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LC-ESI-QTOF/MS Characterization of Phenolic Compounds from Medicinal Plants (Hops and Juniper Berries) and Their Antioxidant Activity

School of Agriculture and Food, Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, VIC 3010, Australia
Author to whom correspondence should be addressed.
Received: 26 November 2019 / Revised: 18 December 2019 / Accepted: 18 December 2019 / Published: 20 December 2019
(This article belongs to the Special Issue Extraction and Characterization of Polyphenols from Food Matrix)


Hops (Humulus lupulus L.) and juniper berries (Juniperus communis L.) are two important medicinal plants widely used in the food, beverage, and pharmaceutical industries due to their strong antioxidant capacity, which is attributed to the presence of polyphenols. The present study is conducted to comprehensively characterize polyphenols from hops and juniper berries using liquid chromatography coupled with electrospray-ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF/MS) to assess their antioxidant capacity. For polyphenol estimation, total phenolic content, flavonoids and tannins were measured, while for antioxidant capacity, three different antioxidant assays including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant assay, the 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical cation decolorization assay and the ferric reducing-antioxidant power (FRAP) assay were used. Hops presented the higher phenolic content (23.11 ± 0.03 mg/g dw) which corresponded to its strong antioxidant activity as compared to the juniper berries. Using LC-ESI-QTOF/MS, a total of 148 phenolic compounds were tentatively identified in juniper and hops, among which phenolic acids (including hydroxybenzoic acids, hydroxycinnamic acids and hydroxyphenylpropanoic acids) and flavonoids (mainly anthocyanins, flavones, flavonols, and isoflavonoids) were the main polyphenols, which may contribute to their antioxidant capacity. Furthermore, the HPLC quantitative analysis showed that both samples had a high concentration of phenolic acids and flavonoids. In the HPLC quantification, the predominant phenolic acids in hops and juniper berries were chlorogenic acid (16.48 ± 0.03 mg/g dw) and protocatechuic acid (11.46 ± 0.03 mg/g dw), respectively. The obtained results highlight the importance of hops and juniper berries as a rich source of functional ingredients in different food, beverage, and pharmaceutical industries.

Graphical Abstract

1. Introduction

Medicinal plants are used in different food, beverage, and pharmaceutical industries. They are rich in bioactive compounds especially polyphenols which can contribute to human health. Polyphenols are secondary bioactive compounds which are classified into several categories consisting of hydroxybenzoic acids (protocatechuic, p-hydroxybenzoic, syringic), hydroxycinnamic acids (caffeic, p-coumaric, ferulic), flavan-3-ols (catechin, epicatechin), flavonoids (catechin, epicatechin, quercetin, apigenin), glycosides, and proanthocyanidins [1]. There is a growing interest in the research of polyphenols due to their antioxidant properties and the evidence for the multiple biological activities, including cardioprotective, anti-inflammatory, anti-carcinogenic, antiviral, and antibacterial properties [2].
Beer is one of the most popular alcoholic beverages in the world [3]. Most of the modern beers are formulated with hops, which contribute bitterness flavors and act as natural preservatives and stabilizers [4]. In addition to hops, some of the brewers normally add a few other flavoring plants such as juniper berries that contain bitter substances, giving the beer a well-rounded, balanced, and tasty bitterness [5]. Hops and juniper berries are rich in phenolic compounds that can contribute to antioxidant capacity and provide a pleasant sensory quality to beverages [4,5,6].
Hops (Humulus lupulus L.) have a unique bitterness and aroma, contain various phenolic compounds, and also one of the indispensable raw materials for beer brewing. About 30% of the polyphenols in beer come from these hops. Dried hops cones possess about 15% polyphenols mainly phenolic acids, prenylated chalcone, flavonoids, and catechins [4]. Some of the common polyphenolic substances that are found in different hops varieties are chlorogenic acid, gallic acid, epicatechin, and kaempferol-3-glucoside [7].
Juniper berries (Juniperus communis L.) have been used as medicinal plants to treat opportunistic infections, a spice for various cuisines, and distinctive flavoring compounds for different beverages [8]. Previous studies have shown that polyphenols in the juniper berries mainly include flavonoids and bioflavonoids that have antioxidant activities, which could scavenge free radicals, prevent the free radical formation and prevent lipid peroxidation [9]. Some of the phenolic compounds such as epicatechin, procyanidin dimer, and epigallocatechin have been determined by liquid chromatography-mass spectrometer (LC-MS) in the Juniperus species found in Portugal [10]. However, relatively less information is available regarding their phenolic profile and antioxidant capacity.
The antioxidant activity of these polyphenols can be assessed by scavenging free radicals or delaying the generation of free radicals using different in vitro methods, including the 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidant assay, the 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical cation decolorization assay, and the ferric reducing-antioxidant power (FRAP) assay [11]. Antioxidant capacity can vary depending upon the sample and the nature and type of solvent extraction. Different types of solvents and their combinations have been used for the extraction of polyphenols from plant materials. Water, aqueous mixtures of ethanol, methanol, and acetone are commonly used solvents to extract compounds with high extraction yields [12]. After an extraction, precise identification and quantitation of these phenolic compounds is a complex task because of their structural diversity.
Liquid chromatography coupled with electrospray-ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF/MS) has been recognized as a powerful analytical tool with high sensitivity and accuracy to determine the phenolic profile of plant materials [13]. Also, high-pressure liquid chromatography (HPLC) has proven to be a very useful tool in the quantitation of targeted polyphenols, in combination with different detectors like ultraviolet–visible (UV) and photodiode array detector (PDA) [2]. These analytical techniques are considered as advanced tools for the characterization, purification, and quantitation of phenolic compounds.
Hops and juniper berries are very important medicinal plants that have strong antioxidant capacity. Therefore, the objective of our study was to identify and characterize the polyphenols from selected medical plants (hops and juniper berries) using LC-ESI-QTOF/MS and quantify through HPLC-PDA. Another objective was to measure the total phenolic content (TPC), total flavonoid content (TFC), and total tannin content (TTC) and further analyze their antioxidant capacity using DPPH, FRAP, and ABTS radical-scavenging activity.

2. Materials and Methods

2.1. Chemical and Reagents

Most of the chemicals used for extraction and characterization were analytical grade and purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Folin–Ciocalteu reagent, gallic acid, aluminum chloride hexahydrate, quercetin, vanillin, catechin, 2,2-diphenyl-1-picrylhydrazyl (DPPH), ascorbic acid, 2, 4, 6-tripyridyl-s-triazine (TPTZ), HCl, 2, 2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and potassium persulfate were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sodium carbonate, ethanol, sodium acetate, sulfuric acid, ferric chloride (Fe [III]Cl3·6H2O), and acetic acid were purchased from the Thermo Fisher (Scoresby, Melbourne, VIC, Australia). HPLC grade methanol, acetic acid, and acetonitrile used for HPLC analyses were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phenolic acid and flavonoid standards (gallic acid, protocatechuic acid, caftaric acid, p-hydroxybenzoic acid, chlorogenic acid, caffeic acid, syringic acid, coumaric acid, catechin, epicatechin gallate, quercetin-3-galactoside, quercetin-3-O-glucuronide (q-3-O-glucuronide), kaempferol-3-O-glucoside, quercetin, and kaempferol) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

2.2. Sample Preparation

Dried hops pellets (AlphAroma) were purchased from a local retail market in Melbourne, Australia. Dried juniper berries (Easten red cedar) were purchased from the Ozspice Store, Melbourne, Australia. Hops pellets and juniper berries were milled into dried powder and were stored at room temperature in a dark area to protect from light exposure, prior to an extraction.

2.3. Extraction of Phenolic Compounds

Two grams of dried powder of hops and juniper berries were macerated in 20 mL of 30% ethanol (w/v). The extraction was carried out in a shaking incubator (ZWYR-240, Labwit, Ashwood, VIC, Australia) at 120 rpm, 4 °C for 12 h. Samples were centrifuged (ROTINA 380R centrifuge, Hettich, Victoria, Australia) at 5000 rpm for 15 min. The supernatant was collected and stored at −20 °C for further analysis.

2.4. Estimation of Polyphenols and Antioxidant Assays

For polyphenol estimation, TPC, TFC and TTC were measured while for antioxidant capacity, three different antioxidant assays, including DPPH, FRAP, and ABTS, were performed using the method of Gu et al. [14]. The data was obtained by the Multiskan® Go microplate photometer (Thermo Fisher Scientific, Waltham, MA, USA).

2.4.1. Determination of Total Phenolic Content (TPC)

The TPC was determined by a spectrophotometric method using Folin–Ciocalteu reagent [15] with some modifications. For determination, 25 µL of the extract was mixed with 25 µL Folin–Ciocalteu reagent solution (1:3 diluted with water) and 200 µL water was added into a 96-well plate (Corning Inc., Midland, NC, USA) followed by incubation at room temperature for 5 min. The reaction mixture was basified by adding 25 µL 10% (w:w) sodium carbonate and incubated again for 60 min in dark area. Then, the absorbance was measured at 765 nm by a spectrophotometer plate reader (Thermo Fisher Scientific, Waltham, MA, USA). The TPC in samples was quantified from a calibration curve prepared with gallic acid standard with different concentrations ranging from 0–200 µg/mL and expressed as mg of gallic acid equivalents (GAE) per g dry weight (dw) (mg GAE/g dw) of the sample.

2.4.2. Determination of Total Flavonoid Content (TFC)

The TFC was determined by the aluminum chloride method [16] with some modifications. An 80 µL of the extract was mixed with 80 µL of 2% aluminum chloride (diluted with ethanol) and 120 µL of 50 g/L sodium acetate solution in a 96-well plate and incubated at 25 °C for 2.5 h. Then, the absorbance of the mixture was subsequently measured at 440 nm. The TFC was calculated as mg of quercetin equivalent per g (mg QE/g dw) of weight of samples using the calibration curve of quercetin (0–50 µg/mL).

2.4.3. Determination of Total Tannins Content (TTC)

The TTC was determined by vanillin and p-dimethylaminocinnamaldehyde methods [16] with some modifications. Twenty-five µL of the extract was mixed with 150 µL of 4% vanillin solution (diluted with methanol) and 25 µL of 32% sulfuric acid in a 96-well plate and incubated at 25 °C for 15 min and the absorbance was measured at 500 nm. The TTC was expressed as mg of catechin equivalent per g (mg CE/g dw) of samples using a calibration curve prepared with catechin solution ranging from 0–1000 µg/ML.

2.4.4. 2,2-Diphenyl-1-Picrylhydrazyl (DPPH) Antioxidant Assay

The DPPH scavenging activity was determined by the DPPH assay method [17] with some modifications. For the DPPH. assay, 40 µL of the extract was added to the 40 µL of DPPH methanolic solution (0.1 mM) in a 96-well plate. The mixtures were shaken vigorously and incubated at 25 °C for 30 min and the absorbance was measured at 517 nm. The DPPH radical-scavenging activity of extracts was expressed as mg of ascorbic acid equivalents per g (mg AAE/g dw) of samples using standard equation, plotted at different concentrations of standard ranges from 0–50 µg/mL.

2.4.5. Ferric Reducing-Antioxidant Power (FRAP) Assay

The FRAP assay was determined based on the method [17] with some modifications. The FRAP method involves assessing the ability of the test material to reduce iron in Fe3+-TPTZ complex (ferric-2,4,6-tripyridyl-s-Triazine) to the Fe2+-TPTZ complex by the test substance [11]. The FRAP dye was prepared by mixing of sodium acetate solution (300 mM), TPTZ (2, 4, 6-tripyridyl-s-triazine) solution (10 mM), and Fe[III] solution (20 mM) in 10:1:1 ratio, respectively. A 20 µL of extract or standard was added to 280 µL of prepared FRAP dye solution in a 96-well plate and incubated at 37 °C for 10 min. The absorbance was measured at 593 nm. The FRAP results were converted to mg of ascorbic acid equivalents per g (mg AAE/g dw) of samples using the standard curve, plotted at different concentrations of standard ranges from 0–50 µg/mL.

2.4.6. 2,2′-Azino-bis-3ethylbenzothiazoline-6-sulfonic Acid (ABTS) Radical Scavenging Assay

The ABTS scavenging activity was carried out by the ABTS+ radical cation decolorization assay with some modification [17]. Here, 5 mL of 7 mmol/L of ABTS solution was mixed with 88 µL of a 140 mM potassium persulfate solution to produce ABTS+. The mixture was placed in the dark at room temperature for 16 h. Then, the prepared ABTS+ solution was diluted with analytical grade ethanol to obtain an initial absorbance of 0.7 at 734 nm. Then, 10 µL of extract or standard was mixed with 290 µL of prepared diluted ABTS solution in a 96-well plate and incubated at room temperature for 6 min in the dark area. Then, the absorbance was measured at 734 nm after incubation. The antioxidant ability was expressed as mg of ascorbic acid equivalents per g (mg AAE/g dw) of samples using the calibration curve prepared for ascorbic acid, plotted at different concentrations of standard ranges from 0–2000 µg/mL.

2.5. LC-ESI-QTOF/MS Analysis

Polyphenol characterization was carried out using the method of Gu et al. [14] and was performed by Agilent 1200 series HPLC (Agilent Technologies, CA, USA) equipped with an Agilent 6520 Accurate-Mass Q-TOF LC/MS (Agilent Technologies, CA, USA). The separation was carried out using a Synergi Hydro-RP 80A, LC column 250 × 4.6 mm, 4 µm (Phenomenex, Torrance, CA, USA). Mobile phase A was prepared in the ratio of water/acetic acid (98:2, v/v), and mobile phase B consisted of acetonitrile/water/acetic acid (100:1:99, v/v/v). Both mobile phase A and B were degassed at 21 °C for 15 min. The extract was filtered using the syringe filters (Kinesis, Redland, QLD, Australia) and transferred into HPLC vials. The flow rate was set to be 0.8 mL/min and the injection volume was 6 μL of each sample. Gradient elution was performed by a mixture of mobile phase A and B in the following program: 0–20 min, 10% B; 20–30 min, 25% B; 30–40 min, 35% B; 40–70 min, 40% B; 70–75 min, 55% B; 75–77 min, 80% B; 77–79 min, 100% B; 79–82 min, 100% B; 82–85 min, 10% B. At the end of program, the eluent composition was back to initial gradient and the column was equilibrated for 3 min before next injection.
Electrospray ionization (ESI) was used as a source in operating both negative and positive modes. Mass spectra in the m/z range 50 to 1300 were obtained. The mass spectrometry conditions were set as follows: nitrogen gas temperature 300 °C with the flow rate 5 L/min, sheath gas temperature 250 °C with the flow rate 11 L/min, nebulizer gas pressure 45 psi. The capillary and nozzle voltage were set at 3.5 kV and 500 V, respectively. Data acquisition and analysis were performed using Agilent LC-MS-QTOF MassHunter data acquisition software version B.03.01.

2.6. HPLC Analysis

The quantification of targeted phenolic compounds was determined by using the method of Gu et al. [14] and carried by Agilent 1200 series HPLC (Agilent Technologies, CA, USA) equipped with a photodiode array (PDA) detector. The same column and conditions were maintained described above in LC-ESI/QTOF/MS except for sample injection volume of 20 μL. Detection was examined by three different wavelengths (280, 320, and 370 nm) for various phenolic compounds. 280 nm wavelength was used for the identification of hydroxybenzoic acids, 320 nm was used for hydroxycinnamic acids, and 370 nm was used for the identification of the flavonol group. Data acquisition and analysis were performed using Agilent LC-ESI/QTOF/MS MassHunter data acquisition software version B.03.01.

2.7. Statistical Analysis

One-way analysis of variance (ANOVA) was used to test for differences in mean values between different samples, followed by Tukey’s honestly significant differences (HSD) multiple rank test at p < 0.05. The results are shown as mean ± standard deviation (SD). ANOVA was performed by Minitab Program for Windows version 18.0 (Minitab, LLC, State College, PA, USA).

3. Results and Discussion

3.1. Polyphenol Estimation (TPC, TFC, and TTC)

Medicinal plants including hops and juniper berries are rich in phenolic compounds. The amount of phenolics content in both samples were determined by the TPC, TFC, and TTC, and the results are expressed as gallic acid equivalents, quercetin equivalent, and catechin equivalent, respectively.
Table 1 shows that the TPC values was significantly higher in hops (23.11 ± 0.03 mg GAE/g dw) as compared to juniper berries (9.08 ± 0.01 mg GAE/g dw; p < 0.05). In the present study, it was found that hops and juniper berries had lower TPC values compared with the previously reported studies, which could be due to the fact that researchers applied ethanol extraction with high concentrations [18] or employed a freeze-drying step before extraction [10]. Regarding the TFC, the juniper (2.25 ± 0.01 mg QE/g dw) contains more flavonoids as compared to hops (1.37 ± 0.01 mg QE/g dw). Previously, only a few flavonoids were detected in hops [7] while flavonoids were considered as the major polyphenol classes in juniper berries [9]. Nasri et al. [19] also reported a higher concentration of total flavonoids (8.90 ± 0.48 mg/g dw) from juniper berries (Juniperus phoenicea), it was found that different juniper varieties have different flavonoids content. Furthermore, Miceli et al. [20] also reported a significant difference (p < 0.05) in TPC and TFC values between different varieties of juniper berries including J. communis L. var. communis and J. communis L. var. saxatilis (Pall). Table 1 shows that hops contained a significantly higher (p < 0.05) amount of tannins as compared to juniper berries. High tannins in our study are in agreement with previous research of Gorjanović et al. [21] in ethanol extracts of hop (Humulus lupulus L.).

3.2. Antioxidant Activities

Antioxidant properties are very important due to the deleterious role of free radicals in foods and biological systems [22]. The antioxidant capacities of extracts were evaluated by the most commonly used antioxidant assays: DPPH, FRAP, and ABTS methods.
The DPPH is a stable and purple free radical that has been widely employed to determine antioxidant capacity and presents a typical absorption band at 517 nm [23]. The method is based on the reduction of the stable free radical DPPH. in the presence of a hydrogen-donating antioxidant, and the formation of the non-radical form DPPH-H as a result of the reaction [22]. Table 1 shows that the free radical scavenging activities of hops extract (9.26 ± 0.02 mg AAE/g dw) was significantly higher (p < 0.05) than that of juniper extract, which was consistent with the result of TPC, indicating that antioxidant activities of samples were related to the TPC. Previously, Elmastaş et al. [24] reported that juniper ethanol extracts (J. communis L.), have stronger DPPH scavenging activity than aqueous extracts. Al-Mustafa et al. [25] also presented a high correlation (R2 = 0.87) between the DPPH scavenging activity and the total phenolic content of different medicinal extracts.
The ferric reducing power determination is based on the reduction of Fe3+ to Fe2+ by electron transfer from the sample or antioxidant and the ability of the extracts to act as antioxidants by donating electrons could increase with increased absorbance [22]. Table 1 shows that there was a significant difference (p < 0.05) in FRAP values between hops and juniper berries, agreeing with the result of TPC, indicating ferric reducing power of samples may be related to the TPC. Previously, Abram et al. [26] reported simlar FRAP antioxidant capacity in different hops varieties using ethanol extraction. In addition, ethanol extract of J. communis showed better scavenging activity in the FRAP assay among five different Juniperus species, including J. communis, J. excelsa, J. foetidissima, J. oxycedrus, and J. sabina [27].
To evaluate the antioxidant capacity of food extracts, ABTS+ radical scavenging activity has been widely applied based on hydrogen-donating antioxidants against nitrogen radicals [28]. Table 1 shows that the hops (49.54 ± 0.04 mg AAE/g dw) had significantly higher ABTS+ radical scavenging activity as compared to the juniper berries (15.18 ± 0.02 mg AAE/g dw). The ABTS values of our juniper berries differ from the studies of Höferl et al. [8], who reported higher ABTS radical scavenging activity for another juniper berry variety using a different solvent extract. However, Kowalczyk et al. [18] reported similar ABTS+ scavenging activity in hops samples extracted with different solvents including aqueous methanol, aqueous ethanol, and water extract.

3.3. Phenolic Identification by LC-ESI-QTOF/MS

The LC-ESI-QTOF/MS has been proved to be an effective tool for tentatively identifying and characterizing phenolic compounds in several plants [13]. Identification and characterization of compounds were carried out by comparison of their retention time (RT), mass error between mass observed, and the theoretical mass (<10 ppm); mass spectrometric (MS) data obtained under both negative and positive electron spray ionization modes (ESI/ESI+; Supplementary Figures S1 and S2) and data identification scores selected were above 80. Table 2 reports all compounds tentatively identified in both hops and juniper berries in positive and negative ionization modes.
A total of 148 different phenolic compounds were characterized in both hops and juniper berries, including 34 phenolic acids, 78 flavonoids, 8 lignans, 3 stilbenes, 1 hydroxybenzaldehyde, and 24 other polyphenols. Additionally, one non-phenolic metabolite (1,3,5-trimethoxybenzene) was also characterized in hops and juniper berries.
In hops, a total of 117 phenolic compounds were identified (Supplementary Table S1) and categorized into six polyphenol classes including 30 phenolic acids, 61 flavonoids, 4 lignans, 2 stilbenes, 1 hydroxybenzaldehydes, and 19 other polyphenols. In juniper berries, a total of 81 compounds in 5 different classes were characterized (Supplementary Table S2), including 18 phenolic acids, 46 flavonoids, 5 lignans, 2 stilbenes, and 10 other polyphenols.

3.3.1. Hydroxybenzoic Acids

In our study, a total of 3 hydroxybenzoic acids derivatives (Compounds 3, 4 and 6) were identified in both hops and juniper berries in negative modes of ionization. Compound 3 with [M − H] at m/z 315.0746 and 315.0739 was tentatively characterized as protocatechuic acid 4-O-glucoside. Compound 4 with [M − H] at m/z 153.0203 and 153.0204 was tentatively identified as 2,3-dihydroxybenzoic acid. Compound 6 with the molecular formula C7H6O3 and having the precursor ion at m/z 137.0249 and 137.0258 in the negative ESI-mode, was tentatively characterized as 2-hydroxybenzoic acid in both hops and juniper berries. Previously, protocatechuic acid 4-O-glucoside and 2-hydroxybenzoic had already been identified in Juniperus communis var. saxatilis [29].
Compounds 1 and 2, only detected in hops, with [M − H] at m/z 331.0693 and 169.0159 were tentatively characterized as galloyl glucose and gallic acid, respectively, while there were two compounds (Compounds 5 and 7) detected only in juniper berries in negative ionization modes and tentatively assigned as 4-O-methylgallic acid and ellagic acid with [M − H] at m/z 183.0306 and 300.9969, respectively. Previously Miceli et al. [30] also found gallic acid present in J. drupacea berries methanol extract by HPLC-DAD-MS. Also, the gallic acid was previously reported in Humulus lupulus L. by HPLC-UV [31].

3.3.2. Hydroxycinnamic Acids

In the present work, we characterized 6 hydroxycinnamic acids (Compounds 9, 11, 16, 19, 22, and 23) in both hops and juniper berries. Among these, two compounds (Compounds 9 and 19) were identified in both hops and juniper berries samples in positive and negative modes of ionization. Compound 9 with [M + H]+ at m/z 399.1291 and [M − H] at m/z 397.1117 was tentatively characterized as 3-sinapoylquinic acid while compound (19) with [M − H] at m/z 326.1042 and [M + H]+ at m/z 328.1172 was tentatively identified as p-coumaroyl tyrosine. Two more compounds (Compounds 11 and 22) were detected in both samples in negative ionization modes. Compound 11 with [M − H] at m/z 337.0949 and 337.0955 was tentatively identified as 3-p-coumaroylquinic acid while compound 22 showing [M − H] at m/z 191.0733 and 191.072 was tentatively identified as p-coumaric acid ethyl ester. However, two compounds (Compounds 16 and 23) were identified in both samples in positive modes of ionization. Compound 16 with [M + H]+ at m/z 149.0587 and 149.0591 (at RT = 20.491 min) was tentatively characterized as cinnamic acid while compound 23 with [M + H]+ at m/z 195.0656 and 195.0668 was tentatively identified as isoferulic acid. In addition, isoferulic acid and p-coumaric acid ethyl ester were previously reported in Juniperus communis var. saxatilis [29].
In addition to the compounds identified above in both plant samples, there were a total of 9 hydroxycinnamic acids derivatives characterized only in hops, including 3-caffeoylquinic acid, caffeic acid 3-O-glucuronide, rosmarinic acid, p-coumaric acid 4-O-glucoside, ferulic acid 4-O-glucuronide, 3-feruloylquinic acid, ferulic acid 4-O-glucoside, caffeoyl glucose, and 1,2-disinapoylgentiobiose. The p-coumaric acid ethyl ester and 3-caffeoylquinic acid were reported as the bioactive compounds in Saaz hops variety from the Czech Republic [32].

3.3.3. Hydroxyphenylpropanoic Acids

There were 2 hydroxyphenylpropanoic acids (Compounds 33 and 34) detected in both hops and juniper berries. Compound 33 with [M − H] at m/z 181.0524 and with [M − H] at m/z 181.0524 was tentatively identified as 3-hydroxy-3-(3-hydroxyphenyl) propionic acid. Also, compound 34 showing [M − H] at m/z 165.0569 and 165.0555 and with the molecular formula C9H10O3 was tentatively characterized as 3-hydroxyphenylpropionic acid. Based on QTOF-MS analysis, compounds 30, 31 and 32 were only detected in hops and tentatively identified as dihydrocaffeic acid 3-O-glucuronide, dihydrosinapic acid, and dihydroferulic acid 4-O-glucuronide, showing [M − H] at m/z 357.0831, 225.076, and 371.0962, respectively.

3.3.4. Anthocyanins

Based on MS data, a total of 4 anthocyanins were identified in both hops and juniper berries in negative ionization modes, including 2 cyanidin derivatives (Compounds 41 and 48) and 2 delphinidin 3-O derivatives (Compounds 36 and 44). Compound 48 with [M − H] at m/z 448.0982 and 448.0985 was tentatively characterized as cyanidin 3-O-galactoside while compound 41 showing [M − H] at m/z 610.1530 and 610.1529 was tentatively identified as cyanidin 3,5-O-diglucoside, all of which were cyanidin derivatives. Additionally, compound 44 showing 39.382 min was tentatively characterized as delphinidin 3-O-glucoside, while compound 36 with the molecular formula C27H31O17 was tentatively identified as delphinidin 3-O-glucosyl-glucoside, which belonged to delphinidin 3-O derivatives.
There were 12 compounds (35, 37, 38, 39, 40, 42, 43, 45, 46, 47, 50, and 51) only identified in hops in negative ionization modes, mostly being cyanidin and its 3-O-glycosides. In juniper berries, compound 49 was tentatively characterized for cyanidin 3-O-(6’’-dioxalyl-glucoside) with [M − H] at m/z 591.0656 and 45.432 min.

3.3.5. Flavones

In the present work, we identified 2 flavones (Compounds 71 and 73) in both hops and juniper berries in negative modes of ionization. Compound 71 was detected in negative ionization modes with [M − H] at m/z 593.1532 and 593.1518 and was tentatively characterized as apigenin 6,8-di-C-glucoside. In addition, compound 73 showed [M − H] at m/z 447.0949 and 447.0941 and was tentatively identified as 6-hydroxyluteolin 7-O-rhamnoside, which was also discussed in previously literature in Juniperus communis var. saxatilis [29]. Apigenin was previously identified in Tuscan berries of Juniperus communis L. by HPLC/DAD/ESI/MS [9].
Compound 78 was only detected in hops with a precursor ion at [M + H]+ m/z 359.1116, tentatively representing the gardenin B. In juniper berries, compounds 70, 76, and 77 with [M + H]+ at m/z 579.1675, 565.1538, and 345.0957, respectively, were tentatively characterized as isorhoifolin, apigenin 7-O-apiosyl-glucoside, and cirsilineol.

3.3.6. Flavonols

In this work, a total of 13 flavonols were detected in both hops and juniper berries in positive and negative ionization modes, including 2 isorhamnetin derivatives (Compounds 94 and 99), 5 kaempferol derivatives (Compounds 79, 81, 83, 88, and 89), 4 myricetin derivatives (Compounds 82, 84, 85, and 93), and 2 quercetin 3-O derivatives (Compounds 90 and 96). Compound 99 with [M − H] at m/z 315.0508 and 315.0520 was tentatively characterized as isorhamnetin. In previously literature, isorhamnetin was already reported in the Saaz hops variety [33]. Among kaempferol derivatives, compound 83 showing [M + H]+ at m/z 757.2133 was observed and tentatively identified as kaempferol 3-O-glucosyl-rhamnosyl-galactoside. Regarding myricetin derivatives, compound 85 showed [M + H]+ at m/z 319.0427 and 319.0435, and at 33.345 min and was tentatively characterized as myricetin.
In hops compounds 86, 95, 97, and 98 with [M − H] at m/z 741.1900, 549.0901, 635.1637, and 533.0944, were tentatively identified to be quercetin 3-O-xylosyl-rutinoside, quercetin 3-O-(6”-malonyl-glucoside), kaempferol 3-O-(6’’-acetyl-galactoside) 7-O-rhamnoside, and 5,4’-dihydroxy-3,3’-dimethoxy-6:7-methylenedioxyflavone 4’-O-glucuronide, respectively. In juniper berries compounds 80 and 92 were detected in negative modes of ionization and tentatively characterized as patuletin 3-O-glucosyl-(1->6)-[apiosyl(1->2)]-glucoside and spinacetin 3-O-glucosyl-(1->6)-glucoside with precursor [M − H] at m/z 787.1965 and 669.1689, respectively.

3.3.7. Isoflavonoids

A total of 4 isoflavonoids (Compounds 104, 105, 107, and 111) were detected in both hops and juniper berries. Among which, in positive and negative ionization modes, compound 111 with [M + H]+ at m/z 301.1069 and with [M − H] at m/z 299.0931, respectively, was tentatively identified as sativanone. In addition, in negative ionization modes, compound 107 with [M − H] at m/z 301.0375 and 301.0364 and at 69.083 min was tentatively characterized as 5,6,7,3’,4’-pentahydroxyisoflavone, which was also detected in the Saaz hops variety [32] and Juniperus communis var. saxatilis [29] in previous literature.
Compounds 101, 102, and 108 were detected only in hops giving [M + H]+ at m/z 303.0847, 459.1279 and 285.0766 and tentatively characterized as 4’-methoxy-2’,3,7-trihydroxyisoflavanone, 6’’-O-acetyldaidzin and 2’-hydroxyformononetin, respectively. In juniper berries compounds 100, 103, 109, 110, and 112 were tentatively identified as 6’’-O-acetylgenistin, 3’-hydroxydaidzein, 2’,7-dihydroxy-4’,5’-dimethoxyisoflavone, 3’-hydroxymelanettin, and dihydrobiochanin A in positive ionization modes, respectively.

3.4. HPLC Analysis

The HPLC is used to study the polyphenol content and chemical composition of various plants, which had been previously shown to be an effective technique for the quantification of polyphenols [2]. In our study, 15 targeted polyphenols mainly phenolic acids and flavonoids were quantified by their UV spectra and by comparing their retention times with reference standards. According to the HPLC-PDA, flavonoids were the main phenolic class with a higher diversity of compounds (Table 3).
Phenolic acids were high in both hops and juniper berries, representing the dominant class of compounds in these selected medicinal plants. In general, chlorogenic acid (16.48 ± 0.03 mg/g dw), gallic acid (3.41 ± 0.02 mg/g dw), caftaric acid (0.72 ± 0.01 mg/g dw), and syringic acid (0.03 ± 0.01 mg/g dw) were the major phenolic acids in hops. However, these compounds were not detected in juniper berries which contained high concentrations of protocatechuic acid (11.46 ± 0.03 mg/g dw), p-hydroxybenzoic acid (3.12 ± 0.01 mg/g dw), and caffeic acid (0.14 ± 0.01 mg/g dw) in comparison with those in hops. Coumaric acid was present only in juniper berries with (0.32 ± 0.01 mg/g dw). Previously, chlorogenic acid and gallic acid were determined in hops (Humulus lupulus L.) with a high content using HPLC [7]. Previously, Keskin et al. [31] quantified the coumaric acid in Humulus lupulus L. using multiple extractions of diethyl ether, ethyl acetate, and methanol. Chlorogenic acid was also reported in Juniperus communis L. one of the native species grown on Romanian southern sub-Carpathian hills using 50% ethanol (w/v) [34]. Also, protocatechuic acid and gallic acid were quantitated by HPLC in Juniperus drupacea berries from Turkey [30].
Regarding flavonoids, catechin was the most abundant flavonoid in both hops and juniper berries with (9.03 ± 0.02 dw) and (8.47 ± 0.02 dw), respectively. In a small amount, epicatechin gallate was determined in hops (0.02 ± 0.01 mg/g dw) but absent in juniper berries. Quercetin-3-O-galactoside and kaempferol were higher in juniper berries as compared to hops. Kaempferol-3-O-glucoside, quercetin, and quercetin-3-O-glucuronide were higher in hops but also detected in juniper berries. In previous literature, catechin, quercetin and kaempferol-3-O-glucoside were higher in hops (Humulus lupulus L.) while catechin was higher in J. drupacea berries from Turkey [30].

4. Conclusions

The LC-ESI-QTOF/MS analysis was applied for the tentative identification and characterization of phenolic compounds from hops and juniper berries. Consequently, a total 148 phenolic compounds were tentatively identified, based on comparison of their mass spectrometric data obtained under both negative and positive electron spray ionization conditions and categorized into several main polyphenol classes including phenolic acids, flavonoids, lignans, stilbenes, and other polyphenols. The quantification of 15 individual polyphenols was achieved through HPLC-PDA by comparing their UV spectra and the retention times with reference standards. Different antioxidant assays were conducted to evaluate and map an overall antioxidant capacity of both samples. The results show that hops contain a significantly higher phenolic content and antioxidant capacity compared to juniper berries. In addition, antioxidant capacity is related to phenolic content, which can also be consistent with the presented HPLC composition. Although these two plant species show significant differences in phenolic content, they all present antioxidant capacity, which supports their wide application in health, nutrition, and medicine.

Supplementary Materials

The following are available online at, Figure S1: LC-ESI-QTOF/MS basic peak chromatograph (BPC) for characterization of phenolic compounds of juniper berries and hops samples, Figure S2: Extracted ion chromatogram and their mass spectrum, Table S1: Phenolic compounds detected and tentatively characterised in hops extracts by using LC-ESI-QTOF/MS in both positive and negative ionisation modes, Table S2: Phenolic compounds detected and tentatively characterised in juniper berries extracts by using LC-ESI-QTOF/MS in both positive and negative ionisation modes.

Author Contributions

Conceptualization, methodology, validation and investigation, H.A.R.S., J.T. and F.R.D.; resources, H.A.R.S. and F.R.D.; writing—original draft preparation, J.T. and H.A.R.S.; writing—review and editing, H.A.R.S. and F.R.D.; supervision, H.A.R.S. and F.R.D.; funding acquisition, H.A.R.S., and F.R.D. All authors have read and agreed to the published version of the manuscript.


This research was funded by the University of Melbourne under the “McKenzie Fellowship Scheme” and the “Faculty Research Initiative Funds” funded by the Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Australia.


We would like to thank Nicholas Williamson, Shuai Nie and Michael Leeming from the Mass Spectrometry and Proteomics Facility, Bio21 Molecular Science and 49 Biotechnology Institute, the University of Melbourne, VIC, Australia for providing access and support for the use of HPLC and LC-ESI-QTOF/MS and data analysis. We would also like to thank for Kate Howell, Chunhe Gu, Rana Dildar Khan, Chao Ma, Danying Peng, Biming Zhong, Yuying Feng, Danwei Yang, Yasir Iqbal and Akhtar Ali from the School of Agriculture and Food, Faculty of Veterinary and Agricultural Sciences, the University of Melbourne for their incredible support.

Conflicts of Interest

The authors declare no conflict of interest.


  1. Manach, C.; Scalbert, A.; Morand, C.; Rémésy, C.; Jiménez, L. Polyphenols: Food sources and bioavailability. Am. J. Clin. Nutr. 2004, 79, 727–747. [Google Scholar] [CrossRef] [PubMed][Green Version]
  2. Šeruga, M.; Novak, I.; Jakobek, L. Determination of polyphenols content and antioxidant activity of some red wines by differential pulse voltammetry, hplc and spectrophotometric methods. Food Chem. 2011, 124, 1208–1216. [Google Scholar] [CrossRef]
  3. Gerhäuser, C. Beer constituents as potential cancer chemopreventive agents. Eur. J. Cancer 2005, 41, 1941–1954. [Google Scholar] [CrossRef]
  4. Arranz, S.; Chiva-Blanch, G.; Valderas-Martínez, P.; Medina-Remón, A.; Lamuela-Raventós, R.M.; Estruch, R. Wine, beer, alcohol and polyphenols on cardiovascular disease and cancer. Nutrients 2012, 4, 759–781. [Google Scholar] [CrossRef] [PubMed][Green Version]
  5. Anjos, O.; Nunes, D.; Caldeira, I. First evaluation of a traditional portuguese alcoholic beverage, prepared with maceration of juniper berries. Ciência Téc. Vitiv. 2013, 28, 59–69. [Google Scholar]
  6. Zhao, H.; Chen, W.; Lu, J.; Zhao, M. Phenolic profiles and antioxidant activities of commercial beers. Food Chem. 2010, 119, 1150–1158. [Google Scholar] [CrossRef]
  7. Wang, X.; Yang, L.; Yang, X.; Tian, Y. In vitro and in vivo antioxidant and antimutagenic activities of polyphenols extracted from hops (humulus lupulus l.). J. Sci. Food Agric. 2014, 94, 1693–1700. [Google Scholar] [CrossRef]
  8. Höferl, M.; Stoilova, I.; Schmidt, E.; Wanner, J.; Jirovetz, L.; Trifonova, D.; Krastev, L.; Krastanov, A. Chemical composition and antioxidant properties of juniper berry (juniperus communis l.) essential oil. Action of the essential oil on the antioxidant protection of saccharomyces cerevisiae model organism. Antioxidants 2014, 3, 81–98. [Google Scholar] [CrossRef][Green Version]
  9. Innocenti, M.; Michelozzi, M.; Giaccherini, C.; Ieri, F.; Vincieri, F.F.; Mulinacci, N. Flavonoids and biflavonoids in tuscan berries of juniperus communis l.:  Detection and quantitation by hplc/dad/esi/ms. J. Agric. Food Chem. 2007, 55, 6596–6602. [Google Scholar] [CrossRef]
  10. Tavares, L.; McDougall, G.J.; Fortalezas, S.; Stewart, D.; Ferreira, R.B.; Santos, C.N. The neuroprotective potential of phenolic-enriched fractions from four juniperus species found in portugal. Food Chem. 2012, 135, 562–570. [Google Scholar] [CrossRef]
  11. Alam, M.N.; Bristi, N.J.; Rafiquzzaman, M. Review on in vivo and in vitro methods evaluation of antioxidant activity. Saudi Pharm. J. 2013, 21, 143–152. [Google Scholar] [CrossRef] [PubMed][Green Version]
  12. Turkmen, N.; Sari, F.; Velioglu, Y.S. Effects of extraction solvents on concentration and antioxidant activity of black and black mate tea polyphenols determined by ferrous tartrate and folin–ciocalteu methods. Food Chem. 2006, 99, 835–841. [Google Scholar] [CrossRef]
  13. Hossain, M.B.; Rai, D.K.; Brunton, N.P.; Martin-Diana, A.B.; Barry-Ryan, C. Characterization of phenolic composition in lamiaceae spices by lc-esi-ms/ms. J. Agric. Food Chem. 2010, 58, 10576–10581. [Google Scholar] [CrossRef] [PubMed]
  14. Gu, C.; Howell, K.; Dunshea, F.R.; Suleria, H.A.R. Lc-esi-qtof/ms characterisation of phenolic acids and flavonoids in polyphenol-rich fruits and vegetables and their potential antioxidant activities. Antioxidants 2019, 8, 405. [Google Scholar] [CrossRef][Green Version]
  15. Samsonowicz, M.; Regulska, E.; Karpowicz, D.; Leśniewska, B. Antioxidant properties of coffee substitutes rich in polyphenols and minerals. Food Chem. 2019, 278, 101–109. [Google Scholar] [CrossRef]
  16. Stavrou, I.J.; Christou, A.; Kapnissi-Christodoulou, C.P. Polyphenols in carobs: A review on their composition, antioxidant capacity and cytotoxic effects, and health impact. Food Chem. 2018, 269, 355–374. [Google Scholar] [CrossRef]
  17. Sogi, D.S.; Siddiq, M.; Greiby, I.; Dolan, K.D. Total phenolics, antioxidant activity, and functional properties of ‘tommy atkins’ mango peel and kernel as affected by drying methods. Food Chem. 2013, 141, 2649–2655. [Google Scholar] [CrossRef]
  18. Kowalczyk, D.; Świeca, M.; Cichocka, J.; Gawlik-Dziki, U. The phenolic content and antioxidant activity of the aqueous and hydroalcoholic extracts of hops and their pellets. J. Inst. Brew. 2013, 119, 103–110. [Google Scholar] [CrossRef]
  19. Nasri, N.; Tlili, N.; Elfalleh, W.; Cherif, E.; Ferchichi, A.; Khaldi, A.; Triki, S. Chemical compounds from phoenician juniper berries (juniperus phoenicea). Nat. Prod. Res. 2011, 25, 1733–1742. [Google Scholar] [CrossRef]
  20. Miceli, N.; Trovato, A.; Dugo, P.; Cacciola, F.; Donato, P.; Marino, A.; Bellinghieri, V.; La Barbera, T.M.; Güvenç, A.; Taviano, M.F. Comparative analysis of flavonoid profile, antioxidant and antimicrobial activity of the berries of juniperus communis l. Var. Communis and juniperus communis l. Var. Saxatilis pall. From turkey. J. Agric. Food Chem. 2009, 57, 6570–6577. [Google Scholar] [CrossRef]
  21. Gorjanović, S.; Pastor, F.T.; Vasić, R.; Novaković, M.; Simonović, M.; Milić, S.; Sužnjević, D. Electrochemical versus spectrophotometric assessment of antioxidant activity of hop (humulus lupulus l.) products and individual compounds. J. Agric. Food Chem. 2013, 61, 9089–9096. [Google Scholar] [CrossRef] [PubMed]
  22. Lahmass, I.; Ouahhoud, S.; Elmansuri, M.; Sabouni, A.; Elyoubi, M.; Benabbas, R.; Choukri, M.; Saalaoui, E. Determination of antioxidant properties of six by-products of crocus sativus l. (saffron) plant products. Waste Biomass Valor. 2018, 9, 1349–1357. [Google Scholar] [CrossRef]
  23. Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. LWT-Food Sci. Technol. 1995, 28, 25–30. [Google Scholar] [CrossRef]
  24. Elmastaş, M.; Gülçin, İ.; Beydemir, Ş.; İrfan Küfrevioğlu, Ö.; Aboul-Enein, H.Y. A study on the in vitro antioxidant activity of juniper (juniperus communis l.) fruit extracts. Anal. Lett. 2006, 39, 47–65. [Google Scholar] [CrossRef]
  25. Al-Mustafa, A.H.; Al-Thunibat, O.Y. Antioxidant activity of some jordanian medicinal plants used traditionally for treatment of diabetes. Pak. J. Biol. Sci. 2008, 11, 351–358. [Google Scholar] [CrossRef] [PubMed]
  26. Abram, V.; Čeh, B.; Vidmar, M.; Hercezi, M.; Lazić, N.; Bucik, V.; Možina, S.S.; Košir, I.J.; Kač, M.; Demšar, L.; et al. A comparison of antioxidant and antimicrobial activity between hop leaves and hop cones. Ind. Crop. Prod. 2015, 64, 124–134. [Google Scholar] [CrossRef]
  27. Orhan, N.; Orhan, I.E.; Ergun, F. Insights into cholinesterase inhibitory and antioxidant activities of five juniperus species. Food Chem. Toxicol. 2011, 49, 2305–2312. [Google Scholar] [CrossRef]
  28. Xiang, J.; Li, W.; Ndolo, V.U.; Beta, T. A comparative study of the phenolic compounds and in vitro antioxidant capacity of finger millets from different growing regions in malawi. J. Cereal Sci. 2019, 87, 143–149. [Google Scholar] [CrossRef]
  29. Vasilijević, B.; Knežević-Vukčević, J.; Mitić-Ćulafić, D.; Orčić, D.; Francišković, M.; Srdic-Rajic, T.; Jovanović, M.; Nikolić, B. Chemical characterization, antioxidant, genotoxic and in vitro cytotoxic activity assessment of juniperus communis var. Saxatilis. Food Chem. Toxicol. 2018, 112, 118–125. [Google Scholar] [CrossRef]
  30. Miceli, N.; Trovato, A.; Marino, A.; Bellinghieri, V.; Melchini, A.; Dugo, P.; Cacciola, F.; Donato, P.; Mondello, L.; Güvenç, A.; et al. Phenolic composition and biological activities of juniperus drupacea labill. Berries from turkey. Food Chem. Toxicol. 2011, 49, 2600–2608. [Google Scholar] [CrossRef]
  31. Keskin, Ş.; Şirin, Y.; Çakir, H.E.; Keskin, M. An investigation of humulus lupulus l.: Phenolic composition, antioxidant capacity and inhibition properties of clinically important enzymes. S. Afr. J. Bot. 2019, 120, 170–174. [Google Scholar] [CrossRef]
  32. Inui, T.; Okumura, K.; Matsui, H.; Hosoya, T.; Kumazawa, S. Effect of harvest time on some in vitro functional properties of hop polyphenols. Food Chem. 2017, 225, 69–76. [Google Scholar] [CrossRef] [PubMed]
  33. Liu, Y.; Gu, X.-H.; Tang, J.; Liu, K. Antioxidant activities of hops (humulus lupulus) and their products. J. Am. Soc. Brew. Chem. 2007, 65, 116–121. [Google Scholar] [CrossRef]
  34. Fierascu, I.; Ungureanu, C.; Avramescu, S.M.; Cimpeanu, C.; Georgescu, M.I.; Fierascu, R.C.; Ortan, A.; Sutan, A.N.; Anuta, V.; Zanfirescu, A.; et al. Genoprotective, antioxidant, antifungal and anti-inflammatory evaluation of hydroalcoholic extract of wild-growing juniperus communis l. (cupressaceae) native to romanian southern sub-carpathian hills. BMC Complement. Altern. Med. 2018, 18, 3. [Google Scholar] [CrossRef][Green Version]
Table 1. Polyphenol content and antioxidant activities in hops and juniper berries.
Table 1. Polyphenol content and antioxidant activities in hops and juniper berries.
Antioxidant AssaysHopsJuniper Berries
TPC/mg GAE/g23.11 ± 0.03 a9.08 ± 0.01 b
TFC/mg QE/g1.37 ± 0.01 a2.25 ± 0.01 a
TTC/mg CE/g25.18 ± 0.07 a3.48 ± 0.03 b
FRAP/mg AAE/g4.17 ± 0.03 a2.02 ± 0.01 b
DPPH/mg AAE/g9.26 ± 0.02 a3.57 ± 0.01 b
ABTS/mg AAE/g49.54 ± 0.04 a15.18 ± 0.02 b
All data are the mean ± SD of three replicates. Means followed by different letters (a, b) within the same column are significantly different (p < 0.05) from each other. Data of hops and juniper berries are reported on a dry weight basis.
Table 2. Qualitative characterization of phenolic compounds in hops and juniper berries by LC-ESI-QTOF/MS.
Table 2. Qualitative characterization of phenolic compounds in hops and juniper berries by LC-ESI-QTOF/MS.
No.Proposed CompoundsMolecular FormulaRetention Time (min)Mode of Lonization (ESI−/ESI+)Molecular WeightTheoretical (m/z)Observed (m/z)Mass Error (ppm)Samples
Phenolic acids
Hydroxybenzoic acids
1Galloyl glucoseC13H16O106.583[M − H]332.0743331.0670331.06936.70Hops
2Gallic acidC7H6O56.749[M − H]170.0215169.0142169.01599.68Hops
3Protocatechuic acid 4-O-glucosideC13H16O99.151[M − H]316.0794315.0721315.07467.97* Hops, juniper berries
42,3-Dihydroxybenzoic acidC7H6O412.348[M − H]154.0266153.0193153.02036.14* Hops, juniper berries
54-O-Methylgallic acidC8H8O514.439[M − H]184.0372183.0299183.03064.71Juniper berries
62-Hydroxybenzoic acidC7H6O319.935[M − H]138.0317137.0244137.02493.69* Hops, juniper berries
7Ellagic acidC14H6O845.283[M − H]302.0063300.9990300.9969−7.08Juniper berries
Hydroxycinnamic acids
83-Caffeoylquinic acidC16H18O912.629[M − H]354.0951353.0878353.08941.84Hops
93-Sinapoylquinic acidC18H22O1013.815[M − H]/* [M + H]+398.1213399.1286399.12910.88* Hops, juniper berries
10Caffeic acid 3-O-glucuronideC15H16O1015.396[M − H]356.0743355.0670355.06803.79Hops
113-p-Coumaroylquinic acidC16H18O817.665[M − H]338.1002337.0929337.09496.22* Hops, juniper berries
12Rosmarinic acidC18H16O817.665[M − H]360.0845359.0772359.07808.44Hops
13p-Coumaric acid 4-O-glucosideC15H18O818.957[M − H]326.1002325.0929325.0920−4.31Hops
14Ferulic acid 4-O-glucuronideC16H18O1019.918[M − H]370.0900369.0827369.08331.54Hops
153-Feruloylquinic acidC17H20O920.481[M − H]368.1107367.1034367.10380.94Hops
16Cinnamic acidC9H8O220.491[M + H]+148.0524149.0597149.0587−6.81* Hops, juniper berries
17Ferulic acid 4-O-glucosideC16H20O922.916[M − H]356.1107355.1034355.10586.08Hops
18Caffeoyl glucoseC15H18O924.076[M − H]342.0951341.0878341.08986.09Hops
19p-Coumaroyl tyrosineC18H17NO527.637* [M − H]/[M + H]+327.1107326.1034326.1042−3.98* Hops, juniper berries
201,2-DisinapoylgentiobioseC34H42O1937.991[M − H]754.2320753.2247753.2281−5.47Hops
21VerbascosideC29H36O1554.046[M − H]624.2054623.1981623.1982−0.85Juniper berries
22p-Coumaric acid ethyl esterC11H12O381.109[M − H]192.0786191.0713191.07339.96* Hops, juniper berries
23Isoferulic acidC10H10O481.881[M + H]+194.0579195.0652195.06562.46*Hops, juniper berries
Hydroxyphenylpentanoic acids
245-(3’-Methoxy-4’-hydroxyphenyl)-gamma -valerolactoneC12H14O419.077[M − H]222.0892221.0819221.08357.82Juniper berries
Hydroxyphenylacetic acids
252-Hydroxy-2-phenylacetic acidC8H8O315.247[M − H]152.0473151.0400151.04086.70* Hops, juniper berries
263,4-Dihydroxyphenylacetic acidC8H8O440.227[M − H]168.0423167.0350167.03679.63* Hops, juniper berries
Hydroxyphenylpentanoic acids
273-Hydroxyphenylvaleric acidC11H14O38.978[M + H]+194.0943195.1016195.1019−0.78Hops
285-(3’,4’-dihydroxyphenyl)-valeric acidC11H14O448.883[M + H]+210.0892211.0965211.0956−4.05Hops
295-(3’,4’,-dihydroxyphenyl)-γ-valerolactoneC11H12O468.437[M − H]208.0736207.0663207.06798.32* Hops, juniper berries
Hydroxyphenylpropanoic acids
30Dihydrocaffeic acid 3-O-glucuronideC15H18O1013.772[M − H]358.0900357.0827357.08311.32Hops
31Dihydrosinapic acidC11H14O515.909[M − H]226.0841225.0768225.0760−5.07Hops
32Dihydroferulic acid 4-O-glucuronideC16H20O1018.957[M − H]372.1056371.0983371.0962−4.96Hops
333-Hydroxy-3-(3-hydroxyphenyl) propionic acidC9H10O448.095[M − H]182.0579181.0506181.05249.61* Hops, juniper berries
343-Hydroxyphenylpropionic acidC9H10O349.139[M − H]166.0630165.0557165.05696.63* Hops, juniper berries
35Cyanidin 3-O-(6’’-p-coumaroyl-glucoside)C30H27O138.140[M − H]595.1452594.1379594.1361−2.65Hops
36Delphinidin 3-O-glucosyl-glucosideC27H31O1732.143[M − H]627.1561626.1488626.1464−4.36* Hops, juniper berries
37Peonidin 3-O-sambubioside-5-O-glucosideC33H41O2032.640[M − H]757.2191756.2118756.2098−2.92Hops
38Cyanidin 3-O-sambubioside 5-O-glucosideC32H39O2033.104[M − H]743.2035742.1962742.1933−2.55Hops
39Pelargonidin 3-O-glucosyl-rutinosideC33H41O1934.644[M − H]741.2242740.2169740.2153−2.68Hops
40Delphinidin 3-O-sambubiosideC26H29O1635.903[M − H]597.1456596.1383596.1363−2.73Hops
41Cyanidin 3,5-O-diglucosideC27H31O1637.079[M − H]611.1612610.1539610.1530−2.01* Hops, juniper berries
42Cyanidin 3-O-(6’’-malonyl-3’’-glucosyl-glucoside)C30H33O1938.189[M − H]697.1616696.1543696.1524−2.24Hops
43Cyanidin 3-O-rutinosideC27H31O1538.355[M − H]595.1663594.1590594.1570−3.45Hops
44Delphinidin 3-O-glucosideC21H21O1239.382[M − H]465.1033464.0960464.0945−3.80* Hops, juniper berries
45Peonidin 3-O-sophorosideC28H33O1641.005[M − H]625.1769624.1696624.1682−1.55Hops
46Delphinidin 3-O-(6’’-acetyl-glucoside)C23H23O1342.678[M − H]507.1139506.1066506.1040−5.07Hops
47Pelargonidin 3,5-O-diglucosideC27H31ClO1542.844[M − H]630.1351629.1278629.1293−0.28Hops
48Cyanidin 3-O-galactosideC21H21O1143.275[M − H]449.1084448.1011448.0982−6.42* Hops, juniper berries
49Cyanidin 3-O-(6’’-dioxalyl-glucoside)C25H20O1745.432[M − H]592.0700591.0627591.06564.65Juniper berries
50Cyanidin 3-O-(6’’-acetyl-glucoside)C23H23O1251.143[M − H]491.1190490.1117490.1083−5.46Hops
51CyanidinC15H11O679.801[M − H]287.0556286.0483286.0468−3.14Hops
52XanthohumolC21H22O582.941[M + H]+354.1467355.1540355.1523−3.79Hops
533-Hydroxyphloretin 2’-O-glucosideC21H24O1118.924[M − H]452.1319451.1246451.1252−1.17*Hops, juniper berries
54PhloridzinC21H24O1050.617[M − H]436.1369435.1296435.13011.77Juniper berries
55Dihydroquercetin 3-O-rhamnosideC21H22O1126.544[M − H]450.1162449.1089449.11033.17Hops
56Dihydromyricetin 3-O-rhamnosideC21H22O1264.802[M + H]+466.1111467.1184467.1164−3.04Hops
57Procyanidin dimer B1C30H26O1214.932[M − H]578.1424577.1351577.13550.25* Hops, juniper berries
58(-)-EpigallocatechinC15H14O716.605[M − H]306.0740305.0667305.06680.88Hops
59Procyanidin trimer C1C45H38O1818.576[M − H]866.2058865.1985865.1966−2.37* Hops, juniper berries
604’-O-MethylepigallocatechinC16H16O724.450[M + H]+320.0896321.0969321.0959−3.17Hops
61(-)-EpicatechinC15H14O625.848* [M − H]/[M + H]+290.0790289.0717289.07366.06* Hops, juniper berries
624’’-O-Methylepigallocatechin 3-O-gallateC23H20O1126.636[M + H]+472.1006473.1079473.1062−3.01Hops
63Cinnamtannin A2C60H50O2429.592[M − H]1154.26901153.26201153.2610−0.97Hops
64(+)-Gallocatechin 3-O-gallateC22H18O1149.606[M − H]458.0849457.0776457.07690.42Juniper berries
653’-O-Methyl-(-)-epicatechin 7-O-glucuronideC22H24O1276.365[M + H]+480.1268481.1341481.13400.19Hops
66EriocitrinC27H32O1521.939[M − H]596.1741595.1668595.16680.00Hops
67Naringenin 7-O-glucosideC21H22O1037.278[M − H]434.1213433.1140433.1121−1.57* Hops, juniper berries
68Hesperetin 3’-O-glucuronideC22H22O1248.476[M − H]478.1111477.1038477.10512.88* Hops, juniper berries
69Apigenin 7-O-glucuronideC21H18O118.564[M + H]+446.0849447.0922447.0908−0.89Hops
70IsorhoifolinC27H30O1416.539[M + H]+578.1636579.1709579.1675−5.75Juniper berries
71Apigenin 6,8-di-C-glucosideC27H30O1542.794[M − H]594.1585593.1512593.15323.11* Hops, juniper berries
72Chrysoeriol 7-O-(6’’-malonyl-apiosyl-glucoside)C30H32O1843.739[M − H]680.1589679.1516679.15211.15Hops
736-Hydroxyluteolin 7-O-rhamnosideC21H20O1145.627[M − H]448.1006447.0933447.09493.41* Hops, juniper berries
74Apigenin 6-C-glucosideC21H20O1046.906[M − H]432.1056431.0983431.09921.55Juniper berries
75Chrysoeriol 7-O-glucosideC22H22O1148.695[M − H]462.1162461.1089461.10951.03Juniper berries
76Apigenin 7-O-apiosyl-glucosideC26H28O1455.335[M + H]+564.1479565.1552565.1538−3.00Juniper berries
77CirsilineolC18H16O780.994[M + H]+344.0896345.0969345.0957−2.40Juniper berries
78Gardenin BC19H18O782.411[M + H]+358.1053359.1126359.1116−2.73Hops
79Kaempferol 3-O-xylosyl-glucosideC26H28O1522.777[M − H]/*[M + H]+580.1428581.1501581.15102.14* Hops, juniper berries
80Patuletin 3-O-glucosyl-(1->6)-[apiosyl(1->2)]-glucosideC33H40O2228.535[M − H]788.2011787.1938787.19651.67Juniper berries
81Kaempferol 3,7,4’-O-triglucosideC33H40O2129.079[M − H]772.2062771.1989771.19940.21* Hops, juniper berries
82Myricetin 3-O-rutinosideC27H30O1731.547[M − H]626.1483625.1410625.14161.20* Hops, juniper berries
83Kaempferol 3-O-glucosyl-rhamnosyl-galactosideC33H40O2031.623[M + H]+756.2113756.2059757.21330.08* Hops, juniper berries
84Myricetin 3-O-glucosideC21H20O1333.220[M − H]480.0904479.0831479.08597.56* Hops, juniper berries
85MyricetinC15H10O833.345[M + H]+318.0376319.0449319.0427−5.24* Hops, juniper berries
86Quercetin 3-O-xylosyl-rutinosideC32H38O2033.419[M − H]742.1956741.1883741.19002.02Hops
87Quercetin 3’-O-glucuronideC21H18O1334.131[M + H]+478.0747479.0820479.0810−1.82Juniper berries
88Kaempferol 3,7-O-diglucosideC27H30O1634.512[M − H]610.1534609.1461609.14955.53* Hops, juniper berries
89Kaempferol 3-O-(2’’-rhamnosyl-galactoside) 7-O-rhamnosideC33H40O1934.644[M − H]740.2164739.2091739.21254.28* Hops, juniper berries
90Quercetin 3-O-glucosyl-xylosideC26H28O1635.920* [M − H]/[M + H]+596.1377595.1304595.13284.33* Hops, juniper berries
91Myricetin 3-O-arabinosideC20H18O1237.063[M + H]+450.0798451.0871451.0850−4.61Juniper berries
92Spinacetin 3-O-glucosyl-(1->6)-glucosideC29H34O1838.027[M − H]670.1745669.1672669.16891.98Juniper berries
93Myricetin 3-O-rhamnosideC21H20O1238.637[M − H]464.0955463.0882463.09126.85* Hops, juniper berries
94Isorhamnetin 3-O-glucoside 7-O-rhamnosideC28H32O1638.762[M − H]/* [M + H]+624.1690625.1763625.17720.78* Hops, juniper berries
95Quercetin 3-O-(6”-malonyl-glucoside)C24H22O1542.695[M − H]550.0959549.0886549.09012.88Hops
96Quercetin 3-O-arabinosideC20H18O1143.599[M − H]/*[M + H]+434.0849435.0922435.0925−0.02* Hops, juniper berries
97Kaempferol 3-O-(6’’-acetyl-galactoside) 7-O-rhamnosideC29H32O1643.705[M − H]636.1690635.1617635.16371.29Hops
985,4’-Dihydroxy-3,3’-dimethoxy-6:7-methylenedioxyflavone 4’-O-glucuronideC24H22O1451.110[M − H]534.1010533.0937533.09441.52Hops
99IsorhamnetinC16H12O753.313[M − H]316.0583315.0510315.05080.56* Hops, juniper berries
1006’’-O-AcetylgenistinC23H22O1110.791[M + H]+474.1162475.1235475.1202−6.50Juniper berries
1014’-Methoxy-2’,3,7-trihydroxyisoflavanoneC16H14O620.839[M + H]+302.0790303.0863303.0847−4.47Hops
1026’’-O-AcetyldaidzinC23H22O1021.965[M + H]+458.1213459.1286459.1279−0.27Hops
1033’-HydroxydaidzeinC15H10O541.172[M + H]+270.0528271.0601271.0592−3.12Juniper berries
