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Article

Phenolic and Volatile Composition of a Dry Spearmint (Mentha spicata L.) Extract

1
Department of Food Science, University of Parma, Parma 43125, Italy
2
Kemin Foods, L.C., 2100 Maury Street, Des Moines, IA 50317, USA
*
Author to whom correspondence should be addressed.
Molecules 2016, 21(8), 1007; https://doi.org/10.3390/molecules21081007
Received: 6 June 2016 / Revised: 25 July 2016 / Accepted: 27 July 2016 / Published: 3 August 2016

Abstract

:
The present paper reports a complete mass spectrometric characterization of both the phenolic and volatile fractions of a dried spearmint extract. Phenolic compounds were analysed by ultra-high performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC-ESI-MSn) and a total of 66 compounds were tentatively identified, being the widest phenolic characterisation of spearmint to date. The analysis suggests that the extract is composed of rosmarinic acid and its derivatives (230.5 ± 13.5 mg/g) with smaller amounts of salvianolic acids, caffeoylquinic acids, hydroxybenzoic acids, hydroxycinnamic acids, flavones, and flavanones. Head space solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) technique, that was applied to characterize the volatile fraction of spearmint, identified molecules belonging to different chemical classes, such as p-cymene, isopiperitone, and piperitone, dihydroedulan II, menthone, p-cymen-8-ol, and β-linalool. This comprehensive phytochemical analysis can be useful to test the authenticity of this product rich in rosmarinic acid and other phenolics, and when assessing its biological properties. It may also be applied to other plant-derived food extracts and beverages containing a broad range of phytochemical compounds.

Graphical Abstract

1. Introduction

Among the family of Lamiaceae (Labiatae), mint represents one of the most popular and cultivated officinal and aromatic plants [1]. The cultivation of mint is principally in temperate regions of Europe and Asia, but also in South Africa, Australia, and the United States.
Spearmint (Mentha spicata L.) is an aromatic plant that can be used fresh or as dried leaves or powder, as a seasoning and flavouring herb, or traditionally as an herbal tea. It is commonly used in traditional medicines as a remedy for gastrointestinal and respiratory problems. In addition, spearmint essential oil has economic relevance due to its use in perfumery, confectionary, and pharmaceutical preparations. Besides its flavouring properties, spearmint is also widely used as an antimicrobial agent and as a preservative in food, mainly on account of the phenolic and terpenoid content [2].
The volatile (non-polar) profile of traditional cultivars of spearmint essential oils is mainly constituted by carvone (22%–73%) and limonene (8%–31%), with smaller quantities of 1,8-cineole (4%–7%), menthone (1%–5%), menthol, eucalyptol, and other minor compounds. The profile varies based on plant variety, growth, climate conditions, and harvest time [3,4,5]. The antimicrobial activity of these spearmint essential oil components has been widely described in the literature. Volatile molecules are indeed produced by the plant, serving as a defence mechanism upon predator attack (i.e., pathogens and insects) [5].
Polar extracts of spearmint leaves are, on the contrary, characterised mainly by a high content of phenolic compounds such as rosmarinic acid, luteolin, and apigenin derivatives [6,7]. Some of these components have been shown to have antioxidant properties; therefore, Mentha spicata could also be considered an antioxidant source [7]. In fact, spearmint and spearmint extracts are often used as preservative agents to delay the oxidative degradation that occurs in food during processing or over time with storage [1]. More intriguingly, the anti-inflammatory properties of spearmint extracts rich in phenolic compounds have been demonstrated in vivo in rats [8].
Aqueous extracts from typical commercially grown spearmint lines reportedly contain 0%–6% rosmarinic acid on a dry weight basis [9,10]. However, based on the reported benefits of rosmarinic acid, spearmint lines were developed through selective-breeding techniques to contain higher levels of phenolic compounds such as rosmarinic acid [11]. Therefore, this study aimed to comprehensively characterise the phytochemical profile of a dried aqueous extract from these proprietary spearmint lines. The phenolic composition was fully examined by means of UHPLC-ESI-MSn, while the composition of the volatile fraction was investigated using head space solid-phase microextraction (HS-SPME)/GC-MS technique.

