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Commercial Propolis Liquid Products: 

Comparison of Physicochemical Properties and 

Antioxidant and Antimicrobial Properties 

 

 

 

 

 

 

 

 

 

 

A thesis presented in partial fulfilment of the requirements  

for the degree of Master of Food Technology  

at Massey University, Auckland, New Zealand 

 

 

 

 

 

 

 

 

 

 

Mengyin Sun 

2019 



   

 

  i 

Summary 

Propolis is a resinous substance, which is well-known for its functional properties (e.g. 

antioxidant, antimicrobial and anti-inflammatory) and collected by honeybees from 

various plants. Due to its various health beneficial properties, propolis is widely used in 

many products (e.g. foods, beverages and toothpastes) and liquid propolis extract 

products are also commercially available as natural healthy supplements. Raw propolis 

has been broadly investigated, while, there has been much less research on the 

physicochemical and functional properties of commercial liquid propolis products. This 

study was thus aimed to evaluate and compare 20 commercial propolis liquid products 

manufactured in 4 different countries (Australia, China, Korea and New Zealand), in 

terms of physiochemical properties (e.g. water and ethanol miscibility, colour, pH), 

chemical composition (e.g. total phenolic and total flavonoid contents) and functional 

properties (e.g. antioxidant capacity and antimicrobial activity). Besides, all propolis 

samples were analysed for the detection of heavy metal (e.g. lead, cadmium, and arsenic) 

and rare earth elements in order to determine the safety and quality of propolis products. 

Also, the content of salicin in propolis was measured as an indicator of the adulteration 

of propolis with poplar tree gum.  

The visual colour of liquid propolis products varied from dark brown, red to green. 

Almost all commercial propolis samples analysed in this study were more soluble in 

ethanol than in water, except a propolis sample containing Tween 20 (emulsifier). Most 

propolis samples were also acidic with pH < 5, whereas, the Korean propolis samples 

containing potassium carbonate had alkaline pH values. The analysed total flavonoid (TF) 

content of 19 propolis products matched their labelled values specified on their product 

packaging. However, some unexpected results were obtained with the TF content being 

measured to be higher than the total phenolic (TP) content from 4 Korean propolis 

samples, in which salicin was also detected. This indicates that those 4 propolis products 

might have been adulterated with poplar tree gum. In terms of the functional properties 

of propolis, it was found that their antioxidant activity highly corresponded to the TP and 

TF contents. On the contrary, there was no linear correlation between TP or TF content 

and antimicrobial activity of the propolis products. Propolis products showed a greater 

effect on Gram-positive bacteria (S. aureus and B. cereus) than the Gram-negative 



   

 

  ii 

bacterium (E. coli). Among all propolis samples from the different regions, the propolis 

samples from New Zealand had a relatively higher TP and TF content and also showed a 

higher antimicrobial activity than the propolis samples from the other countries. 

Nevertheless, the content of heavy metal elements (As and Pb) detected was relatively 

much higher in New Zealand propolis products than that from the other countries. On the 

other hand, liquid propolis products from Australia contained less heavy metal elements 

and had the lower possibility of adulteration by poplar tree gum and the stable antioxidant 

and antimicrobial activities, which seemed to be a better choice among the 20 samples 

studied in this study. 

In conclusion, since there is no proper criterion to monitor the quality of propolis, it is 

necessary to develop a series of indices to evaluate the commercial liquid propolis 

products, for example, sensory (colour and smell), chemical composition (TP and TF 

contents), functional properties (antioxidant and antimicrobial activities) and safety 

properties (heavy metal elements and adulterations).  

  



   

 

  iii 

Acknowledgements 

I would like to express my appreciation to my supervisor, Dr Sung Je Lee, for providing 

this interesting project and his valuable suggestions, advice and support during this 

research work. Also, I really appreciate A/Prof Marie Wong for her training of HPLC 

equipment for this research project. I would like to extend my thanks to the laboratory 

technicians, Rachel Liu, Negah Nikanjam and PC Tong from Massey University, and Hui 

Xiao (Beijing Municipal Centre for Food Safety Monitoring and Risk Assessment), Tao 

He (China Food and Drug Industries Quality and Safety Promotion Association, FDSA) 

and Xuyang Li (Beijing Products Quality Supervision and Inspection Institute) from 

China for their technical support. At last, I would like to thank Roy Wang for his 

assistance in my experiment. 

 

 

 



   

 

  iv 

Table of Content 

Summary ...................................................................................................................... i 

Acknowledgements .................................................................................................... iii 

Chapter 1. Introduction .............................................................................................. 1 

Chapter 2. Literature Review ..................................................................................... 4 

2.1 Introduction ............................................................................................... 4 

2.2 Extractions of Propolis............................................................................... 6 

2.2.1 Preparation of Propolis Extracts ............................................................. 6 

2.2.2 Different Extraction Solvents ................................................................. 6 

2.3 Physical and Chemical Properties .............................................................. 8 

2.3.1 Physical Characteristics ......................................................................... 8 

2.3.2 Chemical Composition ........................................................................... 8 

2.4 Functional Properties of Propolis ............................................................. 13 

2.4.1 Antioxidant Property ................................................................................. 13 

2.4.2 Antimicrobial Property ........................................................................ 14 

2.4.3 Anti-inflammatory Properties............................................................... 15 

2.5 Analyses of Propolis ................................................................................ 15 

2.5.1 Chemical Composition ......................................................................... 15 

2.5.2 Determination of Total Phenolics and Total Flavonoids ....................... 16 

2.5.3 Evaluation of Antioxidant Property ...................................................... 16 

2.5.4 Evaluation of Antimicrobial Activity ................................................... 17 

2.5.5 Determination of Elements in Propolis ................................................. 17 

2.5.6 Identification of Adulteration of Propolis ............................................. 18 

2.6 Conclusions ............................................................................................. 19 

Chapter 3. Commercial Propolis Liquid Products: Comparison of Physical and 

Chemical Properties .................................................................................................. 20 

3.1 Introduction ............................................................................................. 20 

3.2 Materials and Methods............................................................................. 22 

3.2.1 Materials .............................................................................................. 22 

3.2.2 Commercial Propolis Samples ............................................................. 22 

3.3 Analyses of Physicochemical Properties .................................................. 23 

3.3.1 Miscibility ........................................................................................... 23 

3.3.2 Colour ................................................................................................. 24 

3.3.3 pH ....................................................................................................... 24 

3.3.4 Total Phenol (TP) Content ................................................................... 24 



   

 

  v 

3.3.5 Total Flavonoid (TF) Content .............................................................. 25 

3.3.6 Statistical Data Analysis ...................................................................... 25 

3.4 Results and Discussion ............................................................................ 26 

3.4.1 Colour Measurement ............................................................................ 26 

3.4.2 Miscibility of Propolis Liquid Products ................................................ 30 

3.4.3 pH of Propolis Liquid Samples ............................................................ 32 

3.4.4 Total Phenolic and Flavonoid Content in Propolis ................................ 34 

3.5 Conclusions ............................................................................................. 37 

Chapter 4. Commercial Propolis Liquid Products: Comparison of Functional 

Properties .................................................................................................................. 39 

4.1 Introduction ............................................................................................. 39 

4.2 Materials and Methods............................................................................. 40 

4.2.1 Commercial Propolis Samples ............................................................. 40 

4.2.2 Determination of Antioxidant Property ................................................ 40 

4.2.3 Determination of Antimicrobial Property ............................................. 42 

4.3 Results and Discussion ............................................................................ 45 

4.3.1 Antioxidant Capacity ........................................................................... 45 

4.3.2 Antimicrobial Activity ......................................................................... 49 

4.4 Conclusions ............................................................................................. 52 

Chapter 5. Commercial Poplar Type Propolis Liquid Products: Elements and 

Adulteration in Propolis ........................................................................................... 53 

5.1 Introduction ............................................................................................. 53 

5.2 Materials and Methods............................................................................. 55 

5.2.1 Commercial Propolis Samples ............................................................. 55 

5.2.2 Determination of Metal Elements......................................................... 55 

5.2.3 Determination of Salicin for Adulteration of Propolis .......................... 57 

5.3 Results and Discussion ............................................................................ 59 

5.3.1 Heavy Metal and Rare Earth Elements in Propolis ............................... 59 

5.3.2 Salicin Determination in Propolis......................................................... 64 

5.4 Conclusions ............................................................................................. 66 

Chapter 6. Overall Conclusions and Recommendations ......................................... 68 

6.1 Conclusions ............................................................................................. 68 

6.2 Recommendations ................................................................................... 69 

References ................................................................................................................. 71 

Appendix 1. Results of Colour Measurement .......................................................... 78 

Appendix 2. Results of pH ........................................................................................ 79 



   

 

  vi 

Appendix 3. Results of Total Phenolic (TP) and Total Flavonoid (TF) .................. 81 

Appendix 4. Results of DPPH Assay ........................................................................ 85 

Appendix 5. Results of Antimicrobial Properties .................................................... 96 

Appendix 6. Results of Salicin ................................................................................ 102 

Appendix 7. Results of Metal Elements and Rare Earth Elements ....................... 106 

 



   

 

  vii 

List of Figures 

Figure 2.1 Harvest of propolis from nets (Bogdanov & Bankova, 2011) ........................ 6 

Figure 2.2 Flavonoid skeleton structure (Kumar & Pandey, 2013) ............................... 12 

Figure 2.3 Antioxidant effect of quercetin (Alvarez-Suarez, 2017) .............................. 14 

Figure 3.1 Visual appearance (e.g. colour) of 20 different propolis liquid samples. ..... 27 

Figure 3.2 Appearance of 20 different propolis liquid samples after mixing with ethanol 

at 1:9 ratio (v/v) .......................................................................................................... 28 

Figure 3.3 Appearance of 20 different propolis liquid samples after mixing with water at 

1:9 ratio (v/v). ............................................................................................................. 31 

Figure 3.4 Total phenolic (TP) content of propolis samples. The significant difference is 

shown by different letters according to Tukey’s HSD test at 95% CL. ......................... 36 

Figure 3.5 Total flavonoid content (TF) of samples. The significant difference is shown 

by different letters according to Tukey’s HSD test at 95% CL. .................................... 36 

Figure 4.1 Correlation between antioxidant capacity and TP or TF content in (A) New 

Zealand propolis products (S9-S14); (B) Australian propolis products (S1 and S2); (C) 

Korean propolis products (S3, S4, S6 and S18); (D) Chinese propolis products (S7 and 

S8) .............................................................................................................................. 48 

Figure 4.2 Plot of MIC vs TP for 20 commercial liquid propolis samples .................... 51 

Figure 4.3 Plot of MIC vs TF for 20 commercial liquid propolis samples .................... 52 

Figure 5.1 The chromatogram of salicin detected by HPLC (Zhang et al., 2011) ......... 66 

Figure A3.1 Standard curve of TP content ................................................................... 81 

Figure A3.2 Standard curve of TF content ................................................................... 83 

Figure A4.1 Plot of RSA% versus concentration of sample 1-6 ................................... 92 

Figure A4.2 Plot of RSA% versus concentration of sample 6-12 ................................. 93 

Figure A4.3 Plot of RSA% versus concentration of sample 13-18 ............................... 94 

Figure A4.4 Plot of RSA% versus concentration of sample19- 20 ............................... 95 

Figure A5.1 Well diffusion S1-S8 (S. aureus. B. cereus, and E. coli from left to right).96 

Figure A5.2 Well diffusion S9-S20 (S. aureus. B. cereus, and E. coli from left to right)

