Estimation of Flavonoid Content in Propolis by Two Different

Mihai C. M. et. al./Scientific Papers: Animal Science and Biotechnologies, 2010, 43 (1)

Estimation of Flavonoid Content in Propolis by Two Different Colorimetric Methods Cristina Manuela Mihai1, Liviu Al. Mărghitaş1, Otilia Bobiş1, Dan Dezmirean1, Mircea Tămaş2 University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, 400372, Calea Manastur Str., 3-5, Romania 2 University of Medicine and Pharmacy”Iuliu Haţieganu” Cluj-Napoca, 400023, Ion Creanga Str., 12, Romania 1

Abstract Propolis is a natural product with very important properties and its chemical composition was investigated by many researchers. In the present study the polyphenols and flavonoid content was investigated for 10 Romanian propolis samples. Extracts were obtained by using ethanol of different concentrations. The investigation was done spectrophotometrically. Flavonoids content was determined using chrysin as internal standard and zirconium oxychloride as reagent. The second method uses aluminium chloride and 2,4-dinitorphenylhydrazine and as internal standards galangin and pinocembrin. Keywords: flavonoids, propolis, spectrophotometric methods.

1. Introduction

has been done by two different colorimetric methods.

Propolis is a natural product with very important properties such as antioxidant, antimicrobial, antiinflammatory. Over the past few years a special interest has been developed on natural products and theirs beneficial effects. A special attention was attributed to the investigation of propolis chemical composition [1, 2]. Researchers have shown that the chemical composition of propolis depends upon the plant source available to bees. General composition of propolis is: 50% resins and vegetable balsam, 30% wax, 10% essential oils, 5% pollen and 5% other substances [3]. This study is focused on the investigation of propolis chemical composition from different regions in Transylvania. The extraction of active principles with ethanol of different concentrations is being investigated. The analysis of flavonoids

2. Materials and methods 2.1. Propolis origin. Propolis samples were harvested from different regions in Romania (Cluj, Bihor, Sălaj, Sibiu, Alba, Satu Mare and Hunedoara) during 20082009. Samples were kept in the freezer (-20ºC) until analysis. Details regarding propolis samples are shown in table 1. Table 1. Propolis origin Sample Collection site PS 1 Bucea, Cluj county PS 2 Cuiesd, Bihor county PS 3 Bocsita, Salaj county PS 4 Zimbor, Salaj county PS 5 Poenita, Sibiu county PS 6 Ocnisoara, Alba county PS 7 Ciucea, Cluj county PS 8 Padureni, Sibiu county PS 9 Halmeu, Satu-Mare county PS 10 Tiream, Satu-Mare county

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* Corresponding author: Cristina Manuela Mihai, Tel: 0264-596385, Fax: 0264-593792, Email: [email protected]

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The methods reported by Popova, 2004 [4] were used to quantify the flavonoids from propolis samples. All these methods are based on formation of stable colored compounds from flavonoids with specific reagents. Determination of flavones/flavonols was performed using AlCl3, and calibration curve (Y = 1.13397 * X – 0.00443; r2=0.99916) was realized using Galangin as internal standard (4-40µg/ml). The group of flavanone/dihydroflavonols was determined by complexation with 2,4 Dinitro-phenylhydrazine, and Pinocembrin (0,14-1mg/ml) was used for calibration curve (Y = 0.24135 * X + 0.00089; r2=0.99954). The content in total flavonoids of analyzed propolis samples can be calculated by summing up the concentration of flavones/flavonols and also the concentration of flavanone/dihydroflavonols. The procedure has been described in a previous paper [5].

