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QUANTITATIVE ANALYSIS OF SOME
PHENOLIC COMPOUNDS FROM VIOLA
SPECIES TINCTURES
ANCA TOIU*, L. VLASE, ILIOARA ONIGA, M. TĂMAŞ
University of Medicine and Pharmacy „Iuliu Hatieganu”, Faculty of
Pharmacy, 12, Ion Creangă Street, 400023, Cluj-Napoca, Romania,
*
corresponding author ancamaria_toiu@yahoo.com
Abstract
We analysed the 10% tinctures of air-dried flowering aerial parts from three Viola
species (Violaceae) and we have performed spectrophotometric determinations of flavonoids
and polyphenol carboxylic acids and a HPLC study of salicylic acid derivatives. For V.
tricolor L. we have established the content in flavonoids (2.108%), polyphenol carboxylic
acids (0.921%) and salicylic acid (91.83·10–3 %). For V. arvensis Murray and V. declinata
Waldst. et Kit. results obtained were, respectively: 1.846% and 1.694% flavonoids, 0.632%
and 0.505% polyphenol carboxylic acids, 92.21·10–3 % and 137.24·10–3 % salicylic acid. We
observed small differences in the chemical composition of the three Viola species.
Rezumat
Am analizat tincturile 10% preparate din părţile aeriene uscate ale unor specii de
Viola (Violaceae) prin determinarea spectrofotometrică a flavonoidelor şi a acizilor
polifenolcarboxilici, iar derivaţii de acid salicilic printr-o metodă HPLC. Pentru specia
Viola tricolor L. au fost obţinute următoarele rezultate: concentraţia de flavonoide este de
2,108%, cea de acizi polifenolcarboxilici de 0,921%, iar cea de acid salicilic de 91,83·10–3
%. Pentru V. arvensis Murray şi V. declinata Waldst. et Kit. rezultatele obţinute au fost,
respectiv: 1,846% şi 1,694% flavonoide, 0,632% şi 0,505% acizi polifenolcarboxilici şi
92,21·10–3 % şi 137,24·10–3 % acid salicilic. S-au observat mici diferenţe ale compoziţiei
chimice între cele trei specii de Viola analizate.


Viola sp.
Flavonoids


salicylic acid
HPLC
INTRODUCTION
The most important medicinal plants from Viola genus (Violaceae)
are considerered V. tricolor, V. arvensis and V. odorata. Wild pansy (Viola
tricolor L.) is largely spread in Romania’s spontaneous flora. The aerial
parts are used in traditional medicine for their anti-inflammatory,
expectorant and diuretic properties, for treating skin conditions, bronchitis,
cystitis, rheumatism. These properties are due to the presence of the
following active principles: saponins, flavonoids, mucilages, salicylic acid
derivatives, coumarins, carotenoids [1- 5].
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The anti-inflammatory activity is related to depurative and antiallergic effects because some of the skin conditions can be caused by
inflammation. Anti-inflammatory properties are ascribed to salicylic
derivatives, rutin and can be enhanced by saponins [6].
In our previous phytochemical studies we have analysed the
polyphenolic compounds (flavonoids, polyphenol carboxylic acids,
anthocyanins, proanthocyanins) from three Viola species by Thin Layer
Chromatography (TLC), High Performance Liquid Chromatography
(HPLC), High Performance Liquid Chromatography – Mass Spectrometry
(HPLC-MS) and by spectrophotometric methods [7,8]. We continued our
reasearch with the analysis of 10% tinctures of V. tricolor, V. arvensis and
V. declinata by spectrophotometric techniques and by a HPLC method.
MATERIALS AND METHODS
We have analysed the aerial parts of Viola tricolor L. and V.
arvensis Murray harvested from Cluj, Romania and V. declinata Waldst. et
Kit. harvested from Alba, Romania in May 2005. The vegetal products were
air dried and then pulverised. The tinctures were prepared as follows: 10g of
vegetal product was extracted with 100g of ethanol 70C at room
temperature, as described in Romanian Pharmacopoeia Xth Edition [9].
Spectrophotometric determinations were performed with a UVVIS JASCO V-530 spectrophotometer.
