JASMR, 5(2): 141-152 (2010)

http://www.asmr.eg.net

PHYTOCHEMICAL AND BIOLOGICAL STUDIES OF

SOME POLYSACCHARIDES ISOLATED FROM ALOE,
TAMARINDUS, OPUNTIA AND CITRUS
Nagwa M Ammar 1, Abdel Nasser B Singab 2, Sherweit H El-Ahmady 2,
Amira A El-Anssary 1, Eman G Haggag 3 and Rania S Shabban 4*
Pharmacognosy Departments of National Research Center1, Faculty of Pharmacy, Ain Shams
University2, Faculty of Pharmacy, Helwan University3, and Faculty of Pharmaceutical Sciences and
Industries, Future University4, Cairo, Egypt.
*Corresponding author e-mail: rania_samir@hotmail.com.

ABSTRACT
Objectives: The aim of this study is the phytochemical and biological evaluation of polysaccharides isolated from some
medicinal plants belonging to the Genera Aloe, Tamarindus, Opuntia and Citrus. Antihyperglycemic, anti-inflammatory,
immunomodulatory and antioxidant of the polysaccharides were studied. Extraction, isolation and identification of the
biologically active constituents have been carried out aiming at discovery of new active constituents from botanical origin which
can be utilized in drug industry.
Methods: The Polysaccharides from the plants under investigation were isolated using cold and hot extraction methods then
hydrolysed, the polysaccharide hydrolysates were identified by Paper and HPLC chromatographic techniques. The isolated
polysaccharides were tested for their anti-inflammatory, antioxidant, hypoglycemic and immunomodulating activities. The
optimum parameters for the extraction of polysaccharides isolated from the seeds of Tamarindus indica Linn. Family
Leguminosae were examined by using three factor statistical techniques.
Results: Maximum yield of polysaccharides were obtained from Tamarindus indica Linn. seeds (21%), which was increased by
varying temperature and time (55%). The results of the biological activities revealed significant antihyperglycemic,
immunomodulating, anti-inflammatory and antioxidant activities with variable degrees. Chromatographic investigation of the
polysaccharide hydrolysates revealed the presence of hexoses, pentoses and uronic acids with qualitative and quantitative
variations in the chemical composition.
Conclusion: Tamarindus indica Linn. seeds had the highest yield of polysaccharide and the highest significant biological
activities.
Keywords: Polysaccharides, Antiinflammatory, Antioxidant, Hypoglycemic, Immunomodulatory.

