POLYSACCHARIDES IN CENTAURIUM ERYTHRAEA RAFN.

Full text

Stoiko Liliya et al / Int. J. Res. Ayurveda Pharm. 8 (Suppl 2), 2017
252
Research Article
www.ijrap.net
POLYSACCHARIDES IN CENTAURIUM ERYTHRAEA RAFN.
Stoiko Liliya 1, Dakhym Iryna 2*, Pokotylo Olena3, Marchyshyn Svitlana 4
1Assistant Professor, Department of Pharmacy Management, Economics and Technology, І. Horbachevsky Ternopil
State Medical University, Maidan Voli 1, Ternopil, Ukraine
2Associate Professor, Department of Pharmacognosy and Medical Botany, І. Horbachevsky Ternopil State Medical
University, Maidan Voli 1, Ternopil, Ukraine
3Associate Professor, Department of Pharmacy Management, Economics and Technology, І. Horbachevsky Ternopil
State Medical University, Maidan Voli 1, Ternopil, Ukraine
4Professor, Department of Pharmacognosy and Medical Botany, І. Horbachevsky Ternopil State Medical University,
Maidan Voli 1, Ternopil, Ukraine
Received on: 18/02/17 Accepted on: 11/04/17
*Corresponding author
E-mail: iradakhym@gmail.com
DOI: 10.7897/2277-4343.082122
ABSTRACT
We used gas chromatography–mass spectrometry (GC–MS) to determine polysaccharide and monosaccharide content of Centaury herb. The
monosaccharides present in the investigated raw material were derivatized with hydroxylamine hydrochloride in the mixture of pyridine/methanol
(4:1 v/v), identified by their retention time in the gas chromatograms and compared to those of pure reference compounds. The monosaccharides were
quantified by comparing their response signal to that of sorbitol as an internal standard. Six components among free carbohydrates and eight
components among monosaccharides after hydrolysis were identified and quantified in the Centaury herb. Among the monosaccharides after
hydrolysis glucose and pinitol had the highest content at 26.86 mg/g and 15.26 mg/g respectively. Glucose (13.07 mg/g), saccharose (7.16 mg/g) and
pinitol (3.71 mg/g) had the highest content among the free carbohydrates. We used gravimetric analysis to study qualitative content of
polysaccharides in dried Centaury herb. We determined that 11.80±0.01 % of total mass was water-soluble polysaccharides and 2.33±0.05 % pectin
substances.
Keywords: Centaurium erythraea, gas chromatography, gravimetric method, monosaccharides, polysaccharides
INTRODUCTION
Centaury (Centaurium erythraea Rafn.) is a flowering plant in
the family Gentianaceae. This is a biennial plant with basal leaf
rosette, growing to the height of 15–24 cm. Its stems are
branched, bearing clusters of pink or red flowers1. Centaury
grows in dry and sandy places in Europe, Western Asia, North
Africa and North America. It has also naturalized throughout
eastern Australia, where it is an introduced species2. The whole
plant is utilized in herbal medicine.
Centaury is traditionally given as a bitter tonic to increase
gastric secretions, to relieve dyspeptic discomfort and loss of
appetite. The herb is claimed to have antidiabetic, antitumor,
sedative, anticholinesterase, anti-inflammatory and antipyretic
properties3 and possess wound healing4 activity. Centaury is
included in some cosmetic and toiletry preparations for its
allergy soothing and astringent properties.
Previous studies found that centaury herb contains flavonoids,
hydroxycinnamic acids, tannic substances, coumarines, fatty and
organic acids, aminoacids, carotenoids and chlorophylls5,6,7.
However, there is no published information about some primary
metabolites, such as polysaccharides. Polysaccharides are
energy-rich molecules providing energy for life processes as
well as building parts of the cell walls of plants and animals. In
animals, they play crucial role in the pathogenesis, immune
system, and blood clotting responses. Polysaccharides have
expectorant, laxative, demulcent, sorbent, anti-inflammatory,
antiulcer, anti-viral and detoxifying actions. Many
polysaccharides are used as stabilizers, fillers, emulsifiers and
film coating agents in pharmaceutical industry8. Thus, the aim of
our study was to determine the content and monomeric
composition of polysaccharides in aerial parts of C. erythraea
Rafn. collected from natural habitat.
