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FARMACIA, 2009, Vol.LVII, 1
HPLC EVALUATION OF PHENOLIC AND
POLYPHENOLIC ACIDS FROM PROPOLIS
ANDA NATANELA CROCI, BOGDAN CIOROIU, DOINA LAZAR,
ANDREIA CORCIOVA, BIANCA IVANESCU, MIHAI IOAN LAZAR*
University of Medicine and Pharmacy “Gr. T. Popa”, Faculty of
Pharmacy, Department of Drugs Analysis, Iasi, Romania
*
corresponding author: lmihaiioan@yahoo.com
Abstract
The aim of our study was to evaluate phenolic and polyphenolic acids from
different sorts of propolis from Romania and Israel using an HPLC method. We established
the working conditions regarding columns, mobile phase. For each type of propolis we have
determined the percentage of phenolic and polyphenolic acids within the raw material.
Rezumat
Cercetarea a avut drept scop evaluarea cantitativă printr-o metodă HPLC a acizilor
fenolici şi polifenolici din sorturi de propolis recoltate din România şi Israel. S-au determinat
condiţiile de lucru în ceea ce priveşte coloanele, faza mobilă. Pentru fiecare determinare s-a
menţionat conţinutul procentual în acizi fenolici şi polifenolici, raportat la materialul brut.
Keywords: propolis, HPLC, phenolic acids, polyphenolic acids
Introduction
Propolis or “bee glue” is a resinous product that honey bees, Apis
mellifera L., gather from tree buds and accumulate in the hives. Because
bees collect propolis from different species of Populus, Betula, Ulmus,
Quercus, Salix, Aesculus hippocastanum, Picea, Fraxinus etc. its colour and
chemical composition vary considerably [1, 2].
Propolis chemical composition is complex and includes resins, waxes,
volatile oils, flavonic compounds, phenol carboxylic acids, carbohydrates,
amino acids, vitamins, enzymes, minerals and vegetal impurities [3].
Numerous alcoholic extracts obtained from propolis have been
used as antibacterial, antifungal, antiviral, local anaesthetic, astringent,
antispasmodic, anti-inflammatory, antiulcerous, cytostatic, hypotensive,
immunostimulating. The above mentioned pharmacological activities are
attributed to the complex of chemical compounds among which phenolic
acids play an important part [4, 5].
Materials and methods
In this study we identified and quantified phenolic and
polyphenolic acids from 12 sorts of propolis, 3 from Romania (1-3) and 9
from Israel (4-12) (table I).
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FARMACIA, 2009, Vol.LVII, 1
Table I
Commercial sorts of propolis taken into analysis
1. Grajduri
7. Tiros -1 (T-1)
2. Raducaneni
8. Tiros -2 (T-2)
3. Dranceni
9. Nes Tioana (N.S.)
4. Kfar Lin – 1 (K L1)
10.Galileea Sea (G.S.)
5. Kfar Lin - 2 (K L2)
11. Oca-Ierusalim (O.I.)
6. Iad Mordechai (I.A)
12. Tel-Aviv (T.A)
All phenolic and polyphenolic acids listed in table II have a high
degree of purity (99%).
1. benzoic acid
2. 3,4-dimethoxycinnamic acid
3. p-hydroxybenzoic acid
4. protocatechic acid
5. chlorogenic acid
6. caffeic acid
7. o-coumaric acid
8. p-coumaric acid
Table II
Standards of phenolic and polyphenolic acids
9. salicylic acid
10. trans-cinnamic acid
11. syringic acid
12. ferulic acid
13. gentisic acid
14. R-(-)-mandelic acid
15. S (+)-mandelic acid
16. vanillic acid
Samples were prepared by extraction in methanol. 1 mL of each
methanolic extract was diluted to 25 mL with acetonitrile.
Quantitative determination of compounds was performed using an
external standard method. Analyses were carried out on an HPLC ThermoSurveyor system equipped with automatic sample injection and diode array
detector (DAD) (the spectral detection interval was 190-360 nm). The
chromatographic data were processed using ChromQuest software, model
4.2.34, equipped with a spectral identification module of the compounds
separated on the column.