2. Results and Discussion

2.1. Characterization of the Phenolic Profile

The phenolic fraction of spearmint was fully characterised by means of UHPLC-ESI-MS operating in two complementary conditions. The comprehensive evaluation of the sample allowed for the tentative identification of a total of 66 compounds (Table 1), the widest phenolic characterisation of spearmint to date. More than 200 mass spectrum outputs were analysed for each analytical replicate and experimental condition. Among the classes of identified (poly)phenolic compounds in spearmint, rosmarinic acid derivatives and salvianolic acids were the most prevalent (Figure 1). Different flavones, flavanones, flavonols, phenolic acids, and lignans were also detected. In addition, other phytochemicals, such as organic acids were found.
The retention times and mass spectrum data, reported as peak assignments for the identified phytochemicals, are included in Table 1. Twelve of the 66 identified compounds were identified and quantified by comparison with reference standards. The remaining 54 compounds were tentatively identified based on the interpretation of their mass spectral behaviour obtained from MS2 and MS3 experiments, and by comparing with data from the literature.
The 54 compounds tentatively identified according to their mass spectral behaviour were quantified by comparison with reference compounds selected based on structural similarity and considering that the functional groups may strongly affect their ionisation properties (i.e., salvianolic acid J was quantified as salvianolic acid A, salvianolic acid E as salvianolic acid B, danshensu and its derivatives as caffeic acid, etc.). Accordingly, in this case, data reported in Table 2 must be considered as semi-quantification. Nevertheless, some compounds responded to the electro-spray ionisation in a unique manner relative to the reference standards used or did not reach the limit of quantification (LOQ) of the corresponding reference compound; therefore, they were not quantified to avoid miscalculation of the phenolic content of the spearmint extract.
The total amount of phenolic compounds of the evaluated spearmint extract calculated on the basis of UHPLC-ESI-MSn data was 262.97 ± 15.90 mg/g, which was in agreement with Dorman et al. [7], who reported a total phenolic content for Mentha spicata L. (spearmint) extract of 214 mg/g, expressed as gallic acid equivalents. More specifically, the sum of rosmarinic acid and other rosmarinic acid derivatives (such as sagerinic acid) in this extract was about the 88% (230.50 ± 13.50 mg/g) of the total amount of detected phenolics, followed by the sum of salvianolic acids (5.6% of total phenolics, 14.70 ± 1.19 mg/g) and caffeoylquinic acids (1.2% of total phenolics, 3.06 ± 0.27 mg/g). Hydroxycinnamic acids, including caftaric acid (an ester of caffeic and tartaric acids), represented about 1.1% of total phenolics (3.00 ± 0.36 mg/g). All of the other detected phenolic groups, such as flavonols, flavanones, flavones, hydroxybenzoic acids, and hydroxyphenylpropanoic acids represented approximately 1% of the total amount of phenolic compounds (0.01 to 0.99 mg/g).
Among the detected compounds, rosmarinic acid, a caffeic acid dimer, was identified by comparing the mass spectra obtained for the sample with those registered for a rosmarinic acid standard solution. This compound occurred at the highest concentration (173.76 ± 11.52 mg/g) and is approximately four-fold higher than the 4.6 mg/g reported for other water extracted spearmint lines [7]. Differences in the amount of rosmarinic acid of this extract with respect to other spearmint extracts are likely due to the selective-breeding techniques used for its production. However, rosmarinic acid concentrations could vary due to seasonal growth or extraction procedures. Rosmarinic acid is known to exert anti-inflammatory activities mainly due to its ability to inhibit lipoxygenases and cyclooxygenases, but it has also been shown to have anti-acetylcholinesterase, antioxidant, and antibacterial capabilities [28,29,30]. Furthermore, it was possible to observe the presence of several rosmarinic acid derivatives. In particular, significant amounts of sagerinic acid (8.93 ± 1.10 mg/g) and an isomer of sagerinic acid (peak 41; 40.05 ± 2.20 mg/g) were found. This is consistent with results obtained from analysis of lemon balm extracts [25], but have not been reported in the literature in water-extracted spearmint to date.
Other polar compounds in the spearmint extract included additional caffeic acid derivatives, such as salvianolic acids. Among this group of molecules, salvianolic acid A was the most abundant (7.79 ± 0.52 mg/g), followed by salvianolic acid B (1.35 ± 0.16 mg/g). Both were identified by means of reference compounds and served to identify their respective derivatives and isomers. Salvianolic acid D and F (dimers of caffeic acids), salvianolic acid J (a trimer of caffeic acid), and salvianolic acid E (a tetramer of caffeic acid), were all recognised by comparing the obtained fragmentations with those observed following analysis of extracts from Salvia miltiorrhiza roots [14]. All of these compounds displayed the characteristic mass spectra of salvianolic acids: neutral losses of one caffeic acid molecule (m/z 180) and a danshensu unit (m/z 198). Salvianolic acids have been reported in other members of the Lamaciae family although inconsistent between species. Within the Mentha species, data on salvianolic acid concentrations within water extracts is limited, with concentrations of less than 1% observed in some instances and slightly lower than the currently evaluated extract [6]. Danshensu (dyhydroxyphenyllactic acid), another caffeic acid derivative, as well as other danshensu-like compounds (peaks 53, 54, and 55) were identified on the basis of its molecular ion [M − H] (m/z 197) and its MS2 and MS3 fragments (m/z 179, 153 and 135) [14]. Moreover, a considerable amount of lithospermic acid (3.81 ± 0.26 mg/g), a caffeate trimer, was identified using a reference standard.
The presence of different hydroxycinnamic acids was observed in the first part of the chromatogram. This category was mainly represented by caftaric acid (2.18 ± 0.30 mg/g), followed by caffeic acid (0.71 ± 0.06 mg/g) and other minor components, such as dicaffeic acid and coumaric acid. The phenolic profile contained some compounds in the caffeoylquinic acid family, identified by their respective commercial standards (chlorogenic acid and neochlorogenic acid) or its characteristic fragmentation patterns (feruloylquinic acid). Small amounts of hydroxybenzoic acids were detected (0.57 ± 0.07 mg/g) and the presence of salicylic acid (peak 27) was also observed. Hydroxycinnaminic, hydroxybenzoic, and caffeoylquinic acids have been previously reported to be present in Mentha species with concentrations frequently below 1%, as observed for the current water-extracted spearmint [31].
Small amounts of flavones, flavonols, and flavanones were detected. Among the flavones, the most representative compound, in terms of quantity, was apigenin (0.19 mg/g) which was identified by comparing the obtained mass spectra with those reported in the literature [26]. Rutin, narirutin, and orientin were recognised using their respective commercial standards, while other compounds, such as luteolin-rutinoside, luteolin-hexoside, and luteolin-glucuronide, were identified by comparison of their relative mass spectra to those reported for other vegetables or natural extracts [20,24]. Rutin, luteolin, and several additional flavones have been reported previously in commercially available spearmint at levels similar to those reported for the current extract. However, the apigenin levels reported for the extract was four-fold greater than that previously reported, although less than 1% in both cases [7].

2.2. Characterisation of Volatile Composition

The volatile fraction of dried aqueous spearmint extract was characterised using the HS-SPME/GC-MS technique, which involved obtaining 59 different gas-chromatographic peaks (Figure 2). Peak identification was carried out by comparing recorded mass spectra with those present in the instrument libraries (NIST) and by using the LRI (Linear Retention Index) obtained on two different stationary phase columns (SUPELCOWAX 10 and BP5MS). The detected compounds were semi-quantified using toluene as internal standard (IS). All of the results are listed in Table 3.
Quantitatively, the volatile fraction of the spearmint extract examined had 34.64 ± 10.57 µg/100 mg of volatile compounds. In general, since this extract is water-extracted, the volatile fraction analysis yields percentages of components much lower than those reported in the literature for spearmint leaf material. Ketones were the most representative compounds in this fraction, constituting about 32% of the total volatile amount, followed by terpenoids at 20%. Aldehydes, esters, and furans were also detected at 18%–19% of the total volatile fraction. The highest quantitative individual compounds present in the volatile fraction of the tested spearmint were as follows: p-cymene (3.39 ± 0.98 µg/100 mg), isopiperitone and piperitone (2.37 ± 0.94 and 0.69 ± 0.21 µg/100 mg, respectively), dihydroedulan II (two signals: 2.27 ± 0.66 and 0.69 ± 0.09 µg/100 mg), menthone (2.18 ± 0.72 µg/100 mg), p-cymen-8-ol (1.96 ± 0.74 µg/100 mg), and β-linalool (1.52 ± 0.43 µg/100 mg). These molecules confer characteristic aromatic notes to the product, such as minty, phenolic, and floral flavours [32].
Traditional mint presents a really distinctive flavour, mostly due to the presence of a particular alcoholic cyclic terpene: menthol. This molecule, besides being well-known as a primary aromatic compound, is used in medicine for gastro-intestinal disorders [44]. In our sample, menthol was not detected. This can be attributed to the fact that the chemical composition of mint leaves, as the composition of essential oil, can be dependent on different agronomical factors as plant maturity, variety, growth region, climatic conditions, and genetics [3]. In contrast, other typical spearmint volatile fraction components, such as menthone, carvone, eugenol, piperitone, and isopiperitone, were detected. These volatiles have been already reported in peppermint and spearmint essential oils as being responsible for the typical mint notes [45,46].
Carvone and piperitone are two oxygenated terpenoids generated during the biosynthesis of terpenes, which starts from geranyl pyrophosphate, and they are derived from d-limonene. In particular, carvone, with its characteristic aromatic note of mint and liquorice, has different applications, such as repellent, medical, and flavour preparation [5]. However, the carvone level recorded in the spearmint extract is 200-fold lower than that previously reported in an aqueous extract of peppermint (~0.2 vs. 40 µg/100 mg extract), another member of the Lamiaceae family [47]. This low carvone level, in agreement with Narasimhamoorthy et al. [11], may cause lesser mint notes in this line relative to native spearmint lines, which could support its palatability in food and beverage applications.
Among ketones, the most abundant were menthone (2.18 ± 0.72 µg/100 mg) and β-damascenone (0.66 ± 0.17 µg/100 mg), which were consistent with results found by Rohloff et al. [46] and Ka et al. [37] for spearmint and peppermint. The spearmint volatile fraction was also rich in alcohols. In addition to the p-cymen-8-ol (1.96 ± 0.74 µg/100 mg) as identified in Mentha essential oils [4], detectable amounts of 2-acetyl-4-methylphenol, thymol, carvacrol, and p-menthen-1-ol were also observed.
In addition to ketones, terpenoids, and alcohols, several compounds belonging to different chemical classes represented the remaining 18%–19% of the volatile fraction of the dried spearmint powder. Among these minor volatile compounds, dihydroedulan II (two signals: 2.27 ± 0.66 and 0.69 ± 0.09 µg/100 mg) was identified. Dihydroedulan II is a benzopyran compound that has already been detected in the essential oil of Ocimum basilicum (basil), another member of the Lamiaceae family [48] but not previously reported in Mentha spicata. In accordance with data from Rohloff [46] in peppermint, detectable amounts of R-(+)-menthofuran (0.16 ± 0.05 µg/100 mg) were observed. Slight quantities of aldehydes, in particular furfural (0.52 ± 0.12 µg/100 mg) and 5-methyl furfural (0.18 ± 0.03 µg/100 mg), were also detected. Similarly, Ka et al. [37] identified these compounds in distilled extracts from some medicinal plants, such as Angelica tenuissimae, pine needles from Pinus sylvestris, and leaves of sweet flags (Acorus gramineus).

3. Materials and Methods

3.1. Materials

Methanol, acetonitrile, formic acid, toluene, and C8–C20 alcane solution were purchased from Sigma-Aldrich (Milan, Italy). Ultrapure water from MilliQ system (Millipore, Bedford, MA, USA) was used throughout the experiment. The proprietary spearmint extract was manufactured by Kemin Foods, L.C. (Des Moines, IA, USA) as described [11,49]. In brief, the spearmint extract was prepared by microwave drying within one hour of harvest followed by extraction of the dried spearmint leaf with acidified water.

3.2. Characterization and Quantification of Phenolic Fraction by UHPLC-ESI-MSn

The extraction of phenolic compounds was performed on 200 mg of spearmint extract by adding 1 mL of 80% aqueous methanol acidified with formic acid (1%), according to Sánchez-Salcedo et al. (2015) [50]. The solution was shaken in an ultrasonic bath at room temperature for 25 min. The mixture was then centrifuged at 10,480 g for 5 min at room temperature. In order to obtain an exhaustive extraction of the phenolic fraction, two additional extractions were performed on the same sample. The three supernatants were pooled before UHPLC-ESI-MSn analyses. Each sample was extracted in quadruplicate.
Methanolic extracts of spearmint were analyzed using an Accela UHPLC 1250 equipped with a linear ion trap-mass spectrometer (MS) (LTQ XL, Thermo Fisher Scientific Inc., San Jose, CA, USA) fitted with a heated-electrospray ionization probe (H-ESI-II; Thermo Fisher Scientific Inc.). Separations were performed using a BlueOrchid C18 column (50 × 2 mm, 1.8 µm particle size, Knauer, Berlin, Germany). The total volume injected was 5 µL and the column oven temperature was 30 °C. Two MS experiments in negative mode were performed according to a previous protocol [51]. Optimal parameters for epicatechin analysis (Experimental Conditions 1) were carried out using the following conditions. The MS was operated using a capillary temperature equal to 275 °C, while the source heater temperature was set to 200 °C. The sheath gas flow was operated at 40 units, while both auxiliary and sweep gas were set to 5 units. The source voltage was 4 kV. The capillary and tube lens voltages were −42 and −118 V, respectively. Elution was performed at a flow rate of 0.3 mL/min. The gradient started with 99% of 0.1% aqueous formic acid, keeping isocratic conditions for 2 min, followed by a 10 min linear gradient of acetonitrile in 0.1% formic acid which started at 1% and was increased to 40%. The acidified acetonitrile was increased to 80% between minutes 12 and 13 min, and maintained for 3 min, followed by 4 min at the starting conditions to re-equilibrate the column. Analyses were carried out using full scan, data-dependent MS3 scanning from m/z 100–1500, with collision-induced dissociation (CID) equal to 30 (arbitrary units). Pure helium gas was used for CID.
The second experimental framework utilized MS with conditions optimized for rosmarinic acid analysis (Experimental Conditions 2). The capillary temperature was set to 275 °C, while the source heater temperature was 50 °C. The sheath gas flow was operated at 40 units, while auxiliary and sweep gas were set to 5 and 0 units, respectively. The source voltage was operated at 4 kV. The capillary and tube lens voltages were −26 and −78 V, respectively. Analyses were carried out using full scan, data-dependent MS3 scanning from m/z 100–1500, with CID equal to 30 (arbitrary units). The chromatographic conditions were identical to those used for the preliminary phenolic analyses.
Quantification was performed using selected ion monitoring mode (SIM) by selecting the relative base peak at the corresponding mass to charge ratio (m/z) under Experimental Conditions 2, based on rosmarinic acid. Different dilutions of the extract in 0.1% aqueous formic acid (dilution factors ranging from 10–1000) were used to avoid signal saturation and quantify within the linearity range of the reference compounds.

3.3. Volatile Extraction and Characterization by Head Space Solid Phase Microextraction (HS-SPME) Coupled with GC-MS Technique

The volatile fraction of the spearmint sample was characterized following the protocol of Cirlini et al. (2012) [34] with slight modifications. Briefly, 100 mg of spearmint extract were placed in a 30 mL glass vial. For each SPME analysis, 100 µL of an aqueous toluene standard solution (348 mg/L) were added to the sample. The vial was stirred in a warm water bath at 35 °C for 45 min. For each sample, a SPME fibre was inserted in the sample head space and the sample was stirred at constant speed. The fibre was then removed and inserted into the GC-MS injector for 2 min for the desorption of the volatiles. The analysis was done in duplicate.
The silica fibre adopted for the analysis was coated with 50/30 µm of divinylbenzene-carboxen-polymethylsiloxane (DVB/Carboxen/PDMS; Supelco, Bellefonte, PA, USA). Before starting the analyses, the fibre was conditioned by inserting it into the GC/MS injector at 230 °C for at least 10 min. All the analyses were performed on a Thermo Scientific Trace 1300 gas-chromatograph coupled to a Thermo Scientific ISQ mass spectrometer equipped with electronic impact (EI) source. The separation of analytes was performed on a SUPELCOWAX 10 capillary column (Supelco, 30 m × 0.25 mm, f.t. 0.25 µm) using helium as carrier gas. The injector temperature was set at 230 °C and splitless mode was used as the injection modality keeping the valve closed for 2 min. The oven temperature started at 50 °C for 3 min and was increased to 200 °C (5 °C/min). The final oven temperature (200 °C) was maintained for 18 min and the auxiliary temperature was set at 230 °C. Full scan mode was chosen as the acquisition mode (m/z 41–500).
The tentative identification of the volatiles was performed by comparison of the obtained mass spectra with those present in the instrument libraries (NIST). Furthermore, in order to obtain a more confident identification, the linear retention indices (LRI) were calculated on the basis of a C8–C20 alcane solution analyses. The same procedure was repeated utilizing a different stationary phase column, BP5MS (30 m × 0.25 mm, with 0.25 µm film thickness, SGE Analytical Science, Milan, Italy), on which both the alcane standard solution and spearmint sample were analysed maintaining the same extraction and instrumental conditions as previously described. The semi-quantification of all detected gas-chromatographic signals was performed on the basis of the use of an internal standard (toluene).

4. Conclusions

This study reported the comprehensive characterisation of a spearmint extract developed utilizing selective breeding to yield high rosmarinic acid and other phenolic components, with a particular emphasis on the (poly)phenolic and volatile fraction. The use of two different chromatographic techniques, UHPLC, and GC, both coupled to mass spectrometry, allowed for the elucidation of the fingerprint of these two different fractions.
In particular, the use of the UHPLC-ESI-MSn technique allowed us to fully unravel the (poly)phenolic profile of dried spearmint. A total of 66 different molecules were identified on the basis of their characteristic MSn spectra, with 53 of them semi-quantified. The total amount of phenolic compounds was about 260 mg/g extract, which demonstrated that the spearmint extract is a matrix rich in phenolics. The major phenolic compounds in the spearmint extract were represented by rosmarinic acid and its derivatives (88% of the total phenolics). Among the other molecules identified, different salvianolic, caffeoylquinic, hydroxybenzoic, and hydroxycinnamic acids were detected, as well as small amounts of flavones, flavanones, and flavonols. The results of the spearmint extract volatile profile, analysed using the HS-SPME/GC-MS technique, suggested the extract was mainly represented by 59 volatile compounds belonging to different chemical classes, in particular ketones and terpenoids. Attending to the characteristics of plant extracts, the phytochemical composition of this matrix could vary from season to season and even from lot to lot. Regardless of normal variation, these particularly sensitive techniques would allow testing of the authenticity of the product and assist when evaluating its biological and essential properties. On the other hand, the analysis of a higher number of samples, considering factors such as seasonality as well as agricultural practices and crop location would be quite interesting. This fact could be tackled in further studies, although a reductive approach would be needed since it is not feasible to perform this kind of comprehensive identification for large batches of samples.

Acknowledgments

This study was partly supported by Kemin Food, L.C.

Author Contributions

D.D.R., K.A.H. and K.M.N. conceived and designed the experiments; M.C., P.M. and M.T. performed the experiments; M.C., P.M. and M.T. analyzed the data; D.D.R., K.A.H., K.M.N. and C.D. contributed reagents/materials/analysis tools; M.C., P.M., M.T. and D.D.R. wrote the paper. K.M.N is currently with Midwest Center for Metabolic and Cardiovascular Research, 489 Taft Ave., Suite 202, Glen Ellyn, IL, 60137, USA; [email protected]

Conflicts of Interest

The authors declare no conflict of interest. The founding sponsors had no role in the collection, analyses, or interpretation of data, and in the writing of the manuscript.

Abbreviations

The following abbreviations are used in this manuscript:
CIDcollision-induced dissociation
GC-MSgas chromatography-mass spectrometry
LIRlinear retention indices
HS-SPMEhead space solid-phase microextraction
UHPLC-ESI-MSnultra-high performance liquid chromatography-electrospray ionization-mass spectrometry

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  • Sample Availability: Samples are available from the authors.
Figure 1. Main spearmint phenolics identified in the extract. Peak numbers are based on Table 1.
Figure 1. Main spearmint phenolics identified in the extract. Peak numbers are based on Table 1.
Molecules 21 01007 g001
Figure 2. HS-SPME/GC-MS chromatogram of the spearmint extract analyzed. Numbers correspond with the codes indicated at Table 3.
Figure 2. HS-SPME/GC-MS chromatogram of the spearmint extract analyzed. Numbers correspond with the codes indicated at Table 3.
Molecules 21 01007 g002
Table 1. Identification of phytochemical compounds by UHPLC-MSn in negative mode under different MS operating conditions.
Table 1. Identification of phytochemical compounds by UHPLC-MSn in negative mode under different MS operating conditions.
IDCompoundsRT (min)[M − H] (m/z)MS2 Ion Fragments (m/z) aMS3 Ion Fragments (m/z) aExp. 1 cExp. 2 cIdentification d
1Quinic acid0.57191173 b, 111, 127, 85, 93111, 67xxStd
2l-malic acid0.67133115, 87 x[12]
3Citric acid0.77191111, 173111, 67xx[13]
4Dihydroxyphenyllactic acid (Danshensu)2.61197179, 73, 153135x [14]
5Protocatechuic acid hexoside2.75315153, 109, 225109x [15]
6Dihydroxyphenylacetic acid3.35167123 x[16]
7Hydroxybenzoic acid4.12137137, 93 x[17]
8Caftaric acid4.40311149, 179, 243, 135103, 87, 131, 59, 149 xStd
9Hydroxyphenyllactic acid4.47181163, 135, 73119xx[18]
10Luteolin-8-C-glucoside (orientin)4.83447357, 327 Std
113′-Caffeoylquinic (neochlorogenic acid)4.96353191, 179, 135, 173127, 173, 85, 93 xStd
12THDBCHMCA f5.42295163, 113118 x[19]
13Rosmanol5.44345299179, 119, 143, 113, 161 x[20]
14Coumaric acid5.52163119 x [17]
15Salvianolic acid F5.56313269, 203, 159159, 109, 254, 269x [14]
16Dicaffeic acid5.74341281, 251, 179, 221, 323179, 221, 135xx[21]
175′-Caffeoylquinic (chlorogenic acid)6.17353191, 179127, 173, 85, 83 xStd
18Caffeic acid6.25179135135xxStd
19Ferulic acid derivative6.88489193, 235, 295, 265149, 134, 178x Std
20Rosmarinic acid derivative6.92377359161, 179, 197, 223xxStd
21Rosmarinic acid derivative7.08377359161, 179, 197, 223xxStd
22Feruloylquinic acid7.15367173, 193, 19193, 111, 155, 71xx[22]
23Tetrahydroxy-dimethoxyflavone-hexoside7.29507327, 345, 477, 489312, 167, 295x [23]
24Danshensu derivative7.40527197, 179, 483179, 73x [14]
25Rosmarinic acid-O-caffeic acid7.61539359, 495, 341, 179161, 179, 197, 223xx[14]
26Salvianolic acid J/isomer7.82537339229, 295xx[14]
27Salicylic acid7.8513793, 137 x[17]
28Rosmarinic acid-rutinoside7.96667359, 487161, 197, 179, 223x Std
29Quercetin-rutinoside (rutin)8.07609301, 343, 271, 255, 179179, 151, 257, 273xxStd
30Salvianolic acid J/isomer8.08537493, 295, 339295, 313, 383xx[14]
31Luteolin-rutinoside8.16593285241, 285, 175, 199, 217xx[24]
32Rosmarinic acid-O-hexoside8.25521359161, 197, 179, 223xxStd
33Luteolin-hexoside8.26447285285, 241, 199, 175, 217x [24]
34Luteolin-glucuronide8.3461285285, 241xx[20]
35Salvianolic acid B/E/isomer8.43717519, 475, 339, 537475, 339, 365xx[14]
36Narirutin (Naringenin-7-O-rutinoside)8.45625 (579) e579271xxStd
37Salvianolic acid D8.53417373, 175, 273, 399175, 197, 223 x[14]
38Sagerinic acid8.66719359, 539, 521, 341161, 179, 197, 223x [16]
39Salvianolic acid E8.78717519, 537, 555, 673, 339339, 321, 295, 229xx[14]
40Rosmarinic acid8.86359161, 179, 197, 223161, 133xxStd
41Sagerinic acid isomer8.99719359161, 179, 197, 223 x[25]
42Salvianolic acid A derivative9.08897493, 295295, 313, 179 xStd
43Lithospermic acid9.44537493, 359359, 313, 295xxStd
44Salvianolic acid B9.61717519, 321321, 339, 279, 197, 179xxStd
45Dehydro-Rosmarinic acid9.70343161, 179, 135, 223, 197161, 133xxStd
46Salvianolic acid B/E/isomer9.75717519, 357, 555, 673, 321321, 357, 339xx[14]
47Rosmarinic acid-dihexoside9.83683521359, 161, 197, 223x Std
48G(8-O-4)5H9.88373179, 161, 135, 355, 197135, 161x [14]
49Salvianolic acid A10.02493295, 313, 383, 203159, 277, 109, 267xxStd
50Acacetin derivative10.12637591, 283283, 268xx[18]
51Salvianolic acid A isomer10.25493295, 331, 383159, 277, 109, 267xx[19]
52Rosmarinic acid derivative10.70551519, 359, 313339xx[20]
53Danshensu derivative10.87689527, 491197, 179, 347, 161xx[14]
54Danshensu derivative10.90691529, 493, 511197, 179, 349, 151xx[14]
55Danshensu derivative11.07689527197, 179, 347xx[14]
56Rosmarinic acid derivative11.07691359, 511, 341, 529161, 179, 197, 223x Std
57Apigenin11.17269269, 225, 149, 241181, 197, 225, 183x [26]
58Salvianolic acid A isomer11.22493359, 357, 313161, 179, 197, 223xx[19]
59Cyclolariciresinol11.26359345, 161329, 326xx[27]
60Salvianolic acid B derivative11.40879519, 699, 339339x [25]
61Rosmarinic acid derivative12.33571525341, 359, 161, 179, 221 xStd
62Rosmarinic acid derivative12.69525359, 341, 161, 179161, 179, 197, 223x Std
63Rosmarinic acid derivative13.04507359, 341, 179161, 179, 197, 223xxStd
64Rosmarinic acid derivative13.24849359, 687, 669161, 179, 197, 223xxStd
65Acacetin13.54283268, 269268, 269, 240xx[18]
66Rosmarinic acid derivative13.82507359, 341, 179161, 179, 197, 223xxStd
a Fragment ions are listed in order of relative abundances; b MS2 ions in bold were those subjected to MS3 fragmentation; c Exp. 1, detected under experimental condition 1 (epicatechin); Exp. 2, experimental condition 2 (rosmarinic acid); d Identification means identification mode: [Reference number] or Std (compound identified by comparing retention times and MS data with those of reference compounds). Some compounds have been considered “derivatives” since parts of their spectra match those of their corresponding parent compounds but they cannot be fully identified; e The molecular ion is a formic acid adduct (+46); f THDBCHMCA: 1,2,6,7-tetrahydroxy-5H-dibenzo[a,d]cycloheptene-5-methyl-11-carboxylic acid.
Table 2. Quantitative results (mg/g sample) for polyphenolic fraction of the spearmint extract analyzed.
Table 2. Quantitative results (mg/g sample) for polyphenolic fraction of the spearmint extract analyzed.
ID aCompoundsQuantified as…Concentration (mg/g)
4Dihydroxyphenyllactic acid (Danshensu)Caffeic acid0.77 ± 0.09
5Protocatecuic acid hexosideCaffeic acid0.04 ± 0.00
7Hydroxybenzoic acidCaffeic acid0.57 ± 0.07
8Caftaric AcidCaftaric acid2.18 ± 0.30
9Hydroxyphenyllactic acidCaffeic acid0.07 ± 0.00
10Luteolin-8-C-glucoside (orientin)Luteolin-4-glucoside0.02 ± 0.00
113′-Caffeoylquinic (neochlorogenic acid)3′-Caffeoylquinic b1.79 ± 0.22
14Coumaric acidCaffeic acid0.03 ± 0.00
15Salvianolic Acid FCaffeic acid0.01 ± 0.00
16Dicaffeic acidCaffeic acid0.09 ± 0.00
175′-Caffeoylquinic (chlorogenic acid)5′-Caffeoylquinic b1.16 ± 0.08
18Caffeic acidCaffeic acid0.71 ± 0.06
20Rosmarinic acid derivativeRosmarinic acid2.17 ± 0.25
21Rosmarinic acid derivativeRosmarinic acid1.61 ± 0.11
22Feruloylquinic acid3′-Caffeoylquinic0.11 ± 0.00
24Danshensu derivativeCaffeic acid0.01 ± 0.00
25Rosmarinic acid-O-caffeic acidRosmarinic acid0.05 ± 0.00
26Salvianolic acid J/isomerSalvianolic acid A1.84 ± 0.17
28Rosmarinic acid-rutinosideRosmarinic acid0.17 ± 0.00
29Quercetin-rutinoside (rutin)Rutin b0.01 ± 0.00
30Salvianolic acid J/isomerSalvianolic acid A0.36 ± 0.05
31Luteolin-rutinosideLuteolin-4-glucoside0.17 ± 0.01
32Rosmarinic acid-O-hexosideRosmarinic acid0.28 ± 0.03
33Luteolin-hexosideLuteolin-4-glucoside0.02 ± 0.00
34Luteolin-7-glucuronideLuteolin-4-glucoside0.13 ± 0.00
35Salvianolic acid B/E/isomerSalvianolic acid B0.41 ± 0.05
36Narirutin (Naringenin-7-O-rutinoside)Narirutin b0.04 ± 0.01
37Salvianolic Acid DRosmarinic acid0.29 ± 0.02
38Sagerinic AcidRosmarinic acid8.93 ± 1.10
39Salvianolic Acid ESalvianolic acid B0.16 ± 0.02
40Rosmarinic AcidRosmarinic acid b173.76 ± 11.52
41Sagerinic Acid isomerRosmarinic acid40.05 ± 2.20
42Salvianolic Acid A derivativeSalvianolic acid A1.44 ± 0.30
43Lithospermic AcidLithospermic acid b3.81 ± 0.26
44Salvianolic Acid BSalvianolic acid B b1.35 ± 0.16
45Dehydro-Rosmarinic AcidRosmarinic acid0.52 ± 0.01
46Salvianolic acid B/E/isomerSalvianolic acid B0.30 ± 0.03
47Rosmarinic acid-dihexosideRosmarinic acid0.16 ± 0.01
49Salvianolic Acid ASalvianolic acid A b7.79 ± 0.52
51Salvianolic Acid A isomerSalvianolic acid A0.31 ± 0.06
52Rosmarinic acid derivativeRosmarinic acid0.28 ± 0.02
53Danshensu derivativeCaffeic acid0.06 ± 0.00
54Danshensu derivativeCaffeic acid0.03 ± 0.00
55Danshensu derivativeCaffeic acid0.05 ± 0.00
56Rosmarinic acid derivativeRosmarinic acid0.10 ± 0.01
57ApigeninDaidzein0.19 ± 0.01
58Salvianolic Acid A isomerSalvianolic acid A0.69 ± 0.02
60Salvianolic Acid B derivativeSalvianolic acid B0.05 ± 0.00
61Rosmarinic acid derivativeRosmarinic acid0.67 ± 0.04
62Rosmarinic acid derivativeRosmarinic acid0.09 ± 0.00
63Rosmarinic acid derivativeRosmarinic acid0.01 ± 0.00
64Rosmarinic acid derivativeRosmarinic acid1.30 ± 0.16
66Rosmarinic acid derivativeRosmarinic acid0.09 ± 0.00
Hydroxybenzoic acids c0.61 ± 0.08
Hydroxycinnamic acids3.00 ± 0.36
Caffeoylquinic acids3.06 ± 0.27
Hydroxyphenylpropanoic acids0.99 ± 0.10
Rosmarinic acid derivatives230.50 ± 13.5
Salvianolic acids14.70 ± 1.19
Flavones0.53 ± 0.02
Flavonols0.01 ± 0.00
Flavanones0.04 ± 0.01
Total Phenolics262.97 ± 15.90
a See Table 1 for peak assignment; b Quantified by comparison with its corresponding standard; c hydroxybenzoic acids include compound 5 and 7; hydroxycinnamic acids, compounds 8, 14, 16, and 18; caffeoylquinic acids, 11, 17, and 22; hydroxyphenylpropanoic acids, 4, 9, 24, and 5355; rosmarinic acid derivatives, 20, 21, 25, 28, 32, 37, 38, 40, 41, 45, 47, 52, 56, 6164, and 66; salvianolic acids, 15, 26, 30, 35, 39, 42, 44, 46, 49, 51, 58, and 60; flavones, 31, 33, 34, and 57; flavonols, 29; and flavanones, 36. Mean (n = 3) ± SD.
