evaluation of propolis quality from ... › files › teze › en › 2011 › mihai.pdfeng....

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UNIVERSITY OF AGRICULTURAL SCIENCES AND VETERINARY MEDICINE CLUJ-NAPOCA

DOCTORAL SCHOOL

FACULTY OF ANIMAL HUSBANDRY AND BIOTECHNOLOGIES

Eng. Cristina Manuela MIHAI

EVALUATION OF PROPOLIS QUALITY

FROM TRANSYLVANIA WITH

REGARDS TO STANDARDIZATION (SUMMARY OF PhD THESIS)

SCIENTIFIC COORDINATOR

Prof. Eng. Liviu Alexandru MĂRGHITA Ş, PhD

CLUJ-NAPOCA

2011

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CONTENTS

PhD page

Summary page

Introduction 11 28 Objectives 13 29

FIRST PART: LITERATURE REVIEW 14 CHAPTER I. PROPOLIS – GENERALITIES 15 30

1.1. Propolis importance 15 30 1.2. Chemical composition 17 30 1.3. Aspects regarding standardization 19

CHAPTER II. BIOLOGICAL ACTIVE COMPOUNDS FROM PROPOLIS: POLYPHENOLS 24 30

2.1. Phenolic acids 27 2.2. Flavonoids 27 2.3. Methods for polyphenolic compounds determination 29

2.3.1. Spectrophotometric methods 30 2.3.2. Chromatographic methods 32

CHAPTER III. THERAPEUTICAL EFFECTS OF PROPOLIS 34 3.1. Antioxidant capacity 35 3.2. Antimicrobial capacity 39 3.3. Other effects 40

CHAPTER IV. METHODS FOR THE DETERMINATION OF ANTIOXIDANT AND ANTIMICROBIAL CAPACITIES OF PROPOLIS 42

4.1. Methods for antioxidant capacity determination 42 4.1.1. Free radicals scavenging methods 43 4.1.2. Total antioxidant potential method (FRAP) 44

4.2. Methods for antimicrobial capacity determination 44 4.2.1. Diffusion method 45 4.2.2. Serial dilution method 46

SECOND PART: ORIGINAL RESEARCH 48 CHAPTER V. MATERIAL AND METHOD 49 31

5.1. Biologic material 49 31 5.2. Applied experimental methods 52 31

5.2.1. Alcoholic extracts preparation from propolis 52 5.2.2. Phisico –chemical analysis 54

5.2.2.1. Humidity determination 54 5.2.2.2. Wax content determination – Soxhlet method 55 5.2.2.3. Dry residue determination 56

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5.2.5. UV-Vis spectrofotometrical analysis 57 5.2.5.1. Total polyphenols content determination 57 5.2.5.2. Total flavonoid content determination 58 5.2.5.3. Antioxidant capacity determination through DPPH assay 62 5.2.5.4. Total antioxidant potential determination through FRAP assay 64

5.2.6. High performance liquid chromatography analysis (HPLC – PDA) 65

5.2.6.1. Propolis extracts preparation for HPLC – PDA analysis 65 5.2.6.2. HPLC – PDA cromatographic analysis 66

5.2.7. Microbiological analysis 72 5.2.7.1. Antimicrobial activity determination 72 5.2.7.2. Minimum inhibitory concentration (MIC) determination 73

5.3. Statistical analysis 74 CHAPTER VI. RESULTS REGARDING PHISICO – CHEMICAL PARAMETERS OF PROPOLIS 76 31

6.1. Results regarding water content (humidity) of propolis samples 76 6.2. Results regarding wax content of propolis samples 78 6.3. Results regarding dry residue of propolis alcoholic extracts 80 6.4. Discussions 81 6.5. Preliminary conclusions 93

CHAPTER VII. RESULTS REGARDING SPECTROPHOTOMETRIC DETERMINATIONS OF POLYPHENOLS, FLAVONOIDS AND ANTIOXIDANT CAPACITY OF PROPOLIS 95 34

7.1. Results regarding total polyphenols content from propolis 95 7.2. Results regarding total flavonoid content from propolis 97 7.3. Results regarding antioxidant capacity of propolis determined by DPPH method 102 7.4. Results regarding total antioxidant potential of propolis determined by FRAP method 104 7.5.Discussions 106 7.6.Correlations between phisico-chemical and spectrofotometrical analysis 136 7.7. Preliminary conclusions 142

CHAPTER VIII. REULTS REGARDING PHENOLIC COMPOUNDS OF PROPOLIS 144 39 8.1. Results regarding HPLC – PDA analysis of reference compounds 144 8.2. Results regarding HPLC – PDA analysis of propolis samples 147 8.3. Discussions 162

