Skip to content

Advertisement

  • Research article
  • Open Access

UPLC/FT-ICR MS-based high-resolution platform for determining the geographical origins of raw propolis samples

  • 1,
  • 2,
  • 2 and
  • 1, 3Email author
Journal of Analytical Science and Technology201910:8

https://doi.org/10.1186/s40543-019-0168-2

  • Received: 10 December 2018
  • Accepted: 25 January 2019
  • Published:

Abstract

In this study, we demonstrate a high-resolution 15 Tesla Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry coupled with a reverse-phase ultra-performance liquid chromatography (RP-UPLC) system for determining the geographical origins of raw propolis samples. The UPLC/FT-ICR MS-based high-resolution platform was validated on the ethanol-extracted propolis (EEP) from various propolis raw materials originating from different countries (i.e., Argentina, Brazil, China, and Korea) to determine the geographical origins of the propolis and the origin-specific key compounds. Based on approximately 8000 molecular features extracted from UPLC/FT-ICR MS datasets, a partial least squares-discriminant analysis (PLS-DA) plot showed distinct separations among propolis samples from four different origins. Key propolis components contributing to the discrimination of Korean propolis from Brazilian and Chinese propolis were identified and classified into five subgroups (i.e., flavonoids, phenols, terpenoids, fatty acids, and others). This analysis revealed the characteristic features of the different propolis samples, and this analytical platform can be further used to determine the geographical origins and to assess the quality of the commercial products.

Keywords

  • Propolis
  • Ethanol extract
  • FT-ICR
  • Geographical origin
  • Quality control

Introduction

Propolis, the resinous substance collected by honey bees (Apis mellifera) from buds and resins of various plant species, has been used in folk medicine for many years because of its beneficial effects on various symptoms such as wounds, sore throats, and stomach ulcers (Burdock 1998; Huang et al. 2014). Propolis is composed of various inorganic minerals and organic compounds, including vitamins, amino acids, lipids, organic acids, and flavonoids (Huang et al. 2014). Among the chemical components of propolis, phenolic compounds, including flavonoids, are the major constituents and contribute largely to the pharmacological effects of propolis (Banskota et al. 1998).

Propolis is generally classified as poplar- or Baccharis-type according to its botanical origin. The poplar-type propolis, which originates from Populus buds that are primarily found in East Asian countries such as China and Korea, is known to have high phenolic contents because the poplar-type plants have high phenolic contents (Ristivojevic et al. 2015). Baccharis-type propolis originates from Baccharis dracunculifolia, which inhabits South American countries (i.e., Brazil, Bolivia and Argentina) (Park et al. 2004). The Baccharis spp. is a significant source of tropical Brazilian green propolis, and cinnamic acid is one of the most significant constituents in the Baccharis plants. Typically, artepillin C, a cinnamic acid derivative, is the representative phenolic compound present in Brazilian propolis. In addition, low flavonoid and phenolic contents and a high content of volatile compounds are the relevant characteristics of Baccharis-type plants.

Because the compositional diversity of propolis depends on the habitats of the source plants, propolis samples collected from different origins exhibit different characteristics or biological activities such as antitumor, antibacterial, antiviral, antifungal, anti-inflammatory, and antioxidant activities (Burdock 1998). For pharmaceutical and food applications, knowledge of the chemical composition of the propolis raw materials is necessary; however, to date, the quality of propolis has been examined using a subset of the known phenolic constituents. Therefore, more detailed information is required to better understand the complicated substances in propolis and their related functions. Zhou and colleagues reported an HPLC-based determination of the geographical origins of various Chinese propolis (Zhou et al. 2008). Sawaya and coworkers also demonstrated a simple propolis fingerprinting method using easy ambient sonic-spray ionization mass spectrometry (EASI-MS) to characterize their geographical origin (Sawaya et al. 2010). However, these analytical techniques were not suitable for identifying unknown compounds due to its low resolving power and poor mass accuracy. The development of LC/MS-based analytical methods has enabled to more reliably and accurately identify complicated propolis compounds. Pietta et al. showed that the analytical platforms based on atmospheric pressure chemical ionization (APCI) MS and HPLC combined with photodiode array detection can be utilized for reliably identifying a large number of propolis components (Pietta et al. 2002). Gardana and coworkers also applied LC-tandem MS system in order to determine phenolic compounds in different source origins (Gardana et al. 2007).

More recently, high-resolution mass spectrometry has been used to characterize phenolic compounds in propolis with high accuracy. Among various types of high-resolution mass analyzers including Q-Tof and Orbitrap, Fourier transform ion cyclotron resonance (FT-ICR) is mostly considered as a powerful tool to interpret elemental compositions of compounds of interest (Choi et al. 2018). The indisputable resolving power (full width at half maximum, FWHM: > 800,000 at m/z 200) and mass accuracy (< 1 ppm) of FT-ICR enable to identify chemical compositions of extremely complicated mixtures without chromatographic separation. The isotopic fine structure obtained from FT-ICR MS can also be used to determine the elemental formula (Shi et al. 1998; Nakabayashi et al. 2013). da Costa and coworkers showed the direct infusion electrospray ionization (ESI) FT-ICR mass spectrometry-based evaluation of phenolic compounds in plant leaves (da Costa et al. 2016). Gardana and colleagues also used UPLC/MS/MS and high-resolution FT-ICR MS systems to reliably detect propolis allergens in raw propolis materials and commercial formulations (Gardana and Simonetti 2011).

