Lvii, 1 hplc evaluation of phenolic and polyphenolic acids from propolis
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- Bu səhifədəki naviqasiya:
- Keywords
- Materials and methods
- Figure 1
- Standards Quantification limit (mg/mL) Detection limit (mg/mL)
- Results and discussion
- Total 20.13 18.43 18.77
- Total 19.84 20.78 22.31 22.09
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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). Table I Commercial sorts of propolis taken into analysis
All phenolic and polyphenolic acids listed in table II have a high degree of purity (99%). Table II Standards of phenolic and polyphenolic acids
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 Thermo-Surveyor 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 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.
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 Table III Standards retention times
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). Table IV Calibration curves and regression coefficients for all standards
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).
Detection and quantification limits of phenolic and polyphenolic acids
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
Table VII Phenolic and polyphenolic acids content, in Israeli propolis
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%. References
Manuscript received: 28.07.2008 Yüklə 145,75 Kb. Dostları ilə paylaş:
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