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8.4. Preliminary conclusions 170 CHAPTER IX. RESULTS REGARDING ANTIMICROBIAL CAPACITY OF PROPOLIS 172 40 9.1. Results regarding antimicrobial activity 172 9.2. Results regarding minimum inhibitory concentration (MIC) 180 9.3. Discussions 181 9.4. Correlations between microbiological analysis and phisico-chemical and spectrophotometrical analysis 9.5. Preliminary conclusions 196 CHAPTER X. GENERAL CONCLUSSIONS 199 43 ORIGINAL ELEMENTS 201 45 RECOMMENDATIONS AND PERSPECTIVES 201 45 LIST OF PUBLISHED PAPERS 202 BIBLIOGRAPHIC REFERENCES 204 45 APPENDIX 221 Appendix 1 – Abbreviations list 221 Appendix 2 – Calibration curves and UV-Vis spectra for reference compounds regarding HPLC – PDA anlysis 222 Appendix 3 – HPLC – PDA chromatograms for propolis samples 226 Appendix 4 – Antimicrobial activity – original photos 251 Romanian summary 257 English summary

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INTRODUCTION

Lately the interest regarding natural alternative of everything that is obtained

through synthesis is higher and higher. A large number of studies regarding propolis

show that this natural product obtained exclusively by bees and harvested by humans

possesses therapeutical effects. Among these effects are: antioxidant capacity,

antimicrobial capacity, hepatoprotective effect, antifungal activity, local anesthetic anti-

inflammatory effect and others.

Propolis is probably the most complex bee product, having popularity since

ancient times. Without a scientific base, our ancestors have used it as a remedy for

treatment of different diseases. Much more in our days, wehen chemical composition of

propolis is known and its terapeutical effects, we may trust using it.

Very often natural antioxidants are mentioned. Propolis is one of the most rich

natural product in antioxidants (flavonoids, phenolic acids and their esters). Along

antioxidant capacity, this product, propolis, shows a great antimicrobial activity, which is

supported by recent research studies. Due to these properties, propolis will become a

trusted substitute of antibiotics, which nowadays appear to have many side effects.

In the present PhD thesis, there is an important contribution regarding propolis

study by nodern chemical analysis, with the purpose of investigating Transylvanian

propolis quality. Quality control parameters are going to be proposed, which should be

checked when this product is used as a remedy. Propolis quality is determined by

biological active compounds content (phenolic acids and flavonoids), antioxidant

capacity, antimicrobial capacity and also polyphenolic profile. All analysis were

conducted in vitro under laboratory controlled conditions. Experimental procedures were

realized in the Bee Products Quality Control Laboratory, except microbiological

experiments which were conducted in the Microbiology Laboratory, both from

University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca, during

October 2008 – September 2011.

The present PhD thesis “Evaluation of propolis quality from Transylvania with

regards to standardization” is structured in two major parts. First one, “Literature

review” (Chapters I – IV) present the actual state of knowledge with regards to proposed

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objectives, like aspects concerning chemical composition, antioxidant and antimicrobial

capacities, methods used, and also the matter of standardization.

The second part “Original research” (Chapters V – X) presents used biological

material (53 propolis samples from Transylvania region), experimental procedures

(phisico-chemical, spectrophotometrical, chromatographic and microbiological), results

obtained, and general conclusions.

OBJECTIVES

Main objectives of the present PhD thesis are:

� Research regarding propolis composition, by determination of total amounts of

biological active principles (phenolic acids and flavonoids) by means of classical

spectrophotometric methods, and also wax content by authomatic Soxhlet

extraction.

� Research regarding antioxidant capacity of propolis from Transilvania (DPPH

method and FRAP method) evaluated in vitro by means of spectrophotometric

methods

� Research regarding antimicrobial capacity of propolis from Transilvania, by

testing it on internation reference bacterial strains: Staphylococcus aureus ATCC

6538P, Bacillus cereus ATCC 14579, Listeria monocitogenes ATCC 7644,

Escherichia coli ATCC 10536, Pseudomonas aeruginosa ATCC 27853, Candida

albicans ATCC 90028;

� Research regarding polyphenolic profile of propolis by High Performance Liquid

Chromatography (HPLC – PDA) with the purpose of identification and

quantification of specific compounds.

� Research regarding correlations between chemical composition and therapeutic

effects (antioxidant and antimicrobial capacities) of propolis.

� Indication of quality parameters of propolis which should be included when

proposing a standard.