In the present study, ethanol-extracted propolis (EEP) prepared from various propolis samples originating from different countries were analyzed using high-resolution 15 T FT-ICR mass spectrometry coupled with a reverse-phase ultra-high-performance liquid chromatography (RP-UPLC) system to determine the geographical origins of the propolis (Fig. 1). This analysis showed the characteristic features of the different propolis samples and figured out the key molecules discriminating those geographical origins. Furthermore, these results could be further utilized to assess the quality of commercial products.
Fig. 1
Fig. 1

Schematic workflow of UPLC/FT-ICR MS-based high-resolution analytical platform for determining the geographical origins of raw propolis samples

Materials and methods

Chemicals

Caffeic acid, p-coumaric acid, rutin, quercetin, cinnamic acid, kaempferol, chrysin, pinocembrin, caffeic acid phenethyl ester (CAPE), and artepillin C were purchased from Sigma (St. Louis, MO). All other reagents were of analytical grade.

Preparation of ethanol-extracted propolis

Each raw propolis material (10 g) was mixed with ethanol (30 mL) and then incubated for 48 h at room temperature with vigorous shaking. The resultant extract was obtained by filtration. The extracts were finally prepared as 1% of the total flavonoid content based on the quercetin, which was determined as described in the literature (Chang et al. 2002). The resultant EEP samples were stored at − 20 °C until analysis.

UPLC analysis

Chromatography was performed on an ACQUITY HSS T3 column (1.8 μm, 2.1 × 100 mm, Waters, Milford, MA) using an ACQUITY UPLC™ system (Waters) by injecting 2 μL of each EEP sample. The column was maintained at 40 °C. The gradient condition of UPLC started at 10% (v/v) acetonitrile (ACN)/water with 0.1% formic acid and was maintained for 5 min at a flow rate of 300 μL/min; the ACN content was linearly ramped to 45%, where it was maintained at this flow rate for 10 min, then ramped to 90%, where the flow rate was maintained for 3 min. The column was washed with 98% ACN for 4 min and re-equilibrated with 10% ACN for 4 min for the next run. The effluent was monitored at a wavelength of 280 nm.

UPLC/FT-ICR MS analysis

Mass spectrometric analysis of the extracts was performed using a 15 T FT-ICR mass spectrometer (solariX XRTM system, Bruker Daltonics, Billerica, MA) equipped with an electrospray ionization (ESI) source and an ACQUITY UPLC™ system. The UPLC conditions were the same as those previously described. The eluent was introduced into the mass spectrometer to acquire high-resolution MS spectra in positive ion mode within the mass range from m/z 150 to m/z 1000. The MS parameters of the positive ESI mass spectrometer were an ESI voltage of 4.5 kV, a drying gas flow rate of 8.0 L/min, a drying gas temperature of 220 °C, a skimmer voltage of 15 V, a collision gas energy of − 3.0 V, an accumulation time of 50 ms, a transient length of 1.398 s, an acquisition size of 4 MB, and a scan number of 1, with a sine-bell apodization window function applied in the time-domain signal. External calibration was performed with quadratic regression using a 100 μg/mL arginine solution. Data acquisition was controlled by the ftmsControl 2.1 and HyStar 4.1 software (Bruker Daltonics), and data processing for selection of molecular features was performed using the DataAnalysis 4.4 program (Bruker Daltonics). Bucketing of the molecular features was processed using ProfileAnalysis 2.2 software (Bruker Daltonics).

Multivariate analyses

The multivariate data analyses such as principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) with Pareto scaling were performed using the SIMCA-P+ 12.0 software (Umetrics, Umeå, Sweden) to discriminate the different origins. Molecular features containing the retention time and mass-to-charge ratio were extracted from the UPLC/FT-ICR MS raw spectra of four propolis samples and one mixture sample to determine key compounds among different propolis samples. The datasets including the UPLC/FT-ICR MS-based molecular features were used for the multivariate analysis. After the PLS-DA analysis, the interesting variables were selected using the variable importance in projection (VIP) scores over 4 for further identification of key propolis components reflecting the origin. The molecular formulae and corresponding compounds of the selected variables were identified via METLIN (Smith et al. 2005), HMDB (Wishart et al. 2007), and other publicly available database searches.

Results and discussion

Quantitation of ten phenolic components in various EEP samples by UPLC analysis

To investigate the chemical composition of propolis originating from different countries, we prepared EEP using raw materials from four different countries (i.e., Argentina, Brazil, China, and Korea). Four EEP samples were analyzed using UPLC with a UV detector (280 nm), and typical chromatograms were obtained. In general, a diode array detector (DAD) has been used to measure a variety of flavonoids or phenolic compounds at a range of wavelengths (i.e., 200–500 nm) because those molecules have typical UV absorption maximum (Harnly et al. 2007; Pellati et al. 2011; Zhang et al. 2013). Most flavonoids or phenols showed an absorption maximum at around 240–290 or 300–350 nm. However, those phenolic compounds were mostly been detected at a UV wavelength of 280 nm (Seal 2016; Yang et al. 2016), so the UPLC-based detection of the phenolic components in EEP samples was done at a wavelength of 280 nm in this study. As shown in Fig. 2, the chromatographic profiles of EEP samples from Asian countries (China and Korea) were similar (Fig. 2d, e), whereas the UPLC chromatograms of South American propolis (Argentina and Brazil) were apparently distinct from each other (Fig. 2b, c). The UPLC profile of Argentinean propolis appeared more similar to that of Korean propolis.
Fig. 2
Fig. 2

UPLC chromatograms of EEP samples from South America (b Argentina, c Brazil) and Asia (d China, e Korea). a The chromatogram obtained from polyphenol standards. The numbers and the corresponding dotted lines indicate each phenolic standard. 1 = cinnamic acid; 2 = coumaric acid; 3 = caffeic acid; 4 = chrysin; 5 = pinocembrin; 6 = CAPE; 7 = kaempferol; 8 = artepillin C; 9 = quercetin; 10 = rutin. f Bar graph showing the concentrations of ten phenolic compounds in various EEP samples of different origins

We selected ten phenolic compounds (i.e., coumaric acid, quercetin, cinnamic acid, kaempferol, chrysin, pinocembrin, CAPE, artepillin C, caffeic acid, and rutin), which are the most well-known constituents of propolis, and quantitated the compounds in the EEP samples using standards of these phenolic compounds. Each concentration of the key components in the EEP samples is shown in Fig. 2f. The major compound in Brazilian propolis was artepillin C, as previously determined, and the concentration of artepillin C in Brazilian propolis was sixfold higher than that in Chinese propolis; no artepillin C was observed in the Argentinean or Korean propolis. In addition to the comparison of the chromatographic profiles of phenolic compounds monitored at the wavelength of 280 nm, we also compared the EEP profiles using UPLC combined with high-resolution FT-ICR MS analysis.

In-depth analysis of EEP samples using high-resolution UPLC/FT-ICR MS

The investigation of key components contributing to the determination of the geographical origins of propolis is necessary for assessing the quality of propolis and for better use of propolis depending on the biological activities of the key components. Total UPLC/FT-ICR MS ion chromatograms were obtained for four EEP samples (Fig. 3a–d). To confirm the analysis of the EEP samples, the identification and intensity-based quantification were carried out from the raw data. The quantitative analysis results for the ten key phenolic species were compared with those obtained by UPLC analysis. The quantitative results via UPLC/FT-ICR MS agreed well with the UPLC data, supporting the reliability of the FT-ICR MS data. The comparative results are summarized in Table 1.
Fig. 3
Fig. 3

Total ion chromatograms of EEP samples from a Argentina, b Brazil, c China, and d Korea, as obtained by UPLC/FT-ICR MS. PLS-DA models obtained using UPLC and UPLC/FT-ICR MS data. Quantitation data of ten phenolic compounds using UPLC was used for model (e), whereas selected key molecular features (VIP > 4, and fold change ≥ 2 or ≤ − 2 or observed only in each sample) obtained by FT-ICR MS analysis were used to generate model (f)

Table 1

Comparisons of ten phenolic compounds identified from EEP samples using UPLC and UPLC/FT-ICR MS

No.

Compound

RT (min)

MW

UPLCa

UPLC/FT-ICR MSb

Brazil

China

Korea

K/B

K/C

Brazil

China

Korea

K/B

K/C

1

Cinnamic acid

9.8

148.05

N.D

1.7

0.2

N/A

0.1

N.D

N.D

N.D

N/A

N/A

2

Coumaric acid

4.8

164.05

25

0.8

1.5

0.1

1.9

O

O

O

Unique

Unique

3

Caffeic acid

3.5

180.04

7

N.D

4.5

0.6

N/A

O

O

O

Unique

Unique

4

Chrysin

14.5

254.06

1.2

16.7

14.1

11.8

0.8

O

O

O

Unique

0.8

5

Pinocembrin

14.9

256.07

10.4

8.3

6.4

0.6

0.8

O

O

O

1.1

1.1

6

CAPE

15.6

284.10

5.3

3.2

13.9

2.6

4.3

O

O

O

7.8

4.3

7

Kaempferol

11.0

286.05

2.3

1

0.4

0.2

0.4

O

O

O

-0.4

1.1

8

Artepillin C

17.7

300.17

104.5

16.8

N.D

N/A

N/A

O

O

O

1.8

2.2

9

Quercetin

9.5

302.04

1.9

0.6

0.3

0.2

0.5

O

O

O

0.3

2.0

10

Rutin

5.6

610.15

N.D

N.D

N.D

N/A

N/A

N.D

N.D

N.D

2.2

0.6

N.D not detected, N/A not applicable

aThe values were the concentration of each component (μmoL/mL of EEP extract exhibiting 1% of the total flavonoid quantity) estimated using UPLC

bThe values were the log2-transformed intensities of each component observed using FT-ICR MS

To elucidate the key compounds determining the geographical origins of propolis, the molecular features obtained via UPLC/FT-ICR MS analysis were investigated. Approximately 8000 molecular features, including the retention time and m/z values, were extracted from the LC/FT-ICR MS raw data for the four EEP samples. PLS-DA plots of the four propolis samples showed that the UPLC/FT-ICR data well separate the propolis samples by their geographic origin, while the Chinese and Korean propolis were not well resolved by the UPLC-based PLS-DA model, indicating that the selected quantitative information for the ten phenolic compounds in EEP samples was not sufficient to differentiate the samples’ origins (Fig. 3e, f). Given these observations, we speculated that the analysis of a few selected constituents in propolis might be insufficient for the quality control or quality assurance of the propolis products and the accurate determination of their source origins. Interestingly, Argentinean propolis differed from the Brazilian propolis, although the two countries are both located in Latin America, whereas China and Korea are in Asia; the Argentinean propolis was not included for further identification of key compounds.

