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Article

Evaluation of the Antioxidant Activities and Phenolic Profile of Shennongjia Apis cerana Honey through a Comparison with Apis mellifera Honey in China

1
College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China
2
AB Sciex Co., Ltd., Beijing 100102, China
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Molecules 2023, 28(7), 3270; https://doi.org/10.3390/molecules28073270
Submission received: 13 March 2023 / Revised: 31 March 2023 / Accepted: 31 March 2023 / Published: 6 April 2023

Abstract

:
This study evaluates the phenolic profile as well as the antioxidant properties of Shennongjia Apis cerana honey through a comparison with Apis mellifera honey in China. The total phenolic content (TPC) ranges from 263 ± 2 to 681 ± 36 mg gallic acid/kg. The total flavonoids content (TFC) ranges from 35.9 ± 0.4 to 102.2 ± 0.8 mg epicatechin/kg. The correlations between TPC or TFC and the antioxidant results (FRAP, DPPH, and ABTS) were found to be statistically significant (p < 0.01). Furthermore, the phenolic compounds are quantified and qualified by high performance liquid chromatography-high resolution mass spectrometry (HPLC-HRMS), and a total of 83 phenolic compounds were tentatively identified in this study. A metabolomics analysis based on the 83 polyphenols was carried out and subjected to principal component analysis and orthogonal partial least squares-discriminant analysis. The results showed that it was possible to distinguish Apis cerana honey from Apis mellifera honey based on the phenolic profile.

1. Introduction

Honey can serve as a source of natural antioxidants. The antioxidant activity of honey is primarily provided by its polyphenols [1]. Thus, a considerable variation of antioxidant activity and polyphenols profile is found among different honey varieties around the world [2,3,4,5]. This variation is mainly due to different floral and geographical origins as well as the type of bees [6,7]. Therefore, the analysis of phenolic profile has been regarded as a very promising technique for studying the floral, geographical and honeybee origins of honeys.
In general, Apis cerana (A. cerana) honey is produced by Apis cerana grazing on various botanical sources. Traditionally, A. cerana honey is more nutritious than other honey varieties because of its long nectar cycle and the wide variety of nectar source [8]. There are recent research reports that have found various benefits for A. cerana honey, such as its’ anti-inflammatory, anti-oxidant, and beneficial effects with regard to acute alcohol-induced liver damage [9,10,11]. These therapeutic activities have been attributed to the phenolic acid and flavonoids content of A. cerana honey [9,11]. Nonetheless, there is still a lack of understanding about the phenolic profile and antioxidant capacity of A. cerana honey. Until now, most of the studies have focused mainly on the phenolic profile and antioxidant activity in mono-floral honeys from Apis mellifera (A. mellifera) in China [12,13,14,15,16,17]. Therefore, it is necessary to evaluate the phenolic compounds in A. cerana honey here.
In ancient China, “Shen Nong’s Herbal Classic” have recorded the use of A. cerana honey from Shennongjia district as the first use of medicine. The Shennongjia forestry district is the only well-preserved subtropical forest ecosystem in the middle latitudes of the world, located in Hubei province in China. Due to its superior climatic conditions and unique geographical environment, the pollen of abundant resources of bee plants and wild medicinal plants is a good source of honey, and the honey is the “shennong poly-floral honey” with local characteristics. As far as we know, there are no reports about the phenolic profile of Shennongjia A. cerana honey. Thus, the antioxidant activity and polyphenol profile of this honey are analyzed in this study. Moreover, considering that Shennongjia A. cerana honey is poly-floral honey; hence, mono- and poly-floral honey from A. mellifera are selected to systematically evaluate the polyphenol profile between the two honey groups.
Furthermore, the recovery of phenolic compounds from honey varies differently depending on the pre-concentration methods [15,18,19]. Thus, to reduce metabolite information losses due to the different extraction methods, phenolic compounds in honeys were isolated using liquid-liquid extraction and solid-phase extraction (SPE) methods, respectively. Afterwards, based on the profile of phenolic compounds obtained by various extraction methods, a metabolomics analysis was carried out and the honey was subjected to a principal component analysis and an orthogonal partial least squares-discriminant analysis. The secondary aim of the present study was to differentiate A. cerana honey and A. mellifera using multivariate techniques.

2. Results and Discussion

2.1. Total Phenolic Content (TPC), Flavonoid Content (TFC), and Antioxidant Activity

The TPC, TFC, and antioxidant activity levels of A. cerana (A.c_1 to A.c_26, n = 26) and A. mellifera (A.m_p1 to p8 from Wuhan, n = 8; A.m_F from Fangxian) honeys from China and two Manuka honeys from New Zealand were evaluated, and the results are presented in Table 1.
The values for TPC in A. mellifera and A. cerana honeys in China ranged from 104.33 ± 4.21 to 379.20 ± 25.86 mg GAE/kg and from 263.02 ± 2.23 to 680.90 ± 35.80 mg GAE/kg, respectively. The values for TFC in A. mellifera and A. cerana honeys from China ranged from 14.74 ± 0.71 to 42.76 ± 0.29 mg EC/kg, and from 35.87 ± 0.44 to 102.24 ± 0.75 mg EC/kg, respectively. Here, A.c_8 and A.c_6, two A. cerana honeys from the Shennongjia region, had the highest TPC and TFC values, respectively, while the lowest TPC and TFC were measured in A.m_p1 and A.m_p7 honey, respectively. The range of values for the TPC and TFC here reported were in agreement with those previously found in Chinese honeys from A. cerana and A. mellifera [9,20]. In addition, previous reports showed similar TPC and TFC amounts for mono-floral and poly-floral honeys from other geographical origins [2,6,21,22].
Table 1 also showed the FRAP, DPPH and ABTS values for different honey samples in China. Two A. cerana honeys, including A.c_1 and A.c_8, had the highest FRAP (A.c_1), DPPH (A.c_1), and ABTS (A.c_8) values. Interestingly, A.m_p1 and A.m_p7 honeys had the lowest levels of TPC and TFC, and, correspondingly, the lowest values of FRAP, DPPH, and ABTS. Moreover, the correlation analysis results showed that there was a correlation between TPC or TFC and the levels of FRAP, DPPH, and ABTS (p < 0.01), suggesting that phenolic compounds are some of the main species responsible for the antioxidant capacity of honey [2]. The correlation between TPC or TFC and the levels of antioxidant activity here reported was in agreement with the results previously reported by other authors [23,24].
Furthermore, as a control, two Manuka honeys from New Zealand had higher TPC, TFC, and antioxidant activity levels than most of the A. cerana and A. mellifera honeys in China in this study. This means that in addition to the influence of bee species, plant and geographical sources can also affect the content of polyphenols [6,7].

2.2. Quantification of Thirteen Polyphenols in Honeys Using Different Extraction Methods

Several common phenolic compounds and abscisic acid in honey that are reported in the literature were isolated using three different extraction methods and quantified in the present study. The LOD (Limit of Detection), LOQ (Limit of Quantitation), linear range, and MS characteristics of these compounds are listed in Supplementary Table S1.
Table 2 shows the average amount of each compound isolated using different methods in the A. cerana and A. mellifera honeys. As seen, the average content of thirteen polyphenols in samples varied considerably depending on the extraction methods. EAC (ethyl acetate, liquid-liquid extraction) generated higher levels of kampferol (p < 0.0001), quercetin (p < 0.0001), vanillic acid (p < 0.0001), and trans-ferulic acid (p < 0.01), while, SPE (XA and PLS, solid-phase extraction) generated higher levels of rutin (p < 0.0001). Between the two SPE cartridges, the Strata XA cartridge showed lower recoveries of vanillic acid (p < 0.0001) and 4-hydroxybenzoic acid (p < 0.0001) compared to ProElut PLS SPE cartridges. The results suggested that different extraction methods have different extraction efficiency for phenolic acids and flavonoids.
In general, most of the flavonoids showed a lower average content than phenolic acids. Among thirteen compounds, kaempferol and 4-hydroxybenzoic acid were the main flavonoid and phenolic acid found in the A. mellifera and A. cerana honeys in China. It was reported that kaempferol and 4-hydroxybenzoic acid were prevalent in A. mellifera honey of different geographic origins in previous studies [14,25,26,27]. In addition, chrysin was present at the lowest levels in A. cerana honey, while rutin had the lowest content in A. mellifera honey. Furthermore, some flavonoids showed significant distinctions between A. cerana and A. mellifera honeys regardless of extraction methods. For example, the contents of quercetin (p < 0.05), rutin (p < 0.01) and p-coumaic acid (p < 0.05) were higher in A. cerana honeys than those in A. mellifera honeys. Three compounds including pinocembrin (p < 0.01), chrysin (p < 0.01), and galangin (p < 0.001) were considered as propolis-derived flavonoids [22,25], had lower contents in A. cerana honeys.

