded at 80°C for sample in D2O solution. The
signal for the residual water was designated as HOD.
HOD
0
0.2
0.4
0.6
0.8
1
1.2
0.00
0.00
0.07
0.13
0.20
0.26
0.33
0.40
0.46
0.53
0.59
0.66
0.73
0.79
0.86
0.92
0.99
Sug
ar
Co
nce
ntra
tion
(µg/m
L)
Kav.
Fig. 2.10. Elution profile of the hot water extracted fraction (C1) of T. conoides on Superdex-30 column with 500 mM sodium acetate buffer (pH 5.0) at 20 mL/h. Elution of polysaccharide was expressed as a function of the partition coefficient Kav [Kav = (Ve - V0)/(Vt - V0) with Vt and V0 being the total and void volume of the column determined as the elution volume of glucose and blue dextran, respectively, and Ve is the elution volume of the sample].
2-2.7. Structural features of the glucan and alginic acid rich fractions
Glucans are soluble, but their water
solubility depends on the branching level: the
higher the branching degree, the higher the
solubility. The less branched glucans are
soluble only in warm water (60–80°C). The
alginic acid (C2) present in the hot water
extract (C) was removed by taking advantage
of the insolubility of Ca- alginate in water.
The molecular mass of glucans is small (5–10
kDa). Therefore, attempts have been made to
separate it from fucoidan by size exclusion
chromatography. Indeed, SEC
on Superdex-30 separates C1 into two fractions C1F1 and C1F2 (Fig. 2.10). The minor fraction,
C1F2, which accounted for 35% of the
total sugar recovered from the column
consisted of mainly glucose as neutral
sugar. Based on calibration with
standard dextrans, the apparent
molecular weight of this glucan would
be 5 kDa. This glucan containing
fraction (C1F2) does not produce blue
colouration with iodine. Moreover 1H
NMR spectrum of this polymer clearly
shows the presence of only one
anomeric signal at 4.827 ppm indicative
of β-configuration. Finally, methylation
analysis of this glucan revealed
the presence of 1,3,5-tri-O-
acetyl-2,4,6-tri-O-methylglucitol
C1F1
C1F2
ppm
38
(98%) and 1,5-di-O-acetyl-2,3,4,6-tera-O-methyl glucitol (2%) residues. On the basis of the
glycosidic linkage and 1H NMR analysis it may be concluded that the glucan of T. conoides is a
linear polysaccharide and contained β-(1→3)-linked glucopyranosyl residues. Sodium alginates
form insoluble precipitates at acid pH and with calcium salts, but they are soluble in solution
between pH 6 and 9. Therefore, these macromolecules were extracted with Na2CO3. The
fractional product (B) was analysed using FT-IR and 1H NMR spectroscopy. The FT-IR
spectrum of this fraction contains band at 3400 (OH stretching), 2925 (CH stretching), 1675 and
1420 (COO stretching) cm-1 characteristic of alginate. Moreover two bands at approximately
1100 and 1025 cm-1 responsible for mannuronic (M) and guluronic (G) units, respectively, were
also observed. To evaluate the content of G, M and G–G linkage, we have investigated the 1H
NMR spectrum (Fig. 2.11) of sodium alginate using procedures as described by Grasdalen, 1983
and Matsushima, 2005. The relative areas of anomeric protons G-1 (H-1 of G) and M-1 (H-1 of
M) correspond to the mole fractions of G and M, respectively. The peak areas of G-5 (H-5
resonances of G) give the distribution of G in G-block and GM-block, and the algebraic sum of
their intensities accounts for the total G content, i.e., the relative area of G-5 is equal to that of G-
1. From the measurement of peak areas of A (δH 5.13 (G-1)), B (δH 4.72–4.84 (G-5 in GM-
block and M-1)) and C (δH 4.45–4.52 (G-5 in G-block)), the guluronic acid content (G%) and
G–G diad frequency (GG%) were calculated using Eqs. (1) and (2), and the calculated values are
46.19 and 42.12, respectively
2-3. PHARMACOLOGICAL ACTIVITIES OF POLYSACCHARIDES
FROM Turbinaria conoides
2-3.1. Antioxidative activity
Ferric ion reducing/antioxidant power (FRAP) assay. In recent years many different methods
are being used to evaluate antioxidative capacity of foods and biological samples (Huang et al.,
(2) (area of B) + (area of C)
GG% (%) = area of C X 100
(1) (area of B) + (area of C)
G% (%) = area of A X 100
39
Table 2.3. Ferric ion reducing abilitya of polysaccharide containing fractions from T. conoides
4 min 30 min
Fractions µµµµmol
Trolox/g
µµµµmol
Fe(II)/g
µµµµmol
Fe(II)/g
µµµµmol
Trolox/g
AF3b 165 79 190 96
Bb 92 53 137 85
C1F2 b 72 38 82 43
aResults are expressed as equivalent of Fe(II) or Trolox per gram dry weight of fractions in aqueous solution. Each value, which is given to its nearest whole number, was the mean of 2 replicates. bFor a description of fractions obtained see “Materials and methods”.
2005). In some of these protocols, antioxidative assays were performed in alcoholic solutions,
but in this condition polysaccharide
would precipitate. Therefore,
the antioxidative capacity of
sulfated polysaccharides (AF3, B
and C1F2) from Turbinaria
was determined by the FRAP
assay. This assay in which a ferric
salt Fe(III) (TPTZ)2Cl3
(TPTZ=2,4,6-tripyridyl-s-triazine)
is used as oxidant (Benzie &
Strain, 1996) takes advantage of
electron transfer reactions (Huang et al., 2005). FRAP values increased considerably from 4 to
30 min, as it has been described for other vegetable and seaweed samples (Ruperez et al., 2002).
Regarding antioxidative capacity of the polysaccharides of present study it is clear that the
fucoidan (AF3) showed the highest reducing power at 4 and 30 min (165 and 190 mol Fe(II) per
g sample dry weight, respectively). This is followed by alginic acid and glucan (Table 2.3).
