Fitoterapia 82 (2011) 1215–1221

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HPLC quantification of coumarin in bastard balm(Melittis melissophyllum L., Lamiaceae)

F. Maggi a, L. Barboni b,⁎, G. Caprioli a, F. Papa a, M. Ricciutelli c, G. Sagratini a, S. Vittori a

a School of Pharmacy, University of Camerino, Via Sant'Agostino 1, I-62032 Camerino, Italyb School of Science and Technology, Chemistry Division, University of Camerino, Via Sant'Agostino 1, I-62032 Camerino, Italyc HPLC-MS laboratory, University of Camerino, Via Sant'Agostino 1, I-62032 Camerino, Italy

a r t i c l e i n f o

⁎ Corresponding author. Tel.: +39 0737402240; faxE-mail address: luciano.barboni@unicam.it (L. Barb

0367-326X/$ – see front matter © 2011 Elsevier B.V. Adoi:10.1016/j.fitote.2011.08.011

a b s t r a c t

Article history:Received 20 June 2011Accepted in revised form 10 August 2011Available online 6 September 2011

Coumarin occurs in many plants used as flavoring and is known to possess hepatotoxic effects.Despite in the EFSA ‘Compendium of botanicals containing toxic substances’ coumarin isreported to be present in Melittis melissophyllum (bastard balm), a plant traditionally used asbeverage in Italy and Serbia, to the best of our knowledge quantitative data has never beenreported. Thus, the amount of coumarin in bastard balm leaves and its variation during the an-nual phenological cycle were determined. The subsp. melissophyllum resulted to contain highlevels of coumarin (14,392 mg/kg), mainly in the early stages of the plant cycle, suggestingprudence in its use as beverage. Furthermore, coumarin was found to be useful as marker com-pound to differentiate the bastard balm subspecies occurring in Italy, since the subsp. albidacontained a much lower content of this molecule (19–34 mg/kg).

© 2011 Elsevier B.V. All rights reserved.

Keywords:Bastard balm (Melittis melissophyllum)CoumarinPhenological cycleHPLC-DAD-MSSubsp. melissophyllumSubsp. albida

1. Introduction

Coumarin (1,2-benzopyrone), firstly isolated in 1822 fromTonka beans [Dypterix odorata (Aubl.) Willd., also known asCoumarouna odorata Aubl.] [1], is the parent compound ofa large class of naturally occurring phenolic constituents havingfused benzene and α-pyrone rings. For its strong fragrantodor, coumarin is widely used in industry as sweetener andfixative of perfumes, flavor enhancer, and other applications[2].

Coumarin occurs in many plants and in the correspondingessential oils used as flavoring ingredients [3–6]. Concerningplants for food consumption, significant concentrations, up to25,000 mg/kg, were found in cassia cinnamon [7].

Coumarin is stored in the vacuoles of fresh plant as o-coumaric acid glucoside, fromwhich is produced by enzymatichydrolysis and successive lactonisation, as a consequence ofplant tissues damage during harvesting and processing [8].

: +39 0737402297.oni).

ll rights reserved.

Synthetic coumarin was used as food flavoring until 1954,when it was banned because of its hepatotoxicity [9,10]. InEurope, maximum levels for coumarin in foodstuffs are inthe range of 5–50 mg/kg [11], and the tolerable daily intake(TDI) for coumarin is 0.1 mg/kg body weight, based on theno observed adverse effect level (NOAEL) for hepatotoxicityin animals [12] and human data available from the pharma-ceutical use of the substance [13].

Coumarin has been found in bastard balm (Melittismelissophyllum L., Lamiaceae) [14], a perennial herb growingin thermophilous copse broadleafwoodlands.M.melissophyllumoccurs in Italy with two subspecies: subsp. melissophyllum L.,growing mainly in the north-western and central Italy, andsubsp. albida (Guss) P.W. Ball, living mainly in southern Italyand islands [15].

In the folk medicine of central Italy the plant, known alsowith the local names of “Erba Lupa”, “Erba Limona” or “Cedrina”,was used as antispasmodic, against insomnia and eye inflamma-tions [16]. In the Abruzzo region it is still used during transhu-mance by shepherds who drink a warm decoction as digestiveand to treat cough and sore throat [17]. In Serbia it was usedfor its sedative properties, for nervous anxiety, insomnia, and

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hysteria [18]. Because of these properties, people used to makean aromatic tea with the fresh or dry leaves, to be drunk aftermeals as digestive and antispasmodic.

Although coumarin is reported to be present in bastardbalm in the EFSA ‘Compendium of botanicals that have beenreported to contain toxic, addictive, psychotropic or othersubstances of concern’ [19], no information is available re-garding the levels of this molecule, the plant part of possibleconcern, and the changes in content during the phenologicalcycle.