fungal, anti-diabetic and anti-neoplastic

INTRODUCTION
Over the last 20 years there has been an
ever increasing interest in the biological
activity of biomolecules. Polysaccharides
widely exist in plants, microorganisms, algae,
and animals are essential biomacromoleules in
life activities and play important roles in cell–
cell communication, cell adhesion, and
molecular recognition in the immune system(1).
Recently, some bioactive polysaccharides
isolated from natural sources have attracted
much attention in the field of biochemistry and
pharmacology; especially plant poly-
saccharides have shown diverse biological
activities such as wound healing, enhancement
of the reticuloendothelial system, stimulation
of the immune system, treatment of tumors and
effects on the hematopoietic system (2). In folk
medicine plants containing polysaccharides
have been used as hypoglycemic (3,4) and anti-
inflammatory (5, 6).
It was claimed that the polysaccharides in
Aloe vera L. gel had therapeutic properties
such as immunostimulation, anti-inflammatory
effects, wound healing, promotion of radiation
damage repair, anti-bacterial, anti-viral, anti-
activities as well as stimulation of
hematopoiesis and anti-oxidant effects (7, 8, 9).
Animal and human studies have demonstrated
the hypoglycemic properties of the Opuntia
cactus where a reduction in serum glucose
levels was best observed when the cactus
cladodes were consumed in cooked form,
alluding to heating as a necessary step in
attaining its hypoglycemic properties (10, 11).
Other reports showed antioxidant,
anti-inflammatory and immunomodulatory
activities of Opuntia ficus indica Mill. fruits
and cladodes (11, 12). In a recent study, the
aqueous extract of the seeds of Tamarindus
indica L. was found to have potent
hypoglycemic activity that reduces blood
glucose level in streptozotocin-induced
diabetic male rat (13). Polysaccharides isolated
from Tamarindus indica Linn. seeds showed
significant immunomodulating effects (14).
While the pectin isolated from Citrus paradisi
Mac. Fady peels was shown to exert a
favorable effect on a wide spectrum of
pathological conditions, their positive
influence on human health has been explained
by their anti-oxidative, hypocholesterolemic,
anti-cancerous and immunomodulating
effects(15).
 Phytochemical and Biological Studies
The aim of this study is the performance
of phytochemical and biological evaluation of
polysaccharides isolated from the seeds of
Tamarindus indica Linn. Family Leguminosae,
dried leaves of Aloe vera L. Family Aloaceae,
fruits of Opuntia ficus indica Mill. Family
Cactaceae and fruit endocarp of Citrus
paradisi Mac. Fady. Family Rutaceae. The
work included studying the effect of
polysaccharides as antihyperglycemic, anti-
inflammatory, immunomodulatory and
antioxidant. Extraction, isolation and
identification of the biologically active
constituents have been carried out aiming at
discovery of new active constituents from
botanical origin which can be utilized in drug
industry.
MATERIALS AND METHODS
Plant Materials
Samples of the whole leaves of Aloe vera
L. Family Aloaceae, the fresh mature prickly
pear fruits of Opuntia ficus-indica Mill. Family
Cactaceae, the fruits of Citrus paradisi
Mac.Fady. Family Rutaceae and the seeds of
Tamarindus indica Linn. Family Leguminosae
were collected from the Egyptian market.
All plant samples under investigation were
authenticated by Dr. Tereiz Labib Youssef
Consultant of Plant Taxonomy at Alorman
Garden, Giza, Egypt. A voucher specimen was
deposited in the National Research Centre
Herbarium.
Materials for Biological investigation
Animals
Adult Wistar strain male albino rats were
used for the screening of the
antihyperglycemic activity, weighing 130 ± 10
g and housed in colony cages (five rats per
cage), at an ambient temperature of 25 ± 2 ◦C,
while to investigate the immunomodulatory
activity male Swiss mice (20-25 g) were used.
As for the anti-inflammatory activity test,
female Sprague Dawley rats were used in this
study.
Chemicals
Carrageenan, type IV (Sigma, USA) was
freshly prepared for induction of acute
inflammation and alloxan freshly dissolved in
saline was used for induction of diabetes.
While to investigate the antioxidant activity D,
L α-tocopherol, linoleic acid, L-ascorbic acid,
ammonium thiocyanate and ferrous chloride
were purchased from Sigma (USA).
Methods of phytochemical investigation
Extraction of polysaccharides (16)
The Leaves, fresh fruits, seeds and peels of
Aloe vera, Opuntia ficus indica, Tamarindus
indica and Citrus paradisi were respectively
142
dried in an electric oven at a temperature not
exceeding 40 oC, and then ground on an electric
grinder to be obtained in a dry powdered form ,
and then the former three plants were extracted
by cold water and hot water extraction methods
using acidified distilled water to obtain the
mucilage that was precipitated from the
combined aqueous extract by adding slowly
while stirring four volumes of ethanol 95 %.
The precipitate obtained by centrifugation was
washed several times with ethanol till free
from chloride ions. The mucilage was then
vigorously stirred in absolute acetone, filtered
and then dried in a vacuum desiccator over
anhydrous calcium chloride.
The extraction of pectin was carried on
the marc left after extracting the mucilage as
mentioned before in case of the three
previously mentioned plant organs or directly
on the dried powdered peels of Citrus paradisi
as it contains only pectic polysaccharides with
no mucilage (15), primarily that was done using
distilled water, 0.3% ammonium oxalate
solution and 0.05 N hydrochloric acid solution
respectively with occasional stirring, then
filtered through cloth and to the combined
pectin solution resulted from the above three
treatments, 1.5 volumes of 95% ethanol
acidified to pH2 with dilute hydrochloric acid
were added while stirring by hand to
precipitate the pectin. The mixture was
allowed to stand for 30 minutes to allow the
pectin to float on the surface, most of the
pectin free solvent could be separated by
siphoning. The precipitate was strained by
muslin and pressed by hand. The pressed cake
was disintegrated in ethanol and repeatedly
washed with the same solvent until the pectin
became free from chlorides (AgNO3 test). The
precipitate was then pressed on cloth, dried in
a vacuum desiccator over anhydrous calcium
chloride.
The dried polysaccharide (mucilage and
pectin) precipitate isolated from the different
plant organs under investigation were tested
using KOH (17), Ruthenium red (18) and subjected
to gel formation test to confirm their nature (19,
20)
, and separately saved for phytochemical and
biological investigation.
Acid hydrolysis (21).
Ten mg of each of the mucilage and pectin
of each plant organ under investigation were
separately heated in 2 ml of 0.5 M sulphuric
acid in a sealed test tube for 20 hours in a
boiling water bath. At the end of the hydrolysis
a flocculent precipitate was noticed in all
cases. This was filtered off and the filtrate was
freed of sulphate ions by precipitation with
barium carbonate. The hydrolysates were
separately concentrated under vacuum at a
 Nagwa M Ammar et al
temperature not exceeding 40 oC to a syrupy
consistency. It was diluted with 10 %