Results are expressed as mol Fe(II) per g sample dry weight. For comparison of potencies, values
are also calculated as mol Trolox/g sample dry weight from regression equations as described by
Pulido, 2000 of Trolox at 4 and 30 min of reaction with the FRAP reagent.
Scavenging effect on1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. For further insight into
the activation mechanism, we examined whether the protective effect was associated with DPPH
radicals. The proton radical scavenging action is known to be one of the various mechanisms
40
Fig. 2.12. Scavenging effect of polysaccharides isolated from the brown seaweed T. conoides on DPPH radicals. BHA: Butylated hydroxyanisole. AF3: The fucoidan rich fraction. B: The alginic acid containing fraction. C1F2: The glucan fraction.
0 1 2 3 4 5
0
20
40
60
80
100
BHA AF3 B C2F3
Sca
veng
ing
effe
cts
(%)
Concentration (mg/ml)
C1F2
for antioxidation. DPPH is one of the compounds that possess a proton free radical and shows a
characteristic absorption at 517 nm (purple) (Matsukawa et al., 1997). When DPPH encounters
proton radical scavengers, its purple colour would fade rapidly (Yamaguchi et al., 1998). An
excellent scavenging capability on DPPH radicals at a dosage of 1 mg/ml (61%) was found with
the fucoidan fraction AF3 as compared with the control BHA (100%) regarding the low dosage
ranges used (Fig. 2.12). More significant and effective radical scavenging capability (90%) was
also found with the fraction AF3 at a
higher dosage (5 mg/ml).
Comparable results with the fraction
AF3 were found to be 62% at the
dosages of 5.0 mg/ml for the alginate
rich fraction B (Fig. 2.12). In
contrast, the glucan fraction C1F2
demonstrated least scavenging
capability (50%) among three
fractions at the concentration of 5.0
mg/ml. Moreover, the marked
inhibitory effect of these
polysaccharides on DPPH radicals was
found to be concentration dependent (Fig. 2.12), though the activities were low. These results
reveal that the polysaccharides of present study are potent scavenger and their antioxidative
activity may be attributed to their proton-donating ability (Shimada et al., 1992). Probably, these
fractions might contain a higher amount of reductone which could react with radicals to stabilise
and terminate radical chain reaction. Whether the structures can be related to scavenging
capacity for DPPH radicals remains to be determined in future studies. However, our preliminary
results suggest that different bioactivities of these fractions apparently may be, in some respects,
linked to their different molecular structures. Most of earlier data on antioxidants of foods and
biological samples were on phenolic compounds and many researchers have been reported
positive correlation between free radical scavenging activity and total phenolic compound. But
this correlation between structure and activity is not always valid. For example, selected
enzymatic extracts of seaweeds (Ecklonia cava and Sargassum coreanum) did not possess
41
antioxidative activity, although they contained as much phenolic compounds as the other extracts
of E. cava. Feruloyl oligosaccharides showed a higher antioxidative capacity than free ferulic
acid (Yuan et al., 2005). Moreover, the former showed greater antioxidative capacity in vivo than
in vitro when compared with vitamin C (Ou et al., 2007). Therefore, it is believed that other
materials in seaweed extracts, such as small molecular weight polysaccharides, pigments,
proteins or peptides, may influence the activity. Indeed, recent data showed that a number of
polysaccharide containing fractions isolated from various sources such as medicinal mushrooms
(Shimada et al., 1992; Jiang et al., 2008), higher plants (Aguirre et al., 2009), marine algae
(Ruperez et al., 2002; Rocha de Souza, 2007; Wang et al., 2008, 2009a) and even some
enzymatic extracts (Je et al., 2009) possess antioxidative activity. Our results are in agreement
with these studies. In addition, the polysaccharides used in this study were purified and,
therefore, conclusively prove their antioxidative potency. Literature data also shows that some
polysaccharides have higher antioxidative potency than the others. For example, the
antioxidative potential of sulfated polysaccharides from the brown seaweed Fucus vesiculosus
was higher than that of agar-like sulfated galactans from the red seaweed Nori (Ruperez, 2001).
In a more recent study this group (Ruperez et al., 2002) found that antioxidative potency of
fucoidan containing fraction from the brown alga Fucus vesiculosus was higher than that of
glucan and alginic acid containing fractions. The glucan (C1F2) and alginate (B) rich fractions of
present study exhibited lower antioxidative potentials than the fucoidan containing fraction AF3
(Table 2.3). This result is in agreement with that of (Ruperez et al., 2002) except that the
antioxidative potency of the purified polysaccharides of present study is lower. Although the
antioxidative capacity of polysaccharide has been proven, the relationships between structure and
antioxidative capacity have not yet been elucidated. This is primarily due to the interplay of two
important factors. Firstly, the huge structural diversity of these polysaccharides has given a
major hindrance in the structure–activity relationship establishment. However, on the basis of the
accumulated data several common structural motifs emerge that are important for activity.
Secondly, many of the hitherto used sulfated polysaccharides contained a number of other
molecules. These later molecules may have their own activities, and or at least dilute the efficacy
of the sulfated polymer itself. However, the polysaccharides described in this work are purified
and therefore, conclusively proves their antioxidative activities.
42
2-4. CONCLUSIONS
In this study, we found that the soluble polysaccharides of T. conoides possess antioxidative
property. The higher molecular mass fucoidan (50 kDa) exhibits stronger activity than the low
molecular mass glucan (5 kDa) and, therefore, this potency probably be directly related with the
molecular mass of these polysaccharides. These differences can as well be attributed to the
differences in structure. The antioxidative activity of these polysaccharides may be attributed to
their proton-donating activity as evidenced through DPPH radical scavenging results. Because
the polysaccharides tested in this study was basically prepared without toxic chemical reagents, it
can be assumed to be potentially useful as a safe antioxidants for food processing industries.
Furthermore, as the isolation of these polysaccharides involves a few inexpensive and easy steps
it will be of an added advantage.