Therefore, in the present work an HPLC analysis of thecoumarin content in bastard balm leaves has been carriedout. The endogenous levels of total coumarin in one popula-tion of bastard balm have been monitored, in order to estab-lish if there is a link between the chemical variability ofcoumarin levels and the development stages during the an-nual phenological cycle. In addition, the quantitative changesduring the plant processing (e.g. evaporation etc.) have beendetermined by analyzing both fresh and dry samples. Becausein some plants coumarin is known to occur mainly as a gluco-sylated precursor (o-coumaric acid glucoside), an additionalaim of this study was to detect this precursor in the plant ex-tracts by using HPLC-MS. Finally, coumarin was investigatedas a chemotaxonomically useful marker compound to distin-guish the two bastard balm subspecies occurring in Italy.

2. Experimental procedures

2.1. Plant material

M. melissophyllum subsp.melissophyllumwas collected in apopulation growing wild in central Italy (Camerino) at differ-ent stages of the plant phenology (3 May, 18 May, 30 May, 14June, 28 June, 12 July and 2 August 2010), from the beginningof vegetative stage to the end of reproductive stage, in orderto establish if there is a link between the variability of couma-rin levels and the development stages during the annual bio-logical cycle. To avoid the daily variations, the sampling wasdone as far as possible in the same time of the day (from11 am to 1 pm). M. melissophyllum subsp. albida was collect-ed in June–July 2010 in three sites in southern Italy and Sicily(Potenza, Cosenza and Palermo, respectively). Voucher speci-mens were identified by Dr. Maggi using available literatureand deposited in the Herbarium Camerinensis (CAME) and inthe Herbarium of Centro Ricerche Floristiche dell'Appennino(APP) (both acknowledged in the Index Herbariorum, http://sciweb.nybg.org/science2/IndexHerbariorum.asp) of the Schoolof Environmental Sciences, University of Camerino (Italy)

Table 1Collection localities of the investigated bastard balm (Melittis melissophyllum) samp

Subspecies Collection site Date of collection

melissophyllum Piedilapiaggia (Camerino) May-August 2010albida Monte Vulture (Potenza) 6 July 2010albida Celico (Cosenza) 30 June 2010albida Madonie, Monti Balatelli (Palermo) 4 June 2010

a Accession number in: CAME, Herbarium Camerinensis, School of EnvironmentalRicerche Floristiche dell'Appennino, Barisciano, Italy.

(collection localities and voucher codes are reported inTable 1).

2.2. Sample preparation

Fresh leaves were manually excised from plants and thenstored in the dark and at room temperature (~22 °C) untilcompletely dry. Part ofmaterial (only for subsp.melissophyllum)was deep frozen at−20 °C just after its collection and was keptat freezing temperature until extraction, in order to find out thepotential changes of coumarin content depending on the watercontent. Dry leaveswere ground to fine powder in amill (blend-er MFC model DCFH 48 IKA-WERK, Staufen, Germany), andsieved (1 mm pore diameter). Fresh leaves were homogenizedwith 10 ml of liquid nitrogen for 5 min and reduced into a pow-der in a mortar. In order to estimate the water content, freshleaves, were left in a stove at 110 °C for 24 h. Moisture contentwas determined by theweighing of the leaves of each collectionbefore and after drying. The experiment was performed threetimes.Moisture levels in percentages ranged from81% at the be-ginning of vegetative stage (3 May 2010) to 60% at the end ofvegetative stage (2 August 2010).

2.3. Reagents and materials

Coumarin (≥99%), methanol (HPLC grade) and acetoni-trile (HPLC grade) were purchased from Sigma-Aldrich(Milan, Italy); hydrochloric acid (37%) was obtained fromCarlo Erba (Milan, Italy). Individual stock solutions were pre-pared by dissolving 1 g of coumarin in 100 ml (ppm) ofmethanol and storing this solution in glass-stopped bottlesat 4 °C. Deionized water (N8 MΩ cm resistivity) was obtainedfrom a Milli-Q SP Reagent Water System (Millipore, Bedford,MA). Disposable Minisart SRP4 filters with a pore width of0.45 μm (Chromafil PET-20/25) were from Sartorius StedimBiotech GmbH (Goettingn, Germany).

2.4. Extraction procedure

Powdered fresh (2 g) and dry leaves (0.4 g correspondingto 2 g of fresh leaves taking into account a mean water contentof 80%) were placed in a round bottom flask, equipped with amagnetic stirrer, and extracted with 5 ml methanol/water(80:20 v/v) for 30 min at room temperature. Then extractswere centrifuged at 6500 rpm for 10 min. The methanol/water mixture was selected as the most effective for extractionof coumarin according to previous results [3]. For quantifica-tion of total coumarin, an acidic hydrolysis of the coumarin pre-cursor was performed according to reported procedures [4,6].

les.

GPS coordinates Altitude (m) Voucher codesa

N 43°09′10″; E 13°07′18″ 590 CAME 13,430N 40°56′08″; E 15°37′04″ 1300 APP 42,031N 39°18′33″; E 16°23′02″ 1200 APP 42,035N 37°54′29″; E 13°59′45″ 933 APP 42,032

Sciences, University of Camerino, Camerino, Italy; APP, Herbarium of Centro

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Briefