Analysis of Polyphenolic Composition of a Herbal Medicinal Product—Peppermint Tincture

The pharmacological activity of peppermint leaf (Menthae piperitae folium) for medical use is mainly attributed to the presence of essential oil, which, according to the European Pharmacopoeia (Ph. Eur.), should constitute not less than 12 mL/kg of raw material. The content of polyphenols in peppermint-based preparations, except peppermint leaf dry extract, has not yet been considered as an essential parameter in the pharmacopeial assessment of peppermint quality. This study concerns the evaluation of the presence of representatives of polyphenolic compounds in 23 commercial peppermint tinctures (ethanolic extracts) purchased in pharmacies in Poland. The non-volatile polyphenolic fraction was investigated, and the presence of flavonoids and phenolic acids was quantified. High performance liquid chromatography coupled with a diode-array detector (HPLC-DAD) and an electrospray ionization mass spectrometer (U(H)PLC-ESI-MS) were used in the experiment. The study showed that eriocitrin, luteolin-7-O-rutinoside, and rosmarinic acid were the main polyphenolic components of the peppermint tinctures, as previously reported for peppermint leaf. Despite this, the research shows the extremely diverse content of the mentioned compounds in analyzed commercial medicinal products. In light of these results, it seems that the pharmacopeial assessment for the peppermint leaf (Ph. Eur.) and peppermint tincture (Polish Pharmacopoeia (FP)) requires correction and supplementation.


Introduction
The importance of polyphenols (flavonoids, phenolic acids, depsides, stilbenes, lignans, tannins, etc.), common compounds in the plant kingdom, is widely known in health care. These bioactive secondary metabolites have been an inexhaustible source of scientific research including the determination of their chemical structure and diverse biological properties [1]. The most commonly used polyphenols in treatment are flavonoids (2-phenyl-benzo-gamma-pyrone) as well as phenolic acids and their esters (depsides) [2]. Mentha × piperita L. fam. Lamiaceae (syn. Labiatae), a hybrid which originated from crossing the two species, Mentha aquatica L. and Mentha spicata L., has been used in medicine since the 13th century in the form of infusions, tinctures, dry and liquid extracts, and essential oil or its constituents (menthol, menthone, and other monoterpenes) mostly as a cholagogue, choleretic, digestive, carminative, and spasmolytic in gastrointestinal disturbances and against respiratory system diseases [1,3]. This plant is cultivated in Europe (mainly England and Germany), North America, and other countries in the temperate climate zone [4]. It is classified as a traditional herbal medicine with indications based on long-standing use for relief of digestive disorders, gastritis, enteritis biliary disorders, diarrhea, skin disorders and minor wounds, pain and inflammation,

Results
The investigation into peppermint tinctures' (23 commercial medicinal products) polyphenolic composition shows the presence of 35 compounds (among them 12 phenolic acids-caffeetannins, 12 flavones, eight flavanones, two jasmonic acid derivatives, and one lignan), of which 10 were determined quantitatively. Chemical structures of identified peppermint components are presented in Figures 1-3.

Results
The investigation into peppermint tinctures' (23 commercial medicinal products) polyphenolic composition shows the presence of 35 compounds (among them 12 phenolic acids-caffeetannins, 12 flavones, eight flavanones, two jasmonic acid derivatives, and one lignan), of which 10 were determined quantitatively. Chemical structures of identified peppermint components are presented in Figures 1-3.    The electrospray ionization mass spectrometer coupled with ultra high performance liquid chromatography (U(H)PLC-ESI-MS) results together with MS/MS fragmentations are presented in Table 1. This analysis has not only confirmed the presence of well-known polyphenols, but also revealed the occurrence of components in peppermint leaf preparations not previously described.  Table 1. This analysis has not only confirmed the presence of well-known polyphenols, but also revealed the occurrence of components in peppermint leaf preparations not previously described. In addition to caffeic acid (peak 3) and rosmarinic acid (peak 21) this research has shown the presence of ten other caffeic acid esters (peaks 1, 7,15,19,22,24,25,27,30,34) known as lithospermic acids or salvianolic acids. Caffeic acid, rosmarinic acid, lithospermic acid A (peak 22), and lithospermic acid B (peak 24) were identified using authentic standards or herbal reference materials (Salvia miltiorrhiza Bunge, Melissa officinalis L.) and literature data [32,34,35]. Interpretation of data from MS/MS experiments for remaining peppermint caffeetannins is presented in Table 2. Peak 1 with pseudo-molecular ion at m/z 197.0454 (fragment ions at 181 and 137) was identified as salvianic acid A (syn. danshensu). Peak 7 with [M − H] − at m/z 537.1048, isobaric to peak 22 was identified as salvianolic acid J or W (lithospermic acid A isomers) [31].  Table 2) [34]. Proposed MS/MS fragmentation pathway of caffeic acid oligomers (e.g., salvianolic acid E) is showed in Figure 4.
Peaks 2 and 4 with pseudo-molecular ions at m/z 305.0700 and 387.1661 were tentatively characterized as 12-hydroxyjasmonic acid derivatives, tuberonic acid sulphate and tuberonic acid O-glucoside, respectively [40]. Peak 26 (m/z 563.2124, fragment ion: 387) was described as a medioresinol derivative [38,40].    Quantification of principal polyphenols was carried out by the high performance liquid chromatography coupled with a diode-array detector (HPLC-DAD) method described previously by Fecka and Turek [43]. The HPLC-DAD method was re-validated and corresponding data for ten authentic standards are presented in Table 3. Figure 5 shows the heatmap of polyphenol content in peppermint tinctures. Detailed contents of caffeic acid (CA), rosmarinic acid (RA), eriocitrin (Er), luteolin rutinoside (Lr(+Lg), luteolin glucuronide + luteolin glucoside calculated as Lgr), luteolin glucuronide (Lgr), narirutin (Nr), isorhoifolin (Ir), hesperidin (Hr(+Dr), hesperdin + diosmin calculated as Hr), eriodictyol (E), and luteolin (L) and sums of quantified phenolic acids (SPA, CA + RA), flavonoids (SF, Er + Lr + Lgr + Nr + Ir + Hr + E + L), and all polyphenols (SPP) are compared in Table A1. Compound contents are expressed as mg per 1 mL of peppermint tincture. Lg and Lgr as well as Dr and Hr were eluted together, thus the amounts of these components were quantified together. Lithospermic acids, salvianolic acids, and other minor caffeic acid esters were not included in the calculations as their concentrations were below the quantification limit (QL) for rosmarinic acid. Chlorogenic acid was not present at all. Typical HPLC chromatograms of peppermint tincture at 280, 320, and 360 nm are presented in Figure 6.
