Flavonoids and other phenolics in herbs commonly used in Norwegian commercial kitchens

Journal Pre-proofs Flavonoids and other phenolics in herbs commonly used in Norwegian commercial kitchens Rune Slimestad, Torgils Fossen, Cato Brede PII: DOI: Reference:

S0308-8146(19)31805-9 https://doi.org/10.1016/j.foodchem.2019.125678 FOCH 125678

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Food Chemistry

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24 April 2019 7 October 2019 7 October 2019

Please cite this article as: Slimestad, R., Fossen, T., Brede, C., Flavonoids and other phenolics in herbs commonly used in Norwegian commercial kitchens, Food Chemistry (2019), doi: https://doi.org/10.1016/j.foodchem. 2019.125678

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Flavonoids and other phenolics in herbs commonly used in Norwegian commercial kitchens Rune Slimestad1*, Torgils Fossen2 and Cato Brede3

1PlantChem

AS, Eikenveien 334, N-5496 Eiken, Norway

2Department

of Chemistry and Centre for Pharmacy, University of Bergen, Allègaten 41, N-5007 Bergen, Norway 3Stavanger

University Hospital, Department of Medical Biochemistry, Armauer Hansens vei 20, N4011 Stavanger, Norway

*Corresponding author. Phone: +47-995 08 228, e-mail: [email protected]

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ABSTRACT: Significant quantities of several important herbs are processed and consumed from Norwegian commercial kitchens annually although surprisingly the contents of polyphenols have been scarcely characterized. We here report on the qualitative and quantitative content of polyphenolic compounds from ten of the most utilized herbs. From parsley (Petroselinum crispum) var. Darki, isorhamnetin 3-(6’’-malonylglucoside)-7-glucoside (2) and diosmetin 7-(2’’-apiosyl-6’’malonylglucoside) (8) are reported for the first time, in addition to seven known flavonoids, some of which are reported for the first time from this plant species. Oregano, rosemary and thyme contained the highest amounts of total phenolics with maximum levels of 23.8, 24.2 and 23.4 mg GAE g-1 dry matter, respectively. Fresh herbs contained significantly higher quantities of phenolics than processed, dried herbs. Parsley, coriander, dill and thyme were the richest sources of flavonoids among the investigated herbs.

Keywords: Herbs; parsley; flavonoids; UHPLC-MS; NMR; HRMS; phenolic content.

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INTRODUCTION For several millennia, spices and herbs have been important dietary constituents used for the enhancement of flavour and organoleptic properties of food. They play an important role as preservatives, partly because their naturally occurring flavonoids and other phenolic constituents act as reducing agents or free radical terminators (Lindsay, 1996) and partly due to the antimicrobial effects of the major phenolics (Martinéz-Graciá et al., 2015). From a human health perspective, phenolic compounds are vital in defence responses, such as anti-aging, anti-inflammatory, antioxidant and anti-proliferative activities. A flavonoid rich diet is recommended for the reduction of the risks of several acute and chronic diseases including coronary and heart diseases, cancer and diabetes. The underlying defence mechanisms at the molecular level are thought to be related to the management of oxidative stress (Bower, Marquez & Gonzalez de Mejia, 2016; Lin et al., 2016). During the last two decades, a significant number of publications have dealt with favourable biological activities of pure flavonoids rather than those of plant extracts. A lot of effort has been made to relate the observed biological activities to individual flavonoid structures (Xiao, 2017; Raffa et al., 2017; Wang, Li & Bi, 2018). However very few pure individual flavonoids, mainly those which are commercially available in large quantities, have been investigated with respect to their potential biological activities (Tallagavadi et al., 2016; Gullòn et al., 2017; Wu et al., 2019). A prerequisite for the investigation of the wider health promoting potential of plant species, is a proper and correct determination of the molecular composition of the plants. While several plant species have been subjected to multiple investigations, it has still been demonstrated that even major flavonoid constituents of important food plants have only recently been identified as novel constituents (Ibrahim et al. 2019; Rayyan, Fossen & Andersen, 2010; Slimestad, Fossen & Verheul, 2008; Fossen, Slimestad & Andersen, 2003; Fossen, Slimestad & Andersen, 2001). Among the 70 species of culinary herbs and spices most often recognized as useful ingredients for foods (Lindsay, 1996,) only a limited number have been properly characterized with respect to their content of flavonoids and other phenolic compounds. Surprisingly, only a limited number of phenolic 3

compounds have hitherto been characterized from several of the most important herbs used in large scales in Norwegian commercial kitchens. The major objective of this paper is to determine the qualitative and quantitative content of flavonoids and other phenolics in the most popular herbs used in Norwegian large-scale commercial kitchens by state-of-the-art spectroscopic and hyphenated chromatographic methods.

MATERIALS AND METHODS Plant material Ten herbs were investigated in this work: basil (Ocimum basilicum), chive (Allium schoenumprasum), coriander (Coriandrum sativum), dill (Anethum graveolens), oregano (Oreganum vulgare), mint (Mentha x piperita), parsley (Petroselinum crispum), rosemary (Rosmarinus officinalis), tarragon (Artemisa dracunculus), and thyme (Thymus vulgaris). Fresh cut herbs packed in modified atmosphere, were received from Einar Hanasand and Viking Urt, both located at Randaberg, southwestern part of Norway. Potted herbs produced by Rosnes Produksjon AS, Ravnsborg Gård and Snarum Gartneri all located at the southeaster part of Norway, were bought from local groceries. Dried herbs from Hindu (Olaf Ellingsen AS) and Santa Maria (Santa Maria Norge AS) were also bought from local groceries. More sample information is given in Table 4.

Chemicals Gum Arabic, hydrochloric acid, 85% phosphoric acid, gallic acid, potassium phosphate, sodium chloride, chlorogenic acid and formic acid were provided from Merck, Norway. Methanol (Rathburn) and acetonitrile (Rathburn) were provided from Teknolab AS, Norway. Rutin, quercetin 3-glucoside and kaempferol 3-glucoside were provided by PlantChem AS, Norway.

Dry matter (DM) contents

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Fresh herbs, including leaves and stalks, were lyophilized for 72 h using a freeze-dryer (CoolSafe 4 ScanVac, ScanLaf AS, Denmark). Dry matter contents were determined based on the sample weights prior to and after lyophilization.