isopropanol in water to about 10 ml., then the
hydrolyzed samples as well as individual
authentic reference sugars were dissolved in
distilled water, then filtered through
Whatmann filter paper no. 1 and microfilter
(0.45µm) and stored in vials to be used in
Paper and HPLC chromatographic
investigations.
Paper chromatographic analysis (22)
After dissolving the residue of the
hydrolysates of each sample of the isolated
pectin and mucilage of each plant organ under
investigation as well as the reference sugars in
10 % isopropanol / water then spotting on
Whatmann no. 1 sheets the spots were
chromatographed adopting the descending
technique for 18 hours, using the following
system (n-Butanol: acetic acid: water) (4: 1: 5)
(v/v/v). The chromatogram was visualized by
spraying with aniline phthalate reagent and
heating in an oven for 5 min. at 110 0C.
HPLC analysis:
HPLC analysis was used to determine the
free sugars in the isolated polysaccharide from
the different plant organs under investigation
qualititavely and quantitavely. The analysis
was performed on operating system model
Shimadzu (SCL-10AVP) equipped with RI
detector RID-10A and high pressure pump LC-
10ADVP. Separation and determination were
performed on Shodex column sugar SC1011
using distilled water as mobile phase with 1 ml
/ min flow rate.
Methods for biological investigation
Screening of the antihyperglycemic activity
The oral glucose tolerance test was
performed on normoglycemic rats fasted for 15
h. A single dose of saline (control), Aloe vera,
Citrus paradisi, Tamarindus indica and
Opuntia ficus indica isolated polysaccharides
were then orally administered to groups of 5
rats at two dose levels (250 and 500 mg/Kg
b.wt). Thirty minutes later, glucose (1.25 g/Kg
b.wt) was orally administered to each rat.
Blood samples were obtained from each rat by
sinocular puncture under light ether anesthesia
at -30 (before drug administration), 0 (just
before glucose administration, 30, 60, 90 and
180 min. for determination of glucose
according to commercial kits.
For induction of diabetes, rats were given
an intraperitoneal injection of alloxan, freshly
dissolved in saline (100 mg/ml) at a dose of
120 mg/kg b.wt., so as to induce diabetic state
with blood glucose levels > 250 mg/ml. At this
dose of alloxan (120 mg/kg b.wt), diabetic
induction was 90 % with a mortality rate of
143
20%. Blood glucose was monitored after
alloxan treatment to confirm the diabetic state
and the diabetic rats were included in the
experiment ten days after alloxan treatment.
Control and diabetic rats were divided into one
control Group I: rats treated with single i.p.
injection of saline, Group II: alloxan diabetic
rats administered orally with equal volumes of
saline (untreated group), Group III, IV, V and
VI Alloxan-diabetic rats administered orally
with A. vera, C. paradisi, T. indica and O.
ficus indica isolated polysaccharide (500
mg/Kg). Animals were continued with the
administration for 15 days. Five animals were
used to assess the effect of different isolated
polysaccharides from the plant organs under
investigation on the change of blood glucose.
While, another five animals were subjected to
oral glucose tolerance (OGTT) after overnight
fasting. Equal number of control and diabetic
rats were also included and done as previously
mentioned.
The results were statistically analysed by
one way ANOVA followed by Tukey, S.
multiple comparison test using SPSS software
(student's virgin), the minimum level of
significance was set at P < 0.05.
Screening of the immunomodulatory
activity
Erythrocyte and leukocytic counts were
performed using the Improved Neubaur
method, hematocrit value was determined with
the microhematocrit technique; lymphocyte
and the haemoglobin percentages were also
determined (23).
Phagocytic activity was determined using
Candida albicans culture (24), Phagocytosis was
estimated by determining the proportion of
macrophages with contained yeast cells in a
random count of 300 macrophages and
expressed as percentage of phagocytic activity.
The results were compared with a control
group of mice (5 mice), and statistically
analysed by one way ANOVA followed by
Tukey, S. multiple comparison test using SPSS
software (student's virgin), the minimum level
of significance was set at P < 0.05.
Screening of the antioxidant activity (In-
Vitro)
The antioxidant activity of the
polysaccharide isolated from the four different
plants organs under investigation was
determined following the reported method of
Osawa and Namiki (25). A control was
performed with linoleic acid but without the
isolated plant polysaccharide. 50 mg/L of
either D, L α-Tocopherol or ascorbic acid was
used as positive controls. The maximum
peroxidation level observed as (7 days) in the
 Phytochemical and Biological Studies
sample that contained no antioxidant
component was used as a test point. The
percent inhibition of linoleic acid peroxidation,
100 – [(Absorbance of sample at the seventh
day I Absorbance of control at the seventh day)
X 100] was calculated to express the
antioxidant activity. All results were
statistically analysed using student's t-test.
Screening of the anti-inflammatory activity
Thirty female Sprague Dawley rats with
average body weight 100.2 ± 1.377 were
divided into five groups each included 6 rats.
Four test groups were run where the rats were
given one oral dose 500 mg of polysaccharide
isolated from A. vera, C. paradisi, O. ficus-
indica or T. indica / kg rat body weight. A
control group was run where no polysaccharide
treatment was given to rats. 0.05 ml of
carrageenan (1 %w/v in 0.9 %NaCl) type IV
(Sigma, USA) was injected into the subplanter
region of the left hind paw 1 hour after oral
treatment. The thickness of the paw (foot)
was measured with vernier calipers
immediately before the injection of
carrageenan and after 1 hour, 2 and 4 hours of
carrageenan injection. The thickness of
inflammation was calculated by subtracting the
thickness of foot before inflammation from
that of the inflamed foot at the different times
intervals. The mean increase of the hind paw
thickness of rats given the isolated
polysaccharide from the plant organs under
investigation were compared with that of the
control inflamed rats. Statistical analysis was
carried out using student's t-test.
Optimization of parameters for extraction
of polysaccharides from the seeds of
Tamarindus indica (27)
The effects of the extraction parameters of
time, temperature and ratio of water to raw
material of the seeds of Tamarindus indica
were optimized by using three factor statistical
techniques, with a view to maximize the
extraction yield under optimal extraction
parameters combination. On the basis of
single-factor experiment for the
polysaccharides production, proper ranges of
extraction time, extraction temperature, ratio of
water to raw material and extraction number
were preliminarily determined. Extraction
yield of polysaccharides was measured by
weighing the extracted polysaccharides in
relation to the dry raw material in grams.
Extraction yield (%) = (dried polysaccharides
weight / dry raw material weight) x 100%.
RESULTS
Phytochemical results:
The percentage yield of polysaccharides
144
It was obvious from table (1) that all the
plant organs under investigation were rich in