2.3. Identification of Individual Polyphenols

One hundred and eleven honey extracts were subjected to the identification of the flavonoids, phenolic acids and abscisic acid based on the optimization conditions of HPLC-QTOF-MS/MS. A total of 83 compounds were tentatively identified, and 13 of them were qualified by comparing the retention times (RT) and the MS spectra with available standards. In the absence of standards, the identification of a further 70 compounds was based on the search for the [M–H] deprotonated molecule and its fragmentation referred to in the literature. Table 3 summarizes the data obtained for each of the identified compounds with their retention times, error in ppm (between the mass found and the accurate mass), as well as the MS/MS fragment ions.
Hydroxycinnamic acids such as caffeic acid and their derivatives were the main phenolic acids found in the study. Caffeic acid was present in all of the honey samples; in addition, ten caffeic acid derivatives were detected: caffeoylquinic acid isomers (compounds 6, 14 and 17), dicaffeoylquinic acid isomers (compounds 25, 27 and 30), and four ester derivatives of caffeic acid (compound 31, 32, 33 and 34). All of the caffeic acid derivatives showed negative product ions at 179 m/z due to the loss of the deprotonated molecule of caffeic acid. Caffeoylquinic acids and dicaffeoylquinic acids were reported in the European honeydew honey [33] and A. mellifera honey from different botanical and geographical origins [2,25,27,28,37]. Caffeic acid ester derivatives were detected in Chilean propolis [30] and Spanish A. mellifera honey [2]. As shown in Table 3, caffeic acid ester derivatives were commonly present in A. mellifera honey in this study, whereas they were relatively rare in A. cerana honey.
Furthermore, both isomers of abscisic acid previously described in other varieties of honey [2] were detected in all of the honey samples in the study. In addition, 4-ethoxy-3-methoxycinnamic acid (compound 24) was identified only in Manuka honeys in the study. By examining the empirical formula of this compound, it was concluded that it may be an ethylated derivative of ferulic acid. It produced MS2 fragments at 193, 179, 151, and 135 m/z, most probably corresponding to [M−H−C2H5], [M−H−C2H5−CH3], [M−H-C3H3O2] and [M−H-C3H3O2−CH3] fragments, respectively.
Concerning flavonoids, four subclasses of compounds were identified: flavonols, flavanonols, flavanones, and flavones, in addition to some flavanonol ester derivatives and flavonols glycosides. The flavanonol ester derivatives mainly came from pinobanksin (compounds 58, 59, 60 and 61), which showed a negative product ion at 271 m/z due to the loss of the deprotonated molecule of pinobanksin. Pinobanksin and its ester derivatives are characteristic flavonoids of propolis, and were found in Spanish A. mellifera honeys [2], sulla honey from the Sicilian native breed of black honeybee [36], as well as the Chilean propolis [30]. In this study, these compounds were present in almost all A. mellifera honeys, but very few were found in A. cerana honey. For example, pinobanksin-3-O-hexanoate (compound 61) was present in all A. mellifera honeys except for A.m_p7 honey, while it was undetectable in all A. cerana honey samples (Table 3).
The flavonols’ glycosides that were mainly from quercetin, kaempferol, methoxykaempferol, and isorhamnetin were previously described in different types of honey [2,33,34]. Numerous derivatives of flavonols’ glycosides were identified in A. mellifera and A. cerana honey extracts in this study: rhamnosides (loss of 146 Da), hexosides (loss of 162 Da), neohesperidoside, rhamnosylhexoside (loss of 308 Da), and dihexosides (loss of 324 Da). For example, in MS2 spectra of compound 47 at 46.92 min and 431 m/z, base peak fragments at 285 m/z (loss of 146 Da) and additional two fragment ions resulting from the loss of 257 and 151 Da could be observed, and it was then concluded that it could be kaempferol–rhamnosides.
In conclusion, propolis-derived caffeic acid and pinobanksin ester derivatives were widely present in A. mellifera honeys in the study, but rarely in A. cerana honeys.

2.4. Metabolomics Analysis

A PCA was conducted to evaluate the effect of the honey species / extraction method on the 83 phenolic compounds from a descriptive point of view (Figure 1). As shown in a PCA scores plot (Figure 1A), all of the A. cerana honey extracts regardless of extraction method (n = 77) were designed in PC1 negative, and most of the A. mellifera honey extracts (n = 29) were designed in PC1 positive. These results suggested that different honey species, rather than extraction methods, could be distinguished based on the levels or the presence of phenolic compounds.
For A. cerana honeys distributed in PC1 negative, most of the honey extracts (n = 72) clustered tightly, except for five honey extracts which were far away from other honey extracts due to their high level of methoxy kaempferol (Figure 1B). For A. mellifera honeys distributed in PC1 positive, the poly-floral A. mellifera honey extracts (n = 24) clustered tightly and were closest to the A. cerana honey group, followed by mono-floral A. mellifera (A.m_F) honey, and then by Manuka honey. The result indicated that botanical and geographical origins have an effect on the phenolic profile in A. mellifera honeys. Manuka honey was differentiated from other honeys for the high contents of 4-methoxyphenyllactic acid and p-hydroxy-hydrocinnamic acid. Fangxian A. mellifera honey was monofloral honey and characterized by a high content of pinobanksin (Figure 2B). Wuhan A. mellifera honey was polyfloral honey, and thus it may be closer to Shennongjia A. cerana honey in its phenolic acid profile because of the diversity of plant sources.
Furthermore, an orthogonal partial least squares-discriminant analysis (OPLS-DA) was conducted to analyze the differences between A. mellifera and A. cerana honey. Figure 2A showed that A. cerana honey samples were located on the right side of the ellipse and were well separated from A. mellifera honey samples. This result indicated that there were significant differences in the two honey groups. In addition, seven-fold cross-validation and 200 permutations were conducted to further verify the predictability of the OPLS-DA model. As shown in Figure 2B, the intercept of Q2 (−0.223) was negative on the vertical axis, and all blue Q2-values to the left were lower than the original points to the right, indicating that the established model was not overfitted for the experiment.
The variables responsible for discriminating A. cerana from A. mellifera honey were then identified using the OPLS-DA VIP (Figure 2C, VIP > 1) and S-plot (Figure 2D). The red variables (Figure 2C, VIP > 1) were tested using a Student’s t-test and the corresponding VIP and p values (p < 0.01) are listed in Supplementary Table S3. An S-plot (Figure 2D) was used to visualize the covariance and correlation between A. mellifera and A. cerana honey. Here, eight variables (compound 1–8 in Supplementary Table S3, p < 0.01) were far from the origin and were located at the far left of the X-axis. This indicated that the contents of these compounds in A. mellifera honey were higher than those in A. cerana honey. Among these compounds, five propolis-derived flavonoids (pinobanksin, pinobanksin-5-methyl ether, galangin, chrysin and pinocembrin), were commonly present in all A. mellifera honeys in the present study (Table 3). These flavonoids have previously been identified in propolis, European honeydew honey, and mono- and polyfloral honey from A. mellifera [2,27,30,33,38].
As shown in Figure 2D, five variables (compound 9–13 in Supplementary Table S3, p < 0.01) were far from the origin and were located at the far right of the X-axis. The result indicated that the contents of these compounds in A. cerana honey were higher than those in A. mellifera honey. The five compounds have been previously reported in tilia, salvia officinalis L., and chestnut source honey samples [2,25,39]. In this study, they were commonly present in A. cerana and A. mellifera honey. The high content levels of these compounds in Shennongjia A. cerana honey may be due to the abundant sources of wild medicinal plants and nectar plants in this region.
Of course, whether these compounds can be used as appropriate markers to distinguish A. cerana honey from A. mellifera honey requires further study and confirmation by expanding the sample size and selecting A. cerana and A. mellifera honey from different geographical and plant sources in the future.

3. Materials and Methods

3.1. Chemicals

All solvents and phenolic compounds used for HPLC analysis were of HPLC grade, and the rest of the chemicals were of analytical grade. Phenolic compounds including caffeic acid (Cafa), trans-cinnamic acid (Tcina), chrysin (Ch), trans-ferulic acid (Fera), galangin (Gal), p-coumaric acid (Pcoa), vanillic acid (Vana), 2-cis-4-trans-abscisic acid (CTabsa), kaempferol (Kaem), 4-hydroxybenzoic acid (4Hba), and quercetin (Quer) were obtained from Sigma–Aldrich. Rutin (Ru), pinocembrin (Pino), gallic acid and epicatechin were from the Bei Na Chuang Lian Institute of Biotechnology. Folin Ciocalteu’s phenol reagent, 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,4,6-tri(2-pyridyl)-1,3,5-triazine (TPTZ), and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) were purchased from the Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Solid-phase extraction cartridges Strata-X-A (60 mg/3 mL) and ProElut PLS (60 mg/3 mL) were acquired from Phenomenex Inc. (Torrance, CA, USA) and Dikma Technologies Inc. (Beijing, China), respectively.

3.2. Honey Samples

The poly-floral honeys were harvested from A. cerana (Shennongjia; n = 26, 110°40′ E, 31°44′ N) between September and October 2017. The poly-floral honeys from A. mellifera (Wuhan; n = 8; 114°21′ E, 30°28′ N) were purchased from the Kangsinong Bee technology Co. Ltd. (Wuhan, China) in 2017. July mountain flower honey from A. mellifera (A.m_F) in Fangxian (110°44′ E, 32°3′ N) was collected in Hubei province. Two Manuka honeys (MGO100+ and MGO250+) from New Zealand, as controls, were purchased from Amazon.com, Inc. (Seattle, WA, USA) in 2017. The floral origins of A.cerana and A. mellifera honey samples were determined by the melissopalynological analysis, as previously reported [40]. The results are listed in Supplementary Table S2.

3.3. Extraction of Phenolic Compounds

The extraction of phenolic compounds was undertaken by solid-phase extraction. The SPE method was carried out according to the previous study [15] with minor modifications. A total of 10.0 g of honey samples were mixed with 50 mL of ultrapure water, and then the solution was adjusted to pH = 2 with HCl for the PLS cartridges or adjusted to pH = 7 with 5% ammonium (v/v) for the Strata X-A cartridges. After removing the impurity particles by centrifugation (8000 g, 10 min), the supernatants were loaded onto the previously conditioned cartridges (according to the manufacturer’s instructions). After loading, these cartridges were washed with 4 mL of acidified ultrapure water (pH = 2) for the PLS SPE cartridges or washed with 4 mL of ultrapure water (pH = 7) for the Strata X-A SPE cartridges. The phenolic compounds retained on the cartridges were then eluted with 5 mL of formic acid: methanol (1:9, v/v). The extract was evaporated at 40 °C under a stream of nitrogen, and then reconstituted in 1 mL of methanol with 0.1% formic acid. The obtained extracts were filtered and stored at −20 °C until further analysis by high performance liquid chromatography–quadrupole time-of-flight mass spectrometry (HPLC–QTOF-MS).
The extraction of phenolic compounds was undertaken with liquid-liquid extraction. Briefly, 10.0 g of honey samples were mixed with 50 mL of ultrapure water, and the solution was then adjusted to pH = 2 with HCl. The honey solution was extracted three times with 20 mL of ethyl acetate. The extracts were evaporated to dryness on a rotary evaporator at 30–40 °C, and then dissolved in 1 mL of methanol with 0.1% formic acid. The obtained extracts were filtered and stored at −20 °C until further analysis by HPLC–QTOF-MS.

3.4. HPLC–QTOF-MS Conditions

HPLC analyses were performed using a Shimadzu LC-20A system (Shimadzu Corporation, Kyoto, Japan) coupled with a quadrupole time-of-flight mass spectrometer (AB Sciex Triple QTOF5600+, AB Sciex, Redwood, CA, USA). The chromatographic separation was carried out using an Eclipse XDB-C18 column (100 mm × 2.1 mm, 3.5 um) (Agilent, Wilmington, DE, USA). The mobile phase consisted of 0.1% formic acid in water (phase A) and 0.1% formic acid in methanol (phase B). The flow rate was 0.3 mL/min and the injection volume was 10 µL, while the temperature of the column oven was set at 35 °C. The gradient separation was performed as follows: 0–1 min, 0% (B); 1–6 min, 0–6% (B); 6–13 min, 6–10% (B); 13–25 min, 10–20% (B); 25–35 min, 20–40% (B); 35–40 min, 40% (B); 40–55 min, 40–65% (B); 55–60 min, 65% (B); 60–75 min, 65–98% (B); 75–80 min, 98% (B). TOF–MS and the data of ten TOF–MS/MS were collected in negative ion mode using the information-dependent acquisition (IDA) function. The parameters were as follows: dynamic background subtraction (DBS); charge monitoring to exclude multiply charged ions and isotopes; Ion Source Gas1: 55 psi; Ion Source Gas2: 60 psi; Curtain Gas: 30 psi; Temperature: 600 °C; IonSpray Voltage Floating: −4500 V; Declustering Potential: 100 V; Collision Energy: 25 V; Collision Energy Spread: 15 V. In order to ensure the stability of outcomes, the calibration reagent (sodium formate) was detected in every two sample intervals, and methanol was used as a blank control to avoid the misjudgment of characteristic markers. In parallel, quality control (QC) samples were prepared by mixing equal volumes (9 µL) from each sample. An aliquot of this pooled sample was analyzed every fourteen samples in order to provide the measure of the system’s stability and performance. The system operation, data acquisition, and analysis were controlled and processed using Analyst 1.7.1,PeakView 2.2, and MultiQuant 3.0 softwares from AB Sciex Inc. (Vaughan, ON, Canada).

3.5. Determination of Total Phenolic Content (TPC) and Total Flavonoids Content (TFC)

TPC and TFC were measured on a UV-2550 Spectrophotometric Reader (Shimadzu Corporation, Kyoto, Japan). The absorbance was measured at 725 nm and 510 nm, respectively. All of the analyses were performed in triplicate. TPC and TFC analysis were performed using the photocolorimetric method, as described by Mohammed Moniruzzaman [41]. The TPC was expressed as milligrams of gallic acid equivalents per kilogram of honey (mg GAE/kg honey), and the standard curve was generated with gallic acid (10–160 μg. mL−1). The TFC were expressed as milligrams of epicatechin equivalents per kilogram of honey (mg EC/kg honey), and the standard curve was plotted using epicatechin (1–100 μg. mL−1).

3.6. Antioxidant Activity

Antioxidant activity assays including DPPH, ABTS and FRAP were studied as described by Habib et al. [42].
Radical scavenging activity assay (DPPH assay). The aqueous solution of honey (0.2 g. mL−1) was mixed with 3.8 mL of DPPH radical solution (0.25 mM). After incubating in the dark for 30 min, the absorbance of the solution was measured at 515 nm. The percentage of free radical scavenging activity that targeted DPPH was calculated using the following equation: DPPH radical savaging activity
( %   inhibition ) = A 0 A 1 A 0 × 100
where A0 is the absorbance of the DPPH control, and A1 is the absorbance in the sample.
ABTS cation radical scavenging. The cation radical ABTS+ was synthesized by the reaction of a 7 mM ABTS solution with a 2.4 mM potassium persulfate solution. The mixture was kept at room temperature in the dark for 14 h. Afterwards, the ABTS+ solution was diluted with methanol until an absorbance of 0.73 ± 0.01 units at 734 nm was achieved. 1.0 mL of the honey sample (20% w/v) was mixed with 1.0 mL of fresh diluted ABTS solution. After incubation at room temperature for 7 min, the absorbance of the solution was measured to be 734 nm. The percentage inhibition calculated as ABTS radical scavenging activity was according to Equation (1), as provided above.
Ferric reducing/antioxidant power assay. The FRAP reagent was prepared before the test by mixing 100 mL of acetate buffer (300 mM, pH 3.6) with 10 mL of TPTZ solution (10 mM in 40 mM HCl) and 10 mL of ferric chloride (FeCl3, 20 mM). A total of 100 µL of the honey solution (0.2 g·mL−1) was mixed with 900 µL of ultrapure water, followed by adding 2.0 mL of the FRAP reagent. The mixture was then vortexed and incubated at 37 °C for 30 min. The absorbance was then determined to be 593 nm using ferrous sulfate standards (0, 0.1, 0.2, 0.5, 1, 1.5, 2.0 mM). The units used for the FRAP values was µmol of ferrous equivalents/100 g of honey sample.