Investigation of key components discriminating Korean propolis from Brazilian and Chinese propolis

As previously mentioned, the chemical composition of South American propolis differs substantially from that of East Asian propolis because of their botanical origins (i.e., poplar- and Baccharis-type plants). As shown in Fig. 3f, we observed good discrimination of Brazilian and Korean propolis; we therefore decided to determine the key propolis components discriminating Korean and Brazilian propolis. In addition, the molecular features obtained from Korean and Chinese propolis samples were investigated to figure out the key molecules that differentiate these two Asian propolis varieties. To determine the key components distinguishing the different propolis samples among 8164 molecular features obtained from UPLC/FT-ICR MS analysis, approximately 100 features with VIP values greater than 4.0 and fold changes above or below 2 or fold changes observed only in each sample were selected for further investigation.

Finally, we obtained 35 and 43 key components discriminating Korean and Brazilian propolis and Korean and Chinese propolis, respectively. All the significant compounds contributing to the geographical determination of Korean and Brazilian propolis are summarized in Table 2, while the key compounds discriminating Korean and Chinese propolis are listed in Table 3. Identification of the chemical formulae was possible from the accurate molecular masses and their experimental isotopic fine structures (IFSs) of all metabolites. Prediction of the compound was made by searching METLIN and HMDB databases. The extracted ion chromatograms, the corresponding mass spectra, and IFSs of the proposed compounds were summarized in the Additional file 1. Then, the proposed key compounds could be divided into five subgroups based on their chemical class (i.e., flavonoids, phenols, terpenoids, fatty acids, and others). The pie charts of the chemical classes of the key compounds discriminating Korean and Brazilian propolis showed that the flavonoids were the most abundant (61%), followed by phenols (22%), terpenoids (6%), others (6%), and fatty acids (5%) in Korean propolis; meanwhile, the most frequently assigned class was flavonoids (29%) and others (24%), followed by phenols (23%), terpenoids (18%), and fatty acids (6%) in Brazilian propolis (Fig. 4a). Key propolis classes for differentiating Korean and Chinese propolis were divided by flavonoids (52%), terpenoids (24%), phenols (16%), and others (8%) in Korean propolis, whereas Brazil-specific compounds were classified by fatty acids (56%), others (22%), flavonoids (11%), and terpenoids (11%) (Fig. 4b).
Table 2

Key compounds discriminating Korean and Brazilian propolis. The key compounds were selected from key molecular features with VIP > 4, and a fold change ≥ 2 or ≤ − 2 or observed only in each sample

No.

RT (min)

m/z

Formula

Error (ppm)

VIP

Expected compound

Feature

Class (function)

Fold change (log2)

Korea only

Brazil only

1

16.79

161.060

C10H8O2

0.118

5.69

6-Methylcoumarin

N/A

O

 

Benzopyrone (flavoring agent)

2

8.26

209.081

C11H12O4

0.053

7.84

Dimethyl caffeic acid

N/A

O

 

Phenols, caffeic acid derivatives

3

16.75

263.128

C15H18O4

0.236

4.13

Artemisin

N/A

O

 

Sesquiterpene lactone (drug for malaria)

4

15.48

315.087

C17H14O6

1.098

11.64

Rosinidin

N/A

O

 

Anthocyanidins

5

12.89

317.066

C16H12O7

0.091

4.79

Rhamnetin

N/A

O

 

Flavonoids (anti-inflammatory activity)

6

16.90

329.102

C18H16O6

0.352

4.62

Ophiopogonanone A

N/A

O

 

Homoisoflavonoids (antioxidant activity) (Wang et al. 2017)

7

10.83

338.139

C20H19NO4

0.053

4.02

Columbamine

N/A

O

 

Alkaloids (antidiabetic agent)

8

14.45

343.118

C19H18O6

0.691

5.27

Tetramethoxyflavone

N/A

O

 

Flavonoid (anticancer activity)

9

15.49

285.112

C17H16O4

0.039

5.03

CAPE

7.8

  

Phenols, caffeic acid derivatives

10

12.48

285.075

C16H12O5

0.025

7.18

Genkwanin

7.3

  

Flavonoids (anti-inflammatory activity)

11

18.43

295.227

C18H30O3

0.698

11.48

Kamlolenic acid

4.9

  

Fatty acids, Kamala oil's glyceride

12

14.43

255.065

C15H10O4

0.020

17.12

Chrysin

4.6

  

Flavonoids

13

17.98

357.133

C20H20O6

0.319

6.89

(2S)-5,7,3′,4′-tetrahydroxy-6-(1,1-dimethylallyl)flavanone

4.2

  

Flavonoids (anticancer activity)

14

10.66

269.081

C16H12O4

0.033

4.11

Strobochrysin

3.9

  

Flavonoids (anticancer activity)

15

17.69

269.081

C16H12O4

0.331

5.89

Tectochrysin

3.7

  

Flavonoids (antibacterial activity)

16

9.99

317.066

C16H12O7

0.003

4.83

Quercetin 3-methyl ether

3.7

  

Flavonoids (antioxidant activity)

17

10.90

273.076

C15H12O5

-0.029

4.70

Butein

2.8

  

Chalconoids (anti-inflammatory activity)

18

14.83

257.081

C15H12O4

0.144

10.44

Pinocembrin

1.1

  

Flavonoids (anticancer activity)

19

19.30

268.264

C17H33NO

0.276

4.52

(5-heptyl-6-methyloctahydroindolizin-8-yl)methanol

-1.7

  

Semiochemicals

20

20.27

310.310

C20H39NO

0.045

5.89

N-Hexadecanoylpyrrolidine

-2.0

  

Alkaloids

21

15.25

301.071

C16H12O6

0.086

8.27

Kaempferide

-3.4

  

Flavonoids (antibacterial activity)

22

15.75

331.081

C17H14O7

0.103

7.87

Quercetin 3,7′-dimethyl ether

-3.5

  