polysaccharides; T. indica seeds had the
highest polysaccharide content (21 %) which is
mainly mucilage (14.65 %), O. ficus indica
polysaccharide yield is close to that of A. vera
(6.5 % and 6.3 % respectively) while C.
paradisi peels had the lowest polysaccharide
content (6 %). Percentage of mucilage
obtained by C.E.M is always higher than that
obtained by H.E.M.
Tests for identity of the isolated
polysaccharides
The isolated polysaccharides were
odourless, soluble in water and insoluble in
ethanol, ether and chloroform, gave positive
Molish test (21) and did not reduce Fehling's and
Barafoed's solutions. They gave negative test for
proteins (28) and left no ash on ignition. The
identity of the isolated polysaccharides from the
plant organs under investigation was confirmed
by the previously mentioned chemical tests, but
the test of KOH (18) alcoholic solution gave
gelatinous precipitate with the isolated pectin
and gave negative results with the isolated
mucilage from each of the plant organs under
investigation.
Chromatographic investigation
The paper and HPLC chromatographic
analysis of the hydrolysates of the isolated
polysaccharides (mucilage and pectin) from each
of the plant organs under investigation are
represented in tables (2) and (3) respectively.
Qualitative PC analysis of the polysaccharide
hydrolysates of each of the four plant organs was
confirmed by quantitative HPLC analysis which
revealed that A. vera mucilage contained mainly
galactouronic acid, mannose, galactose,
arabinose and glucose in the ratio 89.1, 5.0, 2.7,
1.2 and 1.1 % respectively while the pectin had a
relatively higher content of galactouronic acid,
galactose and glucose 91.5, 3.4, 2.9 %
respectively and both mucilage and pectin
contained minute traces of mannose. The
tabulated results of T. indica polysaccharide
hydrolysates showed that the pectin contained 13
% galacturonic acid and 4.8 % glucouronic acid
while the mucilage contained only 3.8 %
glucouronic acid. Both mucilage and pectin of
T. indica had high content of xylose (35.9 % and
12.4 %) and glucose (34.5 % and 16.5 %)
respectively with no mannose. Furthermore,
mucilage and pectin hydrolysates' results of O.
ficus indica showed a rich content in uronic
acids (88 % and 90 % respectively) and smaller
amounts of galactose and arabinose (4.0 %,
2.9% and 4.1 %, 1.5 % respectively) with no
xylose. The results of the hydrolysates of C.
paradisi pectin showed a high content of uronic
acids (88.1 %) mainly glucouronic acid,
 Nagwa M Ammar et al
moderate amounts of rhamnose (7.2 %),
galactose (4.8 %), small amounts of arabinose
(1.5%) and traces of glucose (0.2 %). All the
previous results revealed that the sugar content
of the isolated mucilage and pectin of each plant
organ under investigation was approximately the
same, but it differed in the amount of uronic
acids (galactouronic and glucouronic) which
were higher in pectin than in mucilage in all
cases.
Optimization of the parameters for
extraction of polysaccharides from the seeds
of Tamarindus indica
By optimizing the extraction parameters
the highest yield of polysaccharides was 55%
obtained at 80oC using 10g dry powder/1 liter
distilled water. Increasing the temperature
from 80 to 90 oC decreased the yield of the
polysaccharides because of the partial
hydrolysis of polysaccharides when extraction
temperature was higher; time was proportional
to the yield of the polysaccharides when
extraction time was between 1 and 4 h. The
yield of the polysaccharide was close to the
peak value (54%) when extraction time was
4h. After this point, the yield of the
polysaccharides didn’t change with increasing
the extraction time. The results showed that,
as the extraction number was 1, the yield of the
polysaccharide was 50%, and that as the
extraction number was 2, the yield was 53%.
However, when extraction number exceeds
twice, its influence on the extraction yield
became insignificant. Therefore, taking the
extraction yield and processing costs into
consideration, twice is sufficient for the
extraction of the polysaccharides.
Biological results:
Antihyperglycemic activity
Tables (4), (5) and (6) illustrate the results
of the antihyperglycemic activity investigation.
In oral glucose tolerance test for
normoglycemic rats, all the isolated
polysaccharides from the plant organs under
investigation significantly decreased the
elevated blood glucose levels in experimental
groups after the administration of glucose
except that of C. paradisi which had no
significant hypoglycemic effect on
normoglycemic rats. Polysaccharides of T.
indica seeds had the most potent significant
antihyperglycemic effect in two dose level.
The results in table (5) showed that blood
glucose levels significantly increased to a
diabetic level after the administration of
145
alloxan in all control and experimental groups,
also these results revealed that seeds of T.
indica, fruits of O. ficus indica and leaves of
A.vera isolated polysaccharides had a
promising antihyperglycemic activity on
experimental rats after 2 weeks of
administration with t-value 9.46, 6.8 and 6.1
respectively. As shown in oral glucose
tolerance test results in table (6), all the
isolated polysaccharides showed a significant
antihyperglycemic effect (P < 0.05) in alloxan
induced diabetic rats. The isolated
polysaccharides of T. indica seeds at a dose of
(500 mg/Kg) had the most potent
antihyperglycemic action amongst other plants,
as it caused 25 % decrease in blood glucose
level after 30 minutes of glucose
administration in diabetic groups.
Immunomodulatory activity
The blood picture in mice (PCV %) as
shown in table (7) in all treated experimental
groups is higher than that of control groups
(P < 0.05) with different extents. While the
results in table (8) showed that the isolated
polysaccharides of T. indica seeds caused the
highest significant increase in lymphocyte
count (15 % increase), although A. vera and T.
indica isolated polysaccharides caused a
significant decrease in eosinophil count (66%
and 44% respectively). Table (9) reveals that
the phagocytic activity and phagocytic index
significantly increased by the administration of
the isolated polysaccharides of all the selected
plant organs (P < 0.05) except that of C.
paradisi which had no potent significant
action, it was obvious that A. vera and T.
indica isolated polysaccharides showed the
most potent phagocytic activity enhancers with
an increase (32 %) and (27 %) in phagocytic
activity and phagocytic index respectively.
Antioxidant activity
As shown in table (10), all the isolated
polysaccharides from the plant organs under
investigation exhibited a significant
antioxidant activity, and A. vera had the most
potent action (56.62 % inhibition) which is
close to other antioxidant drugs as D, L α-
Tocopherol (63.5 % inhibition) and L-Ascorbic
acid (59.2 % inhibition).
Anti-inflammatory activity
By measuring the thickness of the left hind
paw (foot) of all experimental and control rats
groups at the different time intervals, table (11)
revealed that A. vera isolated polysaccharides
had the highest percentage of inhibition (28-
50%) which increased gradually by time while
C. paradisi isolated polysaccharides had no
significant effect.
 Phytochemical and Biological Studies