3
STRUCTURAL FEATURES AND ANTITUSSIVE
ACTIVITIES OF A CARBOHYDRATE POLYMER FROM
Adhatoda vasica
44
3-1. INTRODUCTION
Cough is a protective reflex that is vital to remove foreign material and secretions from the
airways (Nasra & Belvisi, 2009). In the physiological conditions in healthy persons this reflex
serves its function appropriately. However, modulation of the cough reflex pathway can lead to
inappropriate coughing and an augmented cough response. Ineffective cough is associated with
respiratory morbidity such as recurrent pneumonia. However, chronic cough can be troublesome.
It impairs the quality of life in adults (French et al., 2002) and significantly worries the parents
of coughing children (Cornford et al., 1993). Therefore, cough is the most common condition for
which patients seek consultation from a doctor (Schappert & Burt, 2006). Codeine and
dextromethorphan are extensively used for the treatment of cough (Chung, 2009). But current
antitussives, such as opiods, have unwanted side-effects (Belvisi & Hele, 2009). Therefore, new
antitussive agents are needed. In this context, natural products scaffolds have been invaluable as
biologically validated platforms for drug development (Ramallo et al., 2011).
Adhatoda vasica, also known as Basak is a shrub belonging to the Acanthaceae family. This
small evergreen shrub has been used in traditional Indian medicine for more than 2000 years
(Wealth of India, 1988; Kapoor, 1990). Leaves of this shrub are the main source of drug
preparation. The juice from the leaves and a decoction or infusion of the leaves and roots, water
extracts or syrups are used as herbal remedy for asthma, bronchitis, and chronic coughs and
breathlessness (Amin & Mehta, 1959; Dhuley, 1999). Basak contains alkaloids of great interest
to researchers including vasicine, vasicinone, deoxyvasicine, vasicol, adhatodinine, vasicinol and
others (Claeson et al., 2000). Other constituents include the vitamins, saponins, flavonoids as
well as steroids, and fatty acids (Wealth of India, 1988). As for example, roots contain a steroid,
called daucosterol, carbohydrates and alkanes (Huq et al., 1967; Jain et al., 1980; Jain & Sharma,
1982). Flowers were shown to harbor a triterpene α-amyrin; flavonoids (astragalin, kaempferol,
quercetin, etc) and alkanes (Huq et al., 1967; Claeson et al., 2000). Two aliphatic
hydroxyketones were also isolated from the aerial parts of A. vasica, namely, 37-
hydroxyhexatetracont-1-en-15-one and 37-hydroxyhentetracontan-19-one (Singh et al., 1991).
Two new pyrroloquinazoline alkaloids [1,2,3,9-tetrahydropyrrolo(2,1-b-quinazolin-9-one-3R-
hydroxy-3(2'-dimethylamino phenyl (desmethoxyaniflorine) and 7-methoxy-3R-hydroxy-1,2,3,9-
tetrahydropyrrolo[2,1-b]-quinazolin-9-one (7-methoxyvasicinone)] were also isolated from A.
45
vasica and their structures were deduced using spectroscopic and X-ray diffraction analyses
(Thappa et al., 1996). Four quinazoline alkaloids anisotine, 3-hydroxy-anisotine, vasicolinone,
vasicoline and one isoquinoline alkaloid 1-phenyl-2-methyl-6,7-dimethoxy-1,2,3,4-
tetrahydroisoquinoline were also isolated from the leaves of A. vasica. Additionally
unsaponifiable fraction of petrol ether extract of leaves were shown to contain higher levels of
beta-sitosterol and tricontane, whereas saponifiable fraction contains higher levels of linolenic,
arachedonic, linoleic, palmitic and oleic acids (Abd El-Megeed Hashem & Ahmed Elsawi,
1998). A new triterpenoid, 3 α-hydroxyoleanane-5-ene was also isolated from the aerial part of
A. vasica (Sultana et al., 2005). Some of these compounds contribute to the observed medicinal
effect of this plant. For example, the alkaloid vasicine showed bronchodilatory activity both in
vitro and in vivo. Vasicinone showed bronchodilatory activity in vitro but bronchoconstrictory
activity in vivo (Atal, 1980). Notably, these compounds cannot be used during pregnancy.
However, there have been no reports on the high molecular weight bioactive compounds present
in A. vasica although plant extracts consisting of carbohydrate polymers exhibit a large range of
pharmacological activities (Inngjerdingen et al., 2006; Cumashi et al., 2007; Sutovska et al.,
2007, 2009; Mantovani et al., 2008; Ghosh et al., 2009a; Baek et al., 2010; Thakur et al., 2012).
The present study reports isolation and chemical characterization of a water extracted
polysaccharide isolated from A. vasica leaf. Using chemical and chromatographic methods we
have been able to deduce structural features of a pectic arabinogalactan. We have also
investigated the antitussive activity of this polysaccharide in terms of number of cough efforts on
the citric acid-induced cough reflex and the reactivity of the airway smooth muscle in vivo
conditions in guinea pigs.
46
Table 3.1. Sugar composition and protein content of the water extracted polysaccharides containing fraction (WE) isolated from A. vasica leaves, the arabinogalactan protein (AGP) and of fractions (F1 – F3) derived by size exclusion chromatography
WE AGP F1 F2 F3 Yielda - - 16 72 12 Total sugarb 49 ndd 51 49 47
Uronicacidb 7.3 ndd 7.6 7.2 6.9
Proteinb Rhamnosec
21 2
ndd 1
19 3
21 2
22 2
Arabinosec 29 32 29 30 30
Xylosec 1 tre 1 1 tre
Mannosec 1 1 1 tre tre
Galactosec 60 66 60 62 61
Glucosec 7 tre 6 5 7
apercent weight of total neutral sugar recovered. bpercent weight of fraction dry weight. cmol percent of neutral sugars. ndd, not determined. tre, trace.
3-2.CHEMICAL CHARACTERIZATION OF THE PECTIC ARABINOGALACTAN FROM Adhatoda vasica
3-2.1. Isolation and chemical composition
The objectives of this research were to analyze the water extracted polysaccharide generated
from the medicinal plant Adhatoda vasica and to study its antitussive activity. In Indian
Ayurvedic system of medicine a decoction of its leaves in water is used as herbal remedy for
chronic coughs and breathlessness (Amin, 1959), therefore fresh leaves of this evergreen shrub
were extracted with water.
