From functional food to medicinal product: Systematic approach in analysis of polyphenolics from propolis and wine
© Medić-Šarić et al; licensee BioMed Central Ltd. 2009
Received: 17 April 2009
Accepted: 22 July 2009
Published: 22 July 2009
In the last decade we have been working on standardization of propolis extract and determination of active constituents of wine those are rich in polyphenolics and have nutritional as well as therapeutic value. Here we are summarizing our results and providing overview on systematic approach how to analyse natural products rich in flavonoids and phenolic acids.
Chromatographic methods (thin layer chromatography and high performance liquid chromatography) were used for identification, quantification, and characterization of individual flavonoid or phenolic acid. Total content of active constituents and antioxidant activity were determined by spectrophotometry. Pharmacokinetic parameters were determined by high performance liquid chromatography and using appropriate software. Quantitative structure-activity relationship study of antioxidant activity was conducted, as well as assessment of prolonged propolis supplementation on antioxidative status of organism.
Thin layer chromatography-densitometry has been proven as quick and reliable method for standard analysis of propolis and wine; the best mobile phase being chloroform – methanol – formic acid (98–100%) in ratio 44 : 3.5 : 2.5 (v/v). Higher number of polyphenolics was determined by high performance liquid chromatography; 15 compared to 9 by thin layer chromatography. Interactions in situ with acetylsalicylic acid were detected with most of polyphenolics analysed. Plasma protein binding and blood-barrier penetration was greatest for flavone. The interactions with human serum albumin have been grater than 95% for all flavonoids analysed. The prolonged propolis consumption increased superoxide dismutase activity.
The necessity of standardization of natural products and their registration as functional nutraceuticals demand easy, quick and inexpensive methods of analysis. In this work we provided overview of analytical part for polyphenolics that could be used as data for possible registration of final products either as functional food or medicinal product.
This feature introduces the readers to the authors' research through a concise overview of the selected topic. Reference to important work from others in the field is included.
The main causes of death in western countries are cardiovascular diseases and cancer. Over 50 percent of the population has some kind of chronic condition (high blood pressure, high cholesterol, arthritis, diabetes, asthma, osteoporosis...), so the goal of the many researches in the last decades is improving the quality of life. Much of the interested was transfered to homeopathy, alternative, and folk medicine, evincing polyphenols as one of the main nutraceuticals .
The most abundant sources of polyphenols, mainly flavonoids and phenolic acids, are propolis and wine. Flavonoids and phenolic acids have antibacterial, antifungal, antiviral, antineoplastic, hepatoprotective, immunomodulating, and antiinflammatory properties. Their use has been proven beneficial in allergies, asthma, diabetes, hypertension, micro bleeding, etc. Much of these pharmacological effects can be associated with antioxidant activity. Hence, antioxidant activity is the most studied property of polyphenols .
In the last decade, we were working on several projects with the aim of developing standardized extracts of propolis and determination of active constituents of wine, transferring knowledge from the laboratory to everyday-practice for the purpose of analyzing natural products. To accomplish this we used rather simple, reliable, and quick spectrophotometric and liquid chromatography methods for analysis of polyphenols, the results of which are reviewed here. These were used to suggest mixtures of propolis that will provide the best final product.
In this review we wanted to systematize the approach in analysis of polyphenols rich samples providing mostly unpublished data (Published data is marked as reference). Some basic concepts of optimization of chromatographic system, chromatographic parameters describing pharmacokinetic and quantitative structure-activity relationship are described. The use of liquid chromatography for different objectives is thoroughly illustrated: identification, quantification, characterization; ADME study (Absorption, Distribution, Metabolism, Excretion) and interactions. To demonstrate in vivo antioxidant effects of polyphenols from propolis preliminary clinical trial of prolongated propolis is presented.
Flavonoids and Phenolic Acids. Propolis and Wine
Wine is an alcoholic beverage that contains a large amount of different polyphenols extracted from grapes during the processes of vinification. These molecules are responsible for color, acerbity, flavor, and antioxidant properties of wine .
Optimization of Chromatographic Systems 
The purpose of optimization of chromatographic systems is to find the one that shows the greatest difference in identification characteristics between substances e.g. retention factor (R F) value in thin layer chromatography.
To accomplish this numerical taxonomy methods were used. Taxonomic entities are polyphenols and their numerical characteristic is R F value. Matrix of resemblance of R F values is analyzed to find the mobile phase in which the resemblance is the lowest and separation of analyzed substances is optimal. In short, the matrix of N substances and t mobile phases is reduced using different algorithms to form a cluster of similar mobile phases, the procedure is repeated until the final cluster including all mobile phases is formed and the results are presented in form of dendrogram.
The highest DP and the smallest T value are attributed to the most appropriate combination of eluents.