 ................................................................................................................................... 96 

Figure A5.3 Sample 1-8 with different dilutions for investigating MIC of S. aureus, 

after first incubation .................................................................................................... 97 

Figure A5.4 Sample 1-8 with different dilutions for investigating MIC of S. aureus, 

adding resazurin .......................................................................................................... 97 

Figure A5.5 Sample 1-8 with different dilutions for investigating MIC of S. aureus, 

adding resazurin and after secondary incubation ......................................................... 98 

Figure A5.6 Samples 9-20 with different dilutions for investigating MIC of S. aureus, 

adding resazurin and after secondary incubation ......................................................... 98 

Figure A5.7 Samples 1-8 with different dilutions for investigating MIC of E. coli, after 

first incubation ............................................................................................................ 99 



   

 

  viii 

Figure A5.8 Samples 1-8 with different dilutions for investigating MIC of E. coli, 

adding resazurin and after secondary incubation ......................................................... 99 

Figure A5.9 Samples 9-20 with different dilutions for investigating MIC of E. coli, 

adding resazurin and after secondary incubation ......................................................... 99 

Figure A5.10 Samples 1-8 with different dilutions for investigating MIC of B. cereus, 

after first incubation .................................................................................................. 100 

Figure A5.11 Samples 1-8 with different dilutions for investigating MIC of B. cereus, 

adding resazurin and after secondary incubation ....................................................... 100 

Figure A5.12 Samples 9-20 with different dilutions for investigating MIC of B. cereus, 

adding resazurin and after secondary incubation ....................................................... 100 

Figure A6.1 Standard curve of salicin by HPLC-MS ................................................. 103 

Figure A6.2 The concentration of salicin in samples by HPLC-MS ........................... 104 

Figure A6.3 Standard curve of salicin by HPLC ........................................................ 105 

Figure A7.1 The concentration of standards (111Cd, 75As, 208Pb) by ICP-MS ............. 109 

Figure A7.2 The concentration of standards (Rare earth elements) by ICP-MS.......... 111 

 

 



   

 

  ix 

List of Tables 

Table 2.1 The core chemical compounds found in propolis since 2000 (Huang et al., 

2014) .......................................................................................................................... 10 

Table 2.2 Principal constituents of main propolis types from different origins (Zabaiou 

et al., 2017) ................................................................................................................. 11 

Table 2.3 The relationship of total flavonoid content and quality of propolis ............... 13 

Table 3.1 Reagents and standards for determination of chemical properties of commercial 

propolis liquid products .............................................................................................. 22 

Table 3.2 Product information about 20 different commercial propolis liquid products 

used in this study ........................................................................................................ 23 

Table 3.3 CIE L*, a*, b*, C* and h values of propolis liquid samples’ colour ............. 30 

Table 3.4 The pH of 20 different propolis liquid samples including extraction medium 

used ............................................................................................................................ 33 

Table 3.5 The results of TP and TF contents in samples .............................................. 35 

Table 4.1 Dilutions of propolis samples to different concentrations with methanol for 

antioxidant analysis ..................................................................................................... 42 

Table 4.2 Materials and microorganisms used to determine the antimicrobial activity of 

propolis samples ......................................................................................................... 43 

Table 4.3 The results of IC50 of 20 propolis samples. The results of TP, TF and pH are 

dedrived from Table 3.5 in Chapter 3 .......................................................................... 46 

Table 4.4 Antimicrobial activity of 20 propolis liquid products evaluated by well 

diffusion and MIC methods ......................................................................................... 50 

Table 5.1 Reagents and standards for determination of metal elements ........................ 55 

Table 5.2 Digestion program for propolis samples ...................................................... 56 

Table 5.3 Reagents and standards for determination of salicin in propolis ................... 57 

Table 5.4 The conditions used for salicin determination in propolis by HPLC ............. 58 

Table 5.5 The Conditions for salicin determination for HPLC part of HPLC-MS ........ 59 

Table 5.6 The Conditions for salicin determination for MS part of HPLC-MS ............ 59 

Table 5.7 The concentration of heavy metal elements in propolis ................................ 62 

Table 5.8 The concentration of rare earth elements (µg/L) in propolis samples............ 63 

Table 5.9 Salicin in propolis samples determined by HPLC and HPLC-MS ................ 65 

Table A1.1 Results of propolis samples in CIE L* a* b* ............................................. 78 

Table A1.2  Results of propolis samples in CIE L* C* h* ........................................... 78 

Table A2.1 pH of 20 propolis samples ........................................................................ 79 

Table A2.2 Tukey HSD test of pH between samples ................................................... 82 

Table A3.1 Absorbance of gallic acid for standard curve (TP) ..................................... 81 

Table A3.2 Absorbance of samples for TP detection ................................................... 81 



   

 

  x 

Table A3.3 Tukey HSD test of TP between samples ab ................................................ 82 

Table A3.4 Absorbance of quercetin for standard curve .............................................. 83 

Table A3.5 Absorbance of samples for TF detection ................................................... 83 

Table A3.6 Tukey HSD test of TF between samplesab ................................................. 84 

Table A4.1 Absorbance and calculated RSA% of sample 1 ......................................... 85 

Table A4.2 Absorbance and calculated RSA% of sample 2 ......................................... 85 

Table A4.3 Absorbance and calculated RSA% of sample 3 ......................................... 85 

Table A4.4 Absorbance and calculated RSA% of sample 4 ......................................... 86 

Table A4.5 Absorbance and calculated RSA% of sample 5 ......................................... 86 

Table A4.6 Absorbance and calculated RSA% of sample 6 ......................................... 86 

Table A4.7 Absorbance and calculated RSA% of sample 7 ......................................... 87 

Table A4.8 Absorbance and calculated RSA% of sample 8 ......................................... 87 

Table A4.9 Absorbance and calculated RSA% of sample 9 ......................................... 87 

Table A4.10 Absorbance and calculated RSA% of sample 10 ..................................... 88 

Table A4.11 Absorbance and calculated RSA% of sample 11 ..................................... 88 

Table A4.12 Absorbance and calculated RSA% of sample 12 ..................................... 88 

Table A4.13 Absorbance and calculated RSA% of sample 13 ..................................... 89 

Table A4.14 Absorbance and calculated RSA% of sample 14 ..................................... 89 

Table A4.15 Absorbance and calculated RSA% of sample 15 ..................................... 89 

Table A4.16 Absorbance and calculated RSA% of sample 16 ..................................... 90 

Table A4.17 Absorbance and calculated RSA% of sample 17 ..................................... 90 

Table A4.18 Absorbance and calculated RSA% of sample 18 ..................................... 90 

Table A4.19 Absorbance and calculated RSA% of sample 19 ..................................... 91 

Table A4.20 Absorbance and calculated RSA% of sample 20 ..................................... 91 

Table A5.1 Inhibition zone containing the diameter (6mm) of well ............................. 97 

Table A5.2 MIC for S. aureus ................................................................................... 101 

Table A5.3 MIC for E. coli ....................................................................................... 101 

Table A5.4 MIC for B. cereus ................................................................................... 101 

Table A6.1 The number and information of samples in this experiment .................... 102 

Table A7.1 Original weight of samples for digestion ................................................. 106 

Table A7.2 The detected concentration of heavy metal elements by ICP-MS ............ 106 

Table A7.3 The detected concentration of rare earth elements by ICP-MS ................ 107 

 

 
 



   

 

 1 

Chapter 1. Introduction 

Propolis is a well-known resinous substance also called bee glue, which has a dark brown 

colour and is produced by honeybees from various plants buds or resins (Silici & Kutluca, 

2005; Bankova et al., 2019). It is generally used to repair honeycombs and stabilise the 

moisture and temperature of beehives by honeybees (Zabaiou, Fouache, Trousson, Baron, 

& Zellagui, 2017; Bankova et al., 2019). In general, propolis comprises of plant resins 

(50%), beeswax (30%), essences (10%), pollens (5%), and other organic constituents (5%) 

(Gómez-Caravaca, Gómez-Romero, Arráez-Román, Segura-Carretero, & Fernández-

Gutiérrez, 2006; Falcão et al., 2010; Huang, Zhang, Wang, Hu, & Li, 2014). 

Propolis can be generally classified into two types, such as Brazilian type and European 

type, according to the botanic origin of propolis (Markham, Mitchell, Wilkins, Daldy, & 

Lu, 1996; Xu, Luo, Chen, & Fu, 2009). The Brazilian type propolis is basically from the 

countries located in tropical zone, including Amazon, Brazil, Cuba, Tunisia and so forth 

(Markham et al., 1996; Xu et al., 2009). The European type is also named as poplar type, 

as this type of propolis is mainly collected from poplar tree which is widely grown not 

only in Europe, but also in Africa, China, Korea, New Zealand and other temperate area 

around the world (Markham et al., 1996; Bankova, de Castro, & Marcucci, 2000).  

The chemical composition and some physical characteristics of propolis vary according 

to the source of plant and the place of region (Silici & Kutluca, 2005; Zabaiou et al., 

2017). In terms of chemical constituents, poplar propolis contains large amounts of 

flavones and flavanones, compared to phenolic acid and their esters (Markham et al., 1996; 

Bankova et al., 2019). On the other hand, the Brazilian propolis is comprised of a high 

proportion of p-coumaric acid derivatives (Markham et al., 1996; Xu et al., 2009; Huang 

et al., 2014). However, the core composition of propolis is phenolics, especially 

flavonoids (Silici & Kutluca, 2005; Viuda-Martos, Ruiz-Navajas, Fernandez-Lopez, & 

Perez-Alvarez, 2008; Xu et al., 2009; Huang et al., 2014; Oryan, Alemzadeh, & Moshiri, 

2018a). 

Propolis is renowned for its functional properties, including antioxidant (Toreti, Sato, 

Pastore, & Park, 2013; Sforcin, 2016), antimicrobial (Kumazawa, Hamasaka, & 

Nakayama, 2004; Silici & Kutluca, 2005), anti-inflammatory (Kumazawa et al., 2004; 



   

 

 2 

Toreti et al., 2013), and anticancer activity (Kumazawa et al., 2004). Studies have shown 

a correlation between the phenolics contents and some functional properties of propolis, 

such as antioxidant property (Zunini et al., 2010). Due to its identified functional 

properties, propolis has been broadly applied to many products, including foods, 

beverages, toothpaste and etc. (Archaina, Rivero, Sosa, & Coronel, 2015; Kubiliene et al., 

2015; Xavier et al., 2017).  

However, raw propolis needs to be purified before being applied to commercial products. 

Liquid extracts are the most common commercialised products that can be found in the 

market (Gómez-Caravaca et al., 2006; Gardana, Scaglianti, Pietta, & Simonetti, 2007; Xu 

et al., 2009). Ethanol, propylene glycol, water, and edible oils have been used as solvents 

to extract and preserve the bioactive compounds from propolis, as they are non-toxic to 

human (Hu et al., 2005; Kubiliene et al., 2015; Sforcin, 2016). 

Cvek et al. (2008) indicated that propolis tends to be a good source that contains 

undesirable trace elements and heavy metal elements, as it is from various plants in 

different region. Some previous studies have shown that the amounts of elements detected 

in propolis are related to its geographical origin (region) (Bonvehí & Bermejo, 2013; 

Formicki, Gren, Stawarz, Zysk, & Gal, 2013). Nevertheless, the concentration of metals 

found in commercial propolis products might also be influenced by process conditions 

(e.g. types of solvent, extraction procedures, and extraction time) (Tosic, Stojanovic, 

Mitic, Pavlovic, & Alagic, 2017). However, up to date, there is less research focusing on 

the element content in commercial propolis products, on which more attention is needed. 