Flavonoids (vasorelaxant activity)

23

18.26

215.107

C14H14O2

0.558

4.60

Dihydropinosylvin

N/A

 

O

Phenols (antifungal activity)

24

16.24

231.102

C14H14O3

0.169

4.25

Aucuparin

N/A

 

O

Benzenoid (phytoalexin)

25

13.18

233.117

C14H16O3

0.133

7.79

Pterosin E

N/A

 

O

Indanones (anti-inflammatory activity)

26

12.39

249.112

C14H16O4

0.072

4.08

Prenyl caffeate

N/A

 

O

Caffeic acid derivatives (flavoring agent)

27

16.96

271.097

C16H14O4

0.181

4.68

Haginin B

N/A

 

O

Flavonoids

28

18.78

273.258

C20H32

0.285

9.26

Casbene

N/A

 

O

Diterpenoids (antifungal activity) (Sitton and West 1975)

29

17.59

301.180

C19H24O3

0.000

17.75

Artepillin C

N/A

 

O

Cinnamic acid derivatives (anti-inflammatory activity)

30

12.48

311.128

C19H18O4

0.077

5.04

Moracin N

N/A

 

O

Flavonoids

31

17.81

321.243

C20H32O3

0.386

4.27

Hydroxyeicosatetraenoic acid (HETE)

N/A

 

O

Unsaturated fatty acids

32

9.86

363.180

C20H26O6

0.080

4.51

Gibberellin A36

N/A

 

O

Diterpenoids (plant hormones) (Stowe and Yamaki 1959)

33

15.13

391.212

C22H30O6

0.427

6.68

Neoquassin

N/A

 

O

Quassinoids (insecticidal activity) (Dou et al. 2008)

34

7.21

517.134

C25H24O12

0.184

5.04

Apigenin 7-(2′′,3′′-diacetylglucoside)

N/A

 

O

Flavonoids (anti-cancer effect)

35

19.74

527.337

C32H46O6

0.759

4.27

(24E)-3alpha-acetoxy-15alpha-hydroxy-23oxo-7,9(11),24-lanostatrien-26-oic acid

N/A

 

O

Triterpenes

No. 1 to 18: key compounds with a fold change of ≥ 2 and no. 19 to 35: key compounds with a fold change of ≤ − 2

Numbers in italic represent the compounds observed only in each sample

Table 3

Key compounds discriminating Korean and Chinese propolis. The key compounds were selected from key molecular features with VIP > 4, and a fold change ≥ 2 or ≤ − 2 or observed only in each sample

No.

RT (min)

m/z

Formula

Error (ppm)

VIP

Expected compound

Feature

Class (function)

Fold change (log2)

Korea only

China only

1

16.60

283.097

C17H14O4

0.247

4.38

5,7-Dimethoxyisoflavone

N/A

O

 

Flavonoids (anabolic and anti-catabolic compound)

2

16.86

285.185

C19H24O2

0.228

5.22

Alpha-terpinyl cinnamate

N/A

O

 

Cinnamic acid derivatives (flavoring agent)

3

17.89

315.196

C20H26O3

0.216

4.71

Lanugone A

N/A

O

 

Terpenes

4

10.83

338.139

C20H19NO4

0.053

5.01

Ficine

N/A

O

 

Flavonoidal alkaloid

5

15.60

403.118

C24H18O6

0.275

4.72

Calomelanol I

N/A

O

 

Flavonoids (antioxidant activity)

6

13.10

421.128

C24H20O7

0.085

4.70

5-O-Methylhoslundin

N/A

O

 

Flavonoids (antioxidant activity)

7

18.67

453.337

C30H44O3

0.490

4.11

Tyromycic acid

N/A

O

 

Triterpenoids

8

18.62

475.378

C30H50O4

0.587

4.90

Soyasapogenol A

N/A

O

 

Triterpenoids (antibacterial activity) (Rupasinghe et al. 2003)

9

20.67

489.394

C31H52O4

0.366

9.99

1α-hydroxy-2β-(4-hydroxybutoxy) vitamin D3

N/A

O

 

Vitamin D3

10

19.12

491.373

C30H50O5

0.611

4.04

Meliantriol

N/A

O

 

Triterpenoids (insecticidal activity)

11

18.36

501.321

C30H44O6

0.578

7.88

Ganolucidic acid D

N/A

O

 

Terpenoids

12

18.77

503.337

C30H46O6

0.723

7.35

Esculentic acid

N/A

O

 

Triterpenoids

13

14.50

563.168

C28H31ClO10

0.783

7.75

Physalin H

N/A

O

 

Physalins

14

16.79

161.060

C10H8O2

0.000

7.05

6-Methylcoumarin

6.2

  

Benzopyrone (flavoring agent)

15

12.62

329.102

C18H16O6

− 0.079

5.80

Alnetin

5.0

  

Flavonoids (antifungal activity)

16

12.48

285.075

C16H12O5

0.088

6.81

Genkwanin

4.5

  

Flavonoids (anticancer activity)

17

14.22

249.112

C14H16O4

− 0.012

8.24

Pyriculol

4.4

  

Phenylpropanoids (phytotoxin)

18

14.71

285.076

C16H12O5

0.203

4.04

Acacetin

2.9

  

Flavonoids (anti-inflammatory activity)

19

14.45

343.118

C19H18O6

0.344

6.04

Tetramethoxyflavone

2.7

  

Flavonoids (anticancer activity)

20

15.48

315.087

C17H14O6

0.133

13.06

Rosinidin

2.4

  