Analytical samples Dried plant material was minced in a bowl chopper and ground (Bosch KM13, Slovenia). Ready-to-use dried herbs in 100 mL glass containers (from Hindu and SantaMaria) were also ground before further analysis. For determination of total phenols and for UHPLC analysis, about 200 mg of each sample was extracted with 10 mL methanol in a 20 mL test tube at ambient temperature for 48 h in the darkness. Samples were filtered through a 0.4 m syringe filter prior to analysis. Two parallels were prepared for each sample.

Fractionation and isolation Fractionation and isolation of flavonoids and phenolics from parsley were based on extracts obtained from 500 g fresh plant material. The plant material was minced (about 20 mm), and extraction was performed using 2 x 500 mL methanol for 24 h in the dark. The extract was filtered (folded filter quality 315, VWR Norway), concentrated to about 100 mL on a rotavapor (Büchi, Switzerland), and partitioned against equal volumes of dichloromethane in order to remove chlorophylls and other lipophilic content. The water phase was further concentrated, 50 mL, and the extract was applied to a bed of 0.5 kg Amberlite XAD7HP (Sigma) in a 5x60 cm chromatography column, rinsed with 2 L distilled water and eluted by use of 2 L MeOH. The purified extract was finally concentrated to about 50 mL. Fractions and isolates were obtained by use of size-exclusion chromatography over a bed of 500 g Sephadex LH20 (GE Healthcare, Norway) (5x100 cm open-top column) by step elution with increasing concentrations of methanol (0, 20, 40, 60 and 80%, 0.1% TFA). Fractions were re-chromatographed on the same column. 5

Total phenolic contents Total phenolic content was determined in accordance to the method of Price and Butler with stabilization of the Prussian Blue complex as described by Graham (Price & Butler, 1977; Graham, 1992). 100 L of sample was diluted with 3 mL deionized water and mixed with 1 mL of a 0.1 M ferric chloride in 0.1 M hydrochloric acid solution together with 1 mL of 8 mM potassium ferricyanide solution. The reaction was allowed to run for fifteen minutes at ambient temperature. 5 mL of an acidic gum arabic solution was added (1 g gum arabic dissolved in 100 mL hot water. 10 mL of this solution was mixed with 10 mL 85 % phosphoric acid and 30 mL water). Absorption at 700 nm was measured on an Agilent 8453 spectrophotometer (Agilent Technologies, Matriks, Norway). Samples were measured against a standard curve of gallic acid, and outputs are given as gallic acid equivalents, mg GAE g-1.

U(H)PLC-MS An Agilent 1290 Infinity II instrument equipped with a 6120 quadrupole mass detector was used to determine the content of individual flavonoids and other phenolic compounds in all samples. Separation was achieved by utilizing a Ascentis Express C18 column (2.1x100 mm, 2 m, Supelco). Water with 0.02% HCOOH (solvent A) and acetonitrile (solvent B) were used for gradient elution with the following time program (% B in A): from 0 to 10 (1 min), from 10 to 30 (11 min), from 30 to 95 (1 min), from 95 to 0 (1 min), and finally isocratic reconditioning for 1 min. Flow was set to 0.300 mL/min (max back pressure 410 bar), and injections of 5 L were used. UV-detection was performed at 280, 320 and 360 nm at 4 nm band width. Masses in the range 250-800 Da were detected using a scan time of 500 msec, a fragmentor at 70 V, and detection was in both positive and negative mode. Gas source temperature was set to 350 ℃ with flow at 10 L min-1, nebulizer pressure was 35 psi, whereas capillary voltage was 4 kV.

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Standard curves of chlorogenic acid, rutin, quercetin 3-glucoside and kaempferol 3-glucoside were developed in concentration ranges of 0.02-300 g mL-1, with linearities (r2) ≥ 0.9967, and S/N-values ≥ 36. High-resolution mass spectrometry (LC-HRMS) was used for exact mass determination of isolated compounds. An iClass UPLC (Waters) equipped with a C18 BEH column (1.7 um, 2.1 x 50 mm, Waters) was used for introducing the samples to the mass spectrometer. A gradient of A) 0.2% formic acid and B) acetonitrile was used as follows (% B in A): 1 (isocratic for 0.5 min), from 1 to 90 (2 min). The mass spectrometer (timsTOF, Bruker) was used in ESI+-mode with an ionization at 2 kV, and with full scan 100-2000 Da with resolution R = 50 000 (FWHM) at 1000 Da. Exactness at RMS < 1 ppm.

NMR NMR samples were prepared by dissolving the isolated compounds in deuterated dimethylsulfoxide (DMSO-D6; 99.96 atom % D, Sigma-Aldrich). The 1D 1H and the 2D 1H-13C HMBC, the 2D 1H-13C HSQC, the 2D 1H-13C HSQC-TOCSY, the 2D 1H-13C H2BC, 2D 1H-13C 1,1 Adequate, the 2D 1H-1H COSY and 2D 1H-1H

ROESY NMR experiments were obtained at 850 MHz at 298K on a Bruker 850 MHz instrument

equipped with a 1H,13C,15N triple resonance cryogenic probe.

Statistics Excel (Microsoft Office) was used for calculation of mean values and standard derivations. Signal-tonoise ratios (S/N) in UHPLC were calculated by use of OpenLAB Data Analysis ver. 2.1 (Agilent Technologies).