The yield of the water extracted polymer (named as WE), after fractional precipitation with
ethanol, was 10mg per gram of fresh leaves (Fig. 3.1). The use of cold water, in principle, may
exclude the extraction of physiologically inactive starch, present in the leaves. Fraction WE
contains 49% (w/w) neutral sugar along with 21% (w/w) protein. The amino acid composition of
protein associated with
fraction WE showed that
glutamic acid / glutamine
(38.5%), alanine (13.3%),
serine (9.4%), and glycine
(6.5%) were the major
constituents. The uronide
content of this fraction is
7.3% (w/w). Thin layer
chromatographic analysis of
the monosaccharides present
in the hydrolysate indicates
Fig. 3.1. Scheme for the isolation and purification of carbohydrate polymers from Adhatoda vasica.
Water, (25-32)°C WE
Size Exclusion Chromatography
Sephacryl S-100
F1
F2
F3
47
Fig. 3.2. Elution profile of the water extracted polysaccharide obtained from Adhatoda vasica leaves on Sephacryl S-100 column with 500 mM sodium acetate buffer (pH 5.5) at 30 ml/h. Collected fractions were analyzed for total sugar content by phenol-sulfuric acid. Elution of polysaccharide was expressed as a function of the partition coefficient Kav [Kav = (Ve-Vo)/(Vt-Vo) with Vt and Vo being the total and void volume of the column determined as the elution volume of potassium hydrogen phthalate and dextran (500 kDa), respectively and Ve is the elution volume of the sample].
F2
F1
F3
the presence of an uronic acid with Rf values similar to that of galacturonic acid. GC analysis of
the TMS derivatives of the derived methyl glycosides confirmed this result, but it also shows the
presence of traces of glucuronic acid. Sugar compositional analysis revealed that fraction WE
consist mainly of arabinose and galactose as the major neutral sugar together with smaller
amount of glucose, rhamnose, mannose and xylose units (Table 3.1). Considering that the water
extracted polymeric fraction (WE) contained galactosyl and arabinosyl residues as the major
sugars, and its protein content is 21%, we have tested its reactivity with β-glucosyl Yariv reagent
that specifically precipitates AGPs. We found that the major part of WE was Yariv soluble.
Sugar compositional analysis of this precipitate (named AGP) shows that it consisted mainly of
galactose residues and, to a lesser extent, arabinose residues, confirming the presence of AGP
(Table 3.1). This material also contained mannose residues probably originating from N-glycans.
3-2.2. Size exclusion chromatography (SEC)
SEC of WE on Sephacryl S-100 yielded three
overlapping subfractions (F1, F2 and F3; Fig.3.2). The
yield and chemical composition of these subfractions
are given in Table 3.1. They had similar
monosaccharide compositions. The only difference
between these three samples, as judged by size
exclusion chromatography, seems to be the molecular
weight. Based on calibration with standard dextrans,
the apparent average molecular mass of
macromolecules present in subfractions F1, F2
and F3 would be 61, 42 and 9 kDa, respectively.
48
Table 3.2. Partially methylated alditol acetates derived from the water extracted polysaccharide fraction (WE) of A. vasica leaves
a 2,3,5-Ara denotes 1,4-di-O-acetyl-2,3,5-tri-O-methylarabinitol, etc. b Terminal-Araf denotes terminal arabinofuranosyl unit, etc. c Percentage of total area
of the identified peaks. trd = trace.
3-2.3. Linkage analysis
Methylation analysis of the polysaccharide from A. vasica yielded a variety of partially
methylated alditol acetates (Table 3.2). The results suggest that galactopyranosyl residues are
1,3- and 1,3,6-linked, whereas arabinofuranosyl units are 1,5- and 1,3,5- linked. Fig. 3.3 shows
EI-MS fragmentation pattern of selected molecules. The presence of 1,2- and 1,2,4-linked
rhamnopyranosyl residues was also indicated. This result suggests the presence of pectic
arabinogalactan. This fraction also contained 1,4-linked glucopyranosyl residues probably
Fig. 3.3. EI-MS fragmentation pattern of (1) 1,3,5 tri- O-acetyl -2,4,6-tri-O-methyl galactitol; (2) 1,3,5,6-tetra-O-acetyl -2,4-di-O-methyl- galactitol; (3) 1,4,5- tri-O-acetyl-2,3-di-O-methyl arabinitol; (4) 1,3,4,5 tetra-O-acetyl-2-O-methyl arabinitol of A. vasica.
(1)
(2)
(3)
(4)
50
3-3. ANTITUSSIVE ACTIVITY OF THE PECTIC ARABINOGALA CTAN FROM Adhatoda vasica
3-3.1. Assessment on chemically induced cough and airways defense reflexes
The effect of water extracted pectic arabinogalactan from A. vasica (WE) was evaluated on
the citric acid-induced cough reflex and reactivity of airways smooth muscle in vivo conditions.
For comparative purposes, codeine was simultaneously assayed as known reference compound.
The results of antitussive test showed that peroral administration of the pectic arabinogalactan in
a dose 50 mg kg−1 body weight brought about a significant decrease in the number of citric acid
induced cough efforts (NE) in adult healthy awaken guinea-pigs (Fig. 3.4). The first statistically
significant result was observed within 30 min after application. Furthermore, this positive effect
was observed during all the study time intervals. Notably, the suppression of cough efforts by the
polymer was quantitatively similar to that of codeine. Airway resistance is a concept used in
respiratory physiology to describe mechanical factors which limit the access of inspired air to the
pulmonary alveoli, and thus determine airflow. It is dictated by inter alia, the diameter of the
airways.
