XXV ARS SEPARATORIA – Toruń, Poland 2010
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APPLICATION OF CHROMATOGRAPHIC METHOD IN
PHENOLIC COMPOUNDS ANALYSIS IN DIFFERENT PART
FROM CICHORIUM INTYBUS L.
A. FILIPIAK, M. KURZAWA, E. SZŁYK
Nicolaus Copernicus University, Faculty of Chemistry, 7 Gagarin str., 87-100
Toruń, Poland
Abstract
The HPLC – RP- C18 column chromatography with photodiode array
detection is developed for phenolic compounds in different part from Cichorium
Intybus L. is described. The method involving solvent extraction, for determining
the level of quercetin, benzoic and cinamic acid derivatives. The impact of
extraction method (water-bath extraction and ultrasonic extraction), procedure and
various extraction conditions (time and temperature) and acidic and basic
hydrolysis was tested. The developed method was validated for specificity,
repeatability, recovery and accuracy.
1. INTRODUCTION
Cichorium intybus, a typical Mediterranean plant indigenous to Europe,
Western Asia, Egypt and North America, varies in perianth colour from
white, red to blue and the flowering period is from June to September.
Throughout Europe, many varieties of Cichorium intybus L. (Asteraceae)
chicory are quite important agricultural crops, highly appreciated for their
bitter taste. This bitterness is due to the presence of large quantities of
sequiterpene lactones. Apart from terpene, in chicory exist other phenolic
compounds; include flavonoids, coumarins and caffeic acid derivatives [1].
Compounds of this type exhibit a variety of biological activities, e.g.
immunomodulatory, anti-inflammatory, antinociceptive, antihepatotoxic and
antidiabetic activities, inhibitory effects on cAMP phosphodiesterase and
acetylcholinesterase, and stress-reducing activity [2-5].
In the present work, different tissues of the plant - cichorium have been
studied, mostly for the presence of phenolic compounds, such as flavonoids
and phenolic acids derivatives.
2. EXPERIMENTAL
The main aims of our research were improving the sample pretreatment, optimization of extraction procedure and separation of studied
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XXV ARS SEPARATORIA – Toruń, Poland 2010
phenolic acids and flavonols from different part of Cichorium intybus L.
collected in Toruń.
The method involving solvent extraction, HPLC – RP- C18 column
chromatography with photodiode array detection is developed for
identification and determining the level of quercetin derivatives (e.g.,
quercitrin, hyperoside, rutin, rhamnetin, keampherol and miricetine) and
derivatives of benzoic and cinamic acids (e.g., caffeic, chlorogenic, ferulic,
sinapic, coumaric, gallic, salicylic acids). Phenolic compounds were
extracted from the sample matrix with ethanol and water/ethanol mixture at a
various temperature and in a different time. Extraction were performed in
water-bath shaker and bath with ultrasonication and then analyzed before
and after acidic and basic hydrolysis.
The HPLC system equipped with auto sampler SIL-20AC HT and
photodiode multi-wavelength detector (SPD-M20A Prominence Diode
Array Detector), SHIMADZU (Kyoto, Japan) was applied. Phenolic acids
were detected by photodiode array detector at four wavelengths: 254, 295,
310 and 325 nm, using a 45-min program, and 360 nm for flavonols at 50min gradient program. Analyses were carried out on Discovery RP-C18
column (5 µm particle size, 150×4,6 mm, SUPELCO), maintained at 30°C.
Mobile phase for phenolic acids was A: 2% acetic acid whereas phase B was
methanol and for flavonols was A: isopropanol-water (95:5 v/v), B:
isopropanol-water-THF (50:40:10 v/v). Every one of solvents and standards
were of HPLC grade. The rate-flow was 1ml/min in both programs.
The total amount of studied flavonols and phenolic acids were
compared with total phenolic content (TPC) using Folin-Ciocalteu’s method.
The total flavonoids content (TFC) was determined using a colorimetric
method described in Polish Pharmacopoeia. UV-VIS spectra were recorded
on a Spectrophotometer UV Unicam HELIOS α, Spectro-Lab (Warsaw,
Poland). 1 cm quartz cuvette was using.
3. RESULTS
Peaks on the chromatograms were identified by comparison of the
retention times with reference standards and by addition of the individual
reference standard to extracts. Due to complexity of natural samples,
identification of the every peak was impossible.
The average values of studied flavonols and phenolic acids (0.984.12mg/g, 2,13-14,78mg/g, respectively) depending on different extraction
conditions and different part from Cichorium intybus L. TPC was higher
than HPLC-PDA method, because selected phenolic acids and flavonols
constitute small part of phenolic compounds (7.64-32.78%) presented in
studied samples.
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XXV ARS SEPARATORIA – Toruń, Poland 2010
1300000
10
1100000
mAu
900000
700000
2
3
500000
5
300000
1
100000
-100000
0
6
7 8
4
5
10
15
20
9
25
30
35
40
Retention time [min]
Fig. 1. HPLC chromatograms for standards (1-gallic acid, 2-caffeic acid, 3chlorogenic acid, 4-ferulic acid, 5-sinapic acid, 6-coumaric acid, 7-benzoic acid, 8salicylic acid, 9-cinnamic acid, 10-citric acid).
1600000
1400000
Cichorium intybus L.
1200000
CP I
1000000
CP II
mAu
800000
CP III
600000
400000
200000
0
0.00000
5.00267
10.00533
15.00800
20.01067
25.01333
30.01600
35.01867
40.02133
45.02400
-200000
Retention time [min]
Fig. 2. Typical chromatogram for extract from Cichorium intybus L. before
hydrolysis digestion (CP I), after acidic (CP II) and alkaline hydrolysis (CP III)
TPC determined by Folin-Ciocalteau method were higher than total
flavonoids content. The correlation between TPC versus TFC was satisfying
(in every case R2>0.95).
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XXV ARS SEPARATORIA – Toruń, Poland 2010
For the phenolic compounds the highest concentrations were obtained
after acidic hydrolysis (II). Acidic hydrolysis, prior to extraction, caused the
increase of the quantitative yield (e.g., concentration of rutin extracted in
ethanol was 0.23-0.56 mg/g, while after acidic digestion 0.47-1.27 mg/g).
Acidic hydrolysis has been used for measurement of aglycones and phenolic
acids from flavonoid glicosides and phenolic acid esters and other
metabolites presented in plants. Alkaline hydrolysis has been performed for
isolation of the phenolic acids, due to They commonly form covalent bond
with the sample matrix. Hydrolysis is useful tool for the analysis of complex
samples.
4. CONCLUSIONS
Many type of supplement dietary consist extract from Cichorium
intybus L. It is well known that medicinal plants collected at different times
and from different localities may considerably differ in their types and
quantities of chemical components, therefore, resulting in different
therapeutic efficacy. Hence the quality control of herbs, plants and herbal
medicine is an important concern for both the health authorities and the
public.
The developed method was validated for specificity, repeatability,
recovery and accuracy. The results demonstrate that HPLC-PDA method can
be suitable for routine analysis of quality control and quantity evaluation of
botanical products containing phenolic compounds.
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