Professional Documents
Culture Documents
Received: 10 July 2002 ■ Summary Background & Aims chlorogenic (9.9 and 1.7 mg/g), caf-
Accepted: 25 November 2002 Yacon (Smallanthus sonchifolius, feic (14.7 and 0.09 mg/g) and fer-
Asteraceae) is a native Andean ulic (traces) acids were determined
plant, cultivated for its tubers in the two fractions. Both fractions
throughout South America. The showed potent antioxidant activity
leaves are used in folk medicine as in DPPH (IC50 = 16.1 ± 3.4 and
a medicinal tea for hypoglycemia. 24.3 ± 2.7 µg/ml) and
This paper describes the antioxi- xanthine/XOD superoxide radical
dant activity of various extracts scavenging (42.0 ± 20.3 and
from S. sonchifolius leaves for their 34.3 ± 11.4 SOD equivalents
content of phenolic components. (U/mg)) tests, they inhibited the
Methods The dried leaves were ex- lipoperoxidation of rat liver subcel-
K. Valentova () · J. Ulrichova · V. Simanek tracted in several ways. Two frac- lular membranes and they pro-
Institute of Medical Chemistry tions were selected for their high tected rat hepatocytes against ox-
and Biochemistry content of phenolic compounds idative injury. Our results may
Palacky University and analyzed by RP-HPLC. The an- predetermine the use of S. sonchi-
Hnevotinska 3
77515 Olomouc, Czech Republic tioxidant activity of these fractions folius leaves in human diet as a po-
Tel./Fax: +4 20-58/5 63-23 02 was tested in 1,1-diphenyl-2-picryl- tential remedy in the prevention of
E-Mail: dankovak@seznam.cz hydrazyl (DPPH) and xanthine/ chronic diseases caused by radicals,
L. Cvak XOD superoxide radical scavenging e. g., arteriosclerosis.
Research Department assays, as inhibition of lipoperoxi-
IVAX-CR dation of subcellular membranes ■ Key words Smallanthus
Opava, Czech Republic
and as protective activity against sonchifolius – phenolic acids –
A. Muck oxidative injury of rat hepatocytes in vitro antioxidant activity –
Institute of Organic Chemistry
and Biochemistry
in primary cultures. Results and cytoprotective effect – rat
Czech Academy of Sciences conclusions The presence of proto- hepatocyte
Prague, Czech Republic catechuic (2.5 and 0.12 mg/g),
mate. In Japan yacon tubers have become popular as a lic compounds content and analyzed by RP-HPLC. In
dietary supplement for people suffering from diabetes vitro antioxidant activity of these fractions was assessed
62 European Journal of Nutrition, Vol. 42, Number 1 (2003)
© Steinkopff Verlag 2003
ence of protocatechuic, chlorogenic, caffeic and ferulic Table 1 HPLC quantification of phenolic acids in extracts from S. sonchifolius
acids in the fractions were proven by comparison with leaves (mg/g of extract)
standards (data not shown). The HP-TLC results were
Extract Protocatechuic Chlorogenic Caffeic acid
then confirmed by HPLC analysis; ferulic acid was pres-
ent only in traces (Fig. 2, Table 1). SOX 2.5 9.9 14.7
OF9 0.12 1.7 0.09
■ Antioxidant activity
The results of antioxidant activities of extracts from S. (Fig. 3), their cytoprotectivity was further studied up to
sonchifolius leaves determined by different non-cellular a concentration of 100 µg/ml. The cytoprotective effects
tests are shown in Table 2. Since the extracts show mod- of extracts on primary cultures of rat hepatocytes intox-
erate cytotoxicity at 1000 µg/ml after 48 h incubation icated by tert-butylhydroperoxide (tBH, 0.5 mM, 1.5 h)
Fig. 2 HPLC analysis of extracts from S. sonchifolius leaves. Mobile phase: A: CH3CN-H2O-CH3COOH (50:49.5:0.5), B: H2O-CH3COOH (99.5:0.5), v/v, linear gradient 0–60 min
5–75 % A in B, column: Luna (C18) 5 µm, 250x2 mm i. d., 0.2 ml/min, detection: DAD total ion current 190–500 nm and ESI MS, standards: 1 – protocatechuic, 2 – chloro-
genic, 3 – caffeic, 4 – ferulic, 5 – rosmarinic, 6 – o-hydroxycinnamic, 7 – cinnamic acid
K. Valentova et al. 65
Antioxidant activity of leaf extracts of S. sonchifolius
is shown in Fig. 4. In all the tests performed, the activity creased recently in finding naturally occurring antioxi-
order of the tested samples was caffeic acid > chloro- dants for use in foods and dietary supplements.
genic acid > OF9 > SOX. Our results suggest that S. sonchifolius leaves are a
promising source of natural antioxidants, mainly phe-
nolic acids. The fraction OF9, even if it contains accord-
Discussion ing to HPLC analysis 14-fold less phenolic acids, dis-
played only 1.5-fold less antioxidant activity than the
Oxidative stress is an important factor in the develop- SOX fraction. The discrepancy between the antioxidant
ment of many chronic diseases, mainly cardiovascular activity, total phenolic content and quantification of
disorders such as arteriosclerosis.Antioxidants are com- phenolic acid in the two tested fractions may be due to a
pounds that can delay or inhibit the oxidation of lipids variety of factors. The fraction OF9 has been prepared in
and other molecules by inhibiting the initiation or prop- a more economic way. Extraction with only 30 % MeOH
agation of oxidizing chain reactions. The antioxidant ac- permitted us to omit one step from the extraction pro-
tivity of phenolic compounds is mainly due to their redox cedure (removal of chlorophyll). Mild temperature con-
properties, which can play an important role in absorb- ditions probably prevented hydrolysis of phenolic gly-
ing and scavenging free radicals, quenching singlet and cosides and polymers. This would play an important
triplet oxygen or decomposing peroxides [10].Due to the role in total phenolic content and antioxidant activity
lack of antioxidants in daily diet, interest has greatly in- but would not appear in the HPLC analysis.
