Isolation and Identification of Polysaccharide Compound by Qualitative

Test in Aqueous Extract of Safflower Petals (Carthamus tinctorius L.)

from Bone Region.
Rahmawati Syukur
1
, Gemini Alam
2
, Arfiana
3

1
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Hasanuddin
University, Makassar, Indonesia
2
Department of Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Hasanuddin
University, Makassar, Indonesia
3
Laboratory of Phytochemistry, Faculty of Pharmacy, Hasanuddin University, Makassar,
Indonesia

Abstract
Isolation and identification of polysaccharide compound by qualitative test from aqueous extract of safflower
petals (Carthamus tinctorius L.) has been conducted. This research aims to get polysaccharide compound
from aqueous extract of safflower petals, which acquired from Bone Regency. Safflower petals were
extracted by infusion method with aqua destillata. The resultant extracts were centrifuged, and the
supernatants was added with 70% ethanol and placed in the refrigerator overnight. The precipitate were
collected, dissolve in water and adjusted to pH 3 by HCl. Insoluble materials were removed by
centrifugation, and the supernatants were again adjusted to pH 7 by NaOH. After that, the precipitates were
collected by centrifugation with addition 70% ethanol, then dried. The dried precipitate were hydrolyzed with
HCl at 100oC, and identified with TLC multiple development, and identifications by test for polysaccharides
compounds. The isolate obtained about 0.0705 gram, characterized by FTIR. The isolate has characteristic
functional groups such as -CH (aliphatic), -OH (hydroxy), -C=O (carbonil), and -C-O- (ester). The
isolate is a polysaccharide compound.

Key word : Safflower, Carthamus tinctorius L., polysaccharide.

INTRODUCTION

One of the plants used in traditional medicine by the people especially in the
district of South Sulawesi is Safflower (Carthamus tinctorius L.) from the Asteraceae tribe.
Safflower an annual plant, spiked on branches and leaves, the flowers are yellow or
reddish. generally planted in hot, dry climates [1]. Empirically, Kasumba steeping the dried
flowers, used by the community for the treatment of measles. In the Chinese medical
system, in addition to the treatment of measles, Turate Kasumba flowers are also used for
the treatment of menopausal disorders, fever, and improve blood circulation system [2].
Safflower contains a number of compounds such as carthamin, carthamidin,
glycosides, coumarins, fatty acids, steroids, and polysaccharides. Based on the study of
modern pharmacology, it is known that the extract Safflower have several biological
activities, such as anticoagulants, antihypertensives, antioxidants, immunosuppressants,
and antitumor. And most of these effects, there is the water extract [3.4].
It has been reported from several studies, that the bioactive polysaccharide
compounds that have been isolated from fungi, bacteria, algae, and from the plant, has
antitumor and immunomodulatory effects [5]. Polysaccharides are compounds that consist
of a combination of a large number of monosaccharide molecules (containing ten or more
monosaccharide) that these compounds can be hydrolyzed into monosaccharides many
molecules [6]. Safflower polysaccharide composed of xylose, fructose, galactose, glucose,
arabinose, and ramnosa uronik acid residues, was reported to stimulate the immune system
in mice [7].
Based on the information that has been obtained, it can be seen that the water
extract of polysaccharide compounds Safflower can boost the immune system. From the
literature search results, in Indonesia has not been a lot of reports of research on the
polysaccharide content of the active compounds of interest Safflower, and its potential as
munomodulator. Therefore, need to be isolated from the aqueous extract polysaccharide
compound interest Safflower, and identify isolated compounds.
In this study, Safflower used comes from Wempubbu village, in Bone region. The
study was conducted in accordance polysaccharide compound isolation procedure, where
the sample is extracted with infundation method. Matter content was determined by the
method of polysaccharide hydrolysis. Identification of isolated compounds performed thin
layer chromatography, using a reagent identification. Then do the characterization of
isolated compounds using FT-IR.
MATERIALS AND METHODS