3.7. Data Processing and Metabolomics Analysis

The first step of the metabolomics analysis was to collect information on the phenolic compounds in honey from the literature. Then, the extracted ion chromatogram (XIC) manager add-on in PeakView software 2.2 was used for isotope pattern matched peak mining of data files of honey samples. The parameters for the data mining experiments were as follows: RT window, 1–80 min; minimum intensity counts ≥100; S/N ratio ≥3; isotope pattern matching ≥80%. In addition to MS data, the spectra from MS/MS were also analyzed using the Fragments Pane add-on in PeakView software 2.2 to verify the fragmentation pattern of the detected compound and then matched with hits in the literature and the ChemSpider database (http://www.chemspider.com, accessed on 13 February 2023).
The peak areas of tentatively identified phenolic compounds in each honey sample were integrated using MultiQuant 3.0 software. The data set consisting of one hundred and eleven honey extracts from 37 honey samples was then subjected to PCA analysis using the R statistical package (Rx64 4.0.4). Pareto scaling of the data was performed to modify the weights of the respective variables. The validation of the obtained PCA model was performed by QC samples to ensure the performance of the models. In addition, the dataset was also subjected to orthogonal partial least squares-discriminant analysis (OPLS-DA) using SIMCA 14.1.

3.8. Statistical Analysis

The analyses were made in triplicate, and the results were expressed as the average ± standard deviation. Both the difference analysis and the correlation analysis were carried out with SPSS 20.0 software (Chicago, IL, USA).

4. Conclusions

The present study evaluated the antioxidant properties and phenolic profile of Shennongjia A. cerana honey in China. Furthermore, a total of 83 phenolic compounds were tentatively identified by HPLC-QTOF-MS/MS in this study. Among these compounds, the presence and levels of propolis-derived caffeic acid and pinobanksin ester derivatives in A. cerana honeys were lower than those in A. mellifera honeys. Moreover, thirteen compounds were tentatively identified as markers to distinguish between A. cerana and A. mellifera honey by PCA and OPLS-DA analysis. These compounds could be appropriate markers that should be studied further in the future.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules28073270/s1, Table S1: The LOD, LOQ, linear range and MS characteristics of 13 phenolic compounds and flavonoids; Table S2: Botanical origin of honey samples identified by melissopalynology analysis; Table S3: The VIP and p values of the marked variables by OPLS-DA analysis.

Author Contributions

X.G. designed the research, performed the experiments, interpreted the data, and wrote the manuscript. J.G. and Z.Z. performed the experiments and wrote the manuscript. Q.D., Y.Z. and J.H. performed the experiments. Z.Y. interpreted the data. P.Z. helped in editing the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China (No. 81071341). And the APC was funded by Science Foundation of Shennongjia forest in Hubei Province (No. 2060403).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the article and supplementary material.

Acknowledgments

We would like to thank Jinmin Zheng and her colleagues in theShennongjia forest district for their assistance in collecting the honey samples. We would also like to thank Zisis Kozlakidis for revising the English grammar of this paper.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are available from the authors.

References

  1. Pyrzynska, K.; Biesaga, M. Analysis of phenolic acids and flavonoids in honey. TrAC Trends Anal. Chem. 2009, 28, 893–902. [Google Scholar] [CrossRef]
  2. Combarros-Fuertes, P.; Estevinho, L.M.; Dias, L.G.; Castro, J.M.; Tomas-Barberan, F.A.; Tornadijo, M.E.; Fresno-Baro, J.M. Bioactive components and antioxidant and antibacterial activities of different varieties of honey: A screening prior to clinical application. J. Agric. Food Chem. 2019, 67, 688–698. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Kavanagh, S.; Gunnoo, J.; Marques Passos, T.; Stout, J.C.; White, B. Physicochemical properties and phenolic content of honey from different floral origins and from rural versus urban landscapes. Food Chem. 2019, 272, 66–75. [Google Scholar] [CrossRef] [PubMed]
  4. Nascimento, K.S.d.; Gasparotto Sattler, J.A.; Lauer Macedo, L.F.; Serna González, C.V.; Pereira de Melo, I.L.; da Silva Araújo, E.; Granato, D.; Sattler, A.; de Almeida-Muradian, L.B. Phenolic compounds, antioxidant capacity and physicochemical properties of Brazilian Apis mellifera honeys. LWT Food Sci. Technol. 2018, 91, 85–94. [Google Scholar] [CrossRef]
  5. Tuksitha, L.; Chen, Y.-L.S.; Chen, Y.-L.; Wong, K.-Y.; Peng, C.-C. Antioxidant and antibacterial capacity of stingless bee honey from Borneo (Sarawak). J. Asia-Pac. Entomol. 2018, 21, 563–570. [Google Scholar] [CrossRef]
  6. Alvarez-Suarez, J.M.; Giampieri, F.; Brenciani, A.; Mazzoni, L.; Gasparrini, M.; González-Paramás, A.M.; Santos-Buelga, C.; Morroni, G.; Simoni, S.; Forbes-Hernández, T.Y.; et al. Apis mellifera vs. Melipona beecheii Cuban polifloral honeys: A comparison based on their physicochemical parameters, chemical composition and biological properties. LWT Food Sci. Technol. 2018, 87, 272–279. [Google Scholar] [CrossRef]
  7. Boussaid, A.; Chouaibi, M.; Rezig, L.; Hellal, R.; Donsì, F.; Ferrari, G.; Hamdi, S. Physicochemical and bioactive properties of six honey samples from various floral origins from Tunisia. Arab. J. Chem. 2018, 11, 265–274. [Google Scholar] [CrossRef] [Green Version]
  8. Mudasar, M.; Mathivanan, V.; Nabi Shah, G.H.; Mir, G.M.; Selvisabhanayakam. Physico-chemical analysis of honey of Apis ceranaindica and Apis Mellifera from different regions of Anantnag District, Jammu & Kashmir. Int. J. Pharm. Pharm. Sci. 2013, 5, 635–638. [Google Scholar] [CrossRef]
  9. Wang, Y.; Gou, X.; Yue, T.; Ren, R.; Zhao, H.; He, L.; Liu, C.; Cao, W. Evaluation of physicochemical properties of Qinling Apis cerana honey and the antimicrobial activity of the extract against Salmonella Typhimurium LT2 in vitro and in vivo. Food Chem. 2021, 337, 127774. [Google Scholar] [CrossRef]
  10. Yang, C.; Gong, G.; Jin, E.; Han, X.; Zhuo, Y.; Yang, S.; Song, B.; Zhang, Y.; Piao, C. Topical application of honey in the management of chemo/radiotherapy-induced oral mucositis: A systematic review and network meta-analysis. Int. J. Nurs. Stud. 2018, 89, 80–87. [Google Scholar] [CrossRef]
  11. Zhao, H.; Cheng, N.; He, L.; Peng, G.; Xue, X.; Wu, L.; Cao, W. Antioxidant and hepatoprotective effects of A. cerana honey against acute alcohol-induced liver damage in mice. Food Res. Int. 2017, 101, 35–44. [Google Scholar] [CrossRef]
  12. Chang, X.; Wang, J.; Yang, S.; Chen, S.; Song, Y. Antioxidative, antibrowning and antibacterial activities of sixteen floral honeys. Food Funct. 2011, 2, 541–546. [Google Scholar] [CrossRef]
  13. Deng, J.; Liu, R.; Lu, Q.; Hao, P.; Xu, A.; Zhang, J.; Tan, J. Biochemical properties, antibacterial and cellular antioxidant activities of buckwheat honey in comparison to manuka honey. Food Chem. 2018, 252, 243–249. [Google Scholar] [CrossRef]
  14. Shen, S.; Wang, J.; Chen, X.; Liu, T.; Zhuo, Q.; Zhang, S.Q. Evaluation of cellular antioxidant components of honeys using UPLC-MS/MS and HPLC-FLD based on the quantitative composition-activity relationship. Food Chem. 2019, 293, 169–177. [Google Scholar] [CrossRef]
  15. Sun, C.; Tan, H.; Zhang, Y.; Zhang, H. Phenolics and abscisic acid identified in acacia honey comparing different SPE cartridges coupled with HPLC-PDA. J. Food Compos. Anal. 2016, 53, 91–101. [Google Scholar] [CrossRef]
  16. Zhou, J.; Yao, L.; Li, Y.; Chen, L.; Wu, L.; Zhao, J. Floral classification of honey using liquid chromatography-diode array detection-tandem mass spectrometry and chemometric analysis. Food Chem. 2014, 145, 941–949. [Google Scholar] [CrossRef]
  17. Zhu, Z.; Zhang, Y.; Wang, J.; Li, X.; Wang, W.; Huang, Z. Sugaring-out assisted liquid-liquid extraction coupled with high performance liquid chromatography-electrochemical detection for the determination of 17 phenolic compounds in honey. J. Chromatogr. A 2019, 1601, 104–114. [Google Scholar] [CrossRef]
  18. Bertoncelj, J.; Polak, T.; Kropf, U.; Korošec, M.; Golob, T. LC-DAD-ESI/MS analysis of flavonoids and abscisic acid with chemometric approach for the classification of Slovenian honey. Food Chem. 2011, 127, 296–302. [Google Scholar] [CrossRef]
  19. Lv, C.; Yang, J.; Liu, R.; Lu, Q.; Ding, Y.; Zhang, J.; Deng, J. A comparative study on the adsorption and desorption characteristics of flavonoids from honey by six resins. Food Chem. 2018, 268, 424–430. [Google Scholar] [CrossRef]
  20. Zhao, J.; Du, X.; Cheng, N.; Chen, L.; Xue, X.; Zhao, J.; Wu, L.; Cao, W. Identification of monofloral honeys using HPLC-ECD and chemometrics. Food Chem. 2016, 194, 167–174. [Google Scholar] [CrossRef]
  21. Can, Z.; Yildiz, O.; Sahin, H.; Akyuz Turumtay, E.; Silici, S.; Kolayli, S. An investigation of Turkish honeys: Their physico-chemical properties, antioxidant capacities and phenolic profiles. Food Chem. 2015, 180, 133–141. [Google Scholar] [CrossRef] [PubMed]
  22. Devi, A.; Jangir, J.; Anu-Appaiah, K.A. Chemical characterization complemented with chemometrics for the botanical origin identification of unifloral and multifloral honeys from India. Food Res. Int. 2018, 107, 216–226. [Google Scholar] [CrossRef] [PubMed]
  23. Beiranvand, S.; Williams, A.; Long, S.; Brooks, P.R.; Russell, F.D. Use of kinetic data to model potential antioxidant activity: Radical scavenging capacity of Australian eucalyptus honeys. Food Chem. 2020, 342, 128332. [Google Scholar] [CrossRef] [PubMed]
  24. Chua, L.S.; Rahaman, N.L.; Adnan, N.A.; Eddie Tan, T.T. Antioxidant activity of three honey samples in relation with their biochemical components. J. Anal. Methods Chem. 2013, 2013, 313798. [Google Scholar] [CrossRef]
  25. Gašić, U.M.; Natić, M.M.; Mišić, D.M.; Lušić, D.V.; Milojković-Opsenica, D.M.; Tešić, Ž.L.; Lušić, D. Chemical markers for the authentication of unifloral Salvia officinalis L. honey. J. Food Compos. Anal. 2015, 44, 128–138. [Google Scholar] [CrossRef] [Green Version]
  26. Ouchemoukh, S.; Amessis-Ouchemoukh, N.; Gómez-Romero, M.; Aboud, F.; Giuseppe, A.; Fernández-Gutiérrez, A.; Segura-Carretero, A. Characterisation of phenolic compounds in Algerian honeys by RP-HPLC coupled to electrospray time-of-flight mass spectrometry. LWT Food Sci. Technol. 2017, 85, 460–469. [Google Scholar] [CrossRef]