Anthocyanidins

21

8.26

209.081

C11H12O4

− 0.033

8.44

Dimethylcaffeic acid

2.1

  

Phenols, caffeic acid derivatives

22

12.89

317.066

C16H12O7

− 0.035

5.18

Rhamnetin

2.0

  

Flavonoids (anti-inflammatory activity)

23

15.97

285.076

C16H12O5

0.158

7.14

8-C-ethylgalangin

1.6

  

Flavonoids (anti-inflammatory activity)

24

17.98

357.133

C20H20O6

0.381

6.51

Kievitone

1.4

  

Flavonoids (phytoalexin)

25

14.83

257.081

C15H12O4

0.086

12.54

Pinocembrin

1.1

  

Flavonoids (anticancer activity)

26

14.38

315.253

C18H34O4

0.184

4.78

Octadecanedioic acid

− 1.1

  

Long-chain fatty acids

27

16.94

643.419

C36H60O8

0.575

4.09

Fasciculic acid A

− 1.2

  

Triterpenoids (calmodulin antagonist activity)

28

15.03

293.042

C15H10O5

0.198

4.03

Apigenin

− 1.5

  

Flavonoids (anticancer activity)

29

18.45

299.258

C18H34O3

0.478

4.65

Ricinoleic acid

− 1.5

  

Unsaturated fatty acids (anti-inflammatory activity) (Vieira et al. 2000)

30

18.68

297.243

C18H32O3

0.273

5.80

Dimorphecolic acid

− 1.8

  

Fatty acids (antibacterial activity) (Mundt et al. 2003)

31

20.27

310.310

C20H39NO

0.055

8.75

Oleoyl ethyl amide

− 2.4

  

Ethanol amide lipids (analgesic activity)

32

19.51

323.258

C20H34O3

0.121

13.73

(±)5-6E,8Z,11Z-eicosatrienoic acid (HETrE)

− 2.9

  

Unsaturated fatty acids

33

18.86

339.253

C20H34O4

0.363

9.83

Aphidicolin

− 3.1

  

Tetracyclic diterpene antibiotics (Antiviral activity)

34

17.52

463.139

C26H22O8

0.555

5.17

Artomunoxanthentrione epoxide

− 3.2

  

a potential biomarker

35

19.68

325.274

C20H36O3

0.357

6.86

Alchornoic acid

− 3.7

  

Fatty acids

36

18.08

341.269

C20H36O4

0.325

7.18

Thromboxanoic acid skeleton

− 3.7

  

Fatty acids

37

19.86

399.290

C26H38O3

0.073

5.54

Ximaosteroid C

− 3.9

  

Steroids

38

19.68

606.546

C38H71NO4

0.659

6.36

Cer(d16:2(4E,6E)/22:1(13Z)(2OH))

− 4.3

  

Fatty acids

39

9.39

287.091

C16H14O5

0.066

6.66

Gummiferol

N/A

 

O

Polyacetylenic diepoxides (anticancer activity)

40

18.21

321.242

C20H32O3

0.159

4.01

15-Hydroxyeicosatetraenoic acid (HETE)

N/A

 

O

Eicosanoids

41

17.83

343.284

C20H38O4

0.303

6.51

Eicosanedioic acid

N/A

 

O

Eicosanoids

42

17.27

389.102

C23H16O6

0.239

4.12

Pamoate

N/A

 

O

Naphthoic acid derivative

43

16.93

421.128

C24H20O7

0.468

7.29

Glabratephrin

N/A

 

O

Flavonoids (antifeedant activity)

Fig. 4
Fig. 4

Pie charts showing compositional differences of key components discriminating a Korean and Brazilian propolis and b Korean and Chinese propolis

In particular, flavonoid species seemed a most significant contributor differentiating Korean propolis from Brazilian and Chinese propolis. In Brazilian propolis, di- and tri-terpenoids including Gibberrellins, a kind of plant hormones that affect plant growth and developments (Hedden and Sponsel 2015), were significantly observed. Those key components would strongly reflect their botanical differences. Between two Asian propolis origins (Korea versus China), flavonoids and terpenoids species were reliably identified as Korea-specific key molecules, but Chinese propolis appears to possess fatty acid class compounds. The unsaturated fatty acids including ricinoleic acid and eicosanedioic acid, distinctly found in the Chinese propolis could also be utilized as dietary sources as the unsaturated fatty acids present in propolis are considered to be a good source to the diet (Rebiai et al. 2017).

Conclusions

Here, a high-resolution 15 T FT-ICR MS equipped with UPLC system was introduced to investigate the key phenolic compounds responsible for determining the geographical origin of propolis (i.e., Korea versus Brazil and Korea versus China). We then proposed 16 flavonoids, 8 phenolic compounds, 4 terpenoids, 2 fatty acids, and 5 others in Korean and Brazilian propolis, while 15 flavonoids, 10 fatty acids, 8 terpenoids, 4 phenols, and 6 others were proposed in Korean and Chinese propolis. These key compounds can be used as chemical markers to classify and identify the geographical origins of propolis. In the pharmaceutical and food industries, those key propolis components could play a significant role in distinguishing high-quality propolis from inferior or fake propolis. Moreover, the information of the key propolis constituents can be utilized to verify the effects of propolis in the prevention and treatment of various symptoms and diseases. Further characterization and biological evidence of the key compounds could focus on the evaluation of the compounds for quality assessment of propolis products and for standardization of propolis.