At present the relationship between cough and bronchoconstriction is not known with
**
**
** **
0
1
2
3
4
5
6
7
8
9
N 30 60 120 300 min
Nu
mb
er o
f co
ug
h e
ffo
rts
(NE
) control
codeine
WE
**
***
**
***
**
***
**
***
Fig. 3.4.The influence of the pectic arabinogalactan from Adhatoda vasica (WE), codeine (positive control) and saline water (negative control) on the citric acid-induced cough efforts (NE) in guinea-pigs recorded at 30, 60, 120 and 300 min time intervals. All used substances were applied by peroral route of administration: plant polysaccharides in the dose of 50 mg kg-1, codeine in the dose 10 mg kg-1and saline water in the dose 1 ml kg-1body weight. N, Initial values before application of the polysaccharides and codeine. Statistical significance
51
N 30 60 120 300 min0
5
10
15
20
25
R x
V (cm
H2O
s-1) Contr
Codeine
WE
certainty. Although it is generally accepted that bronchodilating substances can cause cough
suppression. Therefore, we have evaluated the changes of specific airway resistance as indictor
of this activity. Our result suggests that the application of arabinogalactan from A. vasica in the
dose which provoked cough suppressive activities did not significantly change the values of
specific airway resistance (Fig. 3.5). It also showed that after 30 min from application of
polysaccharide sample a slightly decreased value of specific airway resistance is registered.
Notably, in this time interval application of codeine provoked the same result. Recent studies
showed that several naturally occurring polysaccharides possess antitussive activity (Nosál’ova
et al., 2005; Sutovska et al., 2007, 2009a; Saha et al., 2011; Sinha et al., 2011b). The most potent
antitussive activity is observed with polysaccharide containing the highest proportion of uronic
acid constituent (Sutovska et al., 2007), although neutral polysaccharides may also have
significant activity (Nosál’ova et al., 2005). Therefore, the fact that the pectic arabinogalactan
from A. vasica modulates chemically induced cough reflex and specific airway resistance is not
surprising.
3-3.2. Mechanism of action
The mechanism behind the antitussive activity of the pectic arabinogalactan of present study
is not known, but many antitussive herbs probably work through an antispasmodic action or as
bronchodilator (Ernst, 1998; Pavord & Chung, 2008). They cause bronchial muscle relaxation in
Fig. 3.5. The influence of water extracted polysaccharide from the medicinal plant Adhatoda vasica and control agents (vehicle and codeine) on citric acid induced changes of specific airway resistance (R x V) in vivo conditions, registered before any agent application (values labelled as N in graphs) and after that in 30, 60, 120 and 300 min time intervals.
52
vitro, or decrease airways resistance in vivo. The arabinogalactan of present study possesses very
high cough suppressive effect and decreases the values of specific airway resistance in vivo
conditions only slightly. Pavord (2004) reported that bronchoconstriction causes or enhances the
sensitivity of cough, while bronchodilation does the opposite. However, the role of other
mechanism including bioadhesive effect of the polysaccharide to the epithelial mucosa (Sutovska
et al., 2009a) cannot be ruled out.
3-4. CONCLUSIONS
This study represents the first account on the in vivo antitussive activity of the water extracted
polysaccharide and its structural features. Biological investigations indicated that the pectic
arabinogalactan of A. vasica displayed promising activity in the antitussive assays. Moreover, as
the macromolecule tested in this study was basically isolated without toxic chemical reagents, it
can be assumed to be potentially useful as a safe antitussive agent for industries. Furthermore, as
the isolation of these polysaccharides involves few inexpensive and easy steps it will be of an
added advantage. Finally, the biological activity observed in A. vasica provides a scientific basis
for the use of the plant in traditional medicines.
EXPERIMENTAL SECTION
54
4
MATERIALS AND
METHODS
55
4-1. PLANT MATERIALS AND EXTRACTION OF POLYSACCHARI DES
4-1.1.Isolation of fucoidan, alginic acid and glucan from Turbinaria conoides by
sequential extraction with inorganic solvents
Plant material and preliminary treatments. Samples of Turbinaria conoides were collected
from Okha coast of Gujarat, India, in August 1995. The collected seaweeds were washed
thoroughly with tap water and dried by forced air circulation, followed by pulverization in a
Waring Blender.
Preparation of depigmented algal powder (DAP). After pulverization in a commercial grinder,
Depigmentation of algal powder (270 g) was done using sequential extraction with petroleum
ether (24 h) and acetone (24 h) as solvent in a Soxhlet apparatus. The unextracted material was
placed in a plastic beaker, and air dried to obtain depigmented algal powder (DAP, 173 g).
Extraction with acid. Extractions of DAP (3 g) with 0.1 M HCl (w/v::1:100) were conducted at
30–35°C for 12 h under constant stirring (twice). Separation of the residue from the extract was
performed by filtration through a glass filter (G-2). The residue was washed with additional 0.1
M HCl and the wash was collected to maximize polysaccharide recovery. The extracts were
combined, dialyzed and lyophilized to give fraction A (yield, 8.8%).
Extraction with alkali. The acid insoluble residue was extracted with 3% Na2CO3 (w/v::1:100) at
45–50°C for 4 h under constant stirring (twice). The extract was carefully acidified with
concentrated HCl to pH~1, the precipitate formed was collected by centrifugation, washed with
0.1 M HCl, suspended in water and dissolved by careful addition of NaOH. The slightly alkaline
solution was dialyzed and diluted with 4.0 M CaCl2 solution to make a final concentration of 2%
CaCl2. The precipitate formed at this stage was isolated by centrifugation, washed with water and
treated with 0.1 M HCl (4 x 50 ml, stirring at room temperature for ~2 h). Then the precipitate
was dissolved in dilute NaOH as above, the solution dialyzed and finally lyophilized to yield
sodium alginate (B, yield, 22.6%).
Extraction with water. The residue left after extraction with alkali was once again extracted with
water at 80°C (twice) and the combined extract (C) dialyzed. Then 4.0 M CaCl2 solution was
added to the extract to make a final concentration of 2% CaCl2, and the suspension kept at 5–9°C
overnight. After separating the precipitate (C2) by centrifugation the supernatant dialyzed,
56
concentrated and lyophilized to give fraction C1 (yield, 6.2%). The resulting insoluble residue
was dialyzed against distilled water and finally lyophilized (INS).