RESULTS AND DISCUSSION Flavonoids from parsley A methanol extract of fresh parsley (Petroselinum crispum) contained fifteen flavonoids as detected by means of UHPLC-DAD-MS analysis (Table 1). Preparative isolation of the flavonoids was achieved 7

by repetitive size-exclusion chromatography (Sephadex LH-20). In total nine flavonoids were isolated. Compounds 1, 3, 4, 5 and 7 were identified as isorhamnetin 3,7-O-β-diglucopyranoside (1), hesperetin 7-O-β-glucopyranoside (3), apigenin 7-O-β-(2’’-apiofuranosylglucopyranoside) (4, apiin), isorhamnetin 3-O-β-glucopyranoside (5), apigenin 7- O-β-(2’’-apiofuranosyl-6’’malonylglucopyranoside) (7) and apigenin 7-O-β-glucopyranoside (9) (Figure 1) by use of a combination of different NMR experiments (Tables 2-3, and supplementary material), in addition to high resolution mass spectrometry (supplementary material). Compounds 1, 3 and 5 are identified in parsley for the first time. Apigenin 7-glucoside (9) was not detected in the crude extracts, but found during fractionation, which may indicate that this compound occurred as a hydrolysis product of apiin (4). The aromatic region of the 1D 1H NMR spectrum of 2 showed a 3H ABX system at δ 7.56 (dd, 2.3 Hz, 8.3 Hz; H-6’), δ 7.83 (d 2.3 Hz; H-2’) and δ 6.92 (d 8.3 Hz; H-5’) and a 2H AX system at δ 6.80 (d 2.2 Hz; H-8) and δ 6.44 (d 2.2; H-6), revealing a flavonol having an asymmetrically substituted B-ring. The 3H singlet at δ 3.83 (OCH3), which was confirmed to be attached to the aglycone at the 3’-position by the crosspeak at δ 3.83/147.2 (3’-OCH3/C-3’) observed in the 2D 1H-13C HMBC spectrum and the crosspeak at δ 3.83/7.83 (3’-OCH3/H-2’) observed in the 2D 1H-1H ROESY spectrum, confirmed the identity of the aglycone to be isorhamnetin. The 16 13C signals belonging to isorhamnetin aglycone were assigned by the 2D 1H-13C HMBC spectrum, the 2D 1H-13C H2BC spectrum and the 2D 1H-13C HSQC spectrum. The aliphatic regions of the 1D 1H and 13C NMR spectra of 2 showed the presence of two glucopyranosyl substituents. The large observed anomeric coupling constants (7.2 Hz and 7.7 Hz for H-1’’ and H-1’’’, respectively) confirmed the β-configurations of the glucosyl units. In theory, such large coupling constant would also be present for an α-L-glucopyranosyl unit. However, this is considered unlikely because it is the β-anomer of this sugar that is invariably found in flavonoid glycosides (Veitch and Grayer, 2006). The crosspeaks at δ 5.47/133.6 (H-1’’/C-3) and δ 5.05/163.3 (H1’’’/C-7) observed in the 2D 1H-13C HMBC spectrum of 2 and the crosspeaks at δ 5.05/6.80 (H-1’’’/H8) and δ 5.05/6.44 (H-1’’’/H-6) confirmed that the glucosyl substituents were attached to the 3- and 8

7-positions of the aglycone, respectively. The individual 1H and 13C resonances of the 3-glucosyl and the 7-glucosyl substituents were assigned by examining the 2D 1H-1H COSY, 2D 1H-13C HSQC-TOCSY, 2D 1H-13C H2BC and 2D 1H-13C HSQC spectra. The 2H singlet at δ 3.22 (H-2’’’’) which correlated to a 13C

signal at δ 40.7 (C-2’’’’) in the HSQC spectrum and to the 13C signals at δ 166.4 (C-1’’’’) and 168.0

(C-3’’’’) in the HMBC spectrum was in accordance with a malonyl substituent. The downfield chemical shift values of H-6A’’ (δ 4.17), H-6B’’ (δ 4.07) and C-6’’ indicated the that the malonyl was attached to the 6-position of the 3-glucosyl. The crosspeaks at δ 4.17/166.4 (H-6A’’/C-1’’’’) and δ 4.07/166.4 (H-6B’’/C-1’’’’) observed in the 2D 1H-13C HMBC spectrum confirmed this linkage. Thus, 2 was identified as the novel flavonol isorhamnetin 3-O-(6’’-O-malonyl-β-glucopyranoside)-7-O-βglucopyranoside. A pseudomolecular ion [M+H+] at m/z 727.1709 corresponding to C31H35O20 (calculated: 727.1723; δ = -1.9 ppm) observed in the HR mass spectrum of 2 confirmed this identity. The aromatic region of the 1D 1H NMR spectrum of 8 showed a 3H ABX system at δ 7.57 (dd, 2.3 Hz, 8.6 Hz; H-6’), δ 7.45 (d 2.3 Hz; H-2’) and δ 7.09 (d 8.6 Hz; H-5’), in addition to a 1H singlet at δ 6.82 (H-3) and a 2H AX system at δ 6.75 (d 2.2 Hz; H-8) and δ 6.42 (d 2.2; H-6), revealing a flavone having an asymmetrically substituted B-ring. The 3H singlet at δ 3.86 (OCH3), which was confirmed to be attached to the aglycone at the 4’-position by the crosspeak at δ 3.86/151.4 (4’-OCH3/C-4’) observed in the 2D 1H-13C HMBC spectrum and the crosspeak at δ 3.86/7.09 (4’-OCH3/H-5’) observed in the 2D 1H-1H

ROESY spectrum, confirmed the identity of the aglycone as diosmetin. The 16 13C signals

belonging to diosmetin aglycone observed in the 1D 13C CAPT spectrum were assigned by the 2D 1H13C

HMBC spectrum. The aliphatic regions of the 1D 1H and 13C NMR spectra of 8 showed the

presence of two glycosyl substituents, which were identified as β-glucopyranosyl and apiofuranosyl, respectively (Table 2 and 3). The crosspeak at δ 5.22/162.5 (H-1’’/C-7) observed in the 2D 1H-13C HMBC spectrum and the crosspeaks at δ 5.22/6.75 (H-1’’/H-8) and δ 5.22/6.42 (H-1’’/H-6) observed in the 2D 1H-1H ROESY spectrum confirmed the linkage between the glucosyl substituent and the aglycone to be at the 7-position. The crosspeaks at δ 5.34/75.6 (H-1’’’/C-2’’) and δ 3.54/108.8 (H2’’/C-1’’’) observed in the 2D 1H-13C HMBC spectrum confirmed that the terminal apiofuranosyl was 9

attached to the glucosyl at the 2’’-position. The acyl group was identified as malonyl by the two signals at δ 3.39 (d 15.8 Hz; H-2A’’’’) and δ 3.35 (d 15.8 Hz; H-2B’’’’) observed in the 1D 1H spectrum and the three 13C signals at δ 167.84 (C-3’’’’), δ 166.86 (C-1’’’’) and δ 41.36 (C-2’’’’) observed in the 1D 13C CAPT spectrum. The downfield chemical shift values of H-6A’’ (δ 4.37), H-6B’’ (δ 4.13) and C6’’ (δ 64.02) belonging to the glucosyl unit indicated that the malonyl was attached to the glucosyl at the 6’’-position. The crosspeaks at δ 4.37/166.9 (H-6A’’/C-1’’’’) and δ 4.13/166.9 (H-6B’’/C-1’’’’) observed in the 2D 1H-13C HMBC spectrum confirmed this linkage. Thus, 8 was identified as the novel flavonoid diosmetin 3-O-(2’’-O-apiofuranosyl-6’’O-malonyl-β-glucopyranoside). A pseudomolecular ion [M+H+] at m/z 681.1648 corresponding to C30H33O18 (calculated: 681.1668; δ = -2.9 ppm) observed in the HR mass spectrum of 8 confirmed this identity. In accordance to the presence of the malonylated diosmetin-derivative (8) and the on-line chromatographic and spectral features, compound 6 was assigned to be diosmetin 7apiosylglucoside (Figure 1). Thus, the four main peaks in the chromatogram of the parsley extract were identified the 7-apiosylglucosides and the 7-malonylapiosylglucosides of apigenin (4 and 7) and diosmetin (6 and 8), respectively. Another compound (* Figure 1) occurred with a lower retention time than 7 but revealed similar UV- and MS-spectral features (Table 1). This might be an isomeric structure of apigenin 7-malonylapiosylglucoside (Kaiser, Carle & Kammerer, 2013). Moreover, a second compound (denoted ** in Figure 1) might be an isomeric structure of diosmetin 7malonylapiosylglucoside. E.g., derivatives of chrysoeriol (3’-methoxy-4’-hydroxy-flavone) have previously been reported from parsley (Kaiser, Carle & Kammerer, 2013). Apiin (4) is a well-known constituent of parsley (Vongerichten, 1901). The acylated derivative (7) has been characterized by use of LC-MS (Lin, Lu & Harnly, 2007; Lin & Harnly, 2008; Kaiser, Karle & Kammerer, 2013). Diosmetin derivatives have been characterized through use of co-chromatography and hydrolysis, and diosmetin 7-apiosylglucoside was detected by use of HPLC-MS. The presence of isorhamnetin derivatives have been reported previously but with no exact structure elucidations (Kasier, Carle & Kammerer, 2013; Mattila, Astola & Kumpulainen, 2000). 10

Flavonoids and other phenolics in popular herbs The ten most popular herbs used in Norwegian canteens including parsley, were analysed with respect to their content of specific flavonoids and other major phenolic compounds as well as to the total phenolic content of the methanol extracts. The content of flavonoids in general and flavones in particular was found to be highest in parsley (2065 mg 100 g-1 DW) (Table 1, sum of mean values). The measurements included the 7-apiosylglycosides and 7-malonylapiosylglucosides of apigenin (compounds 4 and 7) and diosmetin (compounds 6 and 8). The amount was similar to that reported for apiin and 6’’-malonylapiin (Lechtenberg et al., 2007), but only half the amount listed in the USDA Flavonoid database (4855 mg 100-1 g DM (USDA Flavonoid Database ver. 3.1)). The content of total phenolics was also found to be high in mint (760 mg 100-1 g DM), thyme (660 mg 100-1 g DM) and tarragon (590 mg 100 g-1 DM) (sum of mean values in table 4). The main phenolic compound in mint was tentatively identified as diosmetin rhamnosylglucoside (Table 1). Total flavonoid content was 617 mg 100-1 g DM, and the herb was also found to have a high level or rosmarinic acid. The content of apigenin- and luteolin O-glycosides in peppermint has previously been found to be 47-3097 mg 100-1 g DM (Areias et al., 2001; Fecka & Turek, 2007). The number of phenolic compounds was limited in both thyme and tarragon. Thyme was found to contain luteolin glucuronide together with rosmarinic acid as the main constituents, whereas tarragon contained rutin and chlorogenic acid. The content of luteolin was determined to be on average 660 mg 100 g-1, but with higher contents in fresh herbs (1489 mg 100 g-1) which is in agreement with that given in the USDA Flavonoid database (USDA Flavonoid Database ver. 3.1). Basil had a mean phenolic content of 2880 mg GAE 100-1 g DM (Table 4). UHPLC analysis revealed only two phenolic compounds namely rutin and rosmarinic acid with the latter dominating (Table 1). The USDA ARS flavonoid database reports no flavonoids from fresh basil. We found chive to contain 44 mg 100-1 g DM of kaempferol glycosides versus 21 mg reported by the flavonoid database. Two quercetin-derivatives were detected in the extracts of coriander at a concentration of 165 mg 100-1 g 11

DM (53 mg 100-1 g in the Flavonoid database). The content of flavonol glucuronides in dill was found to be as high as 313 mg 100-1 g DM, which is higher than that reported by the USDA flavonoid database (113 mg). Oregano had a low content of flavone glucuronides (143 mg 100-1 g DM) and a rosmarinic acid content that matched that of tarragon (Table 1). Not surprisingly, the rosmarinic acid content was found to be highest in rosemary, 1870 mg 100-1 g DM. The content of flavones in oregano has previously been reported to be 1546 mg (USDA Flavonoid database), whereas that of rosemary has been found to be 54-705 mg 100-1 g DM (del Bãno et al., 2004; Wojdylo, Oszmianski & Czemerys, 2007). Samples of rosemary, oregano and thyme were found to contain high levels of total phenolics with concentrations above 2 g GAE 100-1 g DM (Table 4). Total phenolic content varied strongly among the different available products of the same species found in the supermarket. In most cases fresh herbs had a higher content of total phenolics compared to dried herbs, which are offered as ground products packed in boxes. E.g., dried fresh thyme contained 2340 mg phenolics compared to 1300 and 1790 mg for the two box-products, respectively (Table 4). No systematic difference was detected between potted herbs and fresh herbs packed under a modified atmosphere. Variation of phenolic content within each species might in part be due to processing effects and long-term storage (dried products in box versus fresh products), but also due to the use of different varieties as well as cultivation practises among the producers. Spices and herbs are in general rich sources of phenolic compounds, and their antioxidant activities are on average ten times higher than that of fruit and vegetables (Yashin et al., 2017). As part of our interest in mapping the major sources of phenolic constituents in the public diet, we recently surveyed the use of vegetables and herbs in 450 Norwegian large-scale commercial kitchens with respect to turnover volumes and estimated daily intake (Slimestad, Hansen & Verheul, 2018). On average the top ten herbs offered in 450 Norwegian canteens contained 1088 mg GAE 100-1 g DM (Table 4). Herb consumption per lunch meal has previously been estimated to be 8 g FW which would

