Vol 43, No.

12;Dec 2013

Polyphenolic compounds from propolis highlights by the

chromatographic techniques
Lenuţa-Maria Şuta1, Ionela Belu2*
1
Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Medicine and
Pharmacy, “Victor Babes” 2A Eftimie Murgu Square, Timisoara, 300041, Romania, Phone:
0040722309065
e–mail: sutamarilena@yahoo.com.
2*
Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Medicine and
Pharmacy, 2-4, Petru Rares Street, Craiova, 200349, Romania,
Phone: 0040723513837
e–mail: ionelabelu@gmail.com.

Abstract
The purpose of our study was to evaluate polyphenolic compounds by different types of propolis
by sites of Iron Gates Natural Park (Romania) using a liquid chromatography method. Extracts
and fractions were analyzed by high–performance liquid chromatography and assayed
compounds. In experimental conditions of this sphere, we established calibration curves for
standards and regression coefficients. The linearity of calibration curves for all compounds was
good (R2>0.98). We established the working conditions regarding columns, mobile phase. For
each type of propolis we have determined the percentage by polyphenolic compounds within the
raw material.
The aims of this work were to identify and isolate main polyphenolic compounds in extracts from
popolis, and to quantify the main phenolic compounds from twelve samples by high performance
liquid chromatography (HPLC).
Keywords: liquid chromatography method, propolis, polyphenolic compounds.

Introduction
Propolis is a resinous mixture if honey bees collect by tree buds, sap flows, or other vegetal
sources. Propolis is slimy at and above room temperature, 20°C (68°F). Propolis or “bee glue” is
a resinous outcome if honey bees, Apis mellifera L., gather by tree buds and assemble in hives.
As bees collect propolis by distinct species etc. its colour and chemical characterisation vary
notably [Nie et al., 2013; Mihovsky and Pachev, 2012, Bastos et al., 2011]. Characterisation of
propolis differs by hive to hive, by county to county, and by season to season. Normally it is dark
brown in color, but it can be found in green, red, black, and white hues, according on sources of
resin found in particular hive area. Chemical biocharacterisation of propolis varies considerably
by region to region, along by vegetation. In temperate areas, i.e. bees collect resins by trees, such
as poplars and conifers. Typical northern temperate propolis possesses roughly fifty
bioconstituents, primarily resins and vegetable balsams (50%), waxes (30%), essential oils (10%),
pollen (5%) and vegetal impurities [Król et al., 2013, Mitev and Naydenova, 2012]. In neotropical
areas, to a large variety of trees, bees may also gather resin by flowers, which are only known
plant genera if produce floral resins to attract pollinators. Many alcoholic extracts obtained by
propolis have been used as antibacterial, antifungal, local anaesthetic [Pribac and Caunii, 2013]
antiviral, astringent, anti–inflammatory [Ramanauskiene et al., 2009; Caunii and Pribac, 2013]
antiulcerous, cytostatic, hypotensive [Murase et al., 2008]. Pharmacological effects are attributed
to complex of biocompounds among which polyphenolic play a part [Medić–Sarić et al., 2009;
Samfira et al., 2013]. Given immense revenues generated by folk medicines, and modern
pharmaceutical drugs, it is not startling if medicinal use of propolis has both its supporters/rivals.
Supporters of propolis advocate if it has been used for thousands of years, is unlikely to have

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remained its affinity as a folk medicine if its use was ineffective or associated by severe adverse
reactions. Rivals advocate that propolis characterisation varies geographically, seasonally, by bee
species, if it is irresponsible to promote its use without extensive in vitro, in vivo, clinical assay to
represent both safety [Ahangari et al., 2013; Larki et al., 2013 Zhekova and Petkova, 2010].
For impartial information, it is opportune to consult National Institutes of Health. Usually, there
is inaccurate proof for rate efficaciousness by propolis in treating various and different types of
metabolic deficiencies, or other conditions. Propolis is focus by a lot of research. Some
preliminary research findings (literature), together by their limitations, are contained in this study.
Readers are reminded if following data represents preliminary research and does not constitute
medical advice. Readers are directed to their local physician or healthcare provider for medical
advice.

