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

Materials and Methods Moisture content of the dried ground tubers and roots were determined according AOAC 945.32.

The extraction process of biologically active substances from tubers of Helianthus tuberosus was carried out with three solvents – methanol, 70 % (v/v) ethanol and water under ultrasonic and microwave irradiation.

Thin-layer chromatography (TLC) of the obtained extracts was performed on silica gel 60 F254 plates (Merck) with BuOH:i-PrOH:H2O:CH3COOH (7:5:4:2) (v/v) – mobile phase and detecting reagent–diphenylamine-aniline-H3PO4 acetone (1:1:5:50). The fructan content in the extracts were analyzed spectrophotometrically at 480 nm by resorcinol-thiourea reagent. The total phenolic and flavonoid 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 were analyzed by the following antioxidant methods DPPH, ABTS, FRAP and CUPRAC.

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

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 analyzed by the resorcinol method and TLC analysis. The total phenolic contents and the total flavonoid concentration of the extracts were 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.

Type of extraction Ultrasonic irradiation Microwave irradiation

Solvants for extraction CH3OH 70%

C2H5OH

d. H2O CH3OH 70%

C2H5OH

d. H2O

Fructans (inulin and FOSa)

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

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

Introduction Helianthus tuberosus, also known as topinambour or Jerusalem artichoke, originates from North America and was introduced in Europe in the early 17th century. This plant is used in some countries for small scale industrial production of inulin or it is grown as feed material and as a crop for biomass and ethanol production (both in the form of tubers and of stems). Tubers of Jerusalem artichoke are a rich source of biologically active substances such as vitamins, sesquiterpens, phenolic acids, flavonoids and fructans (inulin and fructooligosaccharides). Inulin is a reserve polysaccharide, member of fructan family. It consists mainly of β-(2→1) fructosyl fructose units (Fm), and usually but not always the chain terminate with α-glucopyranosyl

Key words: inulin, resorcinol assay, total phenols, antioxidant activity, Helianthus tuberosus L.

sucrose

FOSs

inulin

Conclusion The highest yields of inulin and FOSs have been obtained from Helianthus tuberosus L. under ultrasonic irradiation using water as a solvent. The solvent 70 % (v/v) ethanol is proper for extraction of biological active substances. The ethanol extracts obtained after microwave influence showed the highest antioxidant activity determined by the following methods: DPPH, ABTS, FRAP and CUPRAC. On the base of the results from our investigation we can conclude that tubers of topinambour (Helianthus tuberosus L.) contain not only dietary fibers – inulin and fructooligosacharides, but they are perspective source for extraction of biological active substances with antioxidant.

Type of

extraction

Ultrasonic irradiation Microwave irradiation

Solvants for

extraction

CH3OH 70 % C2H5OH H2O CH3OH 70 % C2H5OH H2O

DPPH

(mM TE*/g dw)

73,95±0,12 84,72±0,11 n.a 68,73±0,12 166,25±0,15 3,62±0,17

ABTS

(mM TE/g dw)

48,06±0,23 65,91±0,21 n.a 39,59±0,20 137,60±0,23 47,32±0,19

FRAP

(mM TE/g dw)

31,00±0,14 61,48±0,17 17,27±0,16 31,02±0,16 105,31±0,13 28,17±0,19

CUPRAC

(mM TE/g dw)

105,43±0,21 143,23±0,12 64,91±0,13 83,94±0,24 257,40±0,32 83,50±0,24

Total phenols

(mg GAE**/g dw)

5,67±0,21 6,70±0,19 5,97±0,24 3,73±0,27 10,53±0,24 5,24±0,27

Total flavonoids

(mg EQ***/g dw).

6,15±0,31 16,75±0,27 12,75±0,23 5,63±0,11 25,64±0,20 12,03±0,15

n.a – no activity was observed; *TE – Trolox equivalents, **GAE – Gallic acid equivalents; ***EQ quercetin equivalents

Table 2. Antioxidant activity of the extracts obtained from tubers of topinambur (Helianthus tuberosus L.)

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 obtained extracts. After 15 min at 37 º C in darkness the absorbance was measured spectrophotometrically at 734 nm. FRAP assay: 0.1 ml of investigated extracts were added to 3 ml FRAP reagent (0,3 М acetate buffer (рН 3,6) : 10 mM 2,4,6-tripyridyl-s-triazine (TPTZ): 20 mM FeCl3x6H2O (10:1:1; v/v/v) and allowed to react for 10 min at 37 °С in darkness. The absorbance of the formed colored product were measured at 593 nm. CUPRAC assay: The reaction was started by mixing of 1 ml CuCl2X2H2O, 1 ml Neocuproine (7.5 ml in methanol), 1ml 0.1 M ammonium acetate buffer , 0,1 ml of analyzed extracts and 1 ml 1 ml dd H2O. The reaction time was 20 min at 50 °С in darkness. After cooling the absorbance was measured at 450 nm.

O

H

HO

H

HO

H

O

OHH

H

OH

H2C

CH2OH

HO

HO

O

n

O

CH2OH

CH2OH

HO

HO

O

Table 1. Carbohydrate content in extracts from tubers of Helianthus tuberosus L.

aFOS – fructooligosaccharides; bdw- dry weight

fructose

Aknowledgements This investigation was supported by the European Community`s Seventh Framework Programe (FP7/2007-2013) under grant agreement n. 227118, project BaSe Food.

Aims & Tasks The main aim of the presented research was the determination of fructan content of Heliathus tuberosus tubers growing in Bulgaria, as well as radical scavenging activity of extracts with different polarity.

Figure 1. Chemical structure of inulin

unit (1→2) (GFn). (Figure 1). The degree of polymerization (DP) of inulin varies from 2 to 70 and depends on plant species, harvesting time and post-harvest conditions. Molecules with DP<10 are called oligofructoses or fructooligosaccharides (FOSs) and are a subgroup of inulin. The maximum amount of 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 immunomodulation effects.

UNIVERSITY OF FOOD TECHNOLOGIES Bulgaria, 4002 Plovdiv, 26, bulv. “Maritza“

FOOD RESEARCH AND DEVELOPMENT INSTITUTE - PLOVDIV 50 YEARS FOODRDI INTERNATIONAL SCIENTIFIC & PRACTICAL CONFERENCE „FOOD, TECHOLOGIES & HEALTH” November 8-th, 2012 PLOVDIV

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 microwave irradiation with methanol (18), 70% ethanol (15), water(16); used standards Glu – glucose, Fru - fructose, Suc – sucrose, FOS - CLR - Frutafit CLR (DP 7-9); HD - Frutafit HD (DP 9-12), inulin: TEX - Frutafit TEX (DP 22) and RH - Raftiline HP DP=25 .

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.

The significant yields of fructan (inulin and FOS) were obtained when distilled water and 70% ethanol were used as solvents. The highest was the fructan content in water extract after ultrasound-assisted extraction (Table 1).

GFn Fm