By considering food fraud, due to the similar physical and chemical properties of polar 

tree gum to propolis, some studies reported that low-cost poplar tree gum was mixed with 

commercial propolis products to reduce the manufacturing cost and make more profit 

(Zhang, Ping, Wang, Huang, & Hu, 2015). Poplar tree gum is extracted from populous 

buds, which has the similar colour and chemical compositions to the poplar type propolis 

(Vardar-Ünlü, Silici, & Ünlü, 2008; Zhang, Zheng, Liu, & Hu, 2011). To detect its 

adulteration, some phenolic glycosides (e.g. salicin) which is unique in poplar tree gum 

can be analysed and used as a marker to identify whether propolis products have been 

adulterated or not (Pearl & Darling, 1971; Zhang et al., 2011; Zhang et al., 2015).  



   

 

 3 

The physical and chemical properties of raw propolis have been broadly studied 

(Markham et al., 1996; Falcão et al., 2010; Huang et al., 2014; Archaina et al., 2015; do 

Nascimento et al., 2018), while studies on commercial propolis products are scant. 

Therefore, this project aimed to focus on investigating and comparing the properties and 

the quality of liquid commercial propolis products produced in 4 different countries 

(Australia, China, Korea, and New Zealand) by analysing the physiochemical properties 

(e.g. water and ethanol miscibility, colour, and pH), chemical composition (e.g. total 

phenolic and total flavonoid contents), functional properties (e.g. antioxidant and 

antimicrobial properties), and elements (e.g. heavy metal elements and rare earth 

elements), and investigating the adulteration (e.g. presence of salicin) of propolis. In this 

project, 20 different commercial liquid propolis products were used among which 2 were 

produced in Australia, 2 in China, 10 in Korean, and 6 in New Zealand. 

 

 

 

  



   

 

 4 

Chapter 2. Literature Review 

2.1  Introduction 

The word propolis derived from Greek, in which “pro” means “entrance to”, and “polis” 

stands for “city”, demonstrating its defensive function in hive (Toreti et al., 2013; Zabaiou 

et al., 2017; Bankova et al., 2019). It is a renowned resinous material also called bee glue, 

which is collected by honeybees from various plants organs, including buds, saps, resins 

and other sources (Silici & Kutluca, 2005; Bankova et al., 2019). Due to the physical 

properties and composition of propolis, it is used to repair the comb and stabilize the 

temperature and moisture in the hive by bees (Zabaiou et al., 2017; Bankova et al., 2019) 

and also to cover cracks formed by other invaders (Sforcin, 2016).  

Propolis is generally comprised of plant resins (50%), beeswax (30%), aromatic oil (10%), 

pollens (5%), and other organic constituents (5%) (Huang, Zhang, Wang, Li, & Hu, 2014). 

The composition of propolis differs from the source of plant and the place of origin (Silici 

& Kutluca, 2005; Zabaiou et al., 2017), which significantly influence physical, chemical 

and functional properties of propolis. Generally, the density of propolis ranges from 1.11-

1.14 kg/m3, and its melting point is in the range of 80 to 105ºC (Bogdanov & Bankova, 

2011). Propolis can be classified into two groups by their botanic regions, which are 

European type and Brazilian type (Markham et al., 1996). The European type is also 

called poplar type, as poplar trees are the dominant plant of propolis in Europe, China, 

Korea, Africa, Australasia and other temperate area all over the world. The Brazilian type 

is not only from Brazil but also from Cuba, Amazon, Tunisia and some other countries 

located in the tropical zone with lack of poplar trees (Markham et al., 1996; Bankova et 

al., 2000; Xu, Luo, Chen, & Fu, 2009). This means that the chemical substances in 

propolis may vary from different locations, their main compounds, however, have been 

identified to be flavonoids, cinnamic acids, terpenes, phenolic acids, and aromatic acids 

(Silici & Kutluca, 2005; Popova et al., 2007; Huang et al., 2014; Oryan, Alemzadeh, & 

Moshiri, 2018b).  

Due to its chemical compositions, several studies have reported that propolis has various 

functional properties, including antioxidant (Toreti et al., 2013; Sforcin, 2016), anti-

bacteria (Kumazawa et al., 2004; Silici & Kutluca, 2005; Popova et al., 2007) , antiseptic 



   

 

 5 

(Toreti et al., 2013; Sforcin, 2016), anti-inflammatory (Kumazawa et al., 2004; Toreti et 

al., 2013; Sforcin, 2016), and anticancer (Kumazawa et al., 2004). These properties give 

rise to the consumption of propolis as a functional ingredient or supplement in some 

medicines (Viuda-Martos et al., 2008; Toreti et al., 2013), foods, candies, beverages, 

toothpaste, liquid propolis products, and other commercial products (e.g. cosmetics and 

animal feeds) (Kumazawa et al., 2004). Over 200 new products containing propolis have 

been developed and launched in the world market over the past seven years (Agriculture 

and Agri-Food Canada, 2017). Also, in term of value addition, the products which 

claimed containing propolis are more expensive than other bee products. Hence, it is 

essential to find methods to evaluate the quality or the quantity of functional components 

of propolis products. However, there is no clear standard to evaluate and characterize the 

chemical and functional properties of those products (Alvarez-Suarez, 2017; Bankova et 

al., 2019), because of the variability of propolis’ chemical substances as mentioned above. 

In terms of trace elements in propolis, although the composition is negligible, Cvek et al. 

(2008) suggested that the raw propolis could be a source of trace elements as well as some 

heavy metal elements, since it is a collected by honeybees from diverse plants growing in 

different origins under various conditions. In the same vein, Gong, Luo, Gong, Gao, and 

Xie (2012), Bonvehí and Bermejo (2013), and Formicki et al. (2013) noted in their 

research that elements in propolis are related to their geographic region. However, the 

concentration of metal elements in commercial propolis depends on the type of solvent 

applyed to extract the raw propolis (Tosic et al., 2017). However, it should be noted that 

so far, there is limited research analysing elements in commercial propolis from different 

regions. 

With regard to adulteration, due to the scarcity of resources in nature and the high 

production cost of propolis, some studies indicate that a low cost polar tree gum that has 

similar chemical composition and functional properties to propolis was mixed in some 

poplar type propolis products, viz. some companies used the cheaper material to adulterate 

propolis products to make more profit. (Zhang et al., 2011; Zhang et al., 2015). Polar tree 

gum is the extraction of Populus buds. Although it has similar smell, colour, and chemical 

compounds, including flavonoids, phenolics and cinnamic acid derivatives, to the polar 

type propolis (Vardar-Unlu, Silici & Unlu, 2007; Zhang et al., 2011), it also contains 

some phenolic glycosides, including salicin and its derivatives, which are not found in 



   

 

 6 

propolis, as these compounds have been hydrolysed by enzymes secreted by honey bees 

during propolis production (Pearl & Darling, 1971; Zhang et al., 2011; Zhang et al., 2015). 

Therefore, these unique compounds can be selected as an indicator to identify the 

adulteration of propolis. However, this has not been well investigated. 

2.2  Extractions of Propolis  

2.2.1 Preparation of Propolis Extracts  

Propolis is collected from propolis traps which are plastic nets with small holes through 

which bees drop propolis (Bankova et al., 2019). Freezing the traps is likely to be a better 

way to harvest propolis, since it can make propolis hard and brittle (Bogdanov & Bankova, 

2011; Kubiliene et al., 2015; Bankova et al., 2019). And then it could be easily grounded 

into powder.  

 

 

 
 

 

Figure 2.1 Harvest of propolis from nets (Bogdanov & Bankova, 2011) 

 

2.2.2 Different Extraction Solvents 

Crude propolis has to be purified before its commercialisation. The main purpose of the 

extraction is to purify propolis by removing impurities (e.g. beeswax) and activating the 

functional components, including polyphenolic fractions (Bankova et al., 2019). Ethanol, 

propylene glycol, water and some oils have been commerically used as extraction solvents 



   

 

 7 

as they are non-toxic and safe for human consumption (Krell, 1996; Kubiliene et al., 2015; 

Sforcin, 2016).  

Ethanol extracted propolis (EEP) 

Ethanol extracted propolis (EEP) tends to be widely used in commercial liquid propolis 

products, as it is more likely to be purified and contain  more functional compounds 

(Kubiliene et al., 2015; Sforcin, 2016). Krell (1996) indicated that the maximum ratio of 

propolis-ethanol solution to get a high efficiency of the propolis extraction is 3:10 (w/w). 

This was supported by Ildenize et al. (2004), Gómez-Caravaca et al. (2006), and Monroy 

et al. (2017) that the total phenolic  (TP) and total flavonoid (TF) contents were the highest 

when using ethanol as an extraction solvent among non-toxic extraction solvents. Also, 

60-80% ethanol was shown to have a higher extraction capacity of the main functional 

compounds (e.g. flavonoids) (Ildenize et al., 2004; Gómez-Caravaca et al., 2006; 

Bogdanov & Bankova, 2011; Ramanauskien, Inkeniene, Petrikait, & Briedis, 2013; 

Monroy et al., 2017), and the best extraction of some phenolic compounds was obtained 

when using 70% and 95% ethanol solutions (Bogdanov & Bankova, 2011). The ethanol 

extract of propolis can be used in many applications, including food and cosmetic 

products (Krell, 1996). However, the use of propolis in food applications is limited by its 

undesirable smell and taste during consumption. It cannot also be accepted by some 

people with alcohol intolerance.  

Glycol extracted propolis (GEP) 

Glycol (propylene glycol, E1520) is often used to extract propolis to avoid some 

disadvantages associated with EEP and improve the water extraction dissolution (Sforcin, 

2016). The extraction process is similar to using ethanol as the solvent, but with a higher 

temperature and lower ratio (Krell, 1996). 

Water extracted propolis (WEP) 

Water is the most commonly used sovlent in the food industry. The perparation of WEP 

may take a few days by extracting raw propolis(Krell, 1996). During the extraction 

process, because of the low water solubility of some bioactive compounds, heat treatment 

can be applied to enhace the efficiency of extraction. Also, some compounds cannot be 

fully soluble in water, therefore, heating the water and adding propylene glycol are used 

to make better extraction (Kubiliene et al., 2015; Sforcin, 2016). Moreover, ultrasound 

can also be used to help reduce the time of extraction, thereby more efficient (Sforcin, 



   

 

 8 

2016). However, the yield of TP and TF in WEP is still lower than the former two methods 

(Gómez-Caravaca et al., 2006; Kubiliene et al., 2015; Ramanauskiene et al., 2013). For 

water solubility, only less than 10% of propolis’ weight can be dissolved (Bankova et al., 

2019). It has also been found that WEP has a lower antimicrobial activity against some 

typical microbes, including Staphylococcus aureus, Bacillus subtilis, and Escherichia coli, 

when  compaired with EEP and GEP (Ramanauskiene et al., 2013). This could be related 

to the fact that the solubility of biologically active compounds in water is low (Kubiliene 

et al., 2015).  

 

Oil extracted propolis (OEP) 

Oils (e.g. olive oil) have been tested as an extraction medium for propolis by heating 

(Krell, 1996). However, a research conducted by Kubiliene et al. (2015) showed that the 

concentration of TP in OEP was lower than that in WEP and its antimicrobial activity was 

also lower. Hence, OEP is not a common type of propolis that can be readily 

commercialised in the market. 