Materials and methods

In this study we detected and quantified polyphenolic compounds by twelve types of propolis,

Table 1. Commercial types of propolis taken into analysis

1. Sample types of propolis 1 7. Sample types of propolis 7

2. Sample types of propolis 2
3. Sample types of propolis 3
4. Sample types of propolis 4
5. Sample types of propolis 5
6. Sample types of propolis 6
8. Sample types of propolis 8
9. Sample types of propolis 9
10.Sample types of propolis 10
11. Sample types of propolis 11
12. Sample types of propolis 12

by sites Iron Gates Natural Park (Romania) [Parcul Natural Porțile de Fier] (21º21'–22º 36'
eastern longitude, 44º51'–44º28'30'' northern latitude) notated in table 1and shown in Figure 1.

Figure 1. Iron Gates Natural Park (Romania).

To determine de total content of soluble polyphenols of complete extract and methanolic fraction,
samples were sent to Biochemical laboratory. Samples were prepared by extraction in methanol.
1 mL of each methanolic extract was diluted to 25 mL by acetonitrile. Quantitative determination

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of compounds was performed using an external standard method. Polyphenolic compounds listed
in table 2 have a high degree of purity.
Table 2. Standards of phenolic and polyphenolic acids

1. benzoic acid 9. salicylic acid

2. 3,4–dimethoxycinnamic acid 10. trans–cinnamic acid
3. p–hydroxybenzoic acid 11. syringic acid
4. protocatechic acid 12. ferulic acid
5. chlorogenic acid 13. gentisic acid
6. caffeic acid 14. R–(–)–mandelic acid
7. o–coumaric acid 15. S (+)–mandelic acid
8. p–coumaric acid 16. vanillic acid

Analyses were carried out on an HPLC Thermo–Surveyor system equipped by sample injection,
diode array detector (DAD–interval 190–360 nm). Chromatographic information’s were
processed using ChromQuest software, equipped by a spectral identification module of
biocompounds separated on column. Discovery RP–Amide (C16) column (250 x 4.6 mm i.d., 5 µm
particle) was employed initially. For improvement of separation assay we also used a C18 column.
Column temperature was 25ºC and injection volume was 20 µL. Based on molecular spectra of
standards we used two wave length, these being specific to absorption maximum corresponding
to identify molecular spectra.
Flow rate was 1 mL/min and UV detection was performed at 254/280 nm [Rocha et al., 2013,
Samfira et al., 2013, Rodino et al., 2013]. There were made several modifications for
optimization of separating method, such as choosing column, changing acid used in aqueous
solution and changing acetonitrile proportion in mobile phase. Best pH value for separation
efficiency was found to be between 2 and 3. Mobile phase was a mixture of acetonitrile and acetic
acid 10%. A linear gradient was used for elution as described below. All polyphenolic
biocompounds listed in table 2 have a high degree of purity.

Results and discussion

Our preliminary investigations testing liquid chromatography systems previously reported in
literature for propolis samples resulted in poor resolution of compounds analyzed, or a long run
time. Sample mixture to be separated and analyzed is introduced, in a small volume (microliters),
into stream of mobile phase percolating through column. Biocomponents of sample move through
column at different velocities, which are function of characteristic physical interactions with
stationary phase (sorbent).
Velocity of each biocomponent depends on its biochemical nature, on nature of column
(stationary phase) and on composition of mobile phase. Time at which a characteristic analyte
elutes (emerges from column) is retention time.
Retention time measured under particular conditions is considered an identifying characteristic of
a given analyte. Purification methods, such as extraction and chromatography, were applied to
reduce matrix effects in sample extract, so permitting selective, sensitive detection of target
polyphenolic biocompounds [Thirugnanasampandan et al., 2012, Leelavathi and Kuppan, 2013].
Polyphenolic compounds content by the raw propolis samples determined by liquid
chromatography method is listed in table 6 and 7. Chromatogram (liquid chromatography) of
standards is presented in figure 2.

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Figure 2. Liquid chromatogram for standards

Typical separation of phenolic acids is shown in table 3 and expressed in retention times.