The predominant phenolic acid derivative was rosmarinic acid, with an average concentration of 0.14 ± 0.12 mg/mL. The highest amount, 0.44 mg/mL, was detected in sample no. 1. Average caffeic acid concentration was 0.01 mg/mL, while the highest observed value was 0.02 mg/mL. Among analyzed flavanones, eriocitrin (eriodictyol-7-O-rutinoside) showed the highest concentration, up to 1.77 mg/mL and an average of 0.81 ± 0.60 mg/mL. The highest amount of Er was recorded in sample no. 7. Other flavanone glycosides, like hesperidin (calculated as the sum with diosmin, because those compounds were eluted together) and naringenin-7-O-rutinoside, had respectively 0.04 and 0.05 mg/mL on average. The mean concentration of eriodictyol, eriocitrin aglycone, was 0.03 ± 0.02 mg/mL. The study showed that the most common flavone was luteolin-7-O-rutinoside with a mean concentration of −0.27 ± 0.23 mg/mL on average. The highest Lr level was assessed in product no. 8. The average concentration of luteolin-7-O-β-glucuronide was 0.02 ± 0.01 mg/mL. Due to a similar retention time, the content of luteolin-7-O-β-glucoside was evaluated together with Lgr. The average content of aglycone luteolin was 0.01 ± 0.01 mg/mL. The concentration of isorhoifolin (apigenin-7-O-rutinoside) and other flavones and flavanones was in most cases below QL, but its presence was confirmed by U(H)PLC-ESI-MS analysis. The mean sum of phenolic acids (SPA) in analyzed peppermint tinctures was calculated as 0.15 ± 0.12 mg/mL. The average flavonoid Quantification of principal polyphenols was carried out by the high performance liquid chromatography coupled with a diode-array detector (HPLC-DAD) method described previously by Fecka and Turek [43]. The HPLC-DAD method was re-validated and corresponding data for ten authentic standards are presented in Table 3. Figure 5 shows the heatmap of polyphenol content in peppermint tinctures. Detailed contents of caffeic acid (CA), rosmarinic acid (RA), eriocitrin (Er), luteolin rutinoside (Lr(+Lg), luteolin glucuronide + luteolin glucoside calculated as Lgr), luteolin glucuronide (Lgr), narirutin (Nr), isorhoifolin (Ir), hesperidin (Hr(+Dr), hesperdin + diosmin calculated as Hr), eriodictyol (E), and luteolin (L) and sums of quantified phenolic acids (SPA, CA + RA), flavonoids (SF, Er + Lr + Lgr + Nr + Ir + Hr + E + L), and all polyphenols (SPP) are compared in Table A1. Compound contents are expressed as mg per 1 mL of peppermint tincture. Lg and Lgr as well as Dr and Hr were eluted together, thus the amounts of these components were quantified together. Lithospermic acids, salvianolic acids, and other minor caffeic acid esters were not included in the calculations as their concentrations were below the quantification limit (QL) for rosmarinic acid. Chlorogenic acid was not present at all. Typical HPLC chromatograms of peppermint tincture at 280, 320, and 360 nm are presented in Figure 6.
The predominant phenolic acid derivative was rosmarinic acid, with an average concentration of 0.14 ± 0.12 mg/mL. The highest amount, 0.44 mg/mL, was detected in sample no. 1. Average caffeic acid concentration was 0.01 mg/mL, while the highest observed value was 0.02 mg/mL. Among analyzed flavanones, eriocitrin (eriodictyol-7-O-rutinoside) showed the highest concentration, up to 1.77 mg/mL and an average of 0.81 ± 0.60 mg/mL. The highest amount of Er was recorded in sample no. 7. Other flavanone glycosides, like hesperidin (calculated as the sum with diosmin, because those compounds were eluted together) and naringenin-7-O-rutinoside, had respectively 0.04 and 0.05 mg/mL on average. The mean concentration of eriodictyol, eriocitrin aglycone, was 0.03 ± 0.02 mg/mL. The study showed that the most common flavone was luteolin-7-O-rutinoside with a mean concentration of −0.27 ± 0.23 mg/mL on average. The highest Lr level was assessed in product no. 8. The average concentration of luteolin-7-O-β-glucuronide was 0.02 ± 0.01 mg/mL. Due to a similar retention time, the content of luteolin-7-O-β-glucoside was evaluated together with Lgr. The average content of aglycone luteolin was 0.01 ± 0.01 mg/mL. The concentration of isorhoifolin (apigenin-7-O-rutinoside) and other flavones and flavanones was in most cases below QL, but its presence was confirmed by U(H)PLC-ESI-MS analysis. The mean sum of phenolic acids (SPA) in analyzed peppermint tinctures was calculated as 0.15 ± 0.12 mg/mL. The average flavonoid sum (SF) and average sum of all quantified polyphenols (SPP) was 1.24 ± 0.87 and 1.39 ± 0.97 mg/mL, respectively. Product no. 8 had the highest values of SF and SPP, while the lowest values were observed in product no. 14. sum (SF) and average sum of all quantified polyphenols (SPP) was 1.24 ± 0.87 and 1.39 ± 0.97 mg/mL, respectively. Product no. 8 had the highest values of SF and SPP, while the lowest values were observed in product no. 14.

Discussion
Mentha × piperita L. is among the most popular plants of temperate climate countries, where it is cultivated for medicinal purposes. Peppermint leaf (Menthae piperitae folium) is obtained from crops and used for various types of diseases. As an official medicine peppermint was mentioned for the first time in the 13 th century in the Icelandic Pharmacopeia [24]. Essential oil, whose content in the raw material should not be less than 12 mL/kg, is considered a substance that determines the versatile

Discussion
Mentha × piperita L. is among the most popular plants of temperate climate countries, where it is cultivated for medicinal purposes. Peppermint leaf (Menthae piperitae folium) is obtained from crops and used for various types of diseases. As an official medicine peppermint was mentioned for the first time in the 13th century in the Icelandic Pharmacopeia [24]. Essential oil, whose content in the raw material should not be less than 12 mL/kg, is considered a substance that determines the versatile activity of this plant. In the literature over the last 25 years, attention has been drawn to the non-volatile components of peppermint leaf, mainly polyphenols, primarily flavonoids and phenolic acids-caffeetannins, as pharmacologically active substances. Studies have proved that out of several peppermint extracts prepared with different solvents, aqueous and alcoholic extracts possess the highest polyphenol content and highest antioxidant activity [44,45]. According to Dorman and co-workers [46], eriocitrin and rosmarinic acid constitute the largest percentage (37.6% and 37.4%, respectively) of the peppermint polyphenol fraction. In our study, eriocitrin, luteolin-7-O-rutinoside, and rosmarinic acid had the largest percentages (54.7%, 20.3%, and 8.8%, respectively). Numerous reports on the multidimensional action, important for the proper functioning of the body, of both above-mentioned groups of compounds occurring in Menthae piperitae folium-flavonoids (especially glycosides of eriodictyol and luteolin) and phenolic acids (mainly rosmarinic acid)-are changing the current view on proper assessment of the validity of active substance in the raw material. While flavonoids are a well-known subgroup of polyphenols that have been used in medicine for years, phenolic acids are gradually being studied. In this research, quantitative analysis has shown the presence of 35 compounds (among them flavones, flavanones, phenolic acids-caffeetannins, lignin and jasmonic acid derivatives) in commercial peppermint tinctures, and 10 of them were determined quantitatively. Rosmarinic acid is a widely investigated phenolic acid that has gained great interest among pharmacologists [47]. Amoah and co-workers [16] published a comprehensive review in which they analyzed the entire scope of potential use of rosmarinic acid in various branches of medicine and highlighted its pharmacological and biological activities; among them, antioxidant, anti-inflammatory, anti-infective, and neuroprotective actions were described. In addition to caffeic and rosmarinic acids, 10 other caffeetannins were detected in the analyzed medicinal products by U(H)PLC-ESI-MS: salvianic acid A (danshensu), lithospermic acid A and its isomer, lithospermic acid B (salvianolic acids B) with three isomers, didehydrosalvianolic acid B, salvianolic acid C, and ethyl rosmarinate (an ester of rosmarinic acid with ethanol, the solvent used to prepare the tincture, probably an artefact). Generally oligomeric caffeic acid esters in peppermint tinctures were present at lower concentrations, below 0.45 mg/mL (Table A1), and the highest content was assessed for RA. Lithospermic acids A and B (and their isomers) were previously described in peppermint leaf infusions and other water extracts [38,39,48]. According to the authors' data [38], lithospermic acid B makes up 1.0%-9.7% of polyphenol content. It was reported that this compound was an efficient free-radical scavenger that could be used to treat cardiovascular diseases and play a beneficial role in Alzheimer's treatment [49,50]. The presence of didehydrosalvianolic acid B in peppermint leaf was reported previously by Krzyzanowska and co-workers [36]. On the basis of pseudo-molecular and fragmentation ions and literature data, one representative of lignans, medioresinol glucuronide, was identified in the analyzed samples [38].
Currently peppermint leaf activity is attributed only to essential oil-a group of monoterpenes and their derivatives-as the main chemical parameter conditioning the multidirectional pharmacological action of the raw material and peppermint tincture. Polyphenols are found in the volatile fraction in negligible amounts. The content of essential oil in peppermint leaf is from 0.5%-4.5%, while the polyphenolic compounds constitute 19%-23% [28,51,52]. Olennikov and Tanakhaeva [53] conducted a quantitative determination of polyphenolic compounds using four spectrophotometric methods in Mentha × piperita L. originating in Siberia. Hesperidin and rosmarinic acid were used as standards. The authors concluded that the sum of polyphenols and monoterpenes contributes to the medicinal properties of peppermint [53]. The research suggests that the current pharmacopoeia requirement for Menthae piperitae folium should be corrected. In the pharmacopeial parameters required for admission of peppermint leaf to the market, a lower limit for the sum of polyphenols or the main biologically active components of the non-volatile fraction should be specified. According to our results as well as previous reports, the highest concentration compounds among peppermint polyphenols are eriocitrin, luteolin-7-O-rutinoside, and rosmarinic acid. Our study also showed that all polyphenolic compounds occur in analyzed commercial tinctures in extremely different concentrations. According to this, it is necessary to introduce to the pharmacopeial definition of Menthae piperitae folium (Ph. Eur.) and Menthae piperitae tincture cum Menthae piperitae aetheroleo (FP) corrections regarding the requirement for minimum content not only for rosmarinic acid but also for flavonoids or their leading representatives (e.g., eriocitrin and luteolin glycosides). From a pharmaceutical point of view, the presence of an unspecified, and thus unknown content of the active substance is an unacceptable oversight. Considering our research results, it can be assumed that one patient receives peppermint tincture no. 1 with the maximum contents of rosmarinic acid (0.44 mg/mL) and eriocitrin (1.63 mg/mL), and the other tincture no. 14 with the minimum contents of these compounds (0.01 and 0.09 mg/mL), respectively.