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CHAPTER I. PROPOLIS – GENERALITIES

1.1. PROPOLIS IMPORTANCE

All bee products (honey, propolis, polen and royal jelly) show interes within

research teams, being products with a composition not much influenced by humans. All

these products posses therapeutical effects which are recognized worldwide.

1.2. CHEMICAL COMPOSITION

Propolis chemical composition is very variable, depending greatly of the flora

available to bees during harvesting (Marcucci, 1995; Bankova et al., 2000; Ahn and

colab et al., 2007). The interest of many research teams was and is to fully elucidate the

chemical composition of propolis. Until now more than 300 compounds from propolis

composition are known, from different categories: resins, wax, essential oils, minerals

and other substances (Burdock, 1998; Kosalec et al., 2003; Cunha et al., 2004;

Mărghitaş, 2005).

CHAPTER II. BIOLOGICAL ACTIVE COMPOUNDS FROM PROPOLIS:

POLYPHENOLS

Polyphenols are important antioxidants due to their high redox potential. Theirs

antioxidant capacity is considered to be much more higher than of essential vitamins,

having a significant contribution over therapeutic effects of products which contains them

(Tsao şi Deng, 2004). Among the role of antioxidants, polyphenols (especially

flavonoids) show antimicrobial activity (Boyanova şi colab., 2006). Due to therapeutic

effects of polyphenols, already proven by scientific studies, the interest for their

availability is higher and higher, being preferred natural sources.

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CHAPTER V. MATERIAL AND METHOD

5.1. BIOLOGIC MATERIAL

Biologic material subjected to the present study is constituted of 53 propolis

samples from Transylvania (figure 1). These samples were provided by beekeepers

during March 2009 – March 2010. All samples were kept in the freezer at -20˚C until

analysis. Samples were harvested by scraping propolis using hive tools according to

beekeepers.

Figure 1. propolis samples PS1-PS53 (original photo)

5.2. APPLIED EXPERIMENTAL METHODS

Experimental procedures were conducted using four types of analysis: physico-

chemical, spectrophotometric, chromatographic and microbiological.

CHAPTER VI. RESULTS REGARDING PHISICO – CHEMICAL PARAMETERS

OF PROPOLIS

Phisico-chemical analysis were used as a first indicator of propolis quality with

regards to water content, wax content and dry residue content.

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Water content or humidity of propolis samples was registered around

2.39±0.44%. The lowest value was determined for sample PS 45 (1.46±0.09%), while the

highest for sample PS 2 (3.39±0.11%).

Propolis samples distribution according to water content is shown in figure 2.

Samples show a normal distribution.

Figure 2. Histogram of propolis samples distribution according to their water

content

Wax content of propolis samples ranged between wide limits: 13.49±0.50% (PS

46) and 70.89±1.08% (PS 39), with the average of 34.20%. As for the propolis samples

distribution according to wax content, could be a normal one with the exception of three

samples with showed a very high content (PS 9, PS 30 and PS 39) (figure 3).

Dry residue content of propolis rabged between 37.11±0.35% (PS 30) and

98.06±0.35% (PS 19), with the average of 69.50%. According to this physico-chemical

parameter, two samples stand out with unexpected high values: PS 19 (98.06±0.35%) and

PS 13 (97.82±1.41).

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Figure 3. Histogram of propolis samples distribution according to their wax content

Dry residue content is a parameter with interest due to the fact that it reflects the

amount of extracted active principles from propolis by means of used extraction solvent.

Propolis samples distribution according to dry residue content is shown in figure 4.

Figure 4. Histogram of propolis samples distribution according to their dry residue

content

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CHAPTER VII. RESULTS REGARDING SPECTROPHOTOMETRIC

DETERMINATIONS OF POLYPHENOLS, FLAVONOIDS AND ANTIOXIDANT

CAPACITY OF PROPOLIS

Total polyphenols content of the 53 used propolis samples follows a normal

distribution as shown in figure 5.

Figure 5. Histogram of propolis samples distribution according to total polyphenols

content

The range of total polyphenols content was very wide, values were registered

between 23.25±3.20% (PS 12) and 63.23±2.44% (PS 41), with the average of 43.01%.

The two methods used for total flavonoid content show very different results as

shown in figures 6 and 7.

Antioxidant capacity of propolis was evaluated by means of two different

methods. The first one was DPPH method (radical scavenging activity), which offered

results in the range of 0.29±0.10 mmol Trolox / g propolis (PS 30) and 1.40±0.10 mmol

Trolox / g propolis (PS 19). The second one was FRAP method (Ferric reducing

antioxidant potential). „ FRAP values” obtained for propolis samples were in the range of

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0.57±0.04 mmol FeSO4 / g propolis (PS 17) and 2.55±0.10 mmol FeSO4 / g propolis (PS

29) (figures 8 and 9).