The antioxidant activity of polyphenols is generally stituents other than phenolics may be responsible for
ascribed to their hydroxyl groups, but it is not the only the antioxidant activity. Further work is in progress in
factor in determining the potency of their activities. In our laboratory to identify compounds responsible for
the case of chlorogenic acid (caffeic acid ester with the antioxidant activity.
quinic acid), esterification decreased antioxidant activ-
ity [11]. This is in accordance with our results, in all the
models utilized; chlorogenic acid was a less potent an- Conclusion
tioxidant than caffeic acid.
The relationship between phenolic content and an- Both fractions from the leaves of S. sonchifolius, SOX and
tioxidant activity has been recently evaluated by Veli- OF9, showed a potent antioxidant activity in DPPH and
oglu et al. [12] and Zheng and Wang [10]. Their results xanthine/XOD superoxide radical scavenging tests, in-
indicate that a certain correlation between phenolic hibited the lipoperoxidation of rat liver subcellular
content and antioxidant activity exists, but, in many membranes and protected rat hepatocytes against ox-
cases, there was also antioxidant activity in the herbs idative injury. These results may predetermine S. sonchi-
that may be attributable to other unidentified sub- folius leaves as an active component in dietary supple-
stances or to synergistic interactions. ments for the prevention of chronic diseases.
Our hypothesis concerning polymer polyphenols
and glycosides is in accordance with Lodovici et al. [13] ■ Acknowledgments Financial support of Grant Agency of the
Czech Republic (grant No. 303/01/0171), Ministry of Industry and
who showed that the antioxidant activity of red wine is Commerce (grant No. FD-K/096) and Ministry of Education of the
not restricted to monomeric polyphenols. The fraction Czech Republic (grant No. 1125/2002-G3 and MSM 151100003) is
containing monomers displayed about the same activity greatly acknowledged.
as the polymeric fraction. On the other hand, con-
References
1. Valentova K, Frcek J, Ulrichova J (2001) 5. Buedge JA, Aust SD (1978) Microsomal 9. Bergmeyer HU, Bernt E (1974) Lactate
Yacon (Smallanthus sonchifolius) and lipid peroxidation. Methods Enzymol dehydrogenase: UV-Assay with Pyru-
Maca (Lepidium meyenii), traditional 52:302–310 vate and NADH. In: Bergmeyer HU (ed)
Andean crops as new functional foods 6. Mathiensen L, Malterud K E, Sund RB Methods in Enzymatic Analysis. Aca-
on European market. Chem Listy (1995) Antioxidant activity of fruit exu- demic Press, New York, pp 574–576
95:594–601 date and C-methylated dihydrochal- 10. Zheng W, Wang SY (2001) Antioxidant
2. Singleton VL, Rossi JA Jr. (1965) Col- cones from Myrica gale. Planta Med activity and phenolic compounds in se-
orimetry of total phenolics with phos- 61:515–518 lected herbs. J Agric Food Chem
phomolybdic-phosphotungstic acid 7. Berry MN, Edwards AM, Garrit GJ 49:5165–5170
reagents. Am J Enol Vitic 16:416–417 (1991) Isolated Hepatocytes, Prepara- 11. Chen JH, Ho C-T (1997) Antioxidant
3. Mensor LL, Menezes FS, Leitao GG, Reis tion, Properties and Application. Else- activities of caffeic acid and its related
AS, dos Santos TC, Coube CS, Leitão SG vier, New York, pp 1–441 hydroxycinnamic acid compounds. J
(2001) Screening of Brazilian plant ex- 8. Sieuwerts AM, Klijn JGM, Peters HA, Agric Food Chem 45:2374–2378
tract for antioxidant activity by the use Foekens JA (1995) The MTT tetra- 12. Veioglu ZS, Mazza G, Gao L, Oomah BD
of DPPH free radical method. Phy- zolium salt assay scrutinized: how to (1998) Antioxidant activity and total
tother Res 15:127–130 use this assay reliably to measure meta- phenolics in selected fruits, vegetables,
4. Yinrong L, Yeap Foo L (2001) Antioxi- bolic activity of cell cultures in vitro for and grain products. J Agric Food Chem
dant activities of polyphenols from the assessment of growth characteris- 46:4113–4117
sage (Salvia officinalis). Food Chem tics, IC50-values and cell survival. Eur J 13. Lodovici M, Guglielmi F, Casalini C, Me-
75:197–202 Clin Chem Clin Biochem 33:813–823 oni M, Cheynier V, Dolara P (2001) An-
tioxidant and radical scavenging prop-
erties in vitro of polyphenolic extracts
from red wine. Eur J Nutr 40:74–77