1043’-HydroxygenisteinC15H10O645.660[M − H]286.0477285.0404285.0404−0.09* Hops, juniper berries
1053’,4’,5,7-TetrahydroxyisoflavanoneC15H12O650.083[M − H]288.0634287.0561287.05765.34* Hops, juniper berries
106Irisolidone 7-O-glucuronideC23H22O1251.143[M − H]490.1111489.1038489.10492.04Hops
1075,6,7,3’,4’-PentahydroxyisoflavoneC15H10O769.083[M − H]302.0427301.0354301.03757.30* Hops, juniper berries
1082’-HydroxyformononetinC16H12O574.940[M + H]+284.0685285.0758285.07662.04Hops
1092’,7-Dihydroxy-4’,5’-dimethoxyisoflavoneC17H14O678.145[M + H]+314.0790315.0863315.0846−2.58Juniper berries
1103’-HydroxymelanettinC16H12O678.609[M + H]+300.0634301.0707301.07070.58Juniper berries
111SativanoneC17H16O579.413[M − H]/*[M + H]+300.0998301.1071301.10690.77* Hops, juniper berries
112Dihydrobiochanin AC16H14O582.336[M + H]+286.0841287.0914287.09130.05Juniper berries
113EpisesaminC20H18O613.643[M − H]354.1103353.1030353.1019−4.36Juniper berries
114SecoisolariciresinolC20H26O646.713[M + H]+362.1729363.1802363.1780−5.44Hops
115Anhydro-secoisolariciresinolC20H24O546.747[M + H]+344.1624345.1697345.1678−5.38Hops
1167-HydroxymatairesinolC20H22O749.441[M − H]374.1366373.1293373.12971.93Juniper berries
117Lariciresinol-sesquilignanC30H36O1052.522[M − H]556.2308555.2235555.2231−0.48Juniper berries
118SyringaresinolC22H26O865.952[M − H]418.1628417.1555417.15610.46* Hops, juniper berries
119MatairesinolC20H22O677.250[M + H]+358.1416359.1489359.1470−4.74Juniper berries
120ConidendrinC20H20O677.756[M + H]+356.1260357.1333357.13442.21Hops
121ResveratrolC14H12O338.282[M + H]+228.0786229.0859229.08714.74Hops
122Piceatannol 3-O-glucosideC20H22O949.888[M − H]406.1264405.1191405.12073.00Juniper berries
1234’-Hydroxy-3,4,5-trimethoxystilbeneC17H18O478.253[M + H]+286.1205287.1278287.12871.87* Hops, juniper berries
1244-HydroxybenzaldehydeC7H6O226.826[M − H]122.0368121.0295121.03069.07Hops
Other polyphenols
1254-EthylguaiacolC9H12O255.500[M − H]152.0837151.0764151.07702.75Hops
1264-EthylcatecholC8H10O248.128[M − H]138.0681137.0608137.0607−0.35Hops
127p-AnisaldehydeC8H8O212.662* [M − H]/[M + H]+136.0524135.0451135.04564.06* Hops, juniper berries
1282,3-Dihydroxy-1-guaiacylpropanoneC10H12O513.126* [M − H]/[M + H]+212.0685211.0612211.06226.08* Hops, juniper berries
1294-HydroxycoumarinC9H6O312.589[M + H]+162.0317163.0390163.0375−8.98Hops
130CoumarinC9H6O217.642[M + H]+146.0368147.0441147.0429−1.87Hops
131MelleinC10H10O338.100[M − H]/*[M + H]+178.0630179.0703179.0688−6.79* Hops, juniper berries
132ScopoletinC10H8O456.063[M − H]192.0423191.0350191.03500.49Hops
133EsculetinC9H6O482.958[M + H]+178.0266179.0339179.0332−2.96Hops
134AnetholeC10H12O31.126[M + H]+148.0888149.0961149.0950−7.12Hops
135Acetyl eugenolC12H14O380.666[M − H]206.0943205.0870205.08833.91Juniper berries
Other polyphenols
136ArbutinC12H16O76.785[M − H]272.0896271.0823271.08362.33Juniper berries
137PyrogallolC6H6O36.957[M + H]+126.0317127.0390127.03910.29* Hops, juniper berries
138CatecholC6H6O212.335[M − H]110.0368109.0295109.03059.02Juniper berries
1393,4-DihydroxyphenylglycolC8H10O413.010[M − H]170.0579169.0506169.0503−2.96Hops
140Salvianolic acid GC20H18O1049.457[M − H]418.0900417.0827417.08310.75Juniper berries
141Oleoside 11-methylesterC17H24O119.458[M + H]+404.1319405.1392405.1364−1.45Hops
142Hydroxytyrosol 4-O-glucosideC14H20O810.443[M − H]316.1158315.1085315.1072−4.83Hops
1433,4-DHPEA-EDAC17H20O650.083[M − H]320.1260319.1187319.1179−3.03Hops
144p-HPEA-EDAC17H20O550.133[M − H]304.1311303.1238303.12544.55Hops
1453,4-DHPEA-ACC10H12O454.589[M − H]196.0736195.0663195.06671.86* Hops, juniper berries
Phenolic terpenes
146ThymolC10H14O29.593[M + H]+150.1045151.1118151.1108−6.67Juniper berries
147RosmanolC20H26O580.307[M + H]+346.1780347.1853347.1841−3.49Hops
148Carnosic acidC20H28O484.191[M − H]332.1988331.1915331.19354.86Hops
Non-phenolic metabolites
1491,3,5-TrimethoxybenzeneC9H12O341.900[M − H]168.0786167.0713167.07245.35* Hops, juniper berries
* Data presented in the table are from the sample indicated with an asterisk “*”. Also, the compound showing both modes of ionization [M − H]/[M + H]+), “*” mode of ionization belongs to the “*” sample.
Table 3. Quantification of polyphenolic compounds in hops and juniper berries samples by HPLC-PDA.
Table 3. Quantification of polyphenolic compounds in hops and juniper berries samples by HPLC-PDA.
No.Compounds NameChemical FormulaRT (min)Standard CurveHops (mg/g dw)Juniper (mg/g dw)Polyphenol Class
1Gallic acidC7H6O56.836y = 2531.9x + 122383.41 ± 0.02-Phenolic acids
2Protocatechuic acidC7H6O412.569y = 1824x − 161822.25 ± 0.01 b11.46 ± 0.03 aPhenolic acids
3Caftaric acidC13H12O913.774y = 3500.2x − 438220.72 ± 0.01-Phenolic acids
4p-hydroxybenzoic acidC7H6O320.24y = 1387.5x + 5575.11.87 ± 0.01 b3.12 ± 0.01 aPhenolic acids
5Cholrogenic acidC16H18O920.579y = 3043.6x + 4706.316.48 ± 0.03-Phenolic acids
6Caffeic acidC9H8O425.001y = 5622.4x + 239440.11 ± 0.01 a0.14 ± 0.01 aPhenolic acids
7Syringic acidC9H10O526.739y = 2900.6x + 650910.03 ± 0.01-Phenolic acids
8Coumaric acidC9H8O334.455y = 6418.4x + 60121-0.32 ± 0.01Phenolic acids
9CatechinC15H14O619.704y = 779.41x + 2373.39.03 ± 0.02 a8.47 ± 0.02 bFlavonoids
10Epicatechin gallateC22H18O1038.015y = 22958x − 266570.02 ± 0.01-Flavonoids
11Quercetin-3-O-galactosideC21 H20 O1240.134y = 23472x + 1850010.22 ± 0.01 b0.73 ± 0.01 aFlavonoids
12Quercetin-3-O-glucuronideC21H18O1340.659y = 20578x − 368880.15 ± 0.01 a0.07 ± 0.01 bFlavonoids
13Kaempferol-3-O-glucosideC21H20O1147.111y = 22405x − 337664.33 ± 0.02 a1.47 ± 0.02 bFlavonoids
14QuercetinC15H10O770.098y = 2585.7x − 292671.03 ± 0.01 a0.74 ± 0.01 bFlavonoids
15kaempferolC15H10O680.347y = 4425.8x − 1108410.44 ± 0.01 b3.37 ± 0.01 aFlavonoids
All data are the mean ± SD of three replicates. Means followed by different letters (a, b) within the same column are significantly different (p < 0.05) from each other. Data of hops and juniper berries are reported on a dry weight basis.

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Tang, J.; Dunshea, F.R.; Suleria, H.A.R. LC-ESI-QTOF/MS Characterization of Phenolic Compounds from Medicinal Plants (Hops and Juniper Berries) and Their Antioxidant Activity. Foods 2020, 9, 7.

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Tang J, Dunshea FR, Suleria HAR. LC-ESI-QTOF/MS Characterization of Phenolic Compounds from Medicinal Plants (Hops and Juniper Berries) and Their Antioxidant Activity. Foods. 2020; 9(1):7.

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Tang, Jiafei, Frank R. Dunshea, and Hafiz A. R. Suleria. 2020. "LC-ESI-QTOF/MS Characterization of Phenolic Compounds from Medicinal Plants (Hops and Juniper Berries) and Their Antioxidant Activity" Foods 9, no. 1: 7.

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