Solvents and Chemicals
Reagents used in the analysis were of analytical grade. Acetonitrile (HPLC gradient grade), methanol, and 90%-100% formic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Water was obtained in the process of distillation and deionization with Hydrolab Deionizer HLP20UV. U(H)PLC-ESI-MS grade water and formic acid were from Merck (Darmstadt, Germany); U(H)PLC-ESI-MS grade acetonitrile was from Honeywell (Morris Plains, NJ, USA). Analytical grade methanol for dilution was from POCh (Lublin, Poland).
Stock standard solutions (1 mg/mL) were prepared by dissolving a weighted amount (precisely about 1.0-1.7 mg) of standard in the appropriate methanol volume (1.0-1.7 mL) and filtered through a 0.45 µm membrane filter (Millipore, Burlington, MA, USA). Working standard solutions, with concentrations of 0.02, 0.05, 0.1, 0.15, 0.2, and 0.3 mg/mL, were obtained by diluting stock standard solutions with 50% aq. methanol (v/v). Working standard solutions were used for standard curve preparation.
In addition to the above-mentioned compounds, the following infusions (1:20, m/v) from herbal reference materials Melissa officinalis L. (leaves) and Salvia miltiorrhiza Bunge (roots) were used in U(H)PLC-ESI-MS analysis.  The column was equilibrated for 5 min before the next analysis. Blanks were run after each sample to avoid cross-contamination. Other parameters were: column oven temperature 30 • C, injection volume 5 µL.

Content Measurement
The content of individual polyphenols and the sums of particular phytochemical groups (flavonoids and phenolic acids) were determined using the optimized HPLC-DAD method [43]. Calibration equations for quantified flavonoids and phenolic acids were assessed at six concentration levels, and duplicate injections were performed for each concentration (n = 2 × 6). The linearity of standard curves was confirmed by plotting the peak areas (y, mAU/s) and the corresponding concentration (x, mg/mL). Limits of detection (LOD) and limits of quantitation (LOQ) were calculated from calibration equations based on the signal-to-noise ratio (S/N ≥ 3:1 and S/N ≥ 10:1) and expressed as the concentration of the examined compound (mg/mL). Table 3 presents chromatographic characteristics of standard compounds (calibration equation, r 2 , range, LOD, LOQ, working wavelength).

Conclusions
For the first time it is noted that flavonoids and phenolic acids (caffeetannins), the main components of the non-volatile peppermint fraction which play an important role in the pharmacological action of this medicinal plant, have not yet found a place next to essential oil in the pharmacopeial definition of peppermint leaf (Menthae piperitae folium, Ph. Eur.) and peppermint tincture (Menthae piperitae tinctura cum Menthae piperitae aetheroleo, FP). Based on the results of this research as well as data from the literature, the chemical composition of peppermint tincture should be carefully analyzed and standardized. In the light of these results, it seems that the pharmacopeial assessment for Menthae piperitae folium and Menthae piperitae tinctura cum Menthae piperitae aetheroleo requires correction and supplementation.

Conflicts of Interest:
The authors declare no conflicts of interest. 1 Lgr(+Lg) calculated as Lgr, 2 Hr(+Dr) calculated as Hr, a less than 1% CV b less than 3% CV, c less than 5% CV, d less than 7% CV; n = 3.