This procedure of optimization has been used in the analysis of numerous plant samples: Zizyphus jujuba Mill., Chamomilla recutita (L.) Rauschert, Rosmarini folium, Guiera senegalensis J.F.GMEL., Rhamni cathartici fructus, Lavandulae flos, Sambuci flos, Helleborus atrorubens Waldst. et Kit. etc. [7–14].
Chromatographic Parameters Describing Pharmacokinetics of Polyphenols 
As our samples are consumed orally absorption and distribution of polyphenols are of interest. Absorption through the gastrointestinal tract (GIT) membrane, plasma protein binding and the penetration through blood brain barrier (BBB) was studied using in silico approaches and liquid chromatography.
where b is the coefficient of the slope and Φ is the concentration of the organic modifier.
If the polyphenol shows a binding of over 95% it can potentially interact with other drugs of high affinity to HSA as the concentration of active (free) drug can increase significantly (e.g. 5% to 8% is increase of 60% in active form of drug).
Quantitative Structure-Activity Relationship 
The most commonly used topological indices.
Expression for calculation
D i,j represents off-diagonal elements of matrix which stands for the shortest distance in term on number of bonds between atom i and j
Valence connectivity index
v i and v j are weights (valence delta values) of vertices i and j making up edge in vertex weighted graph G
the average distance sum connectivity; where E is the number of edges, μ is cyclomatic number of G and ds i is a distance sum
modified Shannon's equation where n is the number of different sets of elements, N i is the number of elements in the i-th set of elements and the sum is over all sets of elements
molecular topological index (MTI) is based on adjacency matrix (A), the distance matrix (D) and the valency matrix (v); the of elements e i of the row matrix v [A+D] gives Shultz index
Besides QSAR, evolution in rational design has been achieved by using software for assessing physicochemical properties and parameters of bioavailability.
Thin Layer Chromatography
The mobile phases studied.
toluene – ethyl acetate – formic acid (98–100%)
36 : 12 : 5
cyclohexane – ethyl acetate – formic acid (98–100%)
30 : 15 : 5
toluene – ethyl acetate – glacial acetic acid
36 : 12 : 5
cyclohexane – ethyl acetate – glacial acetic acid
31 : 14 : 5
n-hexane – ethyl acetate – formic acid (98–100%)
31 : 14 : 5
toluene – acetone – formic acid (98–100%)
38 : 10 : 5
n-hexane-ethyl acetate – glacial acetic acid
31 : 14 : 5
petroleum ether (40–70°C) – ethyl acetate – formic acid (98–100%)
30 : 15 : 5
carbon tetrachloride – acetone – formic acid (98–100%)
35 : 10 : 5
n-hexane – ethyl acetate – glacial acetic acid
30 : 20 : 1.5
chloroform – methanol – formic acid (98–100%)
44 : 3.5 : 2. 5
DP and I output date for error factor E = 0.03 for each mobile phase.
The presence of coumaric and cinnamic acids could not be established, as these compounds do not form fluorescent complexes with AlCl3.
Results of validation of HPTLC method .
r > 0.99
repeatability of the sample application and intra-day precision
RSD < 3.8%
repeatability of peak-area measurement
RSD < 2.4%
RSD < 6.5%
limit of detection
PME 7.5 ng/band
IFA 7.5 ng/band
CA 60 ng/band
limit of quantification
PME 22.5 ng/band
IFA 22.5 ng/band
CA 180 ng/band
inter-day precision and stability of standard solutions
RSD < 5% (two days)
stability on the plate
RSD < 3.4% (up to 2 hours)*
effect of temperature
RSD < 4.6% (20/26°C)
absorption spectra matching > 0.99
Phenolic acid and flavonoid content (expressed as mean of mass concentration in mg/mL) of the Croatian propolis samples analyzed .
Mixture of propolis
It is evident that R Mw and Φ 0 are well correlated but are not identical. This is the case with isomers sakuranetin and isosakuranetin that have the same chromatographic lipophilicity R Mw = 3.111, but different Φ 0, 19.61 and 20.22 respectively.
Most of the used programs are "blind" to this difference occurring between structural isomers, and for MLOGP even different classes of flavonoids could not be distinguished: galangin (flavonol) and apigenin (flavone).
Results obtained using ChemSilico software .
plasma protein binding
85.41% (myricetin) – 95.65% (flavone)
- phenolic acids
70.22% (sinapic acid) – 85.99% (cinnamic acid)
blood-brain barrier penetration
negative except flavon and flavanone
BBB penetration was positive only for flavone and flavanone, as these do not have hydroxyl groups. Human absorption was the lowest for myricetin (58%).