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give about 13 mg GAE supposed an average dry matter content of 15%. This is a high number considering the low intake of herbs. The few previously reported phenolic constituents of parsley include apiin (apigenin 7apiosylglucoside) which has been identified for more than a century (Vongerichten, 1901), as well as the structurally related acylated compound 6’’-malonylapiin (Eckey-Kaltenbach et al., 1983). Apiin is reported to act as inhibitor for aldose reductase and possesses anti-arrhythmic, anti-bradykinic, antispasmodic, and E.C. 3.2.1.18 (exo-alpha-sialidase) activities (FooDB version 1.0). The common phenolic compound rosmarinic acid, which is a caffeic acid ester of 3-(3,4-dihydroxyphenyl)lactic acid, occur in several herbs including basil, rosemary, oregano, sage, thyme and mint. The compound has shown potential as an atopical dermatitis-mitigating agent and has shown potential for treatment of patients with seasonal allergic rhinoconjuctivitis (Amoah, et al., 2016). Chlorogenic acid and its closely structurally related derivatives are quantitatively important common phenolic constituents of several important herbs, including coriander, dill, mint and tarragon. Several potential health benefits have been associated with chlorogenic acid, including its reported antidiabetic, anti-carcinogenic, anti-inflammatory and anti-obesity activities (Tajik et al., 2017).

CONCLUSIONS Parsley (Petroselinum crispum) contains a higher number of flavonoid structures than previously reported. In addition to apiin, which was reported as a main constituent more than 100 years ago, the structures of another four flavones, three flavonols and one flavanone are now fully elucidated. Some of the structures are reported for the first time (isorhamnetin 3-(6’’-malonylglucoside)-7glucoside (2) and diosmetin 7-(2’’-apiosyl-6’’-malonylglucoside) (8)). Despite the high content of flavonoids found in parsley, the amount of total phenolics was only half of that detected in oregano, rosemary and thyme. These herbs contained the highest levels of phenolic content among the ten most popular herbs used in Norwegian commercial kitchens. The phenolic content was in general

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much higher in fresh rather than dried herbs, and higher in herbs grown in the field as against potted herbs.

ACKNOWLEDGMENTS The present study is in part supported by the Bionær program of the Research Council of Norway (‘Biofresh’ project no 255613/E50). It has also been supported by the Research Council of Norway through the Norwegian NMR Platform, NNP (226244/F50).

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Rosmarinic Acid –

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Lin, L. & Harnly, J.M. (2008). LC-MS profiling and quantification of food phenolic components using a standard analytical approach for all plants. In: Greco, L.V., Bruno, M.N., editors. Food Science and Technology: New Research. New York, NY: Nova Science Publishers, Inc. p. 1-103. Lin, D., Xiao, M., Zhao, J., Li, Z., Xing, B., Li, X., Kong, M., Li, L., Zhang, Q., Liu, Y., Cheng, H., Qin, W., Wu, H. & Chen, S. (2016). An Overview of Plant Phenolic Compounds and Their Importance in Human Nutrition and Management of Type 2 Diabetes. Molecules, 21, 1374-1393. Martínez-Graciá, C., González-Bermúdez, C.A., Cabellero-Valcárcel, A.M., Santaella-Pascual, M. & Frontela-Saceta, C. (2015).

Use of herbs and spices for food preservation: advantages and

limitations. Curr. Op. Food Sci., 6, 38-43. Mattila, P., Astola, J. & Kumpulainen, J. (2000). Determination of flavonoids in plant material by HPLC with diode-array and electro-arraydetections. J. Agric. Food Chem., 48, 5834-5841.

Price, M.L. & Butler, L.G. (1977). Rapid visual estimation and spectrophotometric determination of tannin content of sorghum grain. J. Agric. Food Chem., 25, 1268-1273. Raffa, D., Maggio, B., Raimondi, M.V., Plescia, F. & Daidone, G. (2017). Recent discoveries of anticancer flavonoids. Eur. J. Med. Chem., 142, 213-228. Rayyan, S., Fossen, T. & Andersen, Ø.M. (2019). Flavone C-glycosides from seeds of Fenugreek, Trigonella foenum-graecum L. J. Food Chem. Agric., 58, 7211-7217. Slimestad, R., Fossen, T. & Verheul, M. (2008). The flavonoids of tomatoes. J. Agric. Food Chem., 56, 2436-2441. Slimestad, R., Hansen, J.S. & Verheul, M. (2018). Intake of vegetables and vegetable pigments during a lunch meal in Norwegian canteens with salad bars. J. Food, Agric. Environ. 16, 14-21. Tajik, N., Tajk, M., Mack, I. & Enck, P. (2017). The potential effects of chlorogenic acid, the main phenolic components in coffee, on health: a comprehensive review of the literature. Eur. J. Nutr., 56, 2215-2244. Tallagavadi, V., Rapisarda, P., Galvano, F., Pellicci, P. & Giorgio, M. (2016). Cyanidin 3-O-β-glucoside and protochatecuic acid activate AMPK/mTOR/S6K pathway and improve glucose homeostasis in mice. J. Funct. Foods., 21, 338-348. 16