Figure 6. Total flavonoid content determined by the two methods applied

(samples PS 1 – PS 25)

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Figure 7. Total flavonoid content determined by the two methods applied


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Figure 8. Comparative study of the two applied antioxidant methods


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Figure 9. Comparative study of the two applied antioxidant methods


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CHAPTER VIII. RESULTS REGARDING PHENOLIC COMPOUNDS OF

PROPOLIS

All propolis samples were subjected to HPLC-PDA analysis, chromatograms were

registered at 280 nm and 340 nm, wavelengths specific to phenolic acids and flavonoids.

UV-Vis spectra corresponding to each signal from chromatograms were recorded in the

range 190-650 nm. Two of the propolis samples outstanded due to identification od 10

reference compounds in theirs chemical composition (figures 10 and 11).

10 20 30 40 50

0

400000

800000

1200000

Abs

orba

nta

[mA

U]

Abso

rbance

[m

AU

]

PS 22

Timp de retentie [min]Retention time [min]

Acid si

ringic

/13,61

3

Acid ca

feic/

14,9

63

Vanilin

a/16,

585

Acid p

-cum

aric/

20,2

14

Acid fe

rulic/

22,0

19

Acid t-

cinam

ic/32

,960

Pino

cembr

ina/4

2,176

Crisina

/43,90

7

Galang

ina/

44,9

30

Pino

strob

ina/47

,980

Figure 10. HPLC – PDA chromatogram of propolis sample PS 22

Both samples, were harvested in Mureş county (PS 22 and PS 24), being the only

ones in which t-cinnamic acid was cuantified in relatively low concentrations, of 0.64

and 0.71 mg t-cinnamic acid / g propolis. Among reference compounds identified in

studied propolis samples, three (caffeic acid, p-coumaric acid and ferrulic acid) were

present in all samples. Flavonoids used as reference compounds for spectrophotometric

determinations resulted to be appropriate according to HPLC – PDA analysis.

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10 20 30 40 50

0

400000

800000

1200000

PS 24

Abs

orba

nta

[mA

U]

Ab

sorb

anc

e [

mA

U]

Timp de retentie [min]Retention time [min]

Acid s

iring

ic/13

,656

Acid c

afeic/

15,02

3

Vanilin

a/16,4

48

Acid p-

cum

aric/

20,28

6

Acid fe

rulic/

22,0

76

Acid t-

cinam

ic/33

,008

Pino

cem

brina/

41,9

45

Crisin

a/43,9

49

Galan

gina/4

4,98

7

Pinostr

obina

/48,0

02

Figure 11. HPLC – PDA chromatogram of propolis sample PS 24

Pinocembrin, the flavanone used for flavanones/ dihydroflavonols content

determination was identified and cuantified in 39 propolis samples, the concentration

range being 0.03 ÷ 2.85 mg / g propolis, with an average of 1.05 mg / g propolis. Among

identified flavonoids in propolis, chrysin was the flavone most aboundant. The

concentration range for chrysin was 0.07 ÷ 3.91 mg / g propolis, with an average of 1.59

mg / g propolis. Regarding the flavonols galangin, it was cuantified in 34 propolis

samples, in the concentration range of 0.31 ÷ 3.20 mg / g propolis, with an average of

1.39 mg / g propolis.

CAPITOLUL IX. RESULTS REGARDING ANTIMICROBIAL CAPACITY OF

PROPOLIS

Antimicrobial activity was investigated on six different bacterial strains as

mentiond in chapter “Material and method”. Propolis samples distribution according to

inhibition zone diameter which represent the antimicrobial activity (Staphylococcus

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aureus, Bacillus cereus, Listeria monocitogenes, Escherichia coli and Candida albicans)

is shown in figures 12 – 16.

Regarding gram-negative bacteria Pseudomonas aeruginosa, samples distribution

is not shown due to the resistance it presented to all tested alcoholic propolis extracts.

Figure 12. Histogram of propolis samples distribution according to antimicrobial activity

against Staphylococcus aureus


against Bacillus cereus

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against Listeria monocitogenes


against Escherichia coli

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against Candida albicans

CHAPTER X. GENERAL CONCLUSSIONS

According to proposed objectives of the present PhD thesis, the following could

be concluded:

1. Chemical composition of propolis from Transylvania was determined, by

evaluation of total amounts of biologic active principles (polyphenols and

flavonoids), wax content, water content and dry residue content.