2.3   Physical and Chemical Properties 

The functional properties of propolis are correlated with its chemical composition and 

physical properties, which have been widely researched. Some factors, such as botanical 

regions, plant sources and type of bees, have impacts on the properties of propolis 

(Bankova et al., 2000; Falcão et al., 2010; Huang et al., 2014). 

2.3.1 Physical Characteristics 

The colour of raw propolis varies from origins to origins, but dark brown colour is the 

most common one (Kasote et al., 2017; do Nascimento et al., 2018). The physical status 

and texture of raw propolis change with temperature. It is sticky and soft when its 

temperature is between 20C and 45C but becomes hard and fragile once the temperature 

is below 15C (Krell, 1996). The solid raw propolis turns into liquid at a temperature 

range from 60C to 70C (Krell, 1996). 

2.3.2 Chemical Composition 

More than 300 chemical substances have been identified in propolis until 2000 (Huang et 

al., 2014). Some core chemical compounds of propolis are shown in Table 2.1. The 



   

 

 9 

various chemical constituents are mainly due to the different botanical and plant origin. 

There are many types of propolis according to the geographic origins. For instance, poplar 

propolis from Europe, North America, Asia (e.g. China and Korea) and Australasia 

(Australia and New Zealand); green propolis mainly from Brazil; birch propolis from 

Russia; red propolis from Cuba, Brazil and Mexico; Clusia propolis from Cuba and 

Venezuela, and pacific propolis from the Pacific region (Okinawa, China, and Indonesia) 

(Dos Santos et al., 2017). Recognising the origins and sources of propolis could contribute 

to characterising and standardising the chemical and functional properties of propolis 

products (Bogdanov & Bankova, 2011). Hence, many studies have been conducted to 

analyse the chemical compositions of different origins of propolis. As shown in Table 2.2, 

the principal constituents of propolis are similar but distinctively diverse between 

different origins and types (Zabaiou et al., 2017; Bankova et al., 2019). This means that 

isoflavonoids tend to be the largest part in red propolis, but the proportion of phenolic 

acids seems to be the highest in green propolis. However, there is no admitted criterion 

for each compound or any origin of propolis. Thus, it is hard to evaluate the quality of 

propolis products. 

 

 

 

 

 

 

 

 

 

 

 



   

 

 10 

Table 2.1 The core chemical compounds found in propolis since 2000 (Huang et al., 

2014) 

 Chemical 

Category 
Compound 

Geographical 

Origin 
Plant Source 

1 Flavonoids Luteolin Australia, Brazil, 

Burma, Canada, 

Chinese, Cuba, 

Egypt, Greece, 

Japan, Kenya, 

Mexico, Nepal, 

Poland, Portugal, 

Solomon Island, 

China (Taiwan) 

Populus, 

Macaranga, 

Dalbergia 

2 Prenylated 

flavanoned 

7-O-prenylpino-

cembrin 

Greece, Japan / 

3 Neo-flavonoids Cearoin Nepal Dalbergia 

4 Monoterpenes 

Sesquiterpenes 

Diterpenes 

Linalool abietic 

acid 

Brazil, Greece, 

Indonesia, Iran, 

Malta, Turkey 

Ferula Pinaceae 

Cupressaceae 

5 Triterpenes Lupeol acetate Burma, Brazil, 

Cuba, Egypt, 

Greece 

/ 

6 Phenylpropanoids 

and esters 

p-

Methoxycinnamic 

acid 

Australia, Brazil, 

Egypt, Uruguay 

Citrus 

7 Prenylated 

phenylpropanoids 

3-Prenyl-4-

hydroxycinnamic 

acid 

Brazilian Green 

propolis 

Baccharies 

8 Stilbenes and 

prenylated 

stilbenes 

3-

Prenylresveratrol 

Australis, Brazil, 

Greece, Indonesia, 

Kenya 

Macaranga 

9 Lignans 6-

Methoxydiphyllin 

Kenya / 

10 Coumarins Prenylated 

coumarin 

suberosin 

Iran / 

 

 

 

 

 

 

 

 



   

 

 11 

Table 2.2 Principal constituents of main propolis types from different origins 

(Zabaiou et al., 2017) 

 Propolis type Geographic origin Principal constituents 

1 Polar Propolis Europe, North America, Asia 

(e.g. China, Korea), New 

Zealand, temperate zone 

Flavones, flavanones, 

cinnamic acids and their 

esters 

2 Green Propolis Brazil Prenylated phenolic acids, 

flavonoids, phenolics 

3 Birch Propolis Russia Flavones and flavonols (not 

the same as in Polar type) 

4 Red Propolis Cuba, Brazil, Mexico Isoflavonoids (isovalvans, 

pterocarpans) 

5 Mediterranean 

Propolis 

Sicily, Greece, Crete, Malta, 

Turkey, Algeria 

Terpenoids, Diterpenes 

(primarily acids of labdane 

type) 

6 Clusia 

Propolis 

Cuba, Venezuela Polyprenylated 

benzophenones 

7 Pacific 

Propolis 

Japan, China (Taiwan), 

Indonesia 

prenylated-flavanones 

 

 

Phenolics 

Phenolics are comprised of flavonoids, and phenolic acids and their derivatives, including 

cinnamic acids, p-coumaric acids, caffeic acids, chicoric acids, and ferulic acids (Huang 

et al., 2014; Zabaiou et al., 2017). A number of studies have proved that these compounds 

are associated with antiviral activities of propolis (Huang et al., 2014; Alvarez-Suarez, 

2017; Zabaiou et al., 2017). Moreover, phenolics could contribute to minimizing the 

damage of deoxyribonucleic acid (DNA) by inhibiting the influence in cultured 

fibroblasts (Darendelioglu, Aykutoglu, Tartik, & Baydas, 2016). That is to say, Brazil 

green propolis which is rich in phenolics, tend to be responsible for antiviral and 

inhibitory activities (Huang et al., 2014; Oryan et al., 2018b). Some caffeic acid 

derivatives (tetradecenyl caffeate) and isoferulic acid derivative (2-methyl-2-butenyl 

ferulate) in polar propolis were also identified (Huang et al., 2014). 

 

 



   

 

 12 

Flavonoids 

Flavonoids is part of polyphenols which has a C6-C3-C6 carbon skeleton structure, as 

shown in Figure 2.2.  There are many groups of flavonoids found in propolis, including 

flavanone, flavonol, flavone, flavan, chalcone, isoflavone, isodihydriflavone, isoflavan, 

dihydrochalcone, and neoflavonoid, according to different substitution of functional 

groups in the skeleton (Huang et al., 2014). According to Huang et.al (2014), the 

pharmacological functions of propolis are attributed to flavonoids which are the main 

substances of propolis, and 112 flavonoids have been detected in different types of 

propolis. Inui et al. (2012) analysed the flavonoids compounds and pointed out that these 

appear to be explicitly linked to the antioxidant and antimicrobial properties of propolis. 

This view was supported by a study reported from De Almeida et al. (2013) who proved 

that the extraction of flavonoids from propolis can help heal burn wounds. Similarly, 

Raghukumar, Seidel, Vali, Watson, and Fearnley (2010) found many prenylated 

flavanones are associated with potent antimicrobial activity. As mentioned previously, 

propolis are rich in flavonoids such as in green propolis, red propolis, and birch propolis. 

Thus, the level of total flavonoid content is suggested to be a quality index to evaluate the 

quality of propolis (Gardana et al. 2007). Additionally, the grade evaluation of propolis 

based on the TF content has been applied by the China Government publishing in Chinese 

regulation of propolis (GB/T 24283-2009) (Lv et al., 2009). (Table 2.3).  

 

 

 

 

Figure 2.2 Flavonoid skeleton structure (Kumar & Pandey, 2013) 

 

 

 



   

 

 13 

Table 2.3 The relationship of total flavonoid content and quality of propolis 

Propolis TF content Quality References 

Raw 

<11% Low Gardana et al., (2007) 

11-14% Acceptable 

14-17% Good 

>17% High 

Raw 
15% First grade China National Standard GB/T 

24283-2009 edited by Lv et al., 

(2009) 

8% Second grade 

EEP 
20% First grade 

17% Second grade 

 

2.4  Functional Properties of Propolis 

The functional properties of propolis have been broadly studied (e.g. antioxidant, 

antimicrobial, anti-inflammatory, and anticancer). Antioxidant and antimicrobial 

capacities are the two core functional properties that have been widely researched. 

Literature revealed that the two properties are mainly attributed to the phenolic 

composition in propolis, especially the flavonoids (Viuda-Martos et al., 2008). 

2.4.1 Antioxidant Property 

Oxidation reactions could be generated by free radicals that stem from the metabolic 

processes in human body (Alvarez-Suarez, 2017). Due to the strong oxidation capacity of 

free redicals, its presence could harm cells and tissues in human body. Some anitoxiants 

(e.g. vitamin C and E) have the ability quency free radicals by donating hydrogen atom 

to free radicals and then become antioxidant free radicals. The antioxidant free radicals 

are however relatively stable through conjugation and electron delocalisation, called 

resonance stabilization. This phenomenon is referred to as free radical scavenging 

commonly reported in the study of antioxidant capacity for propolis (Zunini et al., 2010; 

Socha, Galkowska, Bugaj, & Juszczak, 2015). 

Studies have demonstrated the antioxidant capacity of propolis (Toreti et al., 2013; 

Sforcin, 2016; Alvarez-Suarez, 2017; Bankova et al., 2019), which could help to protect 



   

 

 14 

the human body against free radicals. It has been identified that the main functional 

propolis compound for antioxidant is flavonoids.  

 

 

 

 

 

 

Figure 2.3 Antioxidant effect of quercetin (Alvarez-Suarez, 2017) 

 

 

Figure 2.3 shows how a free radical is quenched by quercetin, a common compound of 

flavonoids in propolis, which donates its hydrogen atoms to the free radical, thus reducing 

the harmful hydroxide radical to water molecules. This means the flavonoid-rich propolis 

products will be a good natural antioxidant supplement to help treat oxidative stress-

related diseases, which is a superior option for consumers as it may be safer than artificial 

synthetic antioxidants.  

2.4.2 Antimicrobial Property 

The most renowned properties of propolis are the antimicrobial properties (Alvarez-

Suarez, 2017). Propolis is an essential part for honeybees to protect their hives from 

pathogenic microorganisms and bacteria (Banskota, Tezuka, & Kadota, 2001; Alvarez-

Suarez, 2017).  

Many researchers claimed that propolis has the antimicrobial activity against both Gram-

positive and Gram-negative bacteria, either aerobic or anaerobic types (Alvarez-Suarez, 

2017). Two experimental approaches have been mainly used for determining the 

antimicrobial activity of propolis, which are agar diffusion and broth dilution methods. 



   

 

 15 

These methods determine the inhibitory effect of propolis on the growth of bacteria (etc. 

S. aureus, B. cereus, E. coli, and P. aeruginosa) (Huang et al., 2014; Alvarez-Suarez, 

2017; Dos Santos et al., 2017). However, some researchers argue that the bacterial 

inhibitory effect of propolis tends to be more effective against Gram-positive bacteria 

than Gram-negative ones (Silici & Kutluca, 2005). Also, the extraction solutions of 

propolis may influence the bacterial activity (Grange & Davey, 1990). Furthermore, some 

recent research revealed the antimicrobial activity could defend against bacteria more 

efficiently than before, such as Listeria monocytogenes (Yang, Chang, Chen, & Chou, 

2006) although the previous study indicated that propolis was less sensitive to Listeria 

spp. (Grange & Davey, 1990). In other words, the antimicrobial activity of propolis has 

been explicitly explored with more advanced technology. 