Surprisingly, correlation of R Mw and calculated log P values was rather poor, and the best correlations were found between Φ 0 and XLOGP values for flavonols, and human intestinal absorption calculated by ChemSilico and chromatographic RMw (for flavanones) and Φ 0 (for phenolic acids) values.
These chemical interactions cannot be replaced in vivo analysis, but rather be used as basis for further research of phenolics in biological systems.
High Performance Liquid Chromatography
Gradient of mobile phases used for HPLC analysis of propolis.
Reverse phase HPLC was used for the lipophilicity/hydrophobicity of flavonoids and phenolic acids. Mobile phase A was 2% acetic acid in water and B 2% acetic acid in methanol. Gradient method started with 95% of mobile phase A and 5% of mobile phase B that was linearly increased to 100% during 20/60 minutes . As shown on chromatogram, phenolic acids as more polar substances were first eluted from the column, followed by more hydrophilic and lipophilic flavonoids. HPLC results were used to identify flavonoids and phenolic acids present in propolis samples, to quantify the content of polyphenols, to explain the antioxidant activity in vitro and in vivo as well as they were used for calculations of pharmacokinetic parameters .
For the IAM-HPLC analysis IAM.PC.DD 2 column was used. Mobile phase was phosphate buffer modified with different volumes of methanol. For the HSA-HPLC analysis Chiral HSA column and gradient of phosphate and 2-propanol were used. IAM-HPLC analysis showed that ionization of polyphenolic under physiological pH was influencing the retention of the substance due to ion-ion interaction. The strongest interactions were in the group of flavonoids (kaempferide log k wIAM = 3.581) and lower for phenolic acids (log k wIAM = 1.35–1.68).
Gradient of mobile phases used for HPLC analysis of wine.
Comparison of the results obtained by HPLC and TLC analysis for Pelješac sample.
The Folin-Ciocalteu method was used for quantification of total polyphenols according to the method described by Slinkard and Singleton . The method is based on the reduction of MoO4+ to MoO3+ that is detected by color change from yellow to blue; measured at 765 nm. The results were expressed as equivalents of gallic acid from the calibration curve.
Quantitative Structure-Activity Relationship
Experimental data used for QSAR analysis was taken from the literature. Wiener index, connectivity index, Balaban index, Balaban-type indices from atomic number, mass, van der Waals, electronegativity and polarizability weighted distance matrix, information-theoretic index, Shultz index, together with molecular weight, n-octanol/water partition coefficient, van der Waals volume, molar refractivity and polar surface area of polyphenols were calculated using TAM , HyperChem 8.0 Evaluation software, PCLIENT, Dragon 3.0. Four groups of 3D descriptors were used: geometrical, GETAWAY (Geometry, Topology and Atom Weights Assembly), 3D-MoRSE and RDF (Radial Distribution Function). Linear, polynomial and multiple linear regression analysis was conducted using Statistica 6.0 .
H 7(p) is an autocorrelation descriptor calculated for 3D-spatial molecular geometry based on lag (topological distance) and weighted by atomic polarizabilities. This descriptor belongs to the group of H-GETAWAY descriptors that have been calculated from the molecular influence matrix H. These descriptors are sensitive to significant conformational changes and to the bond lengths that account for atom types and bond multiplicity.
This QSAR investigation of wine polyphenols could be extended to a larger number of substances, including anthocyanins and dimeric procyanidins as the most common polyphenols in wine.
Clinical Trial 
An in vivo study has been conducted on 47 healthy women and men in order to investigate whether daily intake of powdered propolis extract during 30 days has any influence on antioxidative status based on the following blood parameters: activity of superoxide dismutase, glutathione peroxidase and catalase, concentration of plasma malondialdehyde, total cholesterol, low- and high-density lipoprotein cholesterol, triglycerides, glucose, uric acid, ferritin and transferrin, together with routine red blood cell parameters.
Absence of the same results in women was probably due to uncoordinated menstrual cycles. Hence, estrogens are powerful antioxidants, modulators of antioxidant enzyme expression and levels of lipid peroxidase and lipoproteins.
Summary and conclusion
In the last decade interest on nutraceuticals and natural medicinal products is constantly growing. The market is full of antioxidant formulations of wide variety of sources. For the registration of nutraceutical as natural medicinal product analytical procedures have to be developed, product has to be standardized and their functionality and beneficial effects have to be demonstrated. The goal of our studies was standardization of Croatian propolis extracts as a rich sample of flavonoids and phenolic – active components to which pharmacological (antioxidant) activity is attributed. Parallel research on wine samples was done, as wine represents another sample rich in polyphenolics that has been used as constituents e.g. iron wine, tonics.