2. Antioxidant capacity of propolis was determined by means of two

spectrophotometric methods: radical scavenging activity (DPPH method) and total

antioxidant potential (FRAP method). Both methods confirm that propolis

possesses antioxidant capacity.

3. Antimicrobial capacity of propolis against six bacterial strains was determined:

Staphylococcus aureus ATCC 6538P, Bacillus cereus ATCC 14579, Listeria

monocitogenes ATCC 7644, Escherichia coli ATCC 10536, Pseudomonas

aeruginosa ATCC 27853, Candida albicans ATCC 90028. All propolis samples

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showed in vitro antimicrobial activity against Gram-positive bacteria and selective

activity against Gram-negative bacteria.

4. Polyphenolic profile of propolis was determined by means of HPLC – PDA

chromatographic technique, which was as follows: syringic acid, caffeic acid,

vanillin, p-coumaric acid, ferrulic acid, t-cinnamic acid, pinocembrin, chrysin,

galangin and pinostrobin. The presence of pinocembrin, chrysin, galangin and

caffeic acid in the polyphenolic profile of propolis allowed us to classify it as

poplar propolis.

5. Correlations between chemical composition and studied therapeutic effects were

determined. Flavonoid content correlates in a high manner with antioxidant

capacity of propolis. Flavones/flavonols and flavanones/dihydroflavonols are

responsible for antimicrobial activity of propolis as shown by Pearson correlation

coefficients. Total polyphenols concentration can not be considered a major

criteria when estimating antioxidant and antimicrobial capacities, due to weak

correlations shown between these parameters. Wax content could be used as a first

indicator of propolis quality. A high content of wax correlates with a low content

of active principles (flavonoids and phenolic acids).

6. Parameters that should be considered for quality control of propolis are:

a. Water content – maximum 10%;

b. Dry residue content – minimum 35%;

c. Wax content – maximum 35%;

d. Total flavonoids content – minimum 10%;

e. Total polyphenols content – minimum 20%;

f. Antioxidant capacity;

g. Antimicrobial capacity.

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ORIGINAL ELEMENTS

1. Polyphenolic profile characterization of propolis from Transylvania by means of

chromatographic technique (HPLC – PDA) ant its classification as poplar

propolis.

2. Evaluation of antimicrobial capacity of propolis against six international bacterial

strains.

3. Indication of quality control parameters (wax content, water content, dry residue

content, total polyphenols, total flavonoids, antimicrobial and antioxidant

activities) necessary to be checked before its use as therapeutical agent.

RECOMMENDATIONS AND PERSPECTIVES

1. Extension of the present study on country level and standard proposal requiring

propolis quality parameters.

2. Wax content and total flavonoid content should be checked as first indicators of

propolis quality.

3. Use of propolis for therapeutical pourposses only after quality control.

BIBLIOGRAPHIC REFERENCES

1. Ahn, M. R., S. Kumazawa, Y. Usui, J. Nakamura, M. Matsuka, F. Zhu, T. Nakayama, 2007, Antioxidant activity and constituents of propolis collected in various areas of China, Food Chemistry, 101, 1383-1392.

2. Bankova Vassya, S. L. De Castro, M. C. Marcucci, 2000, Propolis: recent advances in chemistry and plant origin, Apidologie, 31, 3-15.

3. Boyanova Lyudmila, R. Kolarov, Galina Gergova, I. Mitov, 2006, In vitro activity of Bulgarian propolis against 94 clinical isolates of anaerobic bacteria, Anaerobe, 12, 173-177.

4. Burdock, G. A., 1998, Review of the biological properties and toxicity of bee propolis (propolis), Food and Chemical Toxicology 36, 347-363.

5. Cunha, I. B. S., A. C. H. F. Sawaya, F. M. Caetano, M. T. Shimiziu, M. C. Marcucci, F. T. Drezza, G. S. Povia, P. O. Carvalho, 2004, Factors that Influence

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the Yield and Composition of Brazilian Propolis Extracts, Journal of Brazilian Chemical Society, 15:6, 1-xxx.

6. Kosalec, I., Marina Bakmaz, S. Pepeljnjak, 2003, Analysis of propolis from the continental and Adriatic regions of Croatia, Acta Pharmaceutica, 53, 275-285.

7. Marcucci, M., 1995, Propolis, chemical composition, biological properties and therapeutic activity, Apidologie 26, 83-99.

8. Mărghitaş, L. Al. , 2005, Albinele şi produsele lor, ediţia 2, Ed. Ceres, Bucureşti. 9. Tsao, R., Z. Deng, 2004, Separation procedures for naturally occurring antioxidant

phytochemicals. Journal of Chromatography B, 812, 85-99.

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