Table 3. Standards retention times

Standard Retention Standard Retention

time (min) time (min)
1. benzoic acid 4 9. salicylic acid 12.5
2. 3,4–dimethoxycinnamic acid 6 10. trans–cinnamic acid 14
3. p–hydroxybenzoic acid 6.5 11. syringic acid 16
4. protocatechic acid 8 12. ferulic acid 17.5
5. chlorogenic acid 8.5 13. gentisic acid 19
6. caffeic acid 11 14. R–(–)–mandelic acid 21
7. o–coumaric acid 11.5 15. S (+)–mandelic acid 22
8. p–coumaric acid 12 16. vanillic acid 27

In experimental conditions of this study, we established calibration curves for standards and
regression coefficients (table 4). The linearity of calibration curves for all compounds was good
(R2>0.98). 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 by standard deviations at minimum
concentrations and slopes values of each analyzed biocompound (table 5).
Quantification showed a good linear relationship between peak area and concentration (r2 > 0.98)
for all standard solutions (Table 4). The limit of quantification (LOQ) and limit of detection
(LOD) were defined by relative standard deviation (RSD > 5%) and by a signal. 3,4–
dimethoxycinnamic acid (caffeic acid dimethyl ester) is antioxidant, antiproliferative and cytotoxic
properties; ornithine decarboxylase and protein tyrosine kinases inhibitor, and exhibits a strong
inhibitory effect on human platelet aggregation [Shimomura et al., 2012, Barbat et al., 2013,
Kershi et al., 2013].
This biocompounds is a cinnamic acid derivative and were major phenolic acids from fruits and
vegetables.
Liquid chromatogram assay separations have theoretical parameters and equations to describe
separation of biocomponents into signal peaks when detected by instrumentation such as by a UV
detector or mass spectrometer.
Resolution equations relate three factors such that high efficiency and separation factors improve
resolution of biocomponent peaks in a liquid chromatogram separation.

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Table 4. Calibration curves and regression coefficients for all standards

Regression
Standard Calibration curve coefficient (R2)
Benzoic acid y=199237076.64x–158457.37 0.9893
3,4–dimethoxycinnamic acid y=3307807.97010x+662.47537 0.9894
p–hydroxybenzoic acid y=51750716.78x+58043.71 0.9862
Protocatechic acid y=175863578.8530x–158400.3747 0.9882
Chlorogenic acid y=102759211.96x–124287.45 0.9871
Caffeic acid y=138367780.954x+321.016 0.9885
o–coumaric acid y=238714527.417x–163785.627 0.9878
p–coumaric acid y=15677367.8768x–107254.6761 0.9879
Salicylic acid y=234114810.824x–278375.080 0.9872
Trans–cinnamic acid y=105257713.0789x–3890.0812 0.9884
Syringic acid y=145007401.8088x+12875.0680 0.9895
Ferulic acid y=140177319.236x–30673.707 0.990
Gentisic acid y=33264224.725x+87463.408 0.9899
r–(–)–mandelic acid y=51750716.98x+58043.71 0.9862
s–(+)–mandelic acid y=51750916.78x+58043.71 0.9862
Vanillic acid y=175863578.8530x–158400.3777 0.9878

The contents of phenolic compounds in these twelve samples are shown in Table 5 and 6.
Table 5. Detection and quantification limits of polyphenolic compounds
Chemical formula Quantification Detection limit
Standards
(molecular formula) limit (mg/mL) (mg/mL)
Benzoic acid C6H5COOH 0.000178 0.00005754
3,4–dimethoxycinnamic acid C11H12O4 0.008268 0.002481
p–hydroxybenzoic acid C7H6O3 0.00246 0.000738
Protocatechuic acid C7H6O4 0.000243 0.00007271
Chlorogenic acid C16H18O9 0.000176 0.00005273
Caffeic acid C9H8O4 0.000164 0.00004712
o–coumaric acid C9H8O3 0.000065 0.00001751
p–coumaric acid C9H8O3 0.000235 0.0000704
Salicylic acid C7H6O3 0.00012 0.0000360
Trans–cinnamic acid C9H8O2 0.000055 0.0000167
Syringic acid C9H10O5 0.0000865 0.0000257
Ferulic acid C10H10O4 0.000037 0.0000117
Gentisic acid C7H6O4 0.000262 0.00007856
r–(–)–mandelic acid C8H8O3 0.000246 0.0000676
s–(+)–mandelic acid C8H8O3 0.000235 0.0000700
Vanillic acid C8H8O4 0.008457 0.0028877

Syringic acid (4–hydroxy–3,5–dimethoxybenzoic acid) is used as organic chemical intermediates,

fragrance and flavour. Syringic acid is a phenolic compound derived from edible plants and fruits
are used as a sedative and local anesthetic, with antitussive and expectorant effect. It is widely
used as a medicine for bronchitis.
Recently syringic acid has been demonstrated to show strong antioxidant, antiproliferative, anti–
endotoxic, anti–cancer activity and hepatoprotective activity [Berretta et al., 2012, Nakov et al.,
2013].

