ANALYSIS OF BIOLOGICALLY ACTIVE SUBSTANCES IN TUBERS OF JERUSALEM ARTICHOKE (Helianthus tuberosus L.) N. Tr. Petkova, R. Z. Vrancheva I. G. Ivanov, P. P. Denev, A. I. Pavlov, J. N. Aleksieva

1. FOOD RESEARCH AND DEVELOPMENT INSTITUTE - PLOVDIV
UNIVERSITY OF FOOD TECHNOLOGIES 50 YEARS FOODRDI
Bulgaria, 4002 Plovdiv, 26, bulv. “Maritza“ INTERNATIONAL SCIENTIFIC & PRACTICAL CONFERENCE „FOOD, TECHOLOGIES & HEALTH”
November 8-th, 2012 PLOVDIV
Analysis of biologically active substances in tubers of Jerusalem artichoke
(Helianthus tuberosus L.)
N. Tr. Petkova1, R. Z. Vrancheva2, I. G. Ivanov1, P. P. Denev1, A. I. Pavlov1,4, J. N. Aleksieva3
1Department of Organic Chemistry, 2Department of Analytical Chemistry, 3Department of Catering and Tourism,
University of Food Technology, 26, Maritza Blvd., Plovdiv, 4002
4Laboratory of Applied Biotechnologies Plovdiv, The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Science, 139 Ruski Blvd,
4000 Plovdiv, Bulgaria
E-mail: petkovanadejda@abv.bg
Abstract
The ultrasonic and microwave assisted extractions of tubers from the
Jerusalem artichoke (Helianthus tuberosus L.) with water, methanol and 70 %
(v/v) ethanol were carried out. The fructan contents (40-60 % dw) were FOSs
analyzed by the resorcinol method and TLC analysis. The total phenolic fructose
contents and the total flavonoid concentration of the extracts were sucrose
estimated, as their antioxidant activities were determined by DPPH, ABTS,
FRAP and CUPRAC methods. The ethanol extracts obtained by the microwave
extraction showed the greatest antioxidant activity, which was probably due
to the highest content of total phenols. inulin
Key words: inulin, resorcinol assay, total phenols, antioxidant activity, Helianthus tuberosus L. Figure 2. TLC of topinambour tuber extracts (Heliathus tuberosus L.) obtained by ultrasonic
influence with the following solvents methanol (17), 70% ethanol (13), water (14); by
Introduction microwave irradiation with methanol (18), 70% ethanol (15), water(16); used standards
Helianthus tuberosus, also known as topinambour or Jerusalem Glu – glucose, Fru - fructose, Suc – sucrose, FOS - CLR - Frutafit CLR (DP 7-9); HD - Frutafit HD
artichoke, originates from North America and was introduced in (DP 9-12), inulin: TEX - Frutafit TEX (DP 22) and RH - Raftiline HP DP=25
Europe in the early 17th century. This plant is used in some .
countries for small scale industrial production of inulin or it is Table 1. Carbohydrate content in extracts from tubers of Helianthus tuberosus L.
grown as feed material and as a crop for biomass and ethanol Type of extraction Ultrasonic irradiation Microwave irradiation
production (both in the form of tubers and of stems).
Tubers of Jerusalem artichoke are a rich source of biologically Solvants for extraction CH3OH 70% d. H2O CH3OH 70% d. H2O
active substances such as vitamins, sesquiterpens, phenolic acids, C2H5OH C2H5OH
flavonoids and fructans (inulin and fructooligosaccharides). Fructans (inulin and FOSa)
Inulin is a reserve polysaccharide, member of fructan family. It GFn Fm
и fructose, %, dwb 3,9±0,5 46,5±0,4 65,8±1,2 3,1±0,7 44,5±0,3 22,6±2,1
consists mainly of β-(2→1) fructosyl fructose units (Fm), and Figure 1. Chemical structure of inulin aFOS – fructooligosaccharides; bdw- dry weight
usually but not always the chain terminate with α-glucopyranosyl
unit (1→2) (GFn). (Figure 1). The degree of polymerization (DP) of inulin varies from 2 to 70 and The significant yields of fructan (inulin and FOS) were obtained when distilled water and
depends on plant species, harvesting time and post-harvest conditions. Molecules with DP<10 are called 70% ethanol were used as solvents. The highest was the fructan content in water extract
oligofructoses or fructooligosaccharides (FOSs) and are a subgroup of inulin. The maximum amount of after ultrasound-assisted extraction (Table 1).
low- and high-molecular fructans (75-85%) is found in topinambour as the greater part of topinambour
is a low-molecular fraction. Inulin and FOSs are classified as soluble dietary fiber. They act as prebiotic as
stimulate growth of Bifidobacteria, low glucose blood level, improve mineral absorption and possess Table 2. Antioxidant activity of the extracts obtained from tubers of topinambur
immunomodulation effects. (Helianthus tuberosus L.)