Safflower petals (Carthamus tinctorius L.), taken from the District Amali, Bone
regency. Safflower petals (Carthamus tinctorius L.) is obtained in the form of dry, ready to
be extracted. The chemical used in the present study were obtained from the following
source. Cellulose TLC plates, Molisch Reagents, and Seliwanoff Reagent. Chamber
(Camag ), FT-IR (Bruker ), UV light (254 nm and 365 nm), pH meter (Sartorius ),
centrifuged (Sorvall RC 5B Plus), and a set of tools infundation.
Extraction and Isolation
Dried petals of safflower (Carthamus tinctorius L.) were extracted with boiling
water for 15 min, then filtered while still hot. The resultant extracts were centrifuged at
4000 rpm for 20 min, and the supernatants were precipitated with 70% ethanol (4 times the
volume of aqueous extract), and placed in the refrigerator overnight . The precipitates
were collected by centrifugation (4000 rpm, 20 min), The precipitate obtained were
dissolved in water, and the aqueous solution was adjusted to pH 3 by HCl. The insoluble
materials were removed by centrifugation , and the recovered supernatants were again
adjusted to pH 7 by NaOH. Active compounds were collected by 70% ethanol
precipitation. Ethanol precipitate was dried, and then proceed to stages of hydrolysis [5].
Hydrolysis of Polysaccharides Compound
Dried precipitate obtained, weighed and then put into a covered Erlenmeyer
meeting, added 0.25 N HCl, placed in a preheated oven at 100C for 3 to 5 hours. Tubes
are removed from the oven, cooled to room temperature. The filtrate was taken, then added
absolute ethanol, to precipitate saccharide. Depositionthe precipitate was taken, and then
evaporated [8,9].
Identification and Characterization of Isolates
Identification of polysaccharide compound by Thin Layer Chromatography on
cellulose plates with different mobile phase. After that, used a spray reagent, for
polysaccharide compound identification. For qualitative tests, used Seliwanoff, Molisch,
and iodine reagents. Isolates obtained were then characterized by FT-IR.
RESULTS AND DISCUSSION
In the early stages, extraction with infundation, is intended to separate the target
compounds, polar polysaccharide, from other compounds present in the sample. The liquid
extract from the results of infundation, conducted by precipitated polysaccharide
compounds using 70% ethanol. It is based on the nature of the polysaccharide compounds
have very low solubility rate of the ethanol, so the only other compounds with low
molecular weight dissolved, and the polysaccharides were precipitate [10]. The precipitate
polysaccharides, separated from the filtrate using a centrifuge. The precipitate obtained is
dissolved then adjusted pH to pH 3, to release proteins that bind to polysaccharides, which
is then neutralized again by NaOH and precipitated with ethanol [11].
Qualitative and quantitative analysis of carbohydrates by using TLC, is a method
that is faster, easier and cheaper than previous methods, such as HPLC and GC. However,
polysaccharides can not be identified by TLC. However, polysaccharide constituent
monosaccharides can be determined by TLC, after hydrolysis [12].
Qualitative analysis of the polysaccharide compound by TLC and spray reagent,
however, do not support the results obtained. This was confirmed by the results of a
literature search of that has been described previously, that the polysaccharide compound
identification can not be done by TLC. Therefore, carried out using acid hydrolysis of
polysaccharide compounds, polysaccharide compounds to break down into simpler
molecules, such as monosaccharides so that they can test their identification.
On acid hydrolysis, dilute acid solution usually used, where the speed of the reaction is
proportional to the concentration of acid. In general, soluble components contained in the
results of acid hydrolysis of polysaccharides is xylose, glucose, cellobiose, furfuraldehida,
hidroksimetilfurfural, and organic acids such as formic acid, levulinat acid, and acetic acid,
[13,14].
From the results of the test compound identification results of hydrolysis, obtained
that states that the test results seliwanoff hydrolysis compounds do not contain fructose
compounds. Test molisch stated that isolates a carbohydrate compound, and for iodine test,
the test results showed a brown color, which leads to the compound dextrin, which is a
breakdown product of the polysaccharide. However, positive results for iodine test still
requires a more specific test. Isolates obtained from the hydrolysis were identified by TLC.
Carbohydrates are a very hydrophilic compound, so it can bind strongly on TLC plates,
such as silica gel, alumina, and cellulose. Therefore, it takes a mobile phase that more
polar on TLC development [15]. Effective with cellulose TLC plates to separate a mixture
of monosaccharides, including amino acids and sugars. Nevertheless, TLC multi eluent or
two-dimensional TLC needed to confirm the results of isolation, which is then detected by
reagent class.
Based on the literature study, the multi TLC eluent used some kind of comparison
eluent for compounds such as polysaccharides and their derivatives: ethyl acetate:
n-propanol: acetic acid: water (4:2:2:1), acetonitrile: water (3:1), acetone: water (3:1), ethyl
acetate: ethanol: water (2:2:1) and methanol: water (2:1).
There showed IR spectral data interpretation (Table 1). The IR spectrum (Figure 1)
shows a broad absorption band at wave number 3344 cm
-1
, believed to be the-OH
(hydroxyl), which usually appears in the wavenumber region 3600-3200 cm
-1
. Absorption
at 2946 cm
-1
and 2836 cm
-1
are sharp and feeble alleged aliphatic CH groups are common
in the region 3200-2700 cm
-1
uptake, and the uptake in the area of the curve of the wave
number 1453 cm
-1
and 1425 cm
-1
which represents a group-CH, which usually appears in
the catchment area from 1470 to 1430 cm
-1
. Absorption appears at wave numbers 1741,
1647, and 1546 cm
-1
, thought to be the carbonyl group (-C = O). whereas the indentation
area, there is a sharp absorption at wave numbers 1158, 1105, and 1024 cm
-1
is thought to
be group-CO-which usually appear at wave numbers 1300-1000 cm
-1
. Carbonyl group
appears at wave number 1741 cm
-1
, probably derived from esters, which usually appears at
wave number 1750-1725 cm
-1
. This is indicated by the presence of the-CO-in the curve of
the wave number 10241 cm
-1
are sharp and strong, where the absorption in the region
1500-600 cm
-1
is the finger print area, which can provide information about the presence in
the mixture of polysaccharide compounds [16,17].
Table 1. FTIR spectra data of polysaccharide compound isolated from safflower
Wavenumber
(cm
-1
)
Band appears Intensity
Possible
functional group
3344 broad Medium -OH
2946 sharp weak -C-H
2836 sharp weak -C-H
1741 sharp weak -C=O
1647 sharp weak -C=O
1546 sharp weak -C=O
1453 broad weak -C-H
1425 broad weak -C-H
1158 sharp weak -C-O-
1105 sharp weak -C-O-
1024 sharp strong -C-O-











Figure 1.FTIR spectrum of polysaccharide compound from Safflower aqueous extract.

CONCLUSION
From the research that has been done, as many isolates obtained
0.0705 gram. Based on the identification test polysaccharide compound, and compound
characterization by FT-IR showed that isolates containing group -CH (aliphatic),-OH
(hydroxy), -C=O (carbonyl), and -CO- (ester), isolated compounds believed to be
polysaccharides.
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