MATERIALS AND METHODS
Plant Material
Aerial parts of C. erythraea were collected on the outskirts of
Zboriv (Ternopil region, Ukraine) during the flowering period in
July 2013. The raw material was authenticated by TSMU
professor Dr. Svitlana Marchyshyn. A voucher specimen no.
133 is kept at the Department of Pharmacognosy and Medical
Botany, TSMU, Ternopil, Ukraine. The plant material was dried
using conventional methods and then stored in paper bags in dry
place.
Chemicals and Methods
Two conventional methods for quantification of polysaccharides
and determining their monomeric content were used: gas
chromatography with mass spectrometry (GC-MS) and
gravimetry. All reagents were of analytical grade (> 95 %
purity). The chemicals were obtained from Sigma-Aldrich, USA
and were: D-mannose (Man), L-rhamnose (Rha), D-ribose
(Rib), D-galactose (Gal), D-xylose (Xyl), D-arabinose (Ara), D-
fucose (Fuc), D-glucose (Glc), D-fructose (Fru), D-saccharose
(Sac), D-sorbitol; methanol, trifluoroacetic acid, hydroxylamine
hydrochloride, pyridine, dichloroethane, hydrochloric acid,

Stoiko Liliya et al / Int. J. Res. Ayurveda Pharm. 8 (Suppl 2), 2017
253
heptanes, ethyl acetate, picric acid, sodium hydrogen carbonate
and sodium hydroxide.
Sample Preparation and Chromatographic Analysis
Centaury herb was powdered in glass mortar. 500 mg of raw
material was placed into the flask and 5 mL of 2 M
trifluoroacetic acid was added for the extraction of bonded
monosaccharides or monosaccharides after hydrolysis.
Hydrolysis was performed under 100 °C for 6 hours. 2 mL of
hydrolysate was evaporated and 2 mL of internal standard was
added.
For the extraction of free monosaccharides 500 micrograms of
methanol solution with internal standard (sorbitol) was added to
500 mg of powdered centaury herb. The extraction took place in
the ultrasonic water bath at 80 °C for 4 hours.
To obtain acetylated aldonitriles 2 mL of extract was evaporated
to dry and 0.3 mL of derivatization reagent (32 mg/mL of
hydroxylamine hydrochloride in pyridine/ methanol (4:1 v/v))
was added. The extract was incubated at 75 °C for 25 min. For
acetylation of aldonitrile derivatives 1 mL of acetic anhydride
was subsequently added to the samples and incubated at 75 °C
for 15 min. 2 mL of dichloroethane was added and the excess of
the derivatization reagents was removed by the double
extraction with 1 M hydrochloric acid and water.
Dichloroethane layer was dried and dissolved in 300 µL of the
mixture of heptane/ethyl acetate (1:1 v/v)9,10.
GC-MS analysis of monosaccharides was performed using gas
chromatograph Agilent 6890N with 5973inert mass detector
(Agilent Technologies, USA). Samples were analyzed on a
capillary column HP-5MS (30 m×0.25 mm×0.25 µm). The oven
temperature was initially set at 160 °C, held for 8 min, then
raised to 240 °C at the rate of 5 °C/min and finally kept at this
point for 6 min. Injections were made in the split mode 1:50.
The detection was performed in the SCAN mode at the width
range of 38-400 m/z. Helium was used as the carrier gas at a
constant flow rate of 1.2 mL/min.
The polysaccharides were hydrolyzed to produce constituent
monosaccharides, which were subsequently converted into
acetylated aldononitriles.
Identification of monosaccharides was based on their retention
times compared to Sac standard and mass library NIST 02.
Quantification was done by using internal standard of sorbitol
added to the sample.
The quantities of water-soluble polysaccharides and pectin
substances were determined by gravimetric method reported by
Marchyshyn et al11. All samples were analyzed in triplicates and
the results were averaged.
RESULTS
Gas chromatography coupled to mass spectrometry (GC/MS)
was used to identify and measure the monosaccharides content
in C. erythraea herb.