Type of Ultrasonic irradiation Microwave irradiation
Aims & Tasks extraction
The main aim of the presented research was the determination of fructan content of Solvants for CH3OH 70 % C2H5OH H2O CH3OH 70 % C2H5OH H2O
Heliathus tuberosus tubers growing in Bulgaria, as well as radical scavenging activity extraction
of extracts with different polarity. DPPH 73,95±0,12 84,72±0,11 n.a 68,73±0,12 166,25±0,15 3,62±0,17
(mM TE*/g dw)
ABTS 48,06±0,23 65,91±0,21 n.a 39,59±0,20 137,60±0,23 47,32±0,19
Materials and Methods (mM TE/g dw)
Moisture content of the dried ground tubers and roots were determined according AOAC 945.32. FRAP 31,00±0,14 61,48±0,17 17,27±0,16 31,02±0,16 105,31±0,13 28,17±0,19
The extraction process of biologically active substances from tubers of Helianthus tuberosus was (mM TE/g dw)
carried out with three solvents – methanol, 70 % (v/v) ethanol and water under ultrasonic and CUPRAC 105,43±0,21 143,23±0,12 64,91±0,13 83,94±0,24 257,40±0,32 83,50±0,24
microwave irradiation. (mM TE/g dw)
Thin-layer chromatography (TLC) of the obtained extracts was performed on silica gel 60 F254 plates Total phenols 5,67±0,21 6,70±0,19 5,97±0,24 3,73±0,27 10,53±0,24 5,24±0,27
(Merck) with BuOH:i-PrOH:H2O:CH3COOH (7:5:4:2) (v/v) – mobile phase and detecting reagent– (mg GAE**/g dw)
diphenylamine-aniline-H3PO4 acetone (1:1:5:50). The fructan content in the extracts were analyzed Total flavonoids 6,15±0,31 16,75±0,27 12,75±0,23 5,63±0,11 25,64±0,20 12,03±0,15
spectrophotometrically at 480 nm by resorcinol-thiourea reagent. The total phenolic and flavonoid (mg EQ***/g dw).
contents were analyzed by using a Folin–Ciocalteu’s assay and Al(NO3)3 reagents.
The obtained methanol, water and 70 % (v/v) ethanol extracts from Helianthus tuberosus tubers n.a – no activity was observed; *TE – Trolox equivalents, **GAE – Gallic acid equivalents; ***EQ
were analyzed by the following antioxidant methods DPPH, ABTS, FRAP and CUPRAC. quercetin equivalents
DPPH assay: 0.15 ml of each extract was mixed with 2.85 ml freshly prepared DPPH solution (0.1
Mm in methanol). After incubating for 15 minutes at 37 º C in darkness, the reduction of absorbance The highest yields of total phenols and flavonoids were achieved with 70 % (v/v) ethanol
associated with microwave irradiation (Table 2). The same extracts possessed the greatest
antioxidant activities.
at 517 nm was measured by spectrophotometer.
ABTS assay: For the assay, 2.85 ml of diluted with methanol
(1:30; v/v) the ABTS. solution was mixed with 0.15 ml of Conclusion
obtained extracts. After 15 min at 37 º C in darkness the The highest yields of inulin and FOSs have been obtained from Helianthus tuberosus L.
absorbance was measured spectrophotometrically at 734 nm. under ultrasonic irradiation using water as a solvent. The solvent 70 % (v/v) ethanol is
FRAP assay: 0.1 ml of investigated extracts were added to 3 ml proper for extraction of biological active substances. The ethanol extracts obtained after
FRAP reagent (0,3 М acetate buffer (рН 3,6) : 10 mM 2,4,6- microwave influence showed the highest antioxidant activity determined by the
H
OH
tripyridyl-s-triazine (TPTZ): 20 mM FeCl3x6H2O (10:1:1; v/v/v) following methods: DPPH, ABTS, FRAP and CUPRAC. On the base of the results from our
H O
and allowed to react for 10 min at 37 °С in darkness. The investigation we can conclude that tubers of topinambour (Helianthus tuberosus L.)
HO
H
absorbance of the formed colored product were measured at contain not only dietary fibers – inulin and fructooligosacharides, but they are
H
HO
CH 2OH H
OH
O
593 nm. perspective source for extraction of biological active substances with antioxidant.
O
HO
CUPRAC assay: The reaction was started by mixing of 1 ml
CuCl2X2H2O, 1 ml Neocuproine (7.5 ml in methanol), 1ml 0.1
H 2C
HO
n M ammonium acetate buffer , 0,1 ml of analyzed extracts and
CH 2OH
O
O
1 ml 1 ml dd H2O. The reaction time was 20 min at 50 °С in Aknowledgements
HO
darkness. After cooling the absorbance was measured at 450 This investigation was supported by the European Community`s Seventh Framework
CH2 OH
nm. Programe (FP7/2007-2013) under grant agreement n. 227118, project BaSe Food.
HO