USDA Database for the flavonoid content of selected foods. Release 3.1 (May 2014). Available online: https//www.ars.usda.gov/ARSUserFiles/80400525/Data/Flav/Flav_R03-1.pdf (Accessed January 8 2019). Veitch, N. & Grayer, R.J. Chapter 16: Chalcones, Dihydrochalcones, and Aurones p. 1023. IN: Flavonoids: Chemistry, Biochemistry and Applications. Andersen, Ø.M. and Markham, K.R. (Eds.) Taylor & Francis Group, Boca Raton, London, New York. Vongerichten, E. (1901). Ueber Apiin und Apiose. Justus Liebigs Annalen der Chemie, 318, 121-136. Wang, T-y., Li, Q. & Bi, K.-s. (2018). Review: Bioactive flavonoids in medical plants: Structure, activity and biological fate. Asian J. Pharm. Sci., 13, 12-23. Wojdylo, A., Oszmianski, J. & Czemerys, R. (2007). Antioxidant activity and phenolic compounds in 32 selected herbs. Food Chem., 105, 940– 9. Wu, S., Hu, R., Tan, J., He, Z., Liu, M., Li, Y., He, X., Hou, D.-X., Luo, J. & He, J. (2019). Cyanidin 3glucoside and its Metabolites Protect Against Nonalcoholic Fatty Liver Disease: Crosstalk Between Serum Lipids, Inflammatory Cytokines and MAPK/ERK Pathway. Stroke, 20, suppl. 1. Xiao, J. (2017). Dietary flavonoid aglycones and their glycosides: Which show better biological significance? Crit. Rev. Food Sci. Nutr., 57,1874-1905. Yashin, A., Yashin, Y., Xia, X. & Nemzer, B. (2017). Antioxidant activity of spices and their impact on human health: A review. Antioxidants 6, 70-88.

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Figure captions

Figure 1. UHPLC chromatogram (360 nm) of a crude extract of parsley, Petroselinum crispum. Peaks were identified as isorhamnetin 3,7-diglucoside (1), isorhamnetin 3-malonylglucoside-7-glucoside (2), hesperetin 7-glucoside (3), apigenin 7-apiosylglucoside (Apiin) (4), isorhamnetin 3-glucoside (5), diosmetin 7-apiosylglucoside (6), apigenin 7-malonylapiosylglucoside (7) and diosmetin 7malonylapiosylglucoside (8). A compound (*) was detected which exhibited similar spectral characteristics to 7, whereas (**) was similar to 8 (table 1).

Figure 2. Structure of flavonoids isolated from parsley, Petroselinum crispum: isorhamnetin 3,7diglucoside (1), isorhamnetin 3-O-(6’’-O-malonyl-β-glucopyranoside)-7-O-β-glucopyranoside (2), hesperetin 7-glucoside (3), apigenin 7-apiosylglucoside (Apiin) (4), isorhamnetin 3-glucoside (5), diosmetin 7-apiosylglucoside (6), apigenin 7-malonylapiosylglucoside (7), diosmetin 3-O-(2’’-Oapiofuranosyl-6’’O-malonyl-β-glucopyranoside) (8) and apigenin 7-glucoside (9).

18

Table 1. UHPLC-MS characterization and quantification of main flavonoids and other phenolics in methanol extracts of herbs. Characteristic ions (m/z) from mass detection are listed with the pseudomolecular ions ([M+H]+ or [M-H]-) followed by fragments ions including those of the aglycone moieties. Amounts (mg 100 g-1 DM) of flavones and flavonols are reported as equivalents of quercetin 3-glucoside, whereas compound 1 together with rosmarinic- and chlorogenic acid are quantified as equivalents of chlorogenic acid. Herb Basil Chive Coriander Dill

Mint

Oregano Parsley

tR (min) 6.83 9.17 4.76 5.86 4.36 6.83 7.19 4.36 7.19 8.07 8.46 4.36 7.17 7.62 8.60 9.17 7.55 8.74 9.16 4.97 5.29 5.50 5.44 5.61 7.20

max (nm)

300sh, 328 264, 340 266, 324 300sh, 326 254, 354 256, 354 300sh, 326 256, 354 264, 346 263, 352 300sh, 326 254, 348 254, 348 252, 266, 346 300sh, 328 266, 346 266, 336 290sh, 330 256, 352 254, 352 270, 336 254, 352 254, 348 284, 332sh

Pos.ions 611 361 625, 287 611, 287 355 611, 303 479, 303 355 479, 303 463, 287 493, 317 355 595, 287 463, 287 609, 301 361 463, 287 447, 271 361 713 641, 317, 479 595 727, 317 727 465, 303

Neg.ions

Amount

711 639 593 725, 681 725 463, 301

High 16 637 23 37 165 115 207 729 268 155 109 nd 68 141 1567 558 224 177 777 nd nd nd nd nd nd

Compounds Low 15 0 16 13 0 8 0 172 47 25 33 nd 0 0 1 0 0 0 549 nd nd nd nd nd nd

Mean 15 366 19 25 31 66 99 392 153 83 77 nd 11 24 582 192 59 84 655 nd nd nd nd nd nd

Quercetin 3-rutinoside Rosmarinic acid Kaempferol hexoside-glucuronide Kaempferol 3-rutinoside Chlorogenic acid Quercetin 3-rutinoside Quercetin glucuronide Chlorogenic acid Quercetin glucuronide Kampferol glucuronide Isorhamnetin glucuronide Chlorogenic acid Luteolin rhamnosyl-hexoside Luteolin glucuronide Diosmetin rhamnosyl-hexoside Rosmarinic acid Luteolin glucuronide Apigenin glucuronide Rosmarinic acid Isorhamnetin 3,7-diglucoside (1) Apigenin dihexoside Isorhamnetin 3-malonylglucoside-7-glucoside a Isorhamnetin 3-malonylglucoside-7-glucoside b (2) Hesperetin 7-glucoside (3) 19

8.53 8.72 8.79 9.52 9.63 9.96 10.14 10.30 10.37 Rosemary

8.94 9.21 Tarragon 4.41 6.83 Thyme 7.43 9.20 nd=not determined

266, 338 256, 342 256, 346 266, 336 266, 336 266, 336 254, 346 254, 346 266, 340 252, 266, 346 300sh, 328 300sh, 326 254, 354 266, 346 300sh, 328

565, 271, 433 479, 317 595, 301, 433 651, 271 651, 271 651, 271 681, 301 681, 301 737, 651 433, 271 609, 301 361 355 611, 303 463, 287 361