A Discovery RP-Amide (C16) column (250 x 4.6 mm i.d., 5 μm
particle) was employed initially. For the improvement of the separation method
we also used a Zorbax Eclipse XDB – C18 column (250 x 4.6 mm, 5µm)
Agilent Technologies and a Hypersil BDS C18 (250 x 4.6 mm, 5µm) Thermo –
Fisher. Column temperature was 25ºC and injection volume was 20 µl.
Based on molecular spectra of standards we used two wavelengths,
these being specific to the absorption maximum corresponding to the
identified molecular spectra. The flow rate was 1 mL/min and UV detection
was performed at 254 and 280 nm.
There were made several modifications for the optimization of the
separating method, such as choosing the column, changing the acid used in
the aqueous solution and changing acetonitrile (ACN) proportion in the
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FARMACIA, 2009, Vol.LVII, 1
mobile phase. The best pH value for the separation efficiency was found to
be between 2 and 3.
The mobile phase was a mixture of acetonitrile (Merck) and acetic
acid 10% (Merck). A linear gradient was used for elution as described bellow.
time
0
30
35
37
CH3COOH (10%)
92
9
92
92
ACN
8
91
8
8
HPLC chromatogram of standards is presented in figure 1. The
typical separation of phenolic acids is shown in table III and expressed in
retention times.
Figure 1
HPLC Chromatogram for standards
Standard
1. benzoic acid
2. 3,4-dimethoxycinnamic acid
3. p-hydroxybenzoic acid
4. protocatechic acid
5. chlorogenic acid
6. caffeic acid
7. o-coumaric acid
8. p-coumaric acid
Retention
time (min)
4
6
6.5
8
8.5
11
11.5
12
Table III
Standards retention times
Standard
Retention
time (min)
9. salicylic acid
12.5
10. trans-cinnamic acid
14
11. syringic acid
16
12. ferulic acid
17.5
13. gentisic acid
19
14. R-(-)-mandelic acid
21
15. S (+)-mandelic acid
22
16. vanillic acid
27
In the experimental conditions of this study, we established
calibration curves for standards and regression coefficients (table IV). The
linearity of calibration curves for all compounds was very good (R2>0.99).
FARMACIA, 2009, Vol.LVII, 1
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
55
Table IV
Calibration curves and regression coefficients for all standards
Regression
coefficient
Standard
Calibration curve
(R2)
benzoic acid
y = 199237096.64x - 158457.37
0.9993
3,4-dimethoxycinnamic acid y = 3307809.99010x + 662.47539
0.9994
p-hydroxybenzoic acid
y = 51750916.98x + 58043.91
0.9962
protocatechic acid
y = 195863578.8530x - 158400.3749
0.9982
chlorogenic acid
y = 102959211.96x - 124289.45
0.9971
caffeic acid
y = 138369780.954x + 321.016
0.9985
o-coumaric acid
y = 238714527.419x - 163985.627
0.9978
p-coumaric acid
y = 15677369.8768x - 109254.6761
0.9979
salicylic acid
y = 234114810.824x - 298375.080
0.9972
trans-cinnamic acid
y = 105257913.0789x - 3890.0812
0.9984
syringic acid
y = 145009401.8088x + 12875.0680
0.9995
ferulic acid
y = 140197319.236x - 30673.907
1.000
gentisic acid
y = 33264224.725x + 87463.408
0.9999
R-(-)-mandelic acid
y = 51750916.98x + 58043.91
0.9962
S-(+)-mandelic acid
y = 51750916.98x + 58043.91
0.9962
vanillic acid
y = 195863578.8530x - 158400.3777
0.9978
Based on calibration curves and considering the dilutions made, we
established the content in phenolic acids for all propolis samples.
Detection limits were determined in accordance with the standard
deviations at minimum concentrations and slopes values of each analyzed
compound (table V).
Table V
Detection and quantification limits of phenolic and polyphenolic acids
Standards
Quantification limit (mg/mL)
Detection limit (mg/mL)
benzoic acid
0.000198464
0.00005954
3,4-dimethoxycinnamic acid
0.008268
0.002481
p-hydroxybenzoic acid
0.00246
0.000738
protocatechuic acid
0.000243019
0.00007291
chlorogenic acid
0.000176
0.00005273
caffeic acid
0.000164
0.00004912
o-coumaric acid
0.00006504
0.00001951
p-coumaric acid
0.000235
0.0000704
salicylic acid
0.000120165
0.0000360
trans-cinnamic acid
0.000055
0.0000167
syringic acid
0.0000865
0.0000259
ferulic acid
0.000039
0.0000117
gentisic acid
0.000262
0.00007856
R-(-)-mandelic acid
0.000246
0.0000696
S-(+)-mandelic acid
0.000235
0.0000700
vanillic acid
0.008457
0.0028877
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FARMACIA, 2009, Vol.LVII, 1
Results and discussion
The phenolic and polyphenolic acids content from the raw propolis
samples determined by HPLC method is listed in table VI and VII.
