Spectrofotometric determination of flavonic compounds from propolis



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FARMACIA, 2009, Vol.LVII, 1



SPECTROFOTOMETRIC DETERMINATION OF FLAVONIC COMPOUNDS FROM PROPOLIS
Anda Natanela Croci, Doina Lazar, liliana Potorac, 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 by UV-Vis spectrometry flavonic compounds from different sorts of propolis from Romania and Israel. We analyzed the major compounds expressed in flavones, flavonols, and flavanones. Total flavones content in Romanian and Israel propolis samples was determined.



Rezumat

Cercetarea a avut drept scop evaluarea spectrofotmetrică a compuşilor flavonici din sorturi de propolis recoltate din România şi Israel. S-au determinat compuşii majoritari ai acestora exprimaţi în flavone, flavonoli si flavanone. Totalul flavonic s-a raportat la probele din Romania şi Israel.



Keywords: propolis; UV-Vis spectrometry; flavones
Introduction

Flavonic compounds and polyphenolic acids are the main constituents of propolis and they can represent up to 40%. These compounds are involved directly as part of the phytochemical complex in many therapeutic actions. Thus, there are known the following actions: anti-inflammatory, anti-hypertensive, antimicrobial, antifungal, antioxidant, hepatoprotective, cholesterol reducing, immunostimulating action [1,2] etc.

In order to estimate total flavonic content of propolis from Romania and Israel, we improved the isolation method of flavonic fraction [4] and based on reactivity differences we have determined by spectrometry the flavones, flavonols, flavanones, isoflavones and total flavonoid content [3, 6].
Materials and methods

All absorption spectra were registered using a UV-Vis spectrophotometer Jasco V530. All chemicals and reagents used were of analytical grade:



  • Quercetin reference standard (Sigma, Germany)

  • Naringenin reference standard (Sigma, Germany)

  • Aluminium chloride (p.a.)

  • 2, 4-dinitrophenylhydrazine (DNPH)

  • Potassium acetate (p.a.)

  • Potassium hydroxide (p.a.)

Flavones and flavonols form with aluminium chloride complexes that absorb light intensely at 415 nm, while flavanones absorb poorly at this wavelength.

Flavanones form with 2,4-dinitrophenylhydrazine phenylhidrazones that absorb light at 495 nm wavelength.

Following the indications of Woiski and Salatino [7] we proceeded to determine total flavonic content. Samples were prepared by extraction in methanol.

In this study we intended to quantitatively determine flavonic compounds in propolis samples collected from three locations in Romania (1-3) and nine locations in Israel (4-12), as listed in table I. For analysis we used 80% methanol extracts, 1:10 g/v (propolis:solvent).



Table I

Commercial sorts of propolis



Propolis sorts

1. Grajduri

7. Tiros -1

2. Raducaneni

8. Tiros -2

3. Dranceni

9. Nes Tioana

4. Kfar Lin -1

10. Galileea Sea

5. Kfar Lin -2

11. Oca-Ierusalim

6. Iad Mordechai

12. Tel-Aviv


Flavones and flavonols determination

Quantitative analysis is based on the calibration curve in relation to which total flavones and flavonols content can be found. For calibration curve we used quercetin. 10 mg quercetin was dissolved in 80% ethanol and than we obtain concentration range varying between 25-100 µg/mL. 0.5 mL standard solutions were mixed with 1.5 mL 95 % ethanol and than treated with 0.1 mL aluminium chloride solution 10 %, 0.1 mL solution potassium acetate 1M and 2.8 mL distilled water. Absorbance is measured at λ=415 nm and the calibration curve is drawn.



Flavanones determination

Flavanones as naringenin and hesperetin react with 2,4-DNPH yielding hydrazones that absorb light intensely at λ=495 nm. We used the method described by Nagy [6]. 10 mg naringenin was dissolved in methanol and than we obtaining the calibration curve using naringenin solutions of 500 - 2000 µg/mL concentrations. 1 mL of standard solutions is treated with 2 mL solution 2,4-dinitrophenylhydrazine 1% and 2 mL methanol. The mixture is kept at 50o C for 50 minutes. After cooling at room temperature, the mixture is treated with 5 mL solution potassium hydroxide 1% in methanol and maintained at room temperature for 2 minutes. 1 mL of mixture is treated with 5 mL methanol and centrifuged if necessary. The supernatant was brought to 25 mL in a volumetric flask. Absorbance at λ=495 nm is measured and the calibration curve is drawn. All 12 alcoholic extract from propolis are treated similarly.