563 477, 315 593 649, 605 649, 605 649, 605 679, 635 679, 635 431, 269

847 nd 531 nd 120 1044 1345 nd nd nd 41 1955 1667 610 1489 1387

540 nd 311 nd 44 66 17 nd nd nd 41 1785 1547 574 142 94

665 nd 456 nd 75 449 420 nd nd nd 41 1870 1607 592 660 775

Apigenin 7-apiosylglucoside (apiin) (4) Isorhamnetin 3-glucoside (5) Diosmetin 7-apiosylglucoside (6) Apigenin 7-malonylapiosylglucoside Apigenin 7-malonylapiosylglucoside (*) Apigenin 7-malonylapiosylglucoside (7) Diosmetin 7-malonylapiosylglucoside (8) Diosmetin/Chrysoeriol 7-malonylapiosylglucoside Apigenin 7-dimalonylapiosylglucoside Apigenin 7-glucoside (9) Diosmetin rhamnosylglucoside Rosmarinic acid Chlorogenic acid Quercetin 3-rutinoside Luteolin glucuronide Rosmarinic acid

20

Table 2. 1H NMR chemical shift values (δ 1H, ppm) and coupling constants (Hz) of isorhamnetin 3,7-diglucopyranoside (1), isorhamnetin 3-(6’’malonylglucopyranoside)-7-glucopyranoside (2), hesperetin 7-O-β-glucopyranoside (3), apigenin 7-(2’’-apiofuranosylglucopyranoside) (4, apiin), isorhamnetin 3- O-β-glucopyranoside (5), apigenin 7-(2’’-apiofuranosyl-6’’-malonylglucopyranoside) (7), diosmetin 7-(2’’-apiofuranosyl-6’’malonylglucopyranoside) (8), and apigenin 7-O-β-glucopyranoside (9) isolated from parsley, Petroselinum crispum recorded in DMSO-D6 at 298K. 1

2

2 3a 3b 6 8 2’ 3’ 5’

6.44 d 2.0 6.80 d 2.0 7.94 d 2.0

6.44 d 2.2 6.80 d 2.2 7.83 d 2.3

6.94 d 8.4

6.92 d 8.3

6’

7.52 dd 2.0, 8.4 12.59 s

7.56 dd 2.3, 8.3

3.83 s

3.83 s

5-OH 3’-OH 3’-OCH3 4’-OH 4’-OCH3

3 5.48 dd, 3.2, 12.2 3.28 m 2.76 dd 3.2, 17.1 6.12 d, 2.2 6.14 d, 2.2 6.92 d, 2.0 6.93 d, 8.3 6.87 dd, 2.0, 8.3

4 6.83 s

8 6.82 s

6.86 s

6.42 d 2.2 6.75 d 2.2 7.45 d 2.3 7.09 d 8.6

6.43 d 2.2 6.82 d 2.2 7.94 ‘d’ 8.8 6.93 ‘d’ 8.8 6.93 ‘d’ 8.8 7.94 ‘d’ 8.8

6.91 d 8.4

6.41 d 2.2 6.77 d 2.2 7.94 ‘d’ 8.9 6.94 ‘d’ 8.9 6.94 ‘d’ 8.9

7.93 ‘d’ 8.7

7.47 dd 2.2, 8.4

7.94 ‘d’ 8.9

7.57 dd 2.3, 8.6

12.98 s

12.94 s 9.44 s

3.82 s

10.39

10.68 s

5.47 d 7.2 3.25 m 3.26 m 3.14 m 3.35 m

5.55 d 7.5 3.21 m 3.25 m 3.10 m 3.10 m

6A’’

4.17 m

3.57 dd 1.7,

9

6.84 s 6.20 d 2.1 6.43 m 7.93 d 2.2

3-O-β-glucopyranoside 1’’ 5.56 d 7.3 2’’ 3.23 m 3’’ 3.25 m 4’’ 3.11 m 5’’ 3.11 m 3.57 d 11.5

7

6.42 d 2.2 6.80 d 2.2 7.93 ‘d’ 8.7 6.93 ‘d’ 8.7 6.93 ‘d’ 8.7

12.95 s

3.76 s

5

5.18 d 7.7 3.54 dd 7.7, 9.2 3.50 t 9.0 3.21 dd 8.8, 9.9 3.78 ddd 2.1, 7.1, 9.9 4.37 dd 2.1, 12.1

3.86 s 5.22 d 7.7 3.54 m 3.50 m 3.22 m 3.78 ddd 2.2, 6.9, 9.8 4.37 dd 2.2, 12.1 21

6B’’

3.38 dd 3.9, 11.5

7-O-β-glucopyranoside 1’’’ 5.07 d 7.8 2’’’ 3.26 m

5.05 d 7.7 3.26

3’’’ 4’’’

3.31 t 8.9 3.16 t 9.0

3.30 3.16

5’’’

3.43 m

3.43

6A’’’

3.69

3.69

6B’’’

3.44 m

3.47

2’’-O-apiofuranosyl 1’’’ 2’’’ 4A’’’ 4B’’’ 5A’’’ 5B’’’ 6’’-O-malonyl 2A’’’ 2B’’’

12.1 3.38 m

4.06 m

4.13 dd 6.9, 12.1

4.95 d 7.8 3.19 dd 7.8, 9.0 3.24 t 9.0 3.12 dd 9.0, 9.5 3.36 m

5.16 d 7.7 3.53 dd, 7.7, 9.1 3.49 t 9.1 3.20 t 8.9

5.06 d 7.7 3.25 dd 7.7, 9.0

3.48 m

3.65 dd 1.9, 12.0 3.42 dd 6.0, 12.0

3.72

3.44 ddd 9.5, 6.0, 2.2 3.70 dd 2.2, 11.9

3.48 m

3.47 dd 6.0, 11.9

5.35 d 1.2 3.76 d 1.2 3.92 d 9.4 3.66 d 9.4 3.33 d 11.2 3.30 d 11.2 3.22 s

4.10 dd 7.1, 12.1

3.29 t 9.0 3.17 dd 9.0, 9.5

5.34 d 1.5 3.74 d 1.5 3.91 d 9.5 3.66 d 9.5 3.32 d 11.2 3.29 d 11.2

5.34 d 1.5 3.74 d 1.5 3.91 d 9.5 3.66 d 9.5 3.32 d 11.2 3.29 d 11.2

3.29 d 15.4 3.25 d 15.4

3.39 d 15.8 3.35 d 15.8

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Table 3. 13C NMR chemical shift values (δ 13C, ppm) of isorhamnetin 3,7-diglucopyranoside (1), isorhamnetin 3-(6’’-malonylglucopyranoside)-7glucopyranoside (2), hesperetin 7-O-β-glucopyranoside (3), apigenin 7-(2’’-apiofuranosylglucopyranoside) (4, apiin), isorhamnetin 3- O-β-glucopyranoside (5), apigenin 7-(2’’-apiofuranosyl-6’’-malonylglucopyranoside) (7), diosmetin 7-(2’’-apiofuranosyl-6’’-malonylglucopyranoside) (8) and apigenin 7-O-βglucopyranoside (9) isolated from Petroselinum crispum recorded in DMSO-D6 at 298K. 2 3 4 5 6 7 8 9 10 1’ 2’ 3’ 4’ 5’ 6’ 3’-OCH3 4’-OCH3 1’’ 2’’ 3’’ 4’’ 5’’ 6A’’