  27. Rusko, J.; Vainovska, P.; Vilne, B.; Bartkevics, V. Phenolic profiles of raw mono- and polyfloral honeys from Latvia. J. Food Compos. Anal. 2021, 98, 103813. [Google Scholar] [CrossRef]
  28. Gasic, U.; Keckes, S.; Dabic, D.; Trifkovic, J.; Milojkovic-Opsenica, D.; Natic, M.; Tesic, Z. Phenolic profile and antioxidant activity of Serbian polyfloral honeys. Food Chem. 2014, 145, 599–607. [Google Scholar] [CrossRef]
  29. Sergiel, I.; Pohl, P.; Biesaga, M. Characterisation of honeys according to their content of phenolic compounds using high performance liquid chromatography/tandem mass spectrometry. Food Chem. 2014, 145, 404–408. [Google Scholar] [CrossRef]
  30. Castro, C.; Mura, F.; Valenzuela, G.; Figueroa, C.; Salinas, R.; Zuniga, M.C.; Torres, J.L.; Fuguet, E.; Delporte, C. Identification of phenolic compounds by HPLC-ESI-MS/MS and antioxidant activity from Chilean propolis. Food Res. Int. 2014, 64, 873–879. [Google Scholar] [CrossRef]
  31. Fyfe, L.; Okoro, P.; Paterson, E.; Coyle, S.; McDougall, G.J. Compositional analysis of Scottish honeys with antimicrobial activity against antibiotic-resistant bacteria reveals novel antimicrobial components. LWT Food Sci. Technol. 2017, 79, 52–59. [Google Scholar] [CrossRef]
  32. Jandrić, Z.; Frew, R.D.; Fernandez-Cedi, L.N.; Cannavan, A. An investigative study on discrimination of honey of various floral and geographical origins using UPLC-QToF MS and multivariate data analysis. Food Control 2017, 72, 189–197. [Google Scholar] [CrossRef]
  33. Vasic, V.; Gasic, U.; Stankovic, D.; Lusic, D.; Vukic-Lusic, D.; Milojkovic-Opsenica, D.; Tesic, Z.; Trifkovic, J. Towards better quality criteria of European honeydew honey: Phenolic profile and antioxidant capacity. Food Chem. 2019, 274, 629–641. [Google Scholar] [CrossRef] [Green Version]
  34. Truchado, P.; Vit, P.; Heard, T.A.; Tomas-Barberan, F.A.; Ferreres, F. Determination of interglycosidic linkages in O-glycosyl flavones by high-performance liquid chromatography/photodiode-array detection coupled to electrospray ionization ion trap mass spectrometry. Its application to Tetragonula carbonaria honey from Australia. Rapid Commun. Mass Spectrom. 2015, 29, 948–954. [Google Scholar] [CrossRef]
  35. Chen, S.D.; Lu, C.J.; Zhao, R.Z. Identification and quantitative characterization of PSORI-CM01, a Chinese medicine formula for psoriasis therapy, by liquid chromatography coupled with an LTQ Orbitrap mass spectrometer. Molecules 2015, 20, 1594–1609. [Google Scholar] [CrossRef] [Green Version]
  36. Mannina, L.; Sobolev, A.P.; Di Lorenzo, A.; Vista, S.; Tenore, G.C.; Daglia, M. Chemical composition of different botanical origin honeys produced by Sicilian black honeybees (Apis mellifera ssp. sicula). J. Agric. Food Chem. 2015, 63, 5864–5874. [Google Scholar] [CrossRef]
  37. Keckes, S.; Gasic, U.; Velickovic, T.C.; Milojkovic-Opsenica, D.; Natic, M.; Tesic, Z. The determination of phenolic profiles of Serbian unifloral honeys using ultra-high-performance liquid chromatography/high resolution accurate mass spectrometry. Food Chem. 2013, 138, 32–40. [Google Scholar] [CrossRef]
  38. Cui, J.; Duan, X.; Ke, L.; Pan, X.; Liu, J.; Song, X.; Ma, W.; Zhang, W.; Liu, Y.; Fan, Y. Extraction, purification, structural character and biological properties of propolis flavonoids: A review. Fitoterapia 2022, 157, 105106. [Google Scholar] [CrossRef]
  39. Truchado, P.; Ferreres, F.; Tomas-Barberan, F.A. Liquid chromatography-tandem mass spectrometry reveals the widespread occurrence of flavonoid glycosides in honey, and their potential as floral origin markers. J. Chromatogr. A 2009, 1216, 7241–7248. [Google Scholar] [CrossRef]
  40. Bodor, Z.; Kovacs, Z.; Benedek, C.; Hitka, G.; Behling, H. Origin identification of Hungarian honey using melissopalynology, physicochemical analysis, and near infrared spectroscopy. Molecules 2021, 26, 7274. [Google Scholar] [CrossRef]
  41. Mohammed, M.; Sulaiman, S.A.; Khalil, M.I.; Gan, S.H. Evaluation of physicochemical and antioxidant properties of sourwood and other Malaysian honeys: A comparison with manuka honey. Chem. Cent. J. 2013, 7, 138. [Google Scholar] [CrossRef] [Green Version]
  42. Habib, H.M.; Al Meqbali, F.T.; Kamal, H.; Souka, U.D.; Ibrahim, W.H. Bioactive components, antioxidant and DNA damage inhibitory activities of honeys from arid regions. Food Chem. 2014, 153, 28–34. [Google Scholar] [CrossRef] [PubMed]
Figure 1. Principal components analysis (PCA) of A. cerana and A. mellifera honeys. Results of PCA of honeys: scores plot (A) and loadings plot (B).
Figure 1. Principal components analysis (PCA) of A. cerana and A. mellifera honeys. Results of PCA of honeys: scores plot (A) and loadings plot (B).
Molecules 28 03270 g001
Figure 2. Orthogonal partial least squares-discriminant analysis (OPLS-DA) of A. cerana and A. mellifera honey. Score scattering plot of OPLS-DA (A) and corresponding validation plot (B); VIP (C) and S-plot (D) of OPLS-DA.
Figure 2. Orthogonal partial least squares-discriminant analysis (OPLS-DA) of A. cerana and A. mellifera honey. Score scattering plot of OPLS-DA (A) and corresponding validation plot (B); VIP (C) and S-plot (D) of OPLS-DA.
Molecules 28 03270 g002
Table 1. Total phenolic and flavonoid contents and antioxidant activity of A. cerana and A. mellifera honeys.
Table 1. Total phenolic and flavonoid contents and antioxidant activity of A. cerana and A. mellifera honeys.
TPC
(mg GAE/Kg)
TFC
(mg EC/Kg)
FRAP
(uM of Fe2+/100 g)
DPPH
(%)
ABTS
(%)
A.m_p1104.33 ± 4.2115.65 ± 1.5960.13 ± 1.553.63 ± 0.2155.95 ± 0.31
A.m_p2239.12 ± 17.9725.10 ± 0.86175.13 ± 4.446.34 ± 0.1968.78 ± 1.02
A.m_p3225.38 ± 10.3421.21 ± 0.51199.50 ± 3.635.76 ± 0.1475.95 ± 0.43
A.m_p4177.04 ± 24.4627.71 ± 1.5393.25 ± 3.842.80 ± 0.1158.78 ± 0.17
A.m_p5186.88 ± 10.3826.42 ± 1.48133.25 ± 2.563.63 ± 0.1665.68 ± 0.13
A.m_p6158.29 ± 5.0531.57 ± 1.04100.13 ± 5.413.42 ± 0.0857.84 ± 0.97
A.m_p7130.48 ± 3.7814.74 ± 0.7178.25 ± 2.972.17 ± 0.2454.73 ± 1.01
A.m_p8172.91 ± 7.9840.18 ± 1.02111.38 ± 5.133.00 ± 0.3560.27 ± 0.27
A.m_F379.20 ± 25.8642.76 ± 0.29300.75 ± 4.328.93 ± 0.3387.57 ± 0.34
MGO100+622.16 ± 3.72111.24 ± 3.91719.50 ± 9.0319.77 ± 0.5694.73 ± 0.23
MGO250+652.33 ± 9.70110.42 ± 2.19626.38 ± 6.8116.02 ± 0.4694.59 ± 0.13
A.c_1550.70 ± 11.1083.28 ± 0.35607.63 ± 7.9717.65 ± 0.5692.97 ± 0.25
A.c_2265.49 ± 5.5745.8 ± 1.08326.38 ± 4.569.18 ± 0.3777.43 ± 0.48
A.c_3450.61 ± 9.1671.63 ± 0.29375.75 ± 3.608.84 ± 0.1888.11 ± 0.16
A.c_4327.67 ± 3.7949.65 ± 0.77323.25 ± 4.637.72 ± 0.3083.38 ± 0.30
A.c_5271.57 ± 8.9439.33 ± 0.86330.13 ± 3.108.22 ± 0.1082.16 ± 0.51
A.c_6470.01 ± 12.73102.24 ± 0.75405.13 ± 5.7811.60 ± 0.0991.76 ± 0.30
A.c_7340.00 ± 4.1350.48 ± 2.16423.88 ± 3.3210.64 ± 0.3291.49 ± 0.34
A.c_8680.90 ± 35.8087.51 ± 3.83541.38 ± 5.6613.60 ± 0.4193.51 ± 0.42