In the analyzed material Rha, Ara, Man, Glc, Gal, Pinitol,
Inositol and Mannitol were identified after acidic hydrolysis and
derivatization with acetylated aldononitriles (Figure 1). The
following free carbohydrates were identified from the water-
methanol solution of the samples: Glc, Pinitol, Inositol, L-Iditol,
Fru and Sac (Figure 2).
The quantitative content of carbohydrates in mg/g calculated in
reference to the internal standard (sorbitol) is shown in the Table
1.
Among bonded monosaccharides glucose and pinitol had the
highest content − 26.86 mg/g and 15.26 mg/g respectively.
Glucose (13.07 mg/g) was the major compound of free
carbohydrates followed by saccharose (7.16 mg/g) and pinitol
(3.71 mg/g).
The content of water-soluble polysaccharides and pectin
substances in centaury was determined at 11.80±0.1 % and
2.33±0.05 % of total dry weight respectively (Figure 3).
DISCUSSION
The monosaccharides identified in this study have various roles
and can cause a variety of pharmacological effects when
administered to mammals.
We found that in C. erythraea herb the predominant
monosaccharide compound was glucose: 26.86 mg/g and 13.07
mg/g among monosaccharides after hydrolysis and free
monosaccharides respectively. The main function of this
aldohexose is to supply energy for physiological processes. Of
all mammalian organs and tissues, the brain, neurons and
developing red blood cells have the highest energy demand. If
concentration of glucose is low, processes requiring mental
effort are deteriorated12.
Table 1: Content of monosaccharides after hydrolysis and free carbohydrates in C. erythraea Rafn.
No.
Carbohydrate
Content in the plant material [mg/g]
Monosaccharides after hydrolysis
Free carbohydrates
1
Rhamnose (Rha)
1.46
2
Arabinose (Ara)
0.98
3
Manose (Man)
1.08
4
Glucose (Glc)
26.86
13.07
5
Galactose (Gal)
3.50
6
Pinitol
15.26
3.71
7
Inositol
1.02
0.28
8
L-Iditol
0.45
9
Mannitol
0.81
10
Sorbitol
internal standard
11
Fructose (Fru)
0.45
12
Saccharose (Sac)
7.16

Stoiko Liliya et al / Int. J. Res. Ayurveda Pharm. 8 (Suppl 2), 2017
254
Figure 1: GC-MS chromatogram of C. erythraea monosaccharides
after hydrolysis
Figure 2: GC-MS chromatogram of C. erythraea free carbohydrates
Figure 3: Water-soluble polysaccharides (WSPS) and pectin substances (PS) in C. erythraea Rafn.
Glucose is also a precursor for the synthesis of biologically
active molecules such as vitamin C (ascorbic acid) and is
involved in the synthesis of polysaccharides.
The second major identified sugar was pinitol (3-O-methyl-D-
chiro-inositol). Its content in the bonded state was 15.26 mg/g
and in free state 3.71 mg/g. The anti-diabetic action of pinitol
was shown in normal, obese-diabetic (ob/ob) and streptozotocin
(STZ)-diabetic mice when administered orally (p.o.) and
intraperitoneally (i.p.)13.
The anti-tumor effect of D-pinitol against 7,12-
dimethylbenzanthracene (DMBA)-initiated rat mammary
carcinogenesis have been investigated in vivo and its mechanism
of action was determined. D-pinitol mitigates tumor growth by
modulating interleukins and hormones and induces apoptosis in
rat breast cancer cells through inhibition of necrosis factor-α14.
Saccharose in free carbohydrates was present in the analyzed
samples in the amount of 7.16 mg/g. It is an easily assimilated
macronutrient that provides a quick source of energy.
CONCLUSION
In this study, we determined composition and quantified
polysaccharides, monosaccharides and monosaccharide
derivatives in dry herb of Centaurium erythraea Rafn., a plant
used in traditional medicine preparations. We used gas
chromatography coupled to mass spectrometry as a relatively
simple and efficient method to determine these compounds, and
these methods can be adapted to determine poly- and
monosaccharide content in other herbal material. The main
compounds identified in Centaurea herb were glucose, pinitol
and saccharose as well as water-soluble polysaccharides and
pectin substances, which, based on published animal studies, can
be involved in therapeutic properties of the herb.
These results point to the need for further studies of
pharmacological effects of the plant and of poly-and
monosaccharides.
REFERENCES
1. Vaughan JG, Judd PA. The Oxford Book of Health Foods.
Oxford: Oxford University Press; 2006.
2. Kumarasamy Y, Nahar L, Cox P J, Jaspars M, Sarker SD.
Bioactivity of secoiridoid glycosides from Centaurium
erythraea. Phytomedicine 2003; 10: 344-347.
3. Berkan T, Ustunes L, Lermioglu F, Ozer A. Anti-
inflammatory, analgesic, and antipyretic effects of an
aqueous extract of Erythraea centaurium. Planta Med
1991; 57:34-37.
4. Kupeli EA, Suntar I, Keles H, Aktay G. Evaluation of the
wound healing activity of Buxus sempervirens L. and
Centaurium erythraea (L.) Rafn. by using in vivo and in
vitro Methods. Planta Med 2013; 13:1155.
5. Marchyshyn SM, Stoiko LI. Definition of Phenolic
compounds in herbs of Centaurium erythraea Rafn. by
HPLC. Pharmaceutical review 2014; 1:15-17.
6. Marchyshyn SM, Stoiko LI. The composition of lipophilic
fraction from the herbs of Centaurium erythraea Rafn.
Ukr Biopharm J 2015; 1:65-68.
7. Marchyshyn SM, Stoiko LI. Volatile compounds of
Centaurium erythraea Rafn. and Gentiana cruciata L.
Phytotherapy. Review 2016; 3:45-48.
8. Kyslychenko VS, Zhuravel IO, Marchyshyn SM,
Minarchenko VM, Khvorost OP Pharmacognosy. 1st ed.
Kharkiv: Golden Pages; 2015.
9. Chen Y, Xie MY, Wang YX, Nie SP, Li C. Analysis of
the monosaccharide composition of purified
polysaccharides in Ganoderma atrum by capillary gas
chromatography. J Phytochem Anal 2009; 20:503.
10. Kozachok S, Marchyshyn S, Ostapchuk A, Zavyalova L.
Monosaccharide composition of Herniaria glabra L. and

Stoiko Liliya et al / Int. J. Res. Ayurveda Pharm. 8 (Suppl 2), 2017
255
Herniaria polygama J.Gay. Curr Issues Pharm Med Sci
2016; 29: 142-145.
11. Marchyshyn SM, Demydyak OL, Dakhym IS, Berdei TS,
Kozyr HR. Investigation of Polysaccharide complexes of
Asteraceae Family Plants. Sci J ScienceRise 2015; 10(4,
Suppl 15):31-36.
12. Mergenthaler P, Lindauer U, Dienel GA, Meisel A. Sugar
for the brain: the role of glucose in physiological and
pathological brain function. Trends Neurosci. 2013;
36:587-597.
13. Bates SH, Jones RB, Bailey CJ. Insulin-like effect of
pinitol. Br. J. Pharmacol. 2000; 130:1944-1948.
14. Rengarajan T, Nandakumar N. D-pinitol mitigates tumor
growth by modulating interleukins and hormones and
induces apoptosis in rat breast carcinogenesis through
inhibition of NF-κB. J Physiol Biochem 2015; 71:191-
204. Rengarajan T, Nandakumar N, Rajendran P, Ganesh
MK, Balasubramanian MP, Nishigaki I.
Cite this article as:
Stoiko Liliya et al. Polysaccharides in Centaurium erythraea
Rafn. Int. J. Res. Ayurveda Pharm. 2017;8(Suppl 2):252-255
http://dx.doi.org/10.7897/2277-4343.082122
Source of support: Nil, Conflict of interest: None Declared
Disclaimer: IJRAP is solely owned by Moksha Publishing House - A non-profit publishing house, dedicated to publish quality research, while
every effort has been taken to verify the accuracy of the content published in our Journal. IJRAP cannot accept any responsibility or liability
for the site content and articles published. The views expressed in ar ticles by our contributing authors are not necessarily those of IJRAP
editor or editorial board members.