1 157.10 133.49 177.79 161.04 99.59 163.07 94.76 156.23 105.88 121.10 113.73 147.16 149.88 115.45 122.42 55.90

2 157.2 133.6 177.8 161.2 99.7 163.3 95.0 156.3 106.0 121.1 113.6 147.2 150.1 115.5 122.8 56.0

3 78.7 42.4 197.3 163.4 96.7 165.4 95.7 162.9 103.5 131.1 114.4 146.7 148.2 112.2 118.0

4 164.41 103.23 182.13 161.29 99.48 162.83 94.94 157.06 105.52 121.17 128.75 116.14 161.50 116.14 128.75

5 156.4 133.0 n.a. 161.5 99.1 165.0 94.0 156.7 104.0 121.2 113.7 147.1 149.7 115.4 122.2 55.9

7 164.40 103.18 181.99 161.23 99.47 162.50 94.77 157.05 105.61 120.97 128.65 116.14 161.59 116.14 128.65

8 164.23 103.89 182.05 161.25 99.42 162.52 94.78 157.03 105.59 122.97 113.22 146.89 151.38 112.19 118.97

9 164.5 103.2 182.2 161.6 99.7 163.2 95.0 157.2 105.6 121.0 128.8 116.3 161.9 116.3 128.8

55.9 100.89 74.53 76.62 70.01 77.68 60.77

101.4 74.5 76.4 70.0 74.2 64.1

101.0 74.5 76.7 70.0 77.7 60.8

23

1’’’ 2’’’ 3’’’ 4’’’ 5’’’ 6’’’ 2’’-O-apiofuranosyl 1’’’ 2’’’ 3’’’ 4A’’’ 5A’’’ 6’’-O-malonyl 1’’’ 2’’’ 3’’’

99.98 73.29 76.65 69.83 77.40 60.84

100.2 73.4 76.8 69.9 77.5 60.9

99.8 73.3 76.5 69.7 77.3 60.8

98.27 75.89 76.94 69.94 77.17 60.67

98.01 75.63 76.53 69.94 73.83 63.85

97.86 75.57 76.51 69.84 73.67 64.02

108.88 76.22 79.44 74.14 64.36

108.85 76.17 79.39 74.08 64.27

108.82 76.15 79.36 74.07 64.24

167.57 42.53 168.05

166.86 41.36 167.84

166.4 40.7 168.0

100.1 73.3 76.7 69.8 77.4 60.8

Table 4. Overview over the most popular herbs used in Norwegian canteens. The herbs were purchased from the local supermarkets in one of three different packages: Fresh herbs in modified atmosphere (m.a.), as dried, pulverized spices (box), and as potted plants (potted). Dry matter content (DM) is given for herbs available as fresh products, and total phenolic content is given as equivalents of gallic acid (GAE). Number of repetitions (n) is 1-3, and each sample was analysed in duplicate.

Species

Supplier

Basil, Ocimum basilicum

Viking Ravnsborg Gård SantaMaria Viking Urt Rosnes AS

Chive, Allium schoenumprasum

Sample condition m. a. potted box m. a. potted

DM (%) 13.2 5.9 13.1 8.1

GAE, mg 100g-1 950 1480 450 470 490

GAE, mg 100g-1 mean ± stdav 958 ± 407 477 ± 40

24

Coriander, Coriandrum sativum Dill, Anethum graveolens

Oregano, Oreganum vulgare Mint, Mentha x piperita Parsley, Petroselinum crispum Rosemary, Rosmarinus officinalis Tarragon, Artemisa dracunculus Thyme, Thymus vulgaris

Viking Urt Ravnsborg Gård SantaMaria Einar Hanasand Rosnes AS Hindu SantaMaria Hindu SantaMaria Viking Urt Snarum Gartneri Einar Hanasand Santa Maria Viking Urt Viking Urt Hindu Viking Urt Hindu SantaMaria

m. a. potted box m. a. potted box box box box m. a. potted m. a. box m. a. m. a. box m. a. box box

10.3 6.1 12.1 7.9 15.4 10.9 16.1 24.7 27.6 22.8 -

830 540 140 910 930 460 650 1840 2380 1450 320 850 840 1270 2420 840 2340 1300 1790

634 ± 300 737 ± 200

2110 ± 273 1076 ± 702 985 ± 216 2416 ± 68 1807 ± 433

25

Fig 1. 26

OH

O

HO

CH3

O

OH

OH

HO

O

OH

HO

O HO

O

OH

O

HO

O

O

OH OH

O OH

O CH3

1 R=H 2 R=OC-CH2-COOH

O

OH

OH

O

3

RO

O

CH3

OR

OH

HO

O

OH

HO

O

HO

O HO

O

HO

O

OH

HO

4 R=H 7 R=CO-CH2-COOH

OH

HO

O

OR

OH

HO

O

O O

O O

HO

O

OH

OH OH

9

OH

CH3

O

OH O

OH

O

5

HO

HO

HO

O

O

OH OH

OH

O

O

O

6 R=H 8 R=CO-CH2-COOH

HO

O

27

Highlights



The ten most important herbs in Norwegian commercial kitchens has been analysed.



The structures of several novel flavonoids from parsley have been elucidated.



Oregano, rosemary and thyme contain the highest level of total phenolics.



Fresh herbs have a higher content of phenolics than processed, dry herbs.

Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

28