4-1.2.Isolation of polysaccharides from Adhatoda vasica
Plant material and preliminary treatments. Leaves of Adhatoda vasica were collected from the
garden of medicinal plants, The University of Burdwan, West Bengal, India. Collected leaves
(10 g) were washed thoroughly with tap water and then blended with water (800 ml) in a mixer
(Waring Products, Inc., Torrington, CT, USA).
Extraction of polysaccharides. Extraction of polysaccharide was conducted by stirring a
suspension of this paste in water (pH 6.0) at 25–32°C for 12 h. Separation of the residue from the
extract was performed by filtration through a glass filter (G-2). The insoluble material was
extracted twice more under similar condition at a solute to solvent ratio of 1:100 (w/v). The
combined liquid extract was dialyzed extensively against water and lyophilized. The recovered
material was dissolved in water, precipitated by the addition of ethanol (4 volumes) and then
collected by centrifugation (repeated three times). The final pellet was dissolved in water and
lyophilized to yield the water extracted polysaccharide, named WE (100 mg).
Isolation of arabinogalactan protein (AGP) with β-glucosyl Yariv reagent. AGP was isolated
according to Schultz et al., 2000. Briefly to a solution of WE in 1% NaCl (w/v) was added an
equal volume of β-glucosyl Yariv reagent also in 1% NaCl. The mixture was kept at 4°C for 18 h
and then centrifuged. The pellet was washed with 1% NaCl followed by pure methanol (3 times
each), dried and treated with sodium metabisulfite (10%). The resulting solution was dialyzed
and freeze dried to yield the arabinogalactan protein (AGP).
4-2. ANALYTICAL METHODS
4-2.1. General
All the experiments were carried out at least in duplicate, the mean and standard deviation
was directly calculated from the functions present in excel program. Evaporations were executed
under reduced pressure at around 45°C. Dialysis was performed against distilled water with
continuous stirring, toluene being added to inhibit microbial growth. During experiments,
moisture was determined by drying ground material in an air-circulated oven at 110°C for 3
hours. Chemicals used were analytical grade or best available. Except otherwise stated data
57
presented are the mean values of at least three independent analyses. When the standard
deviation was greater than 5%, experiments were repeated.
4-2.2. Sugar analysis
Total sugars were estimated by the phenol-sulphuric acid method (Dubois et al., 1956).
Evaluation was corrected afterwards, depending on sugar composition. Total uronic acids
present, were assayed using m-phenyl phenol color reagent (Ahmed & Labavitch, 1977). For
measurement of individual neutral sugar polysaccharides (10-12 mg) were hydrolyzed with 2 M
trifluoro acetic acid (2 h, 100°C).For the insoluble residues, this hydrolysis was followed by a
treatment with 72% (w/w) H2SO4 for 1 h at room temperature and after that, with 1 M H2SO4 for
2 h at 100°C. Myo-Inositol was used as internal standard, and hydrolytic losses were accounted
for by using an external standard (Chattopadhyay et al., 2007b). Monosaccharides were reduced
with sodium borohydride in anhydrous dimethyl sulfoxide and after reduction, the excess sodium
borohydride was decomposed by the addition of acetic acid. Acetylation was conducted by acetic
anhydride and 1-Methylimidazole was added as catalyst (Blakeney et al., 1983). Derived alditol
acetates were analysed by GC (Blakeney et al., 1983) on columns of 3% SP-2340 on
Supelcoport 100–120 mesh, and DB-225 (JW) and by GC-MS (Ghosh et al., 2009b; Ghosh et
al., 2010) or alternatively, trimethylsilyl (TMS) derivatives of methyl glycosides were analysed
by gas chromatography.
4-2.3. Protein estimation
Proteins present in soluble fractions were estimated by the method of Lowry et al., 1951 using
bovine serum albumin as standard. In the insoluble residues it was obtained by estimated the
total nitrogen and multiplying the value with 6.25.
Alternatively, protein was measured in the soluble material by the micro-Bradford method
using bovine serum albumin as standard, and was detected in some chromatography fractions by
the absorbance at 280 nm. Amino acids were released by hydrolysis with 6 M HCl at 110°C for
22 h in a sealed tube. Protections were done for cysteine, methionine and tyrosine using proper
protecting reagents. The liberated amino acids were analyzed by Pharmacia LKB ALPHA PLUS
amino acid analyzer.
58
4-2.4. Sulfate estimation
Turbidometric method. Samples (90 - 100 mg) were hydrolyzed in of 2 N hydrochloric acid (5
ml) in sealed glass tubes at 100oC (Chattopadhyay et al., 2010). Sulfate was then estimated using
a modified turbidometric barium chloride method (Craigie et al., 1984). The reagent was
prepared by dissolving 600 mg of gelatin in 200 ml of distilled water at 70oC and the solution
was allowed to cool. After 16 h at 4oC the temperature was brought to 30 – 35oC and 2.0 g of
BaCl2 .2H2O was added and then dissolved. AR grade K2SO4 was used here as a standard in the
range of 10 – 50 µg S ml-1. Accurately weighted samples (25-50 mg) were hydrolyzed for 2 h at
100oC with 2N HCl (0.75 ml) in a sealed tube. After that, the contents were transferred and made
to volume in a 10 ml volumetric flask. The humic substances were removed by centrifugation. 2
ml of sample, 18 ml of distilled water and 2 ml of HCl (0.5 N) was mixed in a 50 ml Erlenmeyer
flask. 1 ml of BaCl2-gelatin reagent was added to that and swirled. After 30 minutes swirling
again mixed the contents of the flasks and turbidity was measured at 550 nm against a reagent
blank.
Spectroscopic method. The sulfate content was measured according to the method of Rochas &
coworkers (1986) by considering the ratio of the intensity of the 1252 cm-1 band to the intensity
of the 2920 cm-1 band.
4-2.5. Desulfation
Desulfation of the purified fucoidan (AF3) obtained from T. conoides was carried out
according to the procedure of Falshaw & Furneaux (1998). The pyridinium salt of the sample (1
g) was dissolved in DMSO containing 10% MeOH, and then the solution was kept for 3 h at
100oC. The reaction mixture was cooled, diluted with an equal volume of water, and the pH
adjusted to 9.0 by the addition of sodium hydroxide (0.1 M). The solution was dialyzed against
running water for 20 h and after that lyophilization of the dialyzate gave desulfated sample.