Table VI
Phenolic and polyphenolic acids content, in Romanian propolis
Content % (g)
Phenolic acids
Grajduri
Raducaneni
Dranceni
benzoic acid
1.38
1.25
1.34
3,4-dimethoxycinnamic acid
2.25
2.25
2.50
p-hydroxybenzoic acid
1.30
1.30
1.15
protocatechic acid
2.50
2.00
1.90
chlorogenic acid
1.15
1.00
1.25
caffeic acid
1.56
1.56
1.55
o-coumaric acid
1.12
1.10
1.30
p-coumaric acid
1.10
1.10
0.99
salicylic acid
trans-cinnamic acid
0.89
0.89
1.00
syringic acid
0.38
0.60
0.55
ferulic acid
1.54
1.25
1.15
gentisic acid
1.46
1.46
1.50
R-(-)-mandelic acid
1.45
1.12
1.11
S-(+)-mandelic acid
1.25
1.00
1.05
vanillic acid
0.80
0.55
0.43
TOTAL
20.13
18.43
18.77
Table VII
Phenolic and polyphenolic acids content, in Israeli propolis
Phenolic acids
benzoic acid
3,4-dimethoxycinnamic
acid
p-hydroxybenzoic acid
protocatechic acid
chlorogenic acid
caffeic acid
o-coumaric acid
p-coumaric acid
salicylic acid
trans-cinnamic acid
syringic acid
ferulic acid
gentisic acid
R-(-)-mandelic acid
S-(+)-mandelic acid
vanillic acid
TOTAL
K-L1
1.22
K L2
1.22
I.A
1.50
Content % (g)
T-1
T-2
N.S
1.50
1.53
1.35
.G.S
1.45
O.I
1.76
T.A
1.33
2.25
2.48
2.55
2.30
2.35
1.15
1.17
1.63
1.65
1.30
1.32
1.33
1.70
1.22
1.42
1.32
0.90
1.23
1.02
1.00
1.12
1.49
19.84
1.30
1.45
1.50
1.60
1.24
1.60
1.27
1.09
1.23
1.12
1.19
1.10
1.39
20.78
1.32
1.53
1.70
1.70
1.43
1.64
1.34
1.11
1.32
1.30
1.22
1.15
1.50
22.31
1.40
1.76
1.60
1.60
1.30
1.54
1.50
0.89
1.40
1.33
1.20
1.29
1.48
22.09
1.50
2.10
1.55
1.49
1.90
1.49
1.39
0.90
1.32
1.22
1.19
1.29
1.47
22.69
1.62
1.34
1.43
1.42
1.19
1.35
1.40
0.99
1.37
1.24
1.19
1.22
1.50
19.76
1.32
1.49
1.40
1.39
1.24
1.40
1.66
0.70
1.51
1.22
1.22
1.34
1.50
20.01
1.28
1.22
1.35
1.61
1.27
1.38
1.62
0.65
1.33
1.28
1.18
1.24
1.44
20.24
1.34
1.50
1.30
1.59
1.25
1.44
1.63
0.55
1.39
1.26
1.22
1.26
1.57
20.28
FARMACIA, 2009, Vol.LVII, 1
57
In all sorts of propolis from Romania and Israel were find the
standards used, except salicylic acid. In some of Dranceni (Romania)
samples we identify traces of this acid.
Conclusions
HPLC determination of free phenolic and polyphenolic acids from
alcoholic extracts revealed the importance of a judicious choice of
experimental conditions. Romanian propolis has a phenolic acids content
varying between 18.43 and 20.13%. Israeli propolis has a phenolic and
polyphenolic acids content varying between 19.76 and 22.69%. The
medium content in phenolic acids in Romanian propolis is 19.11% and
Israel’s propolis is 20.88%.
1.
2.
3.
4.
5.
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Manuscript received: 28.07.2008