2.4.3 Anti-inflammatory Properties 

The anti-inflammatory properties of propolis products are extremely valuable in medical 

area, which could be an effective treatment of skin disease or wound healing (Banskota 

et al., 2001; Sforcin, 2016). Many factors may interfere with the anti-inflammatory 

activities of propolis products, including the source and concentration of propolis, and the 

extraction method of propolis used. To obtain significant anti-inflammatory activities, 

several experiments have been carried out to determine the functional chemical 

constituents of propolis in the extraction solutions (Banskota et al., 2001).  

2.5  Analyses of Propolis  

2.5.1 Chemical Composition 

There are various instrumental techniques that have been employed to analyse the 

chemical composition of propolis both quantitatively and qualitatively, including UV-

spectrophotometer, high performance liquid chromatography (HPLC), gas 

chromatography (GC) and thin layer chromatography (TCL) (Gómez-Caravaca et al., 

2006; Falcão et al., 2010). 

 

 



   

 

 16 

2.5.2 Determination of Total Phenolics and Total Flavonoids 

Spectrophotometry 

Spectrophotometry has been broadly used to analyse the contents of total phenolics (TP) 

and total flavonoids (TF) in propolis (Santos-Buelga & González-Paramás, 2017). 

Compared to other chromametric means, its advantages are low cost and simple operation 

(Santos-Buelga & González-Paramás, 2017). Aluminium chloride (AlCl3) reaction is 

generally used to analyse the content of flavone and flavonol compounds (Chang, Yang, 

Wen, & Chen, 2002; Pujirahayu, Uslinawaty, & Ritonga, 2014). Another complementary 

colorimetric method is conducted by 2,4-dinitrophenylhydrazine (DNPH) reaction to 

determine the concentration of flavanones and dihydroflavonols. Chang et al. (2002) 

suggested that the cotent of real total flavoniods is close to the sum of the results from the 

two reactions described above. However, since DNPH reacts with carbonyl groups, it is 

more likely to overestimate the content of TF due to the interference of other compounds 

with carbonyl groups, such as saccharides and quercetin (a flavone compound) in the 

sample. The most common method to evaluate the total phenolic content is Folin-

Ciocalteu method (Gómez-Caravaca et al., 2006). 

HPLC and GC 

Both HPLC and GC can be applied to analyse phenolic compounds. GC is a better option 

to analyse the volatile composition, while, HPLC is more likely to be employed in non-

volatile compounds analysis (Gómez-Caravaca et al., 2006; Santos-Buelga & González-

Paramás, 2017). HPLC is therefore more preferable to determine the TF composition, 

since HPLC has extensive applicability and produces a better result than GC approach 

(Markham et al., 1996).  

2.5.3 Evaluation of Antioxidant Property 

The redox reaction is the principle reaction to analyse the antioxidant property of propolis. 

The methods usually reported in the literature are ferric reducing antioxidant power 

(FRAP) and 2,2’-diphenyl-1-picrylhydarzyl free radial (DPPH•) assays. Antioxidant 

capacity can be expressed as trolox equivalent antioxidant capacity (TEAC) or half 

inhibitory concentration (IC50). UV-Vis spectrophotometry is used to evaluate the 

antioxidant activity by measuring the colour change of the reaction between oxidants and 

reactants (Archaina et al., 2015; Socha et al., 2015).  



   

 

 17 

2.5.4 Evaluation of Antimicrobial Activity 

As mentioned prevously, agar diffusion and broth dilution methods are generally used to 

determine the antimicrobial activity of propolis (Almeida et al., 2017). The diffusion 

method involves the incubation of cultures in an agar well or disk on the Muller-Hinton 

agar plates, and evaluate the strength of antibiotic activity by measuring the diameter of 

the inhibitory halos (Afrouzan, Tahghighi, Zakeri, & Es-Haghi, 2018; do Nascimento et 

al., 2018). The broth dillution is cheaper and more sensitive approch than the diffusion 

methods. This minimum inhibitory concentration (MIC) of antimicrobial activity is 

determined by incubating a specific concentration of microorganism cultures in the 

microtiter plates with different concentrations of propolis (Balouiri, Sadiki, & Ibnsouda, 

2016). The two common methods have been standardised by The European Committee 

on Antimicrobial Susceptibility Testing (EUCAST) and The Clinical & Laboratory 

Standards Institute (CLSI). The representative microorganisms used in literature for these 

tests are E. coli (Gram-negative), S. aureus  and B. cereus (Gram-positive) (Almeida et 

al., 2017; Kasote et al., 2017). 

2.5.5 Determination of Elements in Propolis 

Heavy metal and rare earth elements  

Lead (Pb), arsenic (As), and cadmium (Cd) are the main heavy metal elements, which are 

generally contained in the air, soil and water (Bonvehí & Bermejo, 2013). Lead is 

basically from vehicle exhaust which can always be found in the air, while, cadmium is a 

contaminant from the metal industry. Arsenic is a ubiquitous element in the enviroment, 

including water, soil and air (Bonvehí & Bermejo, 2013). Since propolis is collected from 

plants which grow in various places, the contamination of heavy metals could occur along 

the path from the environment to propolis. It can also be inferred that with different 

mineral compounds in soil, the possible source of heavy metal contamination in propolis 

may vary.  

There are 14 elments classified as rare earth elements (REE) group, including cerium (Ce), 

neodymium (Nd), samarium (Sm), terbium (Tb), erbium (Er), praseodymium (Pr), 

holmium (Ho), thulium (Tm), gadolinium (Gd), dysprosium (Dy), lutetium (Lu), 

ytterbium (Yb), lanthanum (La) and yttrium (Y) (Germund, 2004). They have been 

perferably used as indicators of soil in environmental sciences, and always quantified as 



   

 

 18 

a chemically uniform group (Oliveira et al., 2017). This implies that it can be used to trace 

the geographical origin of propolis by analysing the REE composition. However, so far 

very few studies have been conducted to determine the elements in propolis.  

Elemental analysis technique 

Graphite furnace atomic absorption spectrometry (GFAAS) is a traditional method to 

determine the content of elements in various samples, including propolis (Vardar-Unlu, 

Silici, & Unlu, 2008; Korn et al., 2013; Aksoy, Atabay, Tirasoglu, Koparan, & 

Kekillioglu, 2017). However, inductively coupled plasma-optical emission spectrometry 

(ICP-OES) and inductively coupled plasma-mass spectrometry (ICP-MS) are playing a 

dominant role in elemental analysis (Vardar-Unlu et al., 2008; Korn et al., 2013; Aksoy 

et al., 2017; Tosic et al., 2017; Gonzalez-Martin, Revilla, Betances-Salcedo, & Vivar-

Quintana, 2018). Compared to GFAAS, ICP-OES and ICP-MS are a multi-element 

technique to detect elements, which has the lower limit of detection and high accuracy 

(Tosic et al., 2017). Before the measurement, samples normally need to be digested by 

acid (e.g. nitric acid, perchloric acid and hydrofluoric acid). Traditional approach for 

sample digestion is heating by hot plate or hot block, which requires a large amount of 

acid and long time. Microwave-assisted acid digestion technique has been applied to 

sample preparation for elemental analysis for the last few decades, which not only 

simplifies the process but also enhances the efficiency (Korn et al., 2013). In this project, 

microwave-assisted acid digestion technique and ICP-MS were used to detemine the 

elements in commercial liquid propolis products. 

2.5.6 Identification of Adulteration of Propolis 

It has been reported that the extract of populus buds (e.g.  poplar tree gum) has been  used 

to adulterate propolis, since it has  the physical properties and chemical compositions 

similar to propolis (Zhang et al., 2011; Zhang et al., 2015). Salicin belongs to the class of 

organic compounds known as phenolic glycosides, which has been found in the barks, 

leaves and twigs of poplar trees (Pearl & Darling, 1971; Palo, 1984; Clausen et al., 1989; 

Zhang et al., 2011). Zhang et al. (2011) noted that the phenolic glycosides would be 

hydrolysed during the process of collecting propolis by honeybees. This means that 

phenolic glycosides can be used as a marker compound to evaluate the adulteration of 

propolis with poplar tree gum. 



   

 

 19 

Technically, HPLC is the general technique used to determine TP andTF as well as 

indentifying phenolic glycosides (Gómez-Caravaca et al., 2006; Santos-Buelga & 

González-Paramás, 2017; Zhang et al., 2011). However, mass spectrometry (MS) is more 

preferable in analysing organic compounds, since it has higher selectivity and sensivity 

than other detectors (Stobiecki, 2000; Cuyckens & Claeys, 2004; Medana, Carbone, 

Aigotti, Baiocchi, & Appendino, 2008). Forthermore, the combination of high 

performance liquid chromatography with mass spectrometry (HPLC-MS) has been 

applied to determine phenolics and flavonoids for many years. It indicates HPLC-MS is 

available to be also employed to identify salicin in propolis. Both HPLC and HPLC-MS 

were used to determine the concentration of salicin in this project. 

2.6   Conclusions 

Various studies reported the different extraction methods of propolis and  the basic 

chemical compounds of propolis from many different geographic origins, and showed the 

functional properties of propolis. Spectrophotometry, HPLC, HPLC-MS, DPPH• assay, 

well/disk diffusion method and broth dilution test are generally ujsed to analyse the 

chemical and functional properties of propolis. Also, ICP-OES and ICP-MS are the main 

techinque used to determine the content of metal elements. Salicin can be used as a marker 

compound to investigate the adulteration of propolis, since it only exists in popolar tree 

gum not in propolis. In addition, HPLC is also commonly employed to detect the presence 

of salicin in propolis.Although some studies have been carried out to determine the the 

physicochemical and funcitonal properties of propolis, there are still some knowldege 

gaps that need to be investigated. In addition, no studies  have been performed to 

determine and compare differences in the chemical composition, physical properties and 

functional activities between commercially available propolis liquid products 

manufactured from different countries.  



   

 

 20 

Chapter 3. Commercial Propolis Liquid Products: 

Comparison of Physical and Chemical Properties  

Abstract 

The physical and chemical properties of 20 commercial propolis liquid products 

manufactured in 4 countries (Australia, China, Korea and New Zealand) were 

investigated in this study. The pH of propolis samples ranged from 3.55  0.07 to 9.37  

0.03, and the visual colour of samples varied from brown, red to green. The miscibility 

of most samples tended to be better in ethanol than in water, except some samples added 

with an emulsifier (Tween 20). The content of total phenolic (TP) in propolis products 

ranged from 16.35  0.28 mg/mL to 70.22  0.47 mg/mL, and total flavonoid (TF) content 

was from 6.56  0.37 mg/mL to 58.97  0.59 mg/mL. However, some unexpected results 

were obtained with the TF content being higher than the TP content in some propolis 

samples which needs to be further investigated. 