Table 1: Percentage of mucilage and pectin obtained by extraction from plant organs under
investigation calculated on dry weight basis.
Pectin
Plants Mucilage Pectin ammonium Pectin HCL Total polysaccharide
Mucilage H.E.M aqueous
C.E.M oxalate extract extract g%
extract
Aloe vera 5.2 0.05 0.4 0.3 0.35 6.3
Opuntia ficus indica 4.8 0.6 0.5 0.5 0.1 6.5
Tamarindus indica 13.85 0.8 4.15 1.35 0.9 21
Citrus paradisi 2.2 3.2 0.5 6

Table 2: PC investigation of the polysaccharide hydrolysates of the plant organs under

investigation.
Spot HRFx100 A. vera T. indica O. ficus indica C. paradisi Color with aniline Comparable
no. Mucilage Pectin Mucilage Pectin Mucilage Pectin Pectin phthalate with
1 33.18 +ve +ve Red Xylose
2 36 +ve +ve +ve +ve +ve +ve Red Aarabinose
3 24.4 +ve +ve +ve +ve +ve +ve +ve Yellow brown Galactose
4 27 +ve +ve +ve +ve green Glucose
5 31 +ve Bbrownish yellow Mannose
6 30 +ve +ve Yellow brown Rhamnose
7 25 +ve +ve +ve +ve +ve +ve Yellow brown Galacturonic
acid
8 22.8 +ve +ve +ve +ve +ve Yellow brown Glucouronic
acid

Table 3: Percentages of the major sugar components of hydrolysed polysaccharides from plant
organs under investigation obtained by HPLC.
A. Vera T. indica O. ficus indica C. paradisi
Authentic sugars
Mucilage Pectin Mucilage Pectin Mucilage Pectin Pectin
Xylose traces traces 35.9 12.42
Arabinose 1.2 4 10.2 2.95 1.5 1.56
Galactose 2.75 3.4 9.5 5.35 4.02 4.155 4.85
Glucose 1.1 2.9 34.5 16.5 0.18
Mannose 5 traces
Galactouronic acid 89.153 91.5 13 4.65 6.3 12
Glucuronic acid 3.83 4.8 83.5 83.5 73.5
Rhamnose 9 28 9.072 7.25