Identification and quantification of flavonoids and phenolic acids
Although the methods for identification and quantification of propolis samples using HPLC combined with mass spectrometry (MS) and nuclear magnetic resonance (NMR) have been developed and alternative was provided in capillary electrophoresis coupled with MS, these are rather expensive, not readily available and inappropriate for routine analysis [30, 31]. Thus we have decided for TLC method combined with HPLC-diode array detection (DAD) method. Out of 11 mobile phases available from literature based on numerical taxonomy chloroform : methanol : formic acid in volume ratio 44 : 3.5 : 2.5 was selected as the most appropriate for TLC identification of 9 polyphenols . Better separation for identification was achieved using 2D-TLC chromatography, although for quantification we decided for high performance TLC plates using which clear identification and quantification of 7 polyphenols (namely: p-coumaric acid, caffeic acid, chrysin, tectochrysin, pinocembrine-7-methyl ether, isoferulic acid) was performed [18, 19]. Major limiting factor of TLC is length of the plate, so for better separation HPLC was used.
Total content of polyphenols was determined using Folin-Ciocalteu method. As we have done identification using liquid chromatography we did not separately analyze total content of phenolic acids and flavonoids which can be done using well known procedures described in Ph. Eur. and Christ and Müller, respectively [32, 33].
Pharmacokinetic (ADME) parameters of flavonoids and phenolic acids
Passive transport through membranes is mainly determined by the lipophilicity of the substance. The most commonly used experimental value of lipophilicity is chromatographic parameter R M. This parameter was determined by TLC as well by HPLC and to assess lipophilicity within the group of polyphenolics. As this parameter cannot distinguish isomers, additional parameter Φ 0 was used differentiating sakuranetin and isosakuranetin.
Using ChemSilico software plasma protein binding of flavonoids was found rather high (85–96%) compared to phenolic acids (70–86%). High values of flavonoid binding are in accordance to the experimental date e.g. for quercetin (99%) . Passage through blood-brain barrier was negative which is expected for compounds having acidic phenolic and carboxylic groups. Absorption was mainly greater than 60%, but this has to be taken with dose of doubt, as experimental data is contradictory .
Chemical interaction with most commonly used non-steroidal anti-inflammatory drugs (NSAID) and vitamins where characterized on TLC plate. The interactions were most common with acetylsalicylic acid. At the moment this has no practical value as further in vivo studies have to demonstrate applicability of this model .
Interactions of polyphenolics with artificial membranes were probably based on ion-ion and passive diffusion as interaction showed greater partition coefficient for flavonoids than phenolic acids. Binding on HSA-column was higher compared to ChemSilico predictions and more in accordance to experimental data obtained in vivo.
Antioxidant activity of flavonoids and phenolic acids
Antioxidant activity of polyphenolics is well known. To assay the antioxidant activity different methods are used: electron paramagnetic resonance (EPR) spectroscopy, chemiluminescence, enzyme assays... Most often used and readily available is spectrophotometric method using stable free radicals. DPPH· and ABTS·+ were used for spectrophotometry as well TLC in situ for analysis of total antioxidant capacity of propolis. The most active propolis comes from coastal Croatia .
QSAR analysis of polyphenolics based on Trolox test date from literature pointed out that antioxidant activity of polyphenols as hydrogen donating free radical scavengers, is closely related to their chemical structure, especially with the number and arrangement of free hydroxyl groups of polyphenol skeleton .
In vivo study of propolis prolonged used showed beneficial in male population demonstrating reduction in free-radical-induced lipid peroxidation as well as increase in activity of superoxide dismutase. Production of malonaldehyde (degradation product of peroxidation of polyunsaturated fatty acids) reduced and activity of superoxide dismutase (first and most important line of antioxidant enzyme defense) was increased .
Antioxidant supplements are flooding the market. Asking pharmacist for a new product – extract of the plant coming from exotic country, will usually end with the answer "its antioxidant and thus good for your health". The necessity of standardization natural antioxidant products made us write this minireview-providing basis for standardization of natural antioxidant products rich in polyphenols using simple and readily available techniques based on our research on propolis and wine.
absorption, distribution, metabolism, excretion
blood brain barrier
- 3D descriptors:
two-dimensional thin layer chromatography
diode array detection
- DP :
electron paramagnetic resonance
gallic acid equivalent
geometry, topology and atom weights assembly
human serum albumin
high performance thin layer chromatography
immobilized artificial membrane
nuclear magnetic resonance
non-steroidal anti-inflammatory drugs, OTC: over-the-counter
radial distribution function
- R F :
- R M :
chromatographic parameter of lipophilicity
reverse phase high performance liquid chromatography
reverse phase thin layer chromatography
This work was supported by the Ministry of Science, Education and Sports of the Republic of Croatia (projects No. 006-0061117-1237 – MMŠ and 006-0061117-1239 – ŽM). The authors wish to thank all the associates involved in these as well as antecedent projects.
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