Results and discussion

Results of flavones and flavonols (expressed in quercetin) determination on the alcoholic extracts of Romanian propolis sorts are listed in table II.



Table II

Flavones and flavonols content (%) of Romanian propolis



Propolis sort

Content % (g)

Grajduri

23.13

Raducaneni

22.85

Dranceni

22.35

Flavones and flavonols determination in propolis samples from Israel yielded the following results (table III).



Table III

Flavones and flavonols content (%) of Israels propolis



Propolis sort

Content % (g)

Kfar Lin - 1

24.45

Kfar Lin - 2

24.78

Iad Mordechai

26.02

Tiros - 1

25.79

Tiros - 2

26.87

Nes Tioana

28.98

Galileea Sea

28.34

Oca Ierusalim

29.77

Tel Aviv

29.04

The analyzed Romanian samples of propolis have a flavanones (expressed in naringenine) content between 1.82% and 2.35%. Results are listed in table IV.



Table IV

Flavanones content (%) in Romanian propolis



Propolis sort

Content % (g)

Grajduri

1.82

Raducaneni

1.94

Dranceni

2.35

Flavanones content of propolis samples from Israel are found in table V.

Table V

Flavanones content % in Israelis propolis



Sort

Content % (g)

Kfar Lin - 1

2.28

Kfar Lin - 2

2.11

Iad Mordechai

1.99

Tiros - 1

2.31

Tiros - 2

2.35

Nes Tioana

2.25

Galileea Sea

2.35

Oca Ierusalim

2.34

Tel Aviv

2.37


Total flavonic content

By summing flavones, flavonols and flavanones quantities is yielded the total flavonic content determined by two methods (table VI, VI).


Table VI

Total flavonic content (%) of Romanian propolis



Propolis sort

Content % (g)

Grajduri

24.95

Raducaneni

24.79

Dranceni

24.70


Table VII

Total flavonic content (%) of Israelis propolis



Propolis sort

Content % (g)

Kfar Lin - 1

26.53

Kfar Lin - 2

26.89

Iad Mordechai

28.01

Tiros - 1

28.10

Tiros - 2

29.22

Nes Tioana

31.23

Galileea Sea

30.69

Oca Ierusalim

32.11

Tel Aviv

31.41

For a direct comparison we inscribed in figure 1 the total flavonic values of propolis samples from the three Romanian (1-3) locations and nine Israelis locations (4-12).





Figure 1

Total flavonic content (g%) of propolis types from Romania and Israel


Conclusions

Propolis sorts from Romania have a total flavonic content of 24.70- 24.95%. Israel’s propolis has a total flavonic content ranging between 26.53 and 32.11%. Differences are explained by geographical area and native flora of locations from which the analysed propolis samples were collected.


References

  1. Celso V.N., Nakamura T.U., Bando E., Melo A.F.N., Cortez D.A.G., Filho B.P.D., Antibacterial Activity of Ocimum gratissimum L. Essential Oil, Mem Inst Oswaldo Cruz, Rio de Janeiro, 1999, Vol. 94(5), 675-678

  2. Burdock G.A., Review of the biological properties and toxicity of bee propolis (propolis). Food Chem. Toxicol., 1998, 36, 347-363

  1. 3. Kosalec I., Bakmaz M., Pepeljnak S., Analysis of propolis from the continental and Adriatic regions of Croatia, Acta Pharm., 2003, 53, 275-285

  1. Lazar D., Lazar M.I., Corciovã A., Croci A.N., Flavones content evaluation of some sorts of propolis, Algesia and analgesia, Ed. Gr.T.Popa Iasi, 2005, 219-223

  2. Lazar M.I., Lazar D., Croci A.N., Corciova A., Tamba A., Comparative dates about phenolic acids and flavonoids content of some propolis sorts from Moldavia and Israel, Drugs: use, abuse and dependency, Ed. Gr.T.Popa Iasi, 2008, 213-216

  3. Nagy M., Grancai D., Colorimetric Determination of Flavanones in Propolis, Pharmazie, 1996, 51, 100-101

  4. Woisky R. G., Salatino A., Analysis of propolis: some parameters and procedures for chemical quality control, Journal of apicultural research, 1998, Vol. 37(2), 99-105




Manuscript received: 01.09.2008





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