2.2. Chemicals. Chemicals used were: chrysin, pinocembrin, galangin, absolute ethanol, methanol, sodium carbonate (Na2CO3), zirconium oxychloride (ZrOCl2) and aluminum chloride and 2,4 dinitrophenylhydrazine (DNP). 2.3. Equipments. Spectrophotometric measurements were performed on an UV-1700 Schimadzu Spectrophotometer (double-beam) equipped with 1 cm quartz cuvettes. 2.4. Ethanolic extracts. Propolis was grounded into a fine powder. The active principles were then extracted with ethanol 70% and also ethanol 96%. 1 g of propolis was mixed with 30 ml ethanol (for both concentrations) and left over night with continuous stirring. The suspension was then filtered on qualitative filter paper (ø 90 mm). The extraction was repeated three times, the extracts were combined and the volume was adjusted to 100 ml into a volumetric flask (initial extract, 1% concentration). All extracts were kept in the dark until further analysis. 2.5. Flavonoid content with zirconium oxychloride (method 1) An aliquot of 1 ml of the initial extract was transferred into a volumetric flask and the volume was made up to 100 ml with the solvent used for the extraction (0.01%). 3 ml of each final extract were mixed with 1 ml ZrOCl2 2.5% (2.5 g ZrOCl2 were dissolved with 100 ml methanol) into a 25 ml volumetric flask. The volume was made up with methanol. After 30 minutes the absorbance was measured at 288 nm towards a blanc solution. The blanc was prepared by replacing the respective sample with an equivalent aliquot of methanol and was carried out through all the stages of the procedure. For the calibration curve chrysin was used as reference standard. A stock solution (0.1 mg/ml) of chrysin was prepared (1 mg chrysin was dissolved in 10 ml methanol). Aliquots (0.25, 0.5, 1, 1.5 and 2 ml) of this solution were carried out through the procedure described above and the absorbance was plotted against the concentration in order to obtain the calibration curve. The calibration curves can be observed in figure 1. 2.6. Quantitative determination of flavone/flavonols, flavanone/dihydroflavonols (method 2)

Figure 1. Calibration curve for flavonoids using chrysin(A).

3. Results and discussion The present study shows the flavonoid content of 10 Romanian propolis samples. Most propolis samples were dark brown, with amber – reddish pieces and intense aromatic resin flavor. Flavonoid content was investigated using two different colorimetric methods and also ethanol of two different concentrations 70%, respectively 96%. Over the years a wide range of ethanol concentrations was tested for the extraction of active principles such as: 70% [4], 80% [6, 7], 96% [8, 9] and absolute [10].

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Table 2. Flavonoid content pf propolis samples using two different colorimetric methods. Flavonoid content using Sample zirconium oxychloride[%] Extract with Extract with ethanol 70 % ethanol 96 % PS 1 19.67±0.47 23.33±3.77 PS 2 23.67±0.00 31.00±0.47 PS 3 18.50±0.24 24.67±0.01 PS 4 22.67±0.94 33.50±0.24 PS 5 19.33±0.94 27.00±4.71 PS 6 18.33±0.94 24.17±1.42 PS 7 16.17±1.18 17.67±1.41 PS 8 21.33±0.00 23.67±0.47 PS 9 12.33±0.01 14.17±0.24 PS 10 20.67±0.01 25.50±0.71

The first method was used by other researchers in order to determine the flavonoid content in propolis and in poplar buds [11, 12]. Their findings were similar with our results. Polinicencu reported an average of 17.57% flavonoids in Romanian propolis samples. Also the method using zirconium oxychloride was applied for the estimation of flavonoid content in poplar buds. The amount found was 6.25%. The values obtained for flavonoid content by both methods show that the first method exhibits higher quantities of total flavonoids than those obtained by summing up the flavones/flavonols and flavanones/dihydroflavonols. By using ethanol of two different concentrations we may observe that the most concentrate one (96%) shows better results with regard to flavonoid content. Anyways it is known that flavonoid content in propolis samples depends upon the geographical origin and eco-system (plant source) [13], but our samples show that they do not follow the rule. We have investigated samples from 6 different counties from Romania and the ones from Satu Mare show significant differences between them.

Total flavonoid content using AlCl3 and DNP[%] Extract with Extract with ethanol 70 % ethanol 96 % 4.86±0.18 5.81±0.13 6.06±0.20 6.17±0.28 6.60±0.58 7.49±0.24 16.38±0.67 19.07±0.47 5.64±0.26 4.86±0.16 5.55±0.36 6.73±0.36 4.68±0.30 6.11±0.86 5.13±0.13 4.24±0.24 7.39±0.34 9.85±0.11 12.95±0.11 13.84±0.11

Acknowledgements This paper was supported by Project PN II MRC-PA, grant number 51-070/2007 and by Project IDEI, grant number 390/2007.

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4. Conclusions Methods used for the determination of total flavonoid content in propolis show very different concentrations of flavonoids, fact that requires further investigations in order to establish which results are more reliable. As it can be seen by using ethanol of two different concentrations, we may conclude that ethanol 96% offers a better extraction yield of the active principles in propolis.

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