Table 4: Effect of the administration of the isolated polysaccharides from plant organs under
investigation on oral glucose tolerance test in normoglycemic rats.
Time (min.) after glucose administration
Groups -30 0 30 60 90 180
Mean±S.E Mean±S.E Mean±S.E Mean±S.E Mean±S.E Mean±S.E
Control B B A C B B
(Saline) 59.80±3.41 153.80±3.17 149.40±1.94 130.00±4.18 125.00±5.00 153.80±3.17
A. vera AB E D E E E
(250 mg/Kg) 60.80±3.50 138.40±6.05 128.40±5.88 104.00±6.20 98.00±5.65 138.40±6.05
A. vera B G F F F G
(500 mg /Kg) 59.20±3.48 118.00±8.18 107.00±5.01 95.40±5.35 89.40±3.66 118.00±8.18
C. paradisi C A A A A A
(250 mg/Kg) 57.20±3.68 154.60±2.27 149.00±3.66 146.60±2.91 141.60±2.87 154.60±2.27
C. paradisi C D C B B D
(500 mg/Kg) 56.60±4.31 147.00±2.68 138.60±3.57 136.00±4.28 124.40±5.09 147.00±2.68
T. indica C G G G G G
(250 mg/Kg) 56.80±4.32 118.00±4.64 102.60±4.37 90.00±3.54 84.40±3.30 118.00±4.64
T. indica C H H H H H
(500 mg/Kg) 57.00±3.79 108.20±4.21 91.00±2.92 85.0±2.24 78.00±2.55 108.20±4.21
O. ficus indica A C B D C C
(250 mg/Kg) 61.00±2.92 150.20±2.87 145.20±3.32 125.00±2.37 109.00±5.10 150.20±2.87
O. ficus indica B F E E D F
(500 mg/Kg) 59.60±5.14 122.00±2.55 113.00±3.00 104.00±3.67 102.00±3.39 122.00±2.55
Means within the same column of different letters are significantly different at (P < 0.05). S.E = Standard error

146
 Nagwa M Ammar et al

Table 5: Effect of the administration of the isolated polysaccharides from plant organs under
investigation on blood glucose level in alloxan induced diabetic rats.
Treatments
Week N Control Diabetic A. vera C. paradisi T. indica O. ficus indica
Mean ± S.E Mean ± S.E Mean ± S.E Mean ± S.E Mean ± S.E Mean ± S.E
Basal A A A A A A
5
64.00±2.77 227.40±7.98 226.80±7.53 231.00±7.81 234.00±10.30 237.40±5.46
2nd week B B B B B B
5
57.40±5.08 233.80±8.40 143.00±11.47 213.00±8.46 116.40±6.96 147.00±12.00
Total 10 60.70±2.94 230.60±5.57 184.90±15.39 222.00±6.20 175.20±20.46 192.20±16.30
t-value 2.45* 2.55* 6.10*** 2.56* 9.46*** 6.85***
Means within the same column of different letters are significantly different at (P < 0.05).
S.E = Standard error
*= Significant at (P = 0.05).
*** = Significant at (P < 0.001).

Table 6: Effect of the administration of the isolated polysaccharides from plant organs under
investigation on oral glucose tolerance test in alloxan induced diabetic rats.
Time (Min.) after glucose administration
Groups -30 0 30 60 90 180
Mean±S.E Mean±S.E Mean±S.E Mean±S.E Mean±S.E Mean±S.E
Control diabetic C A A A A A
(Saline) 5 228.00±8.15 233.00±9.17 276.00±11.77 287.00±12.31 276.00±11.22 240.00±11.40
A. vera B B D D C B
(500 mg/Kg) 5 229.00±7.48 230.00±7.58 227.60±6.79 216.00±5.10 214.00±3.67 221.20±6.40
C. paradisi D C B B B C
(500 mg /Kg) 5 225.00±4.47 227.00±4.64 247.60±3.50 233.00±5.39 223.40±3.68 208.00±3.74
T. indica E E E E D E
(500 mg/Kg) 5 218.20±5.68 220.60±7.70 209.00±6.00 200.80±6.06 192.80±4.78 187.40±4.45
O. ficus indica A D C C C D
(500 mg/Kg) 5 230.00±7.07 223.00±3.74 240.40±7.36 227.00±6.24 215.00±4.47 204.00±5.34
Means within the same column of different letters are significantly different at (P < 0.05). S.E = Standard error

Table 7: Effect of the administration of the isolated polysaccharides from plant organs under
investigation on blood picture in mice.
PCV % Hb % WBCs X 103 RBCs X 106
Groups N
Mean ± S.E Mean ± S.E Mean ± S.E Mean ± S.E
A. vera 5 B B B C
(500 mg/Kg) 37.20±1.36 16.71±0.80 6.18±0.57 6.26±0.22
C. paradisi 5 C A B BC
(500 mg /Kg) 36.40±1.47 17.74±0.66 6.44±0.44 6.92±0.45
T. indica 5 A C AB A
(500 mg/Kg) 38.40±2.14 13.77±1.08 5.02±0.23 7.51±0.64
O. ficus indica 5 A B AB BC
(500 mg/Kg) 38.80±1.85 16.55±0.55 5.23±0.8 6.74±0.34
Control 5 D C A D
31.20±1.02 13.00±1.09 5.86±0.44 5.67±0.49
Means within the same column of different letters are significantly different at (P < 0.05). S.E = Standard error