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Table 6. Phenolic and polyphenolic acids content propolis

Biocompound Content % (g) Sample types of propolis
Phenolic acids 1 2 3
Benzoic acid 1.38 1.21 1.37
3,4–dimethoxycinnamic acid 2.25 2.24 2.49
p–hydroxybenzoic acid 1.3 1.29 1.26
Protocatechic acid 1.15 1.97 1.87
Chlorogenic acid 1.14 1.03 1.37
Caffeic acid 1.55 1.49 1.42
o–coumaric acid 1.14 1.23 1.29
p–coumaric acid 1.21 1.17 0.76
Salicylic acid – – –
Trans–cinnamic acid 0.91 0.69 1.09
Syringic acid 0.37 0.6 0.47
Ferulic acid 1.49 1.25 1.21
Gentisic acid 1.47 1.38 1.42
r–(–)–mandelic acid 1.39 1.23 1.26
s–(+)–mandelic acid 1.31 1.01 1.12
Vanillic acid 0.8 0.59 0.39
Total 18.86 18.38 18.79

In addition, it is possible to determine and quantify phenolic compounds in highly complex

biological matrices.

Table 7. Phenolic and polyphenolic acids content propolis

Biocompound Content % (g) in Sample types of propolis

Phenolic acids 4 5 6 7 8 9 10 11 12
Benzoic acid 1.32 1.23 1.51 1.56 1.52 1.385 1.47 1.79 1.37
3,4–dimethoxycinnamic acid 2.35 2.51 2.56 2.29 2.34 1.97 1.19 1.64 1.64
p–hydroxybenzoic acid 1.32 1.32 1.35 1.38 1.52 1.632 1.34 1.27 1.36
Protocatechic acid 1.33 1.47 1.56 1.69 2.14 1.37 1.48 1.25 1.52
Chlorogenic acid 1.34 1.51 1.75 1.63 1.56 1.46 1.41 1.36 1.33
Caffeic acid 1.59 1.59 1.73 1.64 1.48 1.44 1.37 1.63 1.58
o–coumaric acid 1.27 1.27 1.48 1.36 1.91 1.189 1.26 1.28 1.27
p–coumaric acid 1.46 1.59 1.67 1.57 1.48 1.36 1.42 1.39 1.48
Salicylic acid – – – – – – – – –
Trans–cinnamic acid 1.35 1.31 1.38 1.54 1.37 1.42 1.67 1.63 1.62
Syringic acid 0.87 1.11 1.14 0.91 0.91 0.97 0.72 0.66 0.57
Ferulic acid 1.25 1.25 1.36 1.42 1.35 1.35 1.54 1.34 1.38
Gentisic acid 1.07 1.14 1.38 1.37 1.24 1.26 1.25 1.27 1.27
r–(–)–mandelic acid 1.08 1.21 1.29 1.25 1.17 1.17 1.27 1.19 1.25
s–(+)–mandelic acid 1.19 1.11 1.16 1.31 1.28 1.23 1.36 1.26 1.28
Vanillic acid 1.53 1.41 1.53 1.49 1.49 1.51 1.52 1.45 1.59
Total 20.32 21.03 22.85 22.44 22.76 20.71 20.27 20.41 20.51

In all types of propolis by Iron Gates Natural Park (Romania) areas were find the standards used,
except salicylic acid. In some of Sample types of propolis 3 (Romania) samples we identify traces
of this acid.

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Conclusions
Liquid chromatography determination of free polyphenolic compounds by alcoholic extracts
revealed the importance of a judicious choice of experimental conditions. Romanian propolis
possess a syringic acid content varying between 0.37 (sample types of propolis 1) and 1.11%
(sample types of propolis 5). Propolis has a 3,4–dimethoxycinnamic acid content varying between
1.19 (sample types of propolis 10) and 2.49% (sample types of propolis 3). The medium content
in polyphenolic compounds in Romanian (sample types of propolis 2) propolis (sample types of
propolis 6) is 18.38% and propolis is 22.85%.
Furthermore, a precise, accurate and reproducible liquid chromatography determination method
has been developed.

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