A.c_9334.67 ± 3.7661.33 ± 1.65344.50 ± 4.597.88 ± 0.4384.31 ± 0.11
A.c_10264.98 ± 4.7945.78 ± 0.46234.50 ± 3.137.38 ± 0.1373.51 ± 0.32
A.c_11270.66 ± 1.9657.86 ± 1.24245.75 ± 4.226.34 ± 0.0971.76 ± 0.59
A.c_12263.02 ± 2.2344.05 ± 1.49263.25 ± 3.508.22 ± 0.5070.13 ± 0.46
A.c_13382.51 ± 3.9752.24 ± 0.75382.63 ± 6.106.88 ± 0.2084.05 ± 0.36
A.c_14407.30 ± 17.0259.14 ± 0.04391.38 ± 5.669.30 ± 0.2388.92 ± 0.62
A.c_15327.02 ± 3.0962.57 ± 0.59321.38 ± 5.007.55 ± 0.4876.89 ± 0.71
A.c_16296.28 ± 8.1560.85 ± 0.68280.13 ± 2.947.38 ± 0.3480.14 ± 0.58
A.c_17326.52 ± 4.7156.59 ± 2.14387.63 ± 3.479.89 ± 0.3587.84 ± 0.47
A.c_18322.43 ± 7.2743.62 ± 1.20350.75 ± 3.666.88 ± 0.2483.38 ± 0.52
A.c_19329.70 ± 2.7047.94 ± 0.41383.88 ± 4.698.39 ± 0.3384.05 ± 0.17
A.c_20302.61 ± 6.9548.88 ± 5.04303.88 ± 3.177.38 ± 0.2980.54 ± 0.63
A.c_21360.04 ± 10.5548.84 ± 4.56360.75 ± 5.289.47 ± 0.5374.86 ± 0.33
A.c_22331.69 ± 12.1535.87 ± 0.44299.50 ± 2.808.01 ± 0.4182.57 ± 0.46
A.c_23343.23 ± 8.7447.53 ± 1.88335.13 ± 6.417.34 ± 0.1283.24 ± 0.48
A.c_24452.07 ± 9.3258.75 ± 1.55520.13 ± 4.0411.39 ± 0.2290.27 ± 0.40
A.c_25317.91 ± 8.1555.67 ± 0.83362.00 ± 3.976.72 ± 0.3474.46 ± 0.17
A.c_26358.04 ± 2.4442.76 ± 0.29312.00 ± 3.676.26 ± 0.4575.00 ± 0.60
Note: The TPC and TFC results are expressed as mean ± S.D. (n = 3).
Table 2. The average content of phenolic compounds (µg/100 g honey) in A. cerana and A. mellifera honeys.
Table 2. The average content of phenolic compounds (µg/100 g honey) in A. cerana and A. mellifera honeys.
A. mellifera Honey
(n = 9)
A. cerana Honey
(n = 26)
Manuka Honey
(n = 2)
KaemKaem_XA2.94 ± 2.312.25 ± 2.27 b0.51 ± 0.30
Kaem_PLS4.67 ± 3.363.56 ± 2.94 b0.99 ± 0.06
Kaem_EAC27.70 ± 12.5947.72 ± 34.19 a9.92 ± 1.27
QuerQuer_XA *0.05 ± 0.100.85 ± 1.08 b0.19 ± 0.07
Quer_PLS **0.66 ± 0.332.20 ± 1.48 b0.99 ± 0.38
Quer_EAC **4.60 ± 1.6514.85 ± 10.38 a5.88 ± 1.58
PinoPino_XA **16.27 ± 25.180.37 ± 0.62 a22.68 ± 20.94
Pino_PLS ***15.53 ± 21.350.46 ± 0.81 a91.12 ± 1.07
Pino_EAC ***15.65 ± 18.290.60 ± 1.04 a46.73 ± 2.14
GalGal_XA ***2.11 ± 2.450.16 ± 0.26 b2.64 ± 2.44
Gal_PLS ****2.25 ± 1.820.22 ± 0.34 ab7.11 ± 0.35
Gal_EAC ****6.87 ± 5.900.55 ± 0.87 a14.58 ± 0.18
ChCh_XA **8.17 ± 11.920.17 ± 0.29 a34.89 ± 5.13
Ch_PLS ****5.65 ± 5.620.22 ± 0.41 a23.12 ± 2.73
Ch_EAC ****10.86 ± 10.320.48 ± 0.98 a17.52 ± 1.49
CTabsaCTbasa_XA56.67 ± 24.6290.82 ± 60.22 a47.16 ± 17.02
CTbasa_PLS54.22 ± 21.2483.60 ± 56.58 a65.78 ± 15.83
CTbasa_EAC54.05 ± 18.7468.81 ± 45.63 a38.82 ± 15.09
4 Hba4 Hba_XA38.86 ± 26.6029.22 ± 17.70 b10.77 ± 4.89
4Hba_PLS *105.21 ± 63.27186.67 ± 88.91 a40.37± 13.53
4Hba_EAC113.86 ± 69.66183.52 ± 98.30 a51.96 ± 7.39
RuRu_XA **1.41 ± 1.903.50 ± 1.61 a0
Ru_PLS **1.42 ± 1.533.84 ± 1.94 a0
Ru_EAC **0.39 ± 0.571.15 ± 0.67 b0
TcinaTcina_XA3.79 ± 11.361.67 ± 4.30 ab0
Tcina_PLS2.43 ± 7.281.03 ± 3.99 b29.05 ± 4.37
Tcina_EAC10.23 ± 10.815.12 ± 7.16 a41.26 ± 14.91
PcoaPcoa_XA *8.34 ± 3.6919.29 ± 13.13 a5.03 ± 0.20
Pcoa_PLS *13.09 ± 14.2726.28 ± 16.11 a16.18 ± 2.15
Pcoa_EAC*12.30 ± 8.9626.29 ± 17.60 a9.53 ± 2.64
VanaVana_XA ****2.13 ± 2.060 c0
Vana_PLS ****7.56 ± 3.233.80 ± 1.53 b6.00 ± 0.55
Vana_EAC12.28 ± 10.2510.18 ± 5.62 a11.38 ± 0.42
CafaCafa_XA49.18 ± 40.9621.18 ± 37.83 a93.88± 8.32
Cafa_PLS50.45 ± 34.2828.02 ± 64.41 a70.37 ± 7.29
Cafa_EAC51.93 ± 32.8431.26 ± 54.89 a48.43± 7.62
FeraFera_XA3.75 ± 1.922.99 ± 2.80 b0.43 ± 0.61
Fera_PLS *5.23 ± 4.842.71 ± 2.12 b2.05 ± 0.28
Fera_EAC9.21 ± 9.325.30 ± 3.25 a2.76 ± 0.52
“*” represents values that differed significantly between A. cerana and A. mellifera honeys for the same compound using the uniform extraction method, * means p < 0.05, ** means p < 0.01, *** means p < 0.001, **** means p < 0.0001. “abc” letters represent values that differed significantly among different extraction methods for the same compound.
Table 3. High resolution MS data and fragmentation of phenolic compounds identified in A. cerana and A. mellifera honeys.
Table 3. High resolution MS data and fragmentation of phenolic compounds identified in A. cerana and A. mellifera honeys.
NoRT (min)NameFormula[M-H]calculated[M-H]experimentalError
(ppm)
MS/MSReferencesDetected in Honey Samples
Phenolic acids and abscidic acid
14.58c gallic acidC7H6O5169.0142169.0142−0.4125[27]8/11 (A. mellifera), 26/26 (A. cerana)
28.18c protocatechuic acidC7H6O4153.0194153.01930.3109, 108[28]11/11 (A. mellifera), 25/26 (A. cerana)
39.65c homogentisic acidC8H8O4167.0350167.03500.0123, 93[29]5/11 (A. mellifera), 7/26 (A. cerana)
411.52c dihydrocaffeic acidC9H10O4181.0503181.0506−1.8163, 135, 119, 93[6]8/11 (A. mellifera), 23/26 (A. cerana)
511.57a 4-hydroxybenzoic acidC7H6O3137.0244137.0244−0.193stdAll
612.36c caffeoylquinic acid isomer 1C16H18O9353.0874353.0878−1.1191, 179, 135[2]11/11 (A. mellifera), 25/26 (A. cerana)
713.68c dimethoxybenzoic acid isomerC9H10O4181.0505181.0506−1.0137, 121[25]9/11 (A. mellifera), 26/26 (A. cerana)
814.16b ethyl gallateC9H10O5197.0453197.0455−1.2153, 109chemspider3/11 (A. mellifera), 22/26 (A. cerana)
914.73a benzoic acidC7H6O2121.0296121.02951.0108, 92std6/11 (A. mellifera), 20/26 (A. cerana)
1017.08a vanillic acidC8H8O4167.0351167.03501.0152, 108, 123, 91std11/11 (A. mellifera), 25/26 (A. cerana)
1117.64c esculetinC9H6O4177.0191177.0193−1.1149, 133, 105, 89[30]All
1217.93c phenylacetic acidC8H8O2135.0451135.0452−0.7107[27]6/11 (A. mellifera), 22/26 (A. cerana)
1317.97a caffeic acidC9H8O4179.0348179.0350−0.9135stdAll
1418.95c caffeoylquinic acid isomer 2C16H18O9353.0875353.0878−0.8191, 179[25]11/11 (A. mellifera), 25/26 (A. cerana)
1521.6c syringic acidC9H10O5197.0455197.0455−0.3182, 166.9, 153, 138, 123, 95[27]9/11 (A. mellifera), 22/26 (A. cerana)
1622.14b p-hydroxy-hydrocinnamic acidC9H10O3165.0556165.0557−1.0147, 119, 103, 72.9chemspiderAll
1723.98c caffeoylquinic acid isomer 3C16H18O9353.0880353.08780.5191, 179[25]9/11 (A. mellifera), 24/26 (A. cerana)
1824.07a p-coumaric acidC9H8O3163.0399163.0401−1.3119, 93stdAll
1925.22c o-coumaric acid C9H8O3163.0397163.0401−2.0119, 93[27]10/11 (A. mellifera), 23/26 (A. cerana)
2026.43c methyl syringateC10H12O5211.0609211.0612−1.2196, 181, 167, 153[31]4/11 (A. mellifera), 14/26 (A. cerana)
2127.84c 4-methoxyphenyllactic acidC10H12O4195.0660195.0663−1.3177, 134, 162, 149[32]8/11 (A. mellifera), 26/26 (A. cerana)
2227.85c coniferyl aldehydeC10H10O3177.0552177.0557−2.6162, 133, 117, 105[33]3/11 (A. mellifera), 26/26 (A. cerana)
2328.88a ferulic acidC10H10O4193.0507193.05060.4178, 149, 134stdAll
2431.6b 4-ethoxy-3-methoxycinnamic acidC12H14O4221.0818221.0819−0.7193, 151, 179, 135chemspider2/11 (A. mellifera), 0/26 (A. cerana)
2535.5c dicaffeoylquinic acid isomer 1C25H24O12515.1196515.11950.2353, 191, 179, 173[28]9/11 (A. mellifera), 23/26 (A. cerana)
2636.61c 2-trans-4-trans-abscidic acidC15H20O4263.1282263.1289−2.5219, 204, 201[2]All
2737.87c dicaffeoylquinic acid isomer 2C25H24O12515.1197515.11950.4353, 191, 179, 173[28]9/11 (A. mellifera), 20/26 (A. cerana)
2838.73a trans-cinnamic acidC9H8O2147.0451147.0452−0.6119,103std7/11 (A. mellifera), 9/26 (A. cerana)
2939.28a 2-cis-4-trans-abscidic acidC15H20O4263.1280263.1289−3.2219, 204, 163, 152, 139stdAll
3041.78c dicaffeoylquinic acid isomer 3C25H24O12515.1193515.1195−0.4353, 191, 179, 173[28]9/11 (A. mellifera), 9/26 (A. cerana)
3153.27c prenyl caffeate C14H16O4247.0973247.0976−1.3179, 161, 135[2]9/11 (A. mellifera), 1/26 (A. cerana)
3253.33c caffeic acid benzyl ester C16H14O4269.0817269.0819−1.0178,161, 134[2]11/11 (A. mellifera), 2/26 (A. cerana)
3355.35c caffeic acid phenylethyl esterC17H16O4283.0973283.0976−0.9268, 215, 179, 161, 135[2]11/11 (A. mellifera), 4/26 (A. cerana)
3457.83c caffeic acid cinnamyl esterC18H16O4295.0968295.0976−2.6178, 134[2]11/11 (A. mellifera), 8/26 (A. cerana)
Flavonols
3522.25c myricetinC15H10O8317.0302317.0303−0.3299, 255, 206.9, 190.9, 163[27]1/11 (A. mellifera), 17/26 (A. cerana)
3632.17c quercetin-3-O-(2-hexosyl) hexoside C27H30O17625.1415625.14100.8463, 300[33]9/11 (A. mellifera), 16/26 (A. cerana)
3733.68c quercetin-3-O-(2-rhamnosyl)hexosideC27H30O16609.1469609.14611.3300[34]10/11 (A. mellifera), 24/26 (A. cerana)
3833.74c methoxy kaempferol 3-O-(2-hexosyl) hexosideC28H32O17639.1575639.15671.3330, 314, 299[33]10/11 (A. mellifera), 21/26 (A. cerana)
3934.77c 8-O-methoxykaempferol-3-O-neohesperidosideC28H32O16623.1638623.16183.3314, 315, 459, 608[2]9/11 (A. mellifera), 25/26 (A. cerana)
4035.48c quercetin 3-O-glucosideC21H20O12463.0878463.0882−0.9301, 300, 271[33]10/11 (A. mellifera), 26/26 (A. cerana)
4135.57c kaempferol 3-O-(2-rhamnosyl)hexosideC27H30O15593.1523593.15121.9284[33]10/11 (A. mellifera), 26/26 (A. cerana)
4235.91c isorhamnetin-3-o-neohesperosideC28H32O16623.1624623.16181.0314, 315, 459[2]11/11 (A. mellifera), 25/26 (A. cerana)
4336.04a rutinC27H30O16609.1467609.14611.0300, 301std8/11 (A. mellifera), 26/26 (A. cerana)
4438.17c quercetin-3-rhamnoside isomerC21H20O11447.0923447.0933−2.2301, 300, 284, 255[33]10/11 (A. mellifera), 24/26 (A. cerana)
4541.96c quercetin-3-rhamnosideC21H20O11447.0931447.0933−0.4301, 300, 151[2]9/11 (A. mellifera), 23/26 (A. cerana)
4643.25a quercetinC15H10O7301.0351301.0354−1.0179, 151stdAll
4746.92c kaempferol 7-O-rhamnosideC21H20O10431.0979431.0984−1.0285, 257, 151[33]11/11 (A. mellifera), 24/26 (A. cerana)
4847.71c methoxy kaempferol C16H12O7315.0506315.0510−1.3300, 272, 255, 165.9[33]All
4947.91a kaempferolC15H10O6285.0401285.0405−1.3229, 185, 151, 239, 257stdAll
5049.09c isorhamnetinC16H12O7315.0509315.0510−0.5300, 151[2]11/11 (A. mellifera), 23/26 (A. cerana)
5149.72c bis-methylated quercetinC17H14O7329.0666329.0667−0.3314, 299, 271[33]All
5253.14c kaempferidC16H12O6299.0554299.0561−2.5284, 271, 255, 237, 211, 165[33]9/11 (A. mellifera), 5/26 (A. cerana)
5355.6a galanginC15H10O5269.0451269.0455−1.6213, 169std11/11 (A. mellifera), 13/26 (A. cerana)
5456.32c galangin-5-methyl ether isomer C16H12O5283.0609283.0612−0.9268, 239, 211[2]11/11 (A. mellifera), 16/26 (A. cerana)
Flavanonols
5529.56c taxifolinC15H12O7303.0508303.0510−0.9285, 275, 241, 177, 125[35]11/11 (A. mellifera), 25/26 (A. cerana)
5640.72c pinobanksin-5-methyl etherC16H14O5285.0765285.0768−1.3267, 252, 224, 165, 138[2]11/11 (A. mellifera), 7/26 (A. cerana)
5741.99c pinobanksinC15H12O5271.0606271.0612−2.3253, 197[33]All
5853.72c pinobanksin-3-O-acetateC17H14O6313.0709313.0718−2.9253, 271[36]9/11 (A. mellifera), 12/26 (A. cerana)
5960.73c pinobanksin-3-O-butyrateC19H18O6341.1023341.1031−2.1253, 271, 197[2]10/11 (A. mellifera), 4/26 (A. cerana)
6065.35c pinobanksin-3-O-pentanoateC20H20O6355.1180355.1187−2.1253, 271[2]11/11 (A. mellifera), 8/26 (A. cerana)
6168.19c pinobanksin-3-O-hexanoateC21H22O6369.1333369.1344−2.8300, 271, 253[30]10/11 (A. mellifera), 0/26 (A. cerana)
Flavanones
6238.42c eriodictyolC15H12O6287.0552287.0561−3.3151, 135[33]All
6345.15c hesperetin isomerC16H14O6301.0714301.0718−1.1164, 286[29]9/11 (A. mellifera), 26/26 (A. cerana)
6452.06c isosakuranetinC16H14O5285.0766285.0768−1.0165, 119[14]11/11 (A. mellifera), 19/26 (A. cerana)
6552.47a pinocembrinC15H12O4255.0661255.0663−0.6213, 171, 151std11/11 (A. mellifera), 16/26 (A. cerana)
6652.57c sakuranetinC16H14O5285.0766285.0768−1.0165, 119[14]11/11 (A. mellifera), 19/26 (A. cerana)
Flavones
6734.09c isovitexinC21H20O10431.0982431.0984−0.3385, 341, 311, 283, 251[27]2/11 (A. mellifera), 9/26 (A. cerana)
6835.45b vitexinC21H20O10431.0980431.0984−0.9341, 311, 283chemspider8/11 (A. mellifera), 10/26 (A. cerana)
6941.25c luteolin 7-O-rhamnosideC21H20O10431.0977431.0984−1.5285, 255, 227[33]10/11 (A. mellifera), 25/26 (A. cerana)
7044.82c luteolinC15H10O6285.0400285.0405−1.5133, 151, 175, 199[27]All
7148.34c apigeninC15H10O5269.0454269.0455−0.5225, 205, 151, 117[27]11/11 (A. mellifera), 25/26 (A. cerana)
7249.48c luteolin-methyl-etherC16H12O6299.0559299.0561−0.7284, 256, 190.9[30]All
7350.29c tectochrysinC16H12O4267.0657267.0663−2.4252, 224, 180[33]11/11 (A. mellifera), 6/26 (A. cerana)
7454.32c methoxychrysinC16H12O5283.0605283.0612−2.4268, 239, 211[2]7/11 (A. mellifera), 25/26 (A. cerana)
7554.36a chrysinC15H10O4253.0504253.0506−1.0209, 143std11/11 (A. mellifera), 17/26 (A. cerana)
7656.86c ermaninC17H14O6313.0718313.07180.2298, 283, 255, 199[30]11/11 (A. mellifera), 2/26 (A. cerana)
Others
779.91c pantothenic acidC9H17NO5218.1028218.1034−2.7146, 88, 71[27]All
7816.25c UI 1C10H7NO3188.0352188.0353−0.5144[2]11/11 (A. mellifera), 25/26 (A. cerana)
7929.33c UI 2C10H7NO3188.0352188.0353−0.6144[2]8/11 (A. mellifera), 26/26 (A. cerana)
8037.35c anchoic acidC9H16O4187.0967187.0976−4.6169, 125, 97chemspiderAll
8139.01b hydroxyoctanoic acidC8H16O3159.1019159.1027−4.8113chemspiderAll
8241.04c decenedioic acidC10H16O4199.0970199.0976−2.9155, 137, 181[31]All
8359.03b aleuritic acidC16H32O5303.2172303.2177−1.7285, 229chemspiderAll
a confirmed with standard. b confirmed with chemspider. c confirmed with references.
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MDPI and ACS Style

Guo, J.; Ding, Q.; Zhang, Z.; Zhang, Y.; He, J.; Yang, Z.; Zhou, P.; Gong, X. Evaluation of the Antioxidant Activities and Phenolic Profile of Shennongjia Apis cerana Honey through a Comparison with Apis mellifera Honey in China. Molecules 2023, 28, 3270. https://doi.org/10.3390/molecules28073270

AMA Style

Guo J, Ding Q, Zhang Z, Zhang Y, He J, Yang Z, Zhou P, Gong X. Evaluation of the Antioxidant Activities and Phenolic Profile of Shennongjia Apis cerana Honey through a Comparison with Apis mellifera Honey in China. Molecules. 2023; 28(7):3270. https://doi.org/10.3390/molecules28073270

Chicago/Turabian Style

Guo, Jingwen, Qiong Ding, Zhiwei Zhang, Ying Zhang, Jianshe He, Zong Yang, Ping Zhou, and Xiaoyan Gong. 2023. "Evaluation of the Antioxidant Activities and Phenolic Profile of Shennongjia Apis cerana Honey through a Comparison with Apis mellifera Honey in China" Molecules 28, no. 7: 3270. https://doi.org/10.3390/molecules28073270

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