The quantitative analysis of flavonoids was performed using the
method described in the Romanian Pharmacopoeia Xth Edition in the
monography Cynarae folium [9]. This direct method of quantitative
determination needs aluminium thrichloride as reagent and rutoside as
standard. The absorbance was measured at 430 nm. The calibration curve
was linear between 0.1 mg/mL and 0.4 mg/mL rutoside.
The quantitative analysis of polyphenol carboxylic acids
(caffeic acid derivatives) was performed using the method described in the
Romanian Pharmacopoeia IXth Edition in the monography Cynarae folium
[10]. We used Arnow reagent (sodium nitrite and sodium molibdate) and the
results were expressed in caffeic acid. The calibration curve was linear
between 0.05 mg/mL and 0.25 mg/mL
HPLC determinations:
Apparatus and chromatographic conditions: We used an
Agilent 1100 HPLC Series (Agilent, USA) equipped with a degasser
G1322A, HP 1100 Series binary pump, a Zorbax SB-C18 reversed-phase
analytical column 100 mm x 3,0 mm i.d., 3.5 µm particle (Agilent
technologies, USA), and we operated at 45oC. The mobile phase was a
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binary gradient: distillated water with 0.1% (v/v) orthophosphoric acid 85%
and acetonitrile. The linear gradient started at 5% acetonitrile for 2 minutes,
followed by isocratic elution with 25% acetonitrile over the next 3 minutes.
The flow rate was 1 mL/min and the injection volume was 10 μL.
Samples preparation: The tinctures were centrifuged at 4000 rpm.
The solutions were diluted with distilled water in a 10 mL volumetric flask
and filtered through a 0.45 µm filter before injection.
Detection: We used the fluorescence detector: 310 nm as
excitation wavelength and 450 nm as emission wavelength. Salicylic acid
was identified by external standard method and by comparing its retention
time to the one of the standard, in the same chromatographic conditions and
quantified by external standard method.
The wavelengths for fluorescence detection were chosen in order to
give an optimal sensitivity and selectivity to this method.
Salicylic acid standard (Sigma, Germany) was used in order to
perform quantitative determinations in distilled water solutions with
concentrations between 68 - 21960 ng/mL (Table I).
Table I
The concentrations of salicylic acid (ng/mL) for the calibration curve
Concentration
68.625 137.25 274.5 549 1098 2196 5490 10980 21960
(ng/mL)
6.48
12.07 22.8 46.21 85.7 156.5 449.7 918.2 2040.1
Area
The calibration curve for salicylic acid standard was linear between
68.625-21960 ng/mL and it is presented in figure 1.
2500
y = 0.0918x - 20.962
R2 = 0.9973
2000
1500
Series1
Linear (Series1)
1000
500
0
0
5000
10000
15000
20000
25000
Figure 1
The calibration curve of salicylic acid
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The fluorescence spectrum of salicylic acid (obtained at 310 nm as
excitation wavelength and 450 nm as emission wavelength) is presented in
figure 2.
Norm.
Emission
wavelength
Excitation
wavelength
1000
800
600
400
200
0
200
250
300
350
400
450
nm
Figure 2
The fluorescence spectrum of salicylic acid
RESULTS AND DISCUSSION
The results of quantitative determinations of phenolic compounds
from the three Viola species are shown in table II.
Table II
Quantitative determinations results for the phenolic compounds
Species
Flavonoids
g (%
rutoside)
Polyphenol
carboxylic acids
g (% caffeic acid)
Salicylic
acid g (%)
V. tricolor L.
2.108
0.921
91.83·10–3
V. arvensis Murr.
1.846
0.632
92.21·10–3
V. declinata Waldst. et
Kit.
1.694
0.505
137.24·10–3
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The flavonoids are the major polyphenolic compounds in all the
samples, the richest species being V. tricolor L. Caffeic acid derivatives are
present in small quantities, as well as salicylic acid. The highest level of
salicylic acid was found in Violae declinatae herba.
CONCLUSIONS
We have analysed phenolic compounds (flavonoids, caffeic acid
derivatives and salicylic acid) by spectrophotometric methods and by a
HPLC technique from 10% tinctures of dried aerial parts from V. tricolor, V.
arvensis and V. declinata.
We completed the literature data with qualitative and quantitative
determinations of phenolic compounds from three Viola species. Our
phytochemical study showed small quantitative differences among the three
Viola species.
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