 

3.1   Introduction 

Propolis is a resinous substance also called bee glue, which has dark colour collected by 

honeybees from various plants buds or resins (Silici & Kutluca, 2005; Bankova et al., 

2019). The chemical composition and physical characteristics of propolis vary depending 

on the plant sources and geographical regions of propolis (Silici & Kutluca, 2005; 

Zabaiou, Fouache, Trousson, Baron, Zellagui, et al., 2017). According to the composition 

differences among regions, propolis is generally classified into two main types, which are 

Brazilian type and European type (poplar type) (Xu et al., 2009). In terms of chemical 

constituent, poplar propolis contains high amounts of flavones and flavanones compared 

to phenolic acid and their esters (Markham et al., 1996; Bankova et al., 2019). On the 

other hand, Brazilian propolis comprises of a high proportion of p-coumaric acid 

derivatives (Markham et al., 1996; Xu et al., 2009; Huang et al., 2014). However, the core 

chemical compounds identified from propolis are phenolics especially flavonoids (Silici 

& Kutluca, 2005; Xu et al., 2009; Huang et al., 2014; Oryan et al., 2018a). 



   

 

 21 

Before commercialisation, crude raw propolis has to be extracted and it has been shown 

that various solvents have been used as an extraction medium, including ethanol, 

propylene glycol (often referred to as glycol), water and/or oil (Kubiliene et al., 2015; 

Sforcin, 2016). Ethanol and glycol extractions are however more commonly used to 

extract propolis in order to produce commercial liquid propolis products due to the 

relatively high yield of extraction (Kubiliene et al., 2015; Sforcin, 2016). Consequently, 

the most common propolis liquid products on the market are an ethanol or glycol base. 

Traditionally, crude propolis is soaked in a solvent, commonly ethonal, to extract 

bioactive compounds and remove the beewax (Krell, 1996). Then the liquid propolis is 

filtered. o achieve an optimal yield, the soaking could be around two weeks.  

Due to the various functional properties (e.g. antioxidant and antimicrobial), propolis is 

broadly applied to many products, such as food, beverage, toothpaste and dietary 

supplement (Archaina et al., 2015; Kubiliene et al., 2015; Xavier et al., 2017). Propolis 

are usually characterised for the contents of total phenolic (TP) and total flavonoid (TF) 

(Santos-Buelga & González-Paramás, 2017). For the evaluation of TP content, Folin-

Ciocalteu method is one of the most widely used methods (Gómez-Caravaca et al., 2006; 

Papotti, Bertelli, Bortolotti, & Plessi, 2012). Aluminium chloride (AlCl3) assay is one of 

the most commonly used methods for the determination of TF content which is based on 

the reaction between the carbonyl and hydroxyl groups of flavonoid and aluminium ion 

(Al3+) (Gómez-Caravaca et al., 2006; Chang et al., 2002). 

Studies on the physical and chemical properties of raw propolis have been reported 

(Markham et al., 1996; Falcão et al., 2010; Huang et al., 2014; Archaina et al., 2015; do 

Nascimento et al., 2018). However, there has been scant research on the physicochemical 

properties of commercially available propolis liquid products. Therefore, the objective of 

this study was to analyse and compare 20 different commercial liquid propolis products 

(e.g. ethanol or glycol-based poplar type) obtained from 4 different countries (Australia, 

China, Korea and New Zealand). The propolis samples were analysed for the 

determination of water and ethanol miscibility, colour, pH and, TP and TF contents.  



   

 

 22 

3.2  Materials and Methods 

3.2.1 Materials 

The reagents and standards used to determine the chemical properties of propolis are 

listed in Table 3.1. 

 

Table 3.1 Reagents and standards for determination of chemical properties of 

commercial propolis liquid products 

Name Concentration/Grade Supplier 

Folin-Ciocalteu Phenol’s Reagent 2 mol/L Merck 

Gallic acid  98.0% Merck 

Ethanol (Absolute) > 99.8 % HPLC Thermo Fisher 

Methanol (Anhydrous) > 99.8 % HPLC Thermo Fisher 

Potassium acetate (CH3COOK)  99 % Scharlau 

Quercetin > 95 % HPLC Grade Sigma-Aldrich 

Sodium carbonate (Na2CO3)  99.8 Univar 

Aluminium trichloride (AlCl3) 99.99 % Sigma-Aldrich 

 

 

 

 

 

3.2.2 Commercial Propolis Samples 

Twenty commercial liquid propolis products were investigated in this project, which were 

manufactured from four different countries including Australia, China, Korea and New 

Zealand. Details of samples are designated as S1 to S20 respectively which are shown in 

Table 3.2. 

 

 

 



   

 

 23 

Table 3.2 Product information about 20 different commercial propolis liquid 

products used in this study 

A GEP represents glycol-based propolis; Sample code was highlighted in blue colour. 
B EEP represents ethanol extracted propolis; Sample code was highlighted in orange colour. 
C WEEP represents water-soluble base by evaporating ethanol after extraction, and it contains potassium 

carbonate and Tween 20; Sample code was highlighted in green colour. 
D SEEP represents water-soluble ethanol extracted propolis containing Tween 20; Sample code was 

highlighted in purple colour. 
E100 means that the TF content in the product is at least 1% (w/v), and 200 means 2% (w/v);  

3.3  Analyses of Physicochemical Properties 

3.3.1 Miscibility 

All propolis samples were analysed for their miscibility with water and ethanol (>99.8%), 

respectively. Briefly, 1 mL of each propolis sample was added into 9 mL of distilled water 

or ethanol in a glass test tube. The mixture of water and propolis was thoroughly mixed 

by shaking and then visually observed for their appearance and the formation of a 

homogeneous solution.  

Sample 

code 
Country 

Propolis 

liquid base 

Labelled 

concentration 
Source of propolis  

S1 

Australia 

GEPA 
Equal to propolis 

400mg/ml 
Australia 

S2 GEPA 
Equal to propolis 

300mg/ml 

S3 

Korea EEPB 

/ 

China S4 / 

S5 / 

S6 / Australia, Brazil, China 

S7 
China GEPA 

TF 40mg/ml 
China 

S8 TF 32.4mg/ml 

S9 

New 

Zealand 

EEPB TF 30 mg/ml 

New Zealand 

S10 
GEPA 

TF 30 mg/ml 

S11 TF 15 mg/ml 

S12 GEPA TF 30 mg/ml 

S13 EEPB TF 30 mg/ml 

S14 GEPA TF 30 mg/ml 

S15 

Korea 

WEEPC 100E 

China 
S16 EEPB 200E 

S17 EEPB 100E Australia 

S18 EEPB 100E Korea 

S19 SEEPD 100E Brazil 

S20 WEEPC 100E Australia, Brazil, China 



   

 

 24 

3.3.2 Colour 

The colour of all original samples and 10 times ethanolic dilutions was analysed using a 

colour spectrophotometer (CM-5, Konica Minolta). The prepared ethanolic dilutions 

were filtered through 20  m filter to remove any precipitates before measurements. Data 

were collected by SpectraMagic NX ver. 2.6 software. The colour measurement was 

based on CIE L*a*b* and L*C*h colour space systems with illuminate D65 at 10 view 

angle. Prior to the measurement of sample colour, the colour spectrophotometer was 

calibrated using both zero calibration plate (CM-A204) and white calibration (100%) with 

distilled water. 

3.3.3 pH 

All propolis samples were analysed using a pH meter (PB-11, Sartorius) for the 

determination of pH at room temperature (20ºC) directly without any dilution. Before 

measurement, the pH meter was calibrated by the buffer solutions of pH 4 and 7, 

respectively. 

3.3.4 Total Phenol (TP) Content 

Sample preparation 

The content of total phenol (TP) in the propolis samples was analysed in this study 

according to the Folin-Ciocalteu method reported by Papotti et al. (2012) with some 

modifications. All samples were diluted with methanol in triplicate before the 

measurement. Sample 6 specified as S6 in Table 3.2 was diluted to a 100-fold with 

methanol, while the other 19 samples were diluted to a 200-fold with methanol. Also, the 

Folin-Ciocalteu reagent was also diluted to a 10-fold with water. All the diluted propolis 

samples (0.2 mL) were then mixed with the Folin-Ciocalteu reagent dilution (5 mL). A 4 

mL of 5 % aqueous sodium carbonate (Na2CO3) solution was added into the mixtures 

after incubation for 3 minutes and well shaken.  

A series of different concentrations of gallic acid (100, 200, 250, 400 and 500 mg/L) were 

used to construct a standard curve using the same reagents as samples for the 

determination of the TP content in the propolis samples. All the standards and samples 

prepared were incubated for 1 hr under the dark condition and ambient temperature, and 

then measured for their absorbance at 760 nm using a spectrophotometer (UV-1601, 



   

 

 25 

Shimadzu). The spectrophotometer was set to zero using an absolute methanol solution 

as the blank solvent. The TP contents of triplicate samples measured were averaged and 

expressed as mg of gallic acid equivalent (GE) per mL of propolis (mg GE/mL). 

3.3.5 Total Flavonoid (TF) Content 

Sample preparation 

Aluminium chloride method reported by Chang et al. (2002) was modified to analyse the 

TF content of propolis samples. Sample dilutions were prepared in triplicate before the 

measurement. Sample 6 was 100 times diluted with absolute methanol, and the other 19 

samples were diluted to 200-fold with absolute methanol. The diluted samples (0.5 mL) 

were mixed with 0.1 mL of aluminium trichloride (AlCl3) solution (2% w/v), 0.1 mL of 

potassium acetate (CH3COOK) solution (1 mol/L) and 4.3 mL of absolute methanol.  

Standard curve preparation 

Quercetin was chosen as a standard to express TF in the propolis samples. A series of 

concentration applied to construct the standard curve, including 50, 75, 100, 150, 200, 

and 300 mg/L. All standards were added with the same reagents as samples. 

The absorbance was used at 415 nm, and the measurements of all the standards and 

samples were made against absolute methanol solution as the blank by UV-1601 

spectrophotometer after incubation for 30 minutes at room temperature and under dark 

condition. Triplicate samples were measured, and the results were recorded as mg of 

quercetin per mL of propolis (mg QE/mL). 

3.3.6 Statistical Data Analysis 

All experiments were conducted in triplicates for each product. The results were presented 

as mean value +/- standard deviation (SD). The results of TF and TP were analysed by 

the analysis of variance (ANOVA) using IBM SPSS software (Ver. 24) to evaluate the 

difference between samples at 95% confidence level. Results of post-hoc Tukey’s test 

were used to compare the mean differences of the TF and TP contents between propolis 

samples. 



   

 

 26 

3.4  Results and Discussion 

3.4.1 Colour Measurement 

The colour of the liquid propolis products was observed visually, before conducting the 

instrumental colour measurement. Figure 3.1 illustrates the colour varies from brown, red, 

to green. Both Australian (S1 and S2) and Chinese (S7 and S8) propolis showed a dark 

brown colour. For the propolis products produced in Korea, the visual colours were 

observed to be different from dark reddish (e.g. S15 and S20), dark green (S19) and light 

brown (S18).  

The colour differences between samples could be due to difference in the crude raw 

propolis initially used for the extraction of propolis. As mentioned in Table 3.2, the 

Korean propolis products were not only extracted from Korean local raw propolis but also 

made from the raw propolis stock obtained from other countries, such as Australia, Brazil 

and China. Although an overall brown hue was also observed from all the New Zealand 

(NZ) samples (S9 – S14), the intensity of their colours varied from light to dark brown as 

shown in Figure 3.1. The difference in colour intensity and darkness seemed to be related 

to the TP and TF contents in S9 - S14 as shown in Table 3.5. For example, among the NZ 

propolis samples, S12 was very dark and its TP and TF contents were the highest while 

S11 was light and its TP and TF contents were the lowest. In other words, the higher TP 

or TF in the NZ samples was related to a dark and non-vivid brown colour. This colour 

phenomenon from NZ made propolis liquid products indicates that phenolic compounds 

including flavonoids are the dominant colour pigment in propolis. 