4-2.6. Linkage analysis
Methylation was carried out using the method of Blakeney & Stone (1985) as described by
Adhikari & coworkers (2006). Exactly weighed (1-2mg) of the dried samples along with
magnetic bars were taken in tubes fitted with air tight rubber corks. The nitrogen atmosphere was
maintained within the tubes. The samples were dissolved in DMSO and to this solution, lithium
methyl carbanion were added. The mixture was then stirred in nitrogen atmosphere for 1 hr. The
tubes were cooled under ice bath and ice cold methyl iodide (0.4 ml) was added to it. After that
59
the tubes was left for 1 hr at room temperature. The methylation process was repeated once more
and excess methyl iodide was removed under vacuum. The resulting methylated polymer was
then extracted with chloroform-methanol [2:1 (v/v)]. The permethylated polysaccharide thus
obtained was hydrolyzed with trifluoro acetic acid (2.5 M) at 120°C for 75 min, reduced with
NaBD4(1 M) in NH4OH (2 M) for 3 h at room temperature and acetylated with acetic anhydride
using perchloric acid as a catalyst. The partially methylated alditol acetates (PMAA) were then
analyzed by GC and GC/MS as described by Sinha et al., 2010. The PMAA were identified by
(i) measurement of relative retention times (ii) methoxyl substitution pattern as obtained from
GC-MS and (iii) carbohydrate composition of the non methylated polymers.
4-2.7. Chromatography
Thin layer chromatography (TLC). The sugars released by acid hydrolysis (as described above)
were also analyzed by TLC as described by Ray, 2006. Briefly, TLC was then conducted by
ascending technique using silica gel G impregnated with 0.5M NaH2PO4 as the supporting
material. Small amounts of samples were spotted in the plate for identification. The Rf values of
the test material were compared to that with the standard compounds to get an idea about the
components present and their purity. The color of the spot developed also conveys message
about its components.
The solvent system used for developing TLC was the following: (1) Ethanol: Phenol: Pyridine:
0.1 M Phosphoric acid :: 10 : 2 : 2 : 4 and (2) 2-Propanol: Methanol: Water :: 16 : 1 : 3
Detection: By spraying saturated solution of aniline phthalate and heating the plate at 100°C.
Size exclusion chromatography (SEC). SEC was performed as described by Chattopadhyay et
al., 2007a. In brief, solutions (1-2 ml) of different extracts in 0.5 M sodium acetate buffer (pH
5.0), were loaded to columns (i) (2.6 x 50 cm; Amersham Biosciences AB) of Superdex-30 (for
C1 solution of T. conoides), (ii) (90 cm x 2.6 cm, Amersham Biosciences AB) of Sephacryl S-
200 (for the solution of the fucoidan fraction (AF3) of T. conoides ) and (iii) (90 cm x 2.6 cm,
Amersham Biosciences AB) Sephacryl S-100 (for the solution of water extracted polymer (WE)
solution of A. vasica ) equilibrated with the same buffer. The column was then eluted
ascendingly with the same buffer at 0.5 ml min-1 at a temperature of 30–35°C. Elution of
polysaccharide was expressed as a function of the partition coefficient Kav [Kav = (Ve-V0)/(Vt-
V0) where, Vt and V0 are the total and void volume of the column determined as the elution
60
volume of glucose and blue dextran, respectively and Ve is the elution volume of the sample].
Here, the column was calibrated with standard dextrans.
Anion Exchange Chromatography (AEC). A solution (20 ml) of dilute acid extracted polymer
from T. conoides (fraction A) in 50 mM sodium acetate (pH 5.5) was applied to a column (2.6 x
25 cm) of DEAE-Sepharose FF (AcO-1). After that, the column was eluted (0.6 ml min-1)
successively with 0.05 M (fraction AF1), 0.7 M (fraction AF2) and 2.0 M (fraction AF3) NaOAc
buffer in a stepwise manner. Appropriate fractions were pooled, dialyzed and then lyophilized.
Gas chromatography (GC).GC was conducted on (i) GC-17A, Shimadzu and (ii) HP6890 series
Chromatograph.
The gas chromatograph was equipped with a flame ionization detector, the following columns
(a – d) and helium as gas vector.
(a) WCOT fused silica capillary column (length 25 m, i.d. 0.25 mm) and film thickness 0.4µm
with CP-Sil 5 CP as stationary phase and helium as gas vector, (b) 3 % SP-2340 on Supelcoport
100-120 mesh, (c) DB-225 (JW) and (d) SGE BP 225.
The oven temperature program was:
(a) 210°C isothermal or (b) 2 min at 120°C, 10°C/min to 160°C, and 1.5°C/min to 220°C and
then 20°C/min to 280°C or (c)170°C for 15min, 170-210°C at 5°C/min and 210°C for 15 min.
Gas liquid chromatography–Mass Spectrometry (GC-MS): The GC-MS analysis were conducted
using (a) Shimadzu (Japan) GC 17A coupled to QP 5050A MS and (b) GC (HP 6890) coupled to
an Autospec (Micromass, Manchester, UK) MS.
The mass spectra were recorded with MS instrument at 70 eV.
4-2.8. Spectroscopy
NMR Spectroscopy. The 1H NMR spectra of the polysaccharide containing fractions of T.
conoides were recorded on a Bruker 500 spectrometer (Bruker Biospin AG, Fallanden,
Switzerland) operating at 500 MHz, respectively, for 1H. The samples (~10 mg of each) were
heated at 80°C for 30 min with water (1 ml), centrifuged and the resulting supernatant
lyophilised. The freeze-dried sample was deuterium-exchanged by lyophilisation with D2O and
then examined in 99.9% D2O.
61
Scavenging effect (%) =
UV-VIS spectroscopy. UV-VIS spectra were recorded with Shimadzu UV- 1600A
spectrophotometer.
Infra-Red spectroscopy. IR spectra were obtained on a FT-IR spectrophotometer (JASCO FT/IR
- 420) using KBr discs containing finely ground samples.