Table 8: Effect of the administration of the isolated polysaccharides from plant organs under
investigation on differential leucocytes count in mice
Lymphocyte Neutrophil Monocyte Eosinophil
Groups N % % % %
Mean±S.E Mean±S.E Mean±S.E Mean±S.E
A. vera 5 C BC B D
(500 mg/Kg) 61.40±0.68 26.80±1.77 10.80±1.11 1.25±0.25
C. paradisi 5 B C C B
(500 mg /Kg) 64.40±0.81 24.20±1.50 8.40±0.93 3.40±0.68
T. indica 5 A D B C
(500 mg/Kg) 68.20±2.24 18.20±2.35 10.80±2.24 2.40±0.75
O. ficus indica 5 D A C A
(500 mg/Kg) 56.00±2.70 32.20±1.50 8.00±1.05 4.00±0.55
Control 5 D C A A
57.60±1.63 25.20±1.98 12.00±1.10 4.00±0.45
Means within the same column of different letters are significantly different at (P < 0.05). S.E = Standard error

147
 Phytochemical and Biological Studies

Table 9: Effect of the administration of the isolated polysaccharides from plant organs under
investigation on phagocytic activity and phagocytic index on experimental mice.
Phagocytic activity Phagocytic index
Groups N
Mean ± S.E Mean ± S.E
A. vera 5 A A
(500 mg/Kg) 26.00±0.63 2.40±0.16
C. paradisi 5 B D
(500 mg /Kg) 21.40±0.51 1.72±0.22

T. indica 5 A A
(500 mg/Kg) 26.00±0.63 2.40±0.18
O. ficus indica 5 B B
(500 mg/Kg) 21.00±0.45 2.14±0.07
Control 5 C C
19.60±0.51 1.88±0.11
Means within the same column of different letters are significantly different at (P < 0.05). S.E = Standard error

Table 10: Antioxidant activity of the isolated polysaccharides from the plant organs under
investigation using thiocyanate method.
Isolated polysaccharide Antioxidant activity
Aloe vera 56.62
Citrus paradisi 34.27
Opuntia ficus indica 43.07
Tamarindus indica 41.12
D, L α-Tocopherol 63.5
L-Ascorbic acid 59.2

Table 11: Percent of inhibition of the isolated polysaccharides from plant organs under
investigation on the mean thickness of inflammation (mm) induced in different
experimental groups by carrageenan.
Time (hours) (Mean ± SE)
Groups
1 2 3
Control 0.158 ± 0.015 0.217 ±0.011 0.283 ± 0.011
inhibition % ─ ─ ─
A. vera 0.117± 0.017 0.l33* ± 0.011 0.l42* ± 0.015
Inhibition % ─ 38 50
C. paradisi 0.175± 0.011 0.217 ± 0.011 0.25 ± 0.013
Inhibition % ─ 0 12
O. ficus indica 0.15 ± 0.018 0.183 ± 0.021 0.2* ± 0.013
Inhibition % 5 15 29
T. indica 0.142 ± 0.015 0.192 ± 0.015 0.217* ± 0.011
Inhibition % 11 12 24
Values significantly differ from the control: * p < 0.001

In previous studies polysaccarides were

DISCUSSION
Egypt is rich in plants containing
polysaccharides abundantly distributed all over
the country especially Aloe vera which has
been known since Ancient Egyptians (6) , as
well as the seeds of Tamarindus indica which
has been considered as a waste material. In
this study phytochemical and biological
investigations were performed on the four
selected Egyptian plant organs rich in
polysaccharides, aiming the discovery of new
active constituents from botanical origin which
can be utilized in drug industry.
prepared from different plant organs as
aqueous extract without the precipitation of the
pure polysaccharide (23, 24, 25, 26) and this caused
the presence of undesirable constituents that
may affect the biological action or even may
cause undesirable effects such as
anthraquinones in Aloe vera (29) or proteins and
fats in Tamarindus indica seeds. So we
suggested isolating the polysaccharide from
the different plant organs under investigation
by precipitation in order to be obtained in a
pure form.
The results of the phytochemical study of
the isolated polysaccharides from the different