The chemical composition of propolis is strongly linked to vegetation present in the collection area as well as collecting periods and climates (Bankova et al. 2000). However, changes in vegetation on the Chinese continent are as great as changes from southern Argentina to northern Brazil. Yang and coworkers showed that the compositions and concentrations of aroma-active components collected from 23 regions of Chinese continent were significantly different (Yang et al. 2010). Therefore, a more detailed investigation of plant species close to the production area of propolis even in the same country is needed to determine the source origin more accurately. Although the results were obtained with only four different propolis samples from each origin and the key propolis compounds were not confirmed by tandem MS analysis, this UPLC/FT-ICR MS-based high-resolution platform showed the potentials for the comprehensive analysis of highly complicated bioactive compounds.

Furthermore, it could also be used to investigate novel propolis compounds with biological activities and are helpful for the pharmaceutical and food industries, which require an understanding of the chemical composition, botanical origin, and biological properties of propolis.

Abbreviations

ACN: 

Acetonitrile

APCI: 

Atmospheric pressure chemical ionization

CAPE: 

Caffeic acid phenethyl ester

EEP: 

Ethanol-extracted propolis

ESI: 

Electrospray ionization

FT-ICR: 

Fourier transform ion cyclotron resonance

IFS: 

Isotopic fine structures

PCA: 

Principal component analysis

PLS-DA: 

Partial least squares-discriminant analysis

RP-UPLC: 

Reverse-phase ultra-performance liquid chromatography

VIP: 

Variable importance in projection

Declarations

Acknowledgements

This work was supported by KBSI grant (G38110) and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2016R1C1B2006863).

Availability of data and materials

Research data have been provided in the manuscript and supporting information file.

Authors’ contributions

CHK and KSJ are involved in research design and execution of the experiments. MYK, SWL, and KSJ are involved in data interpretation and supervision of the studies, and all authors contributed to writing of the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Authors’ Affiliations

(1)
Biomedical Omics Center, Korea Basic Science Institute, Cheongju, 28119, Republic of Korea
(2)
Propolis Research Institute, Seoul Propolis Co, Daejeon, 34025, Republic of Korea
(3)
Division of Bio-Analytical Science, University of Science and Technology, Daejeon, 34113, Republic of Korea