After 10-fold dilution with ethanol, the visual colour of 20 propolis samples was much 

lighter as expected. As Figure 3.2 illustrates that the colour of diluted propolis samples 

also varied from brown, red, orange and yellow. Propolis from New Zealand (S9-S14) 

were generally lighter than other products, presenting yellow and reddish-orange colour. 

The dilution of S3 (Korean propolis) and S8 (Chinese propolis) showed still dark brown, 

but Australian propolis showed reddish colour after dilution. The bright yellow colour 

was found in dilutions of two New Zealand propolis products (S9 and S11) and one 

Korean propolis (S19). The colour of Australia (S1, S2) and New Zealand (S9-S11) 

propolis product dilutions showed a correlation with their TP contents, which was darker 

with higher concentration of TP content (Table 3.5). 



   

 

 27 

 

 

.  

Figure 3.1 Visual appearance (e.g. colour) of 20 different propolis liquid samples 



   

 

 28 

 

 

 

Figure 3.2 Appearance of 20 different propolis liquid samples after mixing with 

ethanol at 1:9 ratio (v/v) 

 

The instrumental measurement of colour was only analysed from the propolis samples 

diluted 10-fold in ethanol. The reasons were that for certain stock propolis liquid samples, 

especially Australian and Chinese ones (S1 and S2; S7 and S8), the measured CIE values 

tended to zero (very dark black) due to the remarkable colour intensity, which resulted in 

the lack of comparison for colour. And most samples were also not miscible with water, 

which is discussed later in this chapter. 

The results of CIE L*, a*, b*, C* and h are shown in Table 3.3. Firstly, there was no trend 

of colour appearance or difference that could be related to differences in the type of 

extraction solvent applied for producing the commercialised liquid propolis products. The 

lightness L* values ranged from 31.34 to 80.14. Among the 20 samples diluted with 

ethanol, 14 samples were overall appeared to be dark, showing as L* less than 50, while 

the other 6 samples (S1, S2, S4, S5, S6 and S8) had a higher lightness value, especially 



   

 

 29 

S8 sample’s L* value (80.14  0.69). It should be mentioned that unlike the observed dark 

colour of S8 sample visually as shown in Figure 3.2, its lightness L* value was measured 

to be highest.  

For the a*, most of the samples were reddish with positive a* values, except three samples 

(S15, S16 and S20) with negative a* values ranging from -0.16 to -0.07 which indicate 

their colour has slightly greenish tint but not pronounced as its value was close to zero. 

For the b* values, all samples had positive b* values, indicating the colours of the propolis 

liquids were yellowish. By combining the measured a* and b* values, it can be concluded 

that most propolis samples had a brown colour mixed with red and yellow, except for 3 

samples (S15, S16 and S20) that were yellowish with a small green tint. As a result, the 

latter 3 samples had the hue angle values higher than 90 (117.86, 154.97 and 153.88) as 

shown in Table 3.3. It was not easy to correlate between the visual colour observation and 

the instrumental colour measurement as indicated above using S8 sample as an example.  

 

 

 

 

 

 

 

 

 

 

 

 



   

 

 30 

Table 3.3 CIE L*, a*, b*, C* and h values of propolis liquid samples’ colour 

Countries Sample L* a* b* C* h 

Australia 
S1 64.69  0.74 18.36  0.22 49.07  0.51 52.39  0.40 69.48  0.42 

S2 75.06  0.12 11.46  0.33 58.63  0.30 59.68  0.12 79.30  0.99 

Korea 

S3 45.55  0.33 30.19  0.65 24.68  0.65 39.16  0.65 39.00  0.58 

S4 70.45  0.22 25.92  0.81 63.92  0.55 68.96  0.21 67.69  0.40 

S5 54.77  0.54 27.58  0.27 40.20  0.59 48.97  0.71 55.30  0.28 

S6 68.71 0.47 16.57  0.26 54.13  0.48 56.61  0.38 72.98  0.39 

China 
S7 42.53  0.06 18.86  0.05 19.01  0.14 26.77  0.13 45.23  0.15 

S8 80.14  0.69 3.56  0.24 54.13  0.37 55.92  0.59 86.28  0.18 

NZ 

S9 40.98  0.30 28.96  0.23 16.44  0.11 33.05  0.17 30.27  0.44 

S10 34.50  0.33 12.29  0.12 4.66  0.23 13.47  0.16 21.33  0.22 

S11 48.57  0.19 35.49  0.33 29.38  0.18 46.31  0.11 39.57  0.28 

S12 32.06  0.15 0.61  0.06 0.46  0.06 0.90  0.07 36.92  0.63 

S13 35.12  0.31 15.30  0.46 5.38  0.28 13.21  0.29 23.77   0.91 

S14 32.27  0.42 1.29  0.31 0.73  0.24 1.49  0.36 27.89  0.58 

Korea 

S15 31.34  0.09 -0.07  0.07 0.46  0.05 0.26  0.06 117.86  0.80 

S16 31.38  0.15 -0.16  0.05 0.06  0.02 0.17  0.04 154.97  0.70 

S17 31.47  0.16 0.19  0.02 0.23  0.11 0.23  0.07 49.83  0.28 

S18 45.94  0.32 31.60  0.40 24.03  0.47 39.89  0.32 36.59  0.25 

S19 33.28  0.57 3.36  0.19 2.13  0.18 3.68  0.13 31.67  0.41 

S20 31.56  0.21 -0.11  0.02 0.19  0.12 0.20  0.09 153.88  0.88 

Blue colour represents glycol-based propolis;  

Orange colour represents ethanol extracted propolis; 

Green colour represents water-soluble base by evaporating ethanol after extraction, and it contains 

potassium carbonate and Tween 20;  

Purple colour represents water-soluble ethanol extracted propolis containing Tween 20.  

 

3.4.2 Miscibility of Propolis Liquid Products 

The results of miscibility of all the samples with ethanol or water at 1:9 ratios are shown 

in Figures 3.2 and 3.3, respectively. Before mixing, all the original propolis liquid 

samples were homogenous solutions as shown in Figure 3.1. However, when mixed with 

water, most samples, except for 3 samples (S15, S19 and S20), became turbid forming 

aggregates and precipitates, resulting in phase separation. The reason for those 3 samples 

remaining as a clear, uniform dispersion without any aggregation could be due to the fact 



   

 

 31 

that the products were further processed by evaporating ethanol and adding Tween 20 to 

make the products. For the GEP (glycol-based propolis) samples as indicated in Table 3.2, 

when they were added into water, relatively larger pieces of aggregates were formed due 

to some coagulation, leading to precipitation. This phenomenon was observed clearly 

from S1 sample as shown in Figure 3.3. On the other hand, EEP (ethanol extract propolis) 

samples seemed to form smaller pieces of aggregates which were suspended in the 

mixtures (e.g. S9 sample). The results indicate the significant poor water miscibility of 

propolis liquid products which could be a barrier that negatively influences the further 

application of propolis liquids in the food industry, for example, to fortify beverage with 

propolis as a functional ingredient.   

 

 

Figure 3.3  Appearance of 20 different propolis liquid samples after mixing with 

water at 1:9 ratio (v/v) 

 

A totally opposite phenomenon was observed when the propolis liquid products were 

mixed with ethanol. In their 10-fold ethanolic dilutions, all the GEP and EEP samples 

were fully miscible with ethanol forming homogeneous and stable solutions. However, 

the two WEEP samples (S15 and S20) had significant sedimentation although this is not 

seen clearly in Figure 3.2. The precipitant was believed to be K2CO3. The reason is that 

firstly, the SEEP (S19) was fully dissolved in ethanol. The only difference between the 



   

 

 32 

WEEP and SEEP samples is that the WEEP contains K2CO3 while the SEEP does not 

according to descriptions specified on their product labels. Secondly, to confirm whether 

the precipitant was K2CO3, several drops of 6 M of HCl was added to the precipitant. It 

was found clearly that bubbles were forming.  

3.4.3 pH of Propolis Liquid Samples 

The pH values of all the 20 samples are shown in Table 3.4. The pH of all commercial 

propolis liquids (EEPs and GEPs) except for those WEEP and SEEP samples (S15, S19 

and S20) were acidic ranging from 3.55  0.07 to 5.54  0.01. Pujirahayu et al. (2014) 

who also investigated commercialised propolis liquids in their study showed some similar 

results although the pH values of propolis were 6.3  0.1 in ethanol solvent and 5.4  0.2 

in propylene glycol solvent in their study.  

On the other hand, the SEEP and WEEPs had higher pH values. The SEEP (S19) sample 

had its pH close to neutral with 6.77  0.07. The WEEP samples (S15 and S20) were 

alkaline with their pH values being 8.61  0.06 and 9.37  0.03, respectively, which could 

be attributed to the presence of K2CO3.  

 

 

 

 

 

 

 

 

 

 



   

 

 33 

Table 3.4 The pH of 20 different propolis liquid samples including extraction 

medium used 

Countries 
Sample 

code 

Extraction 

solvent 
pH 

Australia 
S1 Glycol 3.55  0.07 

S2 Glycol 5.37  0.03 

Korea 

S3 Ethanol 4.74  0.03 

S4 Ethanol 4.65  0.02 

S5 Ethanol 5.41  0.03 

S6 Ethanol 5.50  0.01 

China 
S7 Glycol 4.36  0.04 

S8 Glycol 4.79  0.04 

NZ 

S9 Ethanol 4.88  0.02 

S10 Glycol 4.67  0.04 

S11 Glycol 4.77  0.03 

S12 Glycol 4.45  0.01 

S13 Ethanol 4.86  0.02 

S14 Glycol 4.55  0.02 

Korea 

S15 
Ethanol (contains potassium 

carbonate and Tween 20) 
8.61  0.06 

S16 Ethanol 5.16  0.07 

S17 Ethanol 5.54  0.01 

S18 Ethanol 4.61  0.04 

S19 Ethanol (contains Tween 20) 6.77  0.07 

S20 
Ethanol (contains potassium 

carbonate and Tween 20) 
9.37  0.03 

Blue colour represents glycol-based propolis;  

Orange colour represents ethanol extracted propolis; 

Green colour represents water-soluble base by evaporating ethanol after extraction, and it contains 

potassium carbonate and Tween 20;  

Purple colour represents water-soluble ethanol extracted propolis containing Tween 20;  

 

 



   

 

 34 

3.4.4 Total Phenolic and Total Flavonoid Contents in Propolis 

The original data and standard curve for the determination of total phenolic (TP) and total 

flavonoid (TF) contents are presented in the Appendix 3. The results of TP and TF 

contents are shown in Table 3.5. The TF content between the propolis products ranged 

from 6.56  0.37 QE mg/mL (S15) to 58.97  0.59 QE mg/mL (S12). The analysed results 

could not be evaluated for their accuracy as the exact concentration of TF or TP was not 

clearly specified on their product packages. Nevertheless, when compared to the TF 

content claimed by the companies, the TF content of most samples was higher than its 

content labelled on their product packages, except for S8, in which the identified TF 

content was 27.19  0.76 mg QE/mL which was less than the claimed 32.4 mg/mL. 

Although it is not sure, the result of the Chinese propolis liquid product (S8) being 

measured to be lower in the TF content may be due to different standards chosen and used 

to represent the TF content. In this experiment, quercetin was used as a standard whereas 

rutin is the legal standard mentioned the China regulation to represent the TF content in 

propolis (China propolis standard). 