Mass spectrometry. EI mass spectra were recorded using Shimadzu QP5050A GC/MS
instrument at 70 eV. The characterization of sugars, including 6-O-methyl galactose, was done
on the basis of previously reported mass spectra (Carpita & Shea, 1988) and relative retention
times as described by Ray & Lahaye, 1995.
4-3. BIOASSAY
4-3.1. FRAP assay
The FRAP assay was conducted according to Benzie & Strain, 1996 as modified by Pulido et
al., 2000. Briefly, the oxidant was prepared by mixing 2.5 ml of a 10 mM TPTZ [2,4,6-tri(2-
pyridyl-5-triazine) Fluka Chemicals, Madrid, Spain] solution in 40 mM HCl with 25 ml of 0.3 M
acetate buffer (pH 3.6) and 2.5 ml of 20 mM FeCl3.6H2O. The final solution has Fe(III) of 1.67
mM and TPTZ of 0.83 mM. To measure FRAP value, 900 µl of freshly prepared FRAP reagent
was warmed to 37°C and a reagent blank reading was taken at 593 nm; then 30 µl of test sample
and 90µl of distilled water were added. Absorbance readings were taken after 0.5 s and every 15
s until 30 min using a Shimadzu UV-1601 (PC) Spectrophotometer. The change of absorbance
(DA = A30 min_ A0min) was calculated and related to DA of an Fe(II) standard solution. Aqueous
solutions of known Fe(II) concentrations (100–2000 µM FeSO4.7H2O) were used for calibration.
4-3.2. Scavenging capability for 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals
The method reported by Shimada et al., 1992 was adopted for measurement of free radical
scavenging capability. To each 4 ml of sample solution, 1 ml of freshly prepared methanolic
DMSO solution of DPPH (0.5 mM) was added, mixed well and then let stand for 30 min at room
temperature in the dark. The absorbance of the resulting was recorded at 517 nm. Butylated
hydroxyanisole (BHA) was used as reference compound. The capability to scavenge the DPPH
radical was calculated using the following equation:
x100
62
4-3.3. Antitussive activity of the arabinogalactan from A. vasica
Animals. Animals used in this experiment were kept in faculty animal house, Department of
Experimental Pharmacology, Slovak Academy of Science, Dobra Voda, Slovakia, with food and
water ad libitum and with a standard air conditioning system. The animals were kept one week in
quarantine before starting the experiment. The experimental protocols were approved by the
Institutional Ethics Committee of the Jessenius Faculty of Medicine, Comenius University in
00005636), complied with Slovakian and European Community regulations for the use of
laboratory animals and follow the criteria of experimental animal’s well fare.
Assessment of chemically induced cough and airways defense reflexes. Awaken guinea-pigs
were individually placed in a body plethysmograph box (HSE type 855, Hugo Sachs Elektronik,
Germany) and restricted so that the head protrudes into the nasal chamber and the neck were
sealed with a soft diaphragm. The cough reflex was induced by aerosol of citric acid in a
concentration 0.3 M. The citric acid aerosol was generated by a jet nebulizer (PARI jet nebulizer,
Paul Ritzau, Pari-Werk GmbH, Germany, output 5 l s−1, particles mass median diameter 1.2µm)
and delivered to the head chamber of the plethysmograph for 3min interval, in which number of
cough efforts was counted. The cough effort was defined as sudden PC-recorded enhancement of
expiratory flow associated with typical cough motion and sound followed by trained observer
(Sutovska et al., 2009a).The reactivity of the airway smooth muscle in vivo conditions was
expressed as values of specific airway resistance calculated according to Pennock et al., 1979 by
time difference between pressure changes in head and chest parts of body plethysmograph during
normal breathing pattern. Both, influence on citric acid-induced cough and specific airway
resistance were registered before any agent application (values labeled as N in graphs) and after
that in 30, 60, 120 and 300 min time intervals. The minimal time interval between two
measurements was 2 h to prevent cough receptors adaptation as well as adaptation of laboratory
animals on kind of irritation. All tested compounds (polysaccharide, codeine and vehicle) were
applied by peroral route of administration, plant polysaccharides in the dose of 50mgkg−1,
codeine in the dose 10 mg kg−1 and saline water (vehicle) in the dose 1ml kg−1 body weight.
Statistics. Student’s t-test was used for the statistical analysis of the obtained results. Data are
presented as mean±standard error of the mean (S.E.M.). p< 0.05 was considered statistically
63
significant. Significance of p < 0.05; p < 0.01 and p < 0.001 is shown by one, two or three
asterisks, respectively.
64
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The novel findings presented in this thesis are:
1. Three families of water soluble polysaccharides, namely, sulfated fucan, alginic acid and
glucan can be easily isolated from the marine alga Turbinaria conoides.
2. A 50 kDa sulfated fucan containing inter alia, α-(1→3)-linked fucopyranosyl residues with a
sulfate group at the position 4, has been identified.
3. The alginic acid contains equimolar proportions of guluronic and mannuronic acid residues.
The guluronic acid content (G %) and G–G diad frequency (GG %) of this polymer are 46.2 and
42.1, respectively.
4. A 5 kDa linear glucan containing β-(1→3)-linked glucopyranosyl residue has also been
isolated.
5. These polymers T. conoides from demonstrated dose dependent antioxidative activity. The
fucoidan showed the highest antioxidative activity followed by the alginate and glucan.
6. The decoction of Adhatoda vasica contains a highly branched arabinogalactan.
7. This branched arabinogalactan containing 1,3- and 1,3,6-linked galactopyranosyl & 1,5- and
1,3,5- linked arabinofuranosyl residue, possesses potent in vivo antitussive activity.
8. Peroral administration of this arabinogalactan (50 mg kg−1 body weight) from A. vasica
inhibited the number of coughs induced by citric acid in guinea pigs and slightly decreased the
values of specific airway resistance.
9. Thus, traditional aqueous extraction methods provide polysaccharides, which stimulate an
intense biological response. This could represent an interesting approach in phytotherapeutic