References

  1. Bankova VS, Solange LC, Marcucci MC. Propolis: recent advances in chemistry and plant origin. Apidologie. 2000;31:3–15.View ArticleGoogle Scholar
  2. Banskota AH, Tezuka Y, Prasain JK, Matsushige K, Saiki I, Kadota S. Chemical constituents of Brazilian propolis and their cytotoxic activities. J Nat Prod. 1998;61:896–900.View ArticleGoogle Scholar
  3. Burdock GA. Review of the biological properties and toxicity of bee propolis (propolis). Food Chem Toxicol. 1998;36:347–63.View ArticleGoogle Scholar
  4. Chang CC, Yang MH, Wen HM, Chern JC. Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J Food Drug Anal. 2002;10:178–82.Google Scholar
  5. Choi M, Choi AY, Ahn SY, Choi KY, Jang KS. Characterization of molecular composition of bacterial melanin isolated from Streptomyces glaucescens using ultra-high-resolution FT-ICR mass spectrometry. Mass Spectrom Lett. 2018;9:81–5.Google Scholar
  6. da Costa MF, Galaverna RS, Pudenzi MA, Ruiz ALTG, de Carvalho JE, Eberlin MN, dos Santos C. Profiles of phenolic compounds by FT-ICR MS and antioxidative and antiproliferative activities of Stryphnodendron obovatum Benth leaf extracts. Anal Methods. 2016;8:6056–63.View ArticleGoogle Scholar
  7. Dou J, McChesney JD, Sindelar RD, Goins DK, Khan IA, Walker LA. A new quassinoid from crude quassin-extract of Quassia amara. Int J Pharmacogn. 2008;34:349–54.View ArticleGoogle Scholar
  8. Gardana C, Scaglianti M, Pietta P, Simonetti P. Analysis of the polyphenolic fraction of propolis from different sources by liquid chromatography-tandem mass spectrometry. J Pharm Biomed Anal. 2007;45:390–9.View ArticleGoogle Scholar
  9. Gardana C, Simonetti P. Evaluation of allergens in propolis by ultra-performance liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2011;25:1675–82.View ArticleGoogle Scholar
  10. Harnly JM, Bhagwat S, Lin LZ. Profiling methods for the determination of phenolic compounds in foods and dietary supplements. Anal Bioanal Chem. 2007;389:47–61.View ArticleGoogle Scholar
  11. Hedden P, Sponsel V. A century of Gibberellin research. J Plant Growth Regul. 2015;34:740–60.View ArticleGoogle Scholar
  12. Huang S, Zhang CP, Wang K, Li GQ, Hu FL. Recent advances in the chemical composition of propolis. Molecules. 2014;19:19610–32.View ArticleGoogle Scholar
  13. Mundt S, Kreitlow S, Jansen R. Fatty acids with antibacterial activity from the cyanobacterium Oscillatoria redekei HUB 051. J Appl Phycol. 2003;15:263–7.View ArticleGoogle Scholar
  14. Nakabayashi R, Sawada Y, Yamada Y, Suzuki M, Hirai MY, Sakurai T, Saito K. Combination of liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry with 13C-labeling for chemical assignment of sulfur-containing metabolites in onion bulbs. Anal Chem. 2013;85:1310–5.View ArticleGoogle Scholar
  15. Park YK, Paredes-Guzman JF, Aguiar CL, Alencar SM, Fujiwara FY. Chemical constituents in Baccharis dracunculifolia as the main botanical origin of southeastern Brazilian propolis. J Agric Food Chem. 2004;52:1100–3.View ArticleGoogle Scholar
  16. Pellati F, Orlandini G, Pinetti D, Benvenuti S. HPLC-DAD and HPLC-ESI-MS/MS methods for metabolite profiling of propolis extracts. J Pharm Biomed Anal. 2011;55:934–48.View ArticleGoogle Scholar
  17. Pietta PG, Gardana C, Pietta AM. Analytical methods for quality control of propolis. Fitoterapia. 2002;73(Suppl 1):S7–20.View ArticleGoogle Scholar
  18. Rebiai A, Belfar ML, Mesbahi MA, Nani S, Tliba A, Ghamem Amara D, Chouikh A. Fatty acid composition of Algerian propolis. J Fundam. Appl Sci. 2017;9:1656–71.Google Scholar
  19. Ristivojevic P, Trifkovic J, Andric F, Milojkovic-Opsenica D. Poplar-type propolis: chemical composition, botanical origin and biological activity. Nat Prod Commun. 2015;10:1869–76.PubMedGoogle Scholar
  20. Rupasinghe HP, Jackson CJ, Poysa V, Di Berardo C, Bewley JD, Jenkinson J. Soyasapogenol A and B distribution in soybean (Glycine max L. Merr.) in relation to seed physiology, genetic variability, and growing location. J Agric Food Chem. 2003;51:5888–94.View ArticleGoogle Scholar
  21. Sawaya AC, Abdelnur PV, Eberlin MN, Kumazawa S, Ahn MR, Bang KS, Nagaraja N, Bankova VS, Afrouzan H. Fingerprinting of propolis by easy ambient sonic-spray ionization mass spectrometry. Talanta. 2010;81:100–8.View ArticleGoogle Scholar
  22. Seal T. Quantitative HPLC analysis of phenolic acids, flavonoids and ascorbic acid in four different solvent extracts of two wild edible leaves, Sonchus arvensis and Oenanthe linearis of North-Eastern region in India. J Appl Pharm Sci. 2016;6:157–66.View ArticleGoogle Scholar
  23. Shi SD, Hendrickson CL, Marshall AG. Counting individual sulfur atoms in a protein by ultrahigh-resolution Fourier transform ion cyclotron resonance mass spectrometry: experimental resolution of isotopic fine structure in proteins. Proc Natl Acad Sci U S A. 1998;95:11532–7.View ArticleGoogle Scholar
  24. Sitton D, West CA. Casbene: an anti-fungal diterpene produced in cell-free extracts of Ricinus communis seedlings. Phytochemistry. 1975;14:1921–5.View ArticleGoogle Scholar
  25. Smith CA, O'Maille G, Want EJ, Qin C, Trauger SA, Brandon TR, Custodio DE, Abagyan R, Siuzdak G. METLIN: a metabolite mass spectral database. Ther Drug Monit. 2005;27:747–51.View ArticleGoogle Scholar
  26. Stowe BB, Yamaki T. Gibberellins: stimulants of plant growth. Science. 1959;129:807–16.View ArticleGoogle Scholar
  27. Vieira C, Evangelista S, Cirillo R, Lippi A, Maggi CA, Manzini S. Effect of ricinoleic acid in acute and subchronic experimental models of inflammation. Mediators Inflamm. 2000;9:223–8.View ArticleGoogle Scholar
  28. Wang Y, Liu F, Liang Z, Peng L, Wang B, Yu J, Su Y, Ma C. Homoisoflavonoids and the antioxidant activity of Ophiopogon japonicus root. Iran J Pharm Res. 2017;16:357–65.PubMedPubMed CentralGoogle Scholar
  29. Wishart DS, Tzur D, Knox C, Eisner R, Guo AC, Young N, Cheng D, Jewell K, Arndt D, Sawhney S, Fung C, Nikolai L, Lewis M, Coutouly MA, Forsythe I, Tang P, Shrivastava S, Jeroncic K, Stothard P, Amegbey G, Block D, Hau DD, Wagner J, Miniaci J, Clements M, Gebremedhin M, Guo N, Zhang Y, Duggan GE, Macinnis GD, Weljie AM, Dowlatabadi R, Bamforth F, Clive D, Greiner R, Li L, Marrie T, Sykes BD, Vogel HJ, Querengesser L. HMDB: the human metabolome database. Nucleic Acids Res. 2007;35:D521–6.View ArticleGoogle Scholar
  30. Yang C, Luo L, Zhang H, Yang X, Lv Y, Song H. Common aroma-active components of propolis from 23 regions of China. J Sci Food Agric. 2010;90:1268–82.View ArticleGoogle Scholar
  31. Yang Y, Sun X, Liu J, Kang L, Chen S, Ma B, Guo B. Quantitative and qualitative analysis of flavonoids and phenolic acids in snow chrysanthemum (Coreopsis tinctoria Nutt.) by HPLC-DAD and UPLC-ESI-QTOF-MS. Molecules. 2016;21:1307.Google Scholar
  32. Zhang A, Wan L, Wu C, Fang Y, Han G, Li H, Zhang Z, Wang H. Simultaneous determination of 14 phenolic compounds in grape canes by HPLC-DAD-UV using wavelength switching detection. Molecules. 2013;18:14241–57.View ArticleGoogle Scholar
  33. Zhou J, Li Y, Zhao J, Xue X, Wu L, Chen F. Geographical traceability of propolis by high-performance liquid-chromatography fingerprints. Food Chem. 2008;108:749–59.View ArticleGoogle Scholar

Copyright

© The Author(s). 2019

Advertisement