With regards to the TP content, as shown in Table 3.5, among all the samples, the TP 

content of S12 was the highest (70.22  0.47 GE mg/mL) while it was the lowest (16.35 

 0.28 GE mg/mL) for S4, followed by S15 (17.39  0.37 GE mg/mL), S18 (22.00  0.42 

GE mg/mL), S17 (24.03  0.42 GE mg/mL), and S20 (25.35  0.40 GE mg/mL), which 

had the TP content being lower than 30 GE mg/mL. It should be pointed that the 

commercial propolis liquids are generally made by extraction and dilution with suitable 

carrier solvent, such as ethanol and propylene glycol, with a certain confidential ratio. 

Hence, neither the analysed TP nor TF values can reveal or correlate back to the TP or 

TF content in their raw propolis. Thus, the results of TP or TF cannot be compared directly 

with any literature which studied based on raw propolis. Also, as aforesaid, there is few 

research which focused on the commercial propolis liquid products. Furthermore, 

commercial products could also be manufactured by mixing propolis from different 

sources (e.g. various countries and botanic areas). For instance, S20 was informed to be 

made by blending propolis from Australia, Brazil and China.  

 



   

 

 35 

Table 3.5 The results of TP and TF contents in samples 

Countries 
Sample 

code 

TP 

(mg GE/mL) 

TF 

(mg QE/mL) 

Labelled 

content 
TF/TP (%) 

Australia 

S1 40.50  0.45 29.82  0.27 Equal to 

propolis 
400mg/ml 

73.63  1.49 

S2 31.44  0.51 22.86  0.24 Equal to 

propolis 

300mg/ml 

72.70  1.84 

Korea 

S3 31.17  0.63 21.80  0.64 / 69.93  2.60 

S4 16.35  0.28 6.56  0.37 / 40.14  2.48 

S5 31.35  0.35 35.08  0.60 / 111.92  4.43 

S6 25.35  0.05 18.97  0.52 / 74.82  3.02 

China 

S7 48.99  0.31 51.29  0.12 TF 40mg/ml 104.70  2.25 

S8 48.87  0.49 27.19  0.76 TF 

32.4mg/ml 
55.63  1.68 

NZ 

S9 53.60  0.53 41.30  0.65 TF 30 mg/ml 77.05  1.64 

S10 64.85  0.72 53.87  0.82 TF 30 mg/ml 83.06  1.66 

S11 35.95  0.49 24.74  0.22 TF 15 mg/ml 68.83  1.46 

S12 70.22  0.47 58.97  0.59 TF 30 mg/ml 83.97  1.31 

S13 68.29  0.62 54.86  0.42 TF 30 mg/ml 80.34  1.12 

S14 68.11  0.47 54.63  0.14 TF 30 mg/ml 80.21  0.97 

Korea 

S15 17.39  0.37 20.84  0.37 1% (w/v) 119.86  8.53 

S16 37.36  0.47 54.09  0.50 2% (w/v) 144.79  5.77 

S17 24.03  0.42 29.68  0.40 1% (w/v) 123.52  6.57 

S18 22.00  0.49 12.86  0.19 1% (w/v) 58.43  1.77 

S19 35.04  0.60 27.72  0.25 1% (w/v) 79.10  1.92 

S20 25.35  0.40 24.43  0.16 1% (w/v) 96.39  3.72 

Blue colour represents glycol-based propolis;  

Orange colour represents ethanol extracted propolis; 

Green colour represents water-soluble base by evaporating ethanol after extraction, and it contains 

potassium carbonate and Tween 20;  

Purple colour represents water-soluble ethanol extracted propolis containing Tween 20. 

 

Based on the content of TP shown in Figure 3.4, the 20 samples can be statistically 

identified into 11 groups with the TP content increasing from letters a to k at 95% 

confidence level (CL). Similarly, the content of TF in the 20 products could be divided 

into 12 groups, as shown in Figure 3.5, with increasing with the marked letter from a to l. 



   

 

 36 

The samples in the same group means that there was no significant statistical difference 

of TP or TF at 95% CL by ANOVA test.  

 

Figure 3.4 Total phenolic (TP) content of propolis samples. The significant 

difference is shown by different letters according to Tukey’s HSD test at 95% CL 

 

 

Figure 3.5 Total flavonoid content (TF) of samples. The significant difference is 

shown by different letters according to Tukey’s HSD test at 95% CL 



   

 

 37 

However, the ratios of TF to TP which were greater than 100% were unexpected which 

was observed from some samples (S5, S7, S15, S16, S17 and S20) as shown in Table 3.5. 

For instance, the ratio was 144.79  5.77 % for S16. It should be mentioned that 

flavonoids are defined as a type of polyphenol compounds that have a special C6-C3-C6 

skeleton structure whereas polyphenols are a subgroup of phenolics (Lee and Wong, 

2014). Hence, the TF content should not be greater than the corresponding TP content. 

This phenomenon has not been indicated in the literature. Although it is not sure, the 

TF/TP ratio of > 100% observed from some samples could be due to adulteration of 

propolis by poplar tree gum. According to Zhang et al. (2015), among 66 commercial 

propolis products they analysed, 44 samples were identified to be adulterated by poplar 

tree gum, as the cost of poplar tree gum is only around one-tenth of propolis but it has the 

functional properties similar to propolis. In terms of safety, it is believed that there is no 

harm by replacing propolis with poplar tree gum. The results of the suspected adulteration 

by adding poplar tree gum into some propolis products will be discussed in Chapter 5. 

 

3.5  Conclusions 

The chemical composition of TP and TF and physical properties (miscibility, colour and 

pH) of 20 commercially available propolis liquid products were analysed in this study. 

Visually, most samples had a brownish or yellowish colour in appearance with some 

variations in colour hue, lightness and intensity. The pH of most samples was acidic 

ranging from 3.55  0.07 to 6.77  0.07, while 2 propolis samples (S15 and S20) 

containing potassium carbonate were alkaline with pH 8.61 0.06 and 9.37 0.03, 

respectively. In terms of water or ethanol miscibility, the majority of propolis samples 

were not miscible with water.  However, 3 samples (S15, S19 and S20) which contained 

an emulsifier, Tween 20, showed high water solubility. On the other hand, almost all 

samples were well mixed with ethanol although some small floccules formed in S8, S12 

and S14 (GEP samples), and larger amount of sediments separated out in the samples 

containing potassium carbonate (S15 and S20).  

The TF content of almost all samples ranged from 20 to 59 mg QE/mL and seemed to 

agree reasonably with their labelled content even though the content of TF in S8 was 

slightly lower than its value stated on the product package. Among all the samples, S4 



   

 

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had the lowest contents of TP and TF that were far less than the contents of TP and TF in 

other samples. It also confirmed that a higher TF content corresponded to a higher TP 

content in poplar type propolis. However, the TF contents of 6 samples (S5, S7, S15, S16, 

S17 and S20) were higher than their TP content which was unexpected. This might be 

resulting from the adulteration of propolis by poplar tree gum.  



   

 

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Chapter 4. Commercial Propolis Liquid Products: 

Comparison of Functional Properties  

Abstract 

The antioxidant and antimicrobial capabilities of 20 liquid propolis products from 4 

countries (Australia, China, Korea and New Zealand) were investigated in this chapter. 

In terms of antioxidant property, the IC50 value of products ranged from 0.24  0.02% to 

0.93  0.03%, except samples with alkaline pH (S15 and S20). Two Korean propolis 

samples (S15 and S20) with high pH levels had higher IC50 values (4.34  0.43% and 

1.78  0.06%) indicating their antioxidant activity being lower than the other propolis 

samples. For the antimicrobial activity, Gram-positive bacteria (S. aureus and B. cereus) 

were more sensitive to all propolis products than Gram-negative bacteria (E. coli). Among 

all propolis samples from different countries, New Zealand products had both relatively 

higher antioxidant and antimicrobial activities.  

4.1   Introduction 

Propolis is natural substance of honeybee product, which is also named bee glue. It is a 

resin-like material gathered from various organs from plants, which can contribute to the 

different chemical compositions in propolis (Xavier et al., 2017; Afrouzan et al., 2018; 

Liben, Atlabachew, & Abebe, 2018; Bankova et al., 2019). These compounds vary from 

the region, source of plant, even seasons for collecting and species of honeybees 

(Markham et al., 1996; Silici & Kutluca, 2005; Zabaiou, Fouache, Trousson, Baron, 

Zellagui, et al., 2017; Bankova et al., 2019). The main compounds, such as phenolics and 

flavonoids, are associated with the functional properties of propolis (Huang et al., 2014; 

Oryan et al., 2018a). Various functional properties have been widely studied, including 

antioxidant (Kumazawa et al., 2004; Zunini et al., 2010; Toreti, Sato, Pastore, & Park, 

2013; Sforcin, 2016), antimicrobial (Silici & Kutluca, 2005; Viuda-Martos et al., 2008; 

Al-Ani, Zimmermann, Reichling, & Wink, 2018) and anticancer (Kumazawa et al., 2004).   

The assay of antioxidant capacity can be conducted by DPPH• (2,2-diphenyl-1-

picrylhydrazyl) assay (Archaina et al., 2015; Socha et al., 2015). The principle of this 



   

 

 40 

approach is the redox reaction between free radicals and reductant, which is evaluated by 

measuring the change of absorbance by UV-spectrophotometry at 517 nm (Xu et al., 2009; 

Socha et al., 2015). The antioxidant property of samples measured as the radical 

scavenging activity (RSA %) is calculated and expressed as the half inhibitory 

concentration (IC50). 

For the antimicrobial activity, broth dilution and agar well diffusion are the most common 

approaches (Almeida et al., 2017). For the agar well diffusion method, according to 

Afrouzan et al. (2018) and do Nascimento et al. (2018), the antimicrobial activity is 

evaluated by measuring the diameter of the inhibitory halo on the agar plate around the 

agar well after a standard incubation. The strength of the antimicrobial property evaluated 

by the broth dilution approach can be applied to determine the minimum inhibitory 

concentration (MIC) of each propolis product by incubating different concentrations in 

microtiter plates. Previous studies showed that Gram-positive bacteria are more sensitive 

to propolis than Gram-negative bacteria (Alvarez-Suarez, 2017; Kasote et al., 2017).  

The antioxidant and antimicrobial activity of 20 different commercial propolis liquid 

products obtained from 4 different countries as described in Chapter 3 were analysed and 

compared in this chapter. The antioxidant capacity was evaluated by the DPPH• assay. 

The strength of the antioxidant capacity was ranked by the RSA%. Both agars well 

diffusion and broth dilution methods were used to assess the antimicrobial activity of the 

propolis samples. The evaluation of strength for the property was expressed as diameter 

of inhibitory halo and MIC value, respectively. 

4.2   Materials and Methods 

4.2.1 Commercial Propolis Samples 

Twenty commercial liquid propolis products, which were manufactured in 4 different 

countries (Australia, China, Korea and New Zealand) as shown in Chapter 3 (Table 3.1) 

were analysed for their antioxidant and antimicrobial properties. 

4.2.2 Determination of Antioxidant Property 

The antioxidant property of propolis samples was analysed by the DPPH• (2,2-diphenyl-

1-picrylhydrazyl) assay reported by Xavier et al. (2017) with some modifications. DPPH 



   

 

 41 

was purchased from Sigma-Aldrich Co. (St. Louis, MO, USA) and used as a free radical 

reagent to determine the antioxidant activity of propolis samples by measuring their free 

radical scavenging capacity. Initially, all propolis samples were diluted to a series of