148
 Nagwa M Ammar et al
selected plant organs under investigation
obtained by paper and HPLC chromatographic
techniques matched the previously reported
results, as for Aloe vera the acid hydrolysis of
the isolated polysaccharide furnished
galacturonic acid (85 %) along with small
amounts of galactose, rhamnose, arabinose,
xylose, glucose and mannose (mainly in
mucilage) (30, 31). While for the seeds of
Tamarindus indica polysaccharide hydrolysate
showed a high content of xylose, glucouronic
acid and galacturonic acid (32, 33). The results
of the analysis of the isolated polysaccharide
of Opuntia ficus indica fruits confirmed the
reported data as it showed that it contained
mainly arabinose, galactose, galacturonic and
glucouronic acids (34, 35) and the hydrolysate of
the pectin of Citrus paradisi contained mainly
glucouronic acid (36).
From the results of this work, it is evident
that all the four selected plant organs are rich
in polysaccharides especially the seeds of
Tamarindus indica, which is also the most
promising biologically active as
antihyperglycemic, anti-inflammatory,
immunomodulatory and antioxidant amongst
other plant organs under investigation, also
Aloe vera leaves isolated polysaccharide
showed remarkable promising action in all the
fore mentioned biological activities.
From the previous studies concerning
various plants containing mucilage and pectin,
it was obvious that some of the isolated
polysaccharides (mucilage and/ or pectin) from
different plant organs showed significant
biological activities as a whole isolated
polysaccharide (13, 14), while upon separating
the different polysaccharide in each plant
organ by fractionation chromatographic
techniques or even by using the mucilage or
pectin separately and testing the biological
activity, the results showed that some of the
separated polysaccharides had significant
biological activities (11, 12, 37 and 38) while other
showed no biological action (29). So we
suggested making use of the synergistic action
of mucilage and pectin in each plant organ
under investigation by combining them
together and testing the different suggested
biological activities.
The results of the antihyperglycemic
activity tests of the polysaccharides isolated
from the selected plant organs revealed that the
polysaccharides isolated from Tamarindus
indica seeds and Aloe vera whole leaves
posses the highest hypoglycemic activity in
normoglycemic and alloxan-induced diabetic
rats, followed by the fruits of Opuntia ficus
indica. Theoretically, antihyperglycemic or
antidiabetic plants act through a variety of
149
mechanisms, at present juncture, it is not
possible either to pin point the exact
mechanism of the hypoglycemic effect of the
isolated polysaccharides or to identify the
active principle(s) responsible for such effect.
At present level, some hypothetical
suggestions can be made. Insulin is the main
regulator of glycogenesis in muscle and liver
(39)
, and as alloxan is a toxic glucose analogue
(40)
which selectively destroys insulin-
producing cells in the pancreas, this focus the
one possible way of antidiabetogenic action of
these extracts by improvement of glycogenesis
process in muscle and liver as it was reported
before that polysaccharides extracts in diabetic
rats resulted graded and significant elevation in
both the liver and muscle glycogen levels. (13).
From the results of the immunomodulating
activity investigation of plant polysaccharides
under investigation, its obvious that all extracts
had a significant immunomodulating action but
with different degrees and mechanisms of
action. Preliminary these polysaccharides
positively affect haematopoietic system (2), as
they all increased the PCV % in experimental
mice. A significant increase in leucocyte count
is observed in all cases along with a decrease
in eosinophil count indicated that these
polysaccharides especially the isolated from
Aloe vera leaves and Tamarindus indica seeds
could depress allergic reactions. Its very
important to mention that all the selected
polysaccharides could significantly increase
phagocytic activity along with phagocytic
index, that can drive us to a suggestion that
polysaccharides could stimulate macrophage
chemotaxis immune defensive mechanisms,
these findings are supported by previous
studies that revealed that plant polysaccharides
especially xyloglucans and uronic acid
containing arabinogalactans could activate
innate immune system especially macrophage
activity (41, 42, 43, 44, 45).
The results of the investigation of the
antioxidant activity revealed that the
polysaccharides isolated from all the selected
plant organs possesed a significant antioxidant
action and Aloe vera is the most potent (56%)
amongst others. There are so many suggested
mechanisms reported in earlier studies such as
scavenging O2 and H2O2 radicals (46, 47, and 48)
which can be useful in the inhibition of the
damage of DNA chain induced by hydroxyl
radical (49) and cancer cell proliferation
inhibition (50).
Results of the anti-inflammatory activity
tests showed that the four selected plant organs
isolated polysaccharides had a potent anti-
inflammatory action; these results confirm
previously reported data (51, 52, 53). Several
 Phytochemical and Biological Studies
reports suggested that both the antioxidant and
the anti-inflammatory activities contribute to
the immunomodulating action of the tested
polysaccharides (54).
In this study we selected the most
promising biologically active and highest
polysaccharide yielding plant organ to
optimize the extraction parameters,
Tamarindus indica seeds were significantly
active in all the tested biological activities. As
mentioned before the yield of the
polysaccharide isolated from the seeds of
Tamatindus indica using cold and hot water
extraction and pectin preparation methods (17)
was (21 %), but upon testing all the extraction
parameters of the polysaccharide of T. indica
seeds the results revealed that taking the
extraction yield and processing costs into
consideration, extraction using 10g dry powder
/ 1 liter distilled water at 80oC for 4 hrs and
twice were optimum for the extraction of the
polysaccharides giving the highest reported
yield (55%). Consequently these results can be
used as a starting point for the application of
biologically active plant polysaccharides in
drug industry.
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 Phytochemical and Biological Studies

 


  

   
  
  
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