Table 3. Identification of volatile compounds from the spearmint extract, with relative aromatic notes, calculated LRIs, identification methods, references, and relative amounts.
Table 3. Identification of volatile compounds from the spearmint extract, with relative aromatic notes, calculated LRIs, identification methods, references, and relative amounts.
IDIdentificationFlavour Note [32]LRI-WaxLRI-BP5 aIdentification MethodRef.Concentration (µg/100 mg)
1EthylbenzenePrunus1127871MS + LRI[33]0.04 ± 0.01
2d-LimoneneSweet, citrus and peely12001024MS + LRI[34]0.04 ± 0.01
3CosmeneDahlia, Laurus nobilis12191006MS + LRINIST0.24 ± 0.08
4Cosmene (isomer) 12521142MS + LRINIST0.41 ± 0.03
5o-cymeneLavander and cypress oil12741022MS + LRI[35]0.06 ± 0.01
6Methyl-heptenoneFruity, apple, musty, ketonic and creamy1343 MS 0.05 ± 0.01
7(z)-3-hexen-1-olGreen, grassy, melon rind-like1387853MS + LRI[36]0.07 ± 0.01
8Amyl ethyl carbinolEarthy1395996MS 0.29 ± 0.09
9p-cymenenePhenolic14441090MS + LRI[35]3.39 ± 0.98
10Amyl vinyl carbinolEarthy1453979MS + LRI[34]0.46 ± 0.11
11FurfuralBready1473828MS + LRI[20]0.52 ± 0.12
12α-ionenePlum1485 MS 0.13 ± 0.01
13Dihydroedulan II(not reported)14961292MS + LRI[37]0.69 ± 0.09
14Dihydroedulan II(not reported)15261297MS + LRI[37]2.27 ± 0.66
15β-linaloolFloral15511099MS + LRI[38]1.52 ± 0.43
16(R)-(+)-menthofuranMinty15651159MS + LRI[39]0.16 ± 0.05
175-methylfurfuralCaramellic1582957MS + LRI[38]0.18 ± 0.03
18α-iononeFloral15901428MS + LRI[33]0.14 ± 0.02
19(not identified) 1602 0.27 ± 0.08
20HotrienolSweet tropical16151105MS + LRI[40]0.38 ± 0.19
21trans-p-metha-2,8-dienolMinty16321121MS + LRI[35]0.12 ± 0.03
22SafranalWoody, spicy, phenolic, camphoreous16531196MS 0.53 ± 0.13
233-furanmethanolTobacco1667851MS + LRI[41]0.18 ± 0.01
24Tetramethyl-indane(not reported)1676 MS 0.42 ± 0.09
25(not identified) 1686 0.33 ± 0.04
26Ethyl cyclopentenoloneCaramellic16911087MS 0.75 ± 0.18
27p-menthen-1-olFloral, minty, eucalyptus1701 MS 0.65 ± 0.19
284,7-dibenzofuran(not reported)1714 MS 0.33 ± 0.06
29MenthoneMentholic17351148MS + LRI[35]2.18 ± 0.72
30CamphorCamphoreous17481145MS + LRI[35]0.20 ± 0.02
312-piperidin methenamine(not reported)1759 MS 0.19 ± 0.08
321-(1-butenyl)pyrrolidine(not reported)1783 MS 0.17 ± 0.05
33Methyl salicylateMinty17851205MS + LRI[33]0.21 ± 0.13
34trans-geraniolFloral18041377MS + LRINIST0.10 ± 0.03
35TeresantalolMagnolia18161205MS 0.52 ± 0.12
36β-damascenoneWoody, sweet, fruity, earthy18281381MS + LRI[38]0.66 ± 0.17
375-isoproprenyl-2-methylcyclopent-1-enecarboxaldehyde(not reported)1834 MS 0.43 ± 0.08
38CalameneneHerbal18391525MS + LRI[33]0.34 ± 0.11
39PiperitenoneHerbal, minty18491268MS + LRI[35]0.69 ± 0.21
40p-cymen-8-olSweet, fruity, cherry, coumarin18571175MS + LRI[33]1.96 ± 0.74
41Exo-2-hydroxy cineoleEucalyptus, basilicum1864 MS 0.36 ± 0.01
423,6-dimethyl-phenyl-1,4-diol(not reported)1868 MS 0.44 ± 0.02
43LongipineneHinoki, cypress18841350MS + LRI[42]0.74 ± 0.01
44IsopiperitenoneMinty19321340MS + LRINIST2.37 ± 0.94
45Damascenone (isomer) 1948 MS 0.56 ± 0.12
46Mint lactoneSweet, creamy, coumarinic and coconut1967 MS 0.46 ± 0.03
47α,β-dihydro-β-iononeWoody19791406MS 1.17 ± 0.69
48SeudenoneNutty19901050MS + LRINIST0.50 ± 0.19
49Dihydroxy-durene(not reported)19981322MS 0.31 ± 0.23
50CinerolonMyrthus20111403MS 0.64 ± 0.43
51CarvoneMinty, licorice20541239MS + LRI[33]0.18 ± 0.07
521-acetoxy-p-menth-3-oneMinty2114 MS 0.16 ± 0.05
532,6-diisopropyl naphtalene(not reported)2144 MS 0.33 ± 0.08
54(naphtalene derivative) 2158 MS 0.15 ± 0.05
55EugenolSpicy21641354MS + LRI[35]0.75 ± 0.44
564-ethylphenolPhenolic21711175MS + LRI[38]0.17 ± 0.01
57ThymolHerbal21791289MS + LRI[35]0.62 ± 0.29
582-acetyl-4-methylphenolSweet heavy floral herbal21901180 [43]0.95 ± 0.41
59CarvacrolSpicy22041298MS + LRI[35]0.12 ± 0.03
a No value means not found in literature. Mean (n = 2) ± SD.

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Cirlini, M.; Mena, P.; Tassotti, M.; Herrlinger, K.A.; Nieman, K.M.; Dall’Asta, C.; Del Rio, D. Phenolic and Volatile Composition of a Dry Spearmint (Mentha spicata L.) Extract. Molecules 2016, 21, 1007. https://doi.org/10.3390/molecules21081007

AMA Style

Cirlini M, Mena P, Tassotti M, Herrlinger KA, Nieman KM, Dall’Asta C, Del Rio D. Phenolic and Volatile Composition of a Dry Spearmint (Mentha spicata L.) Extract. Molecules. 2016; 21(8):1007. https://doi.org/10.3390/molecules21081007

Chicago/Turabian Style

Cirlini, Martina, Pedro Mena, Michele Tassotti, Kelli A. Herrlinger, Kristin M. Nieman, Chiara Dall’Asta, and Daniele Del Rio. 2016. "Phenolic and Volatile Composition of a Dry Spearmint (Mentha spicata L.) Extract" Molecules 21, no. 8: 1007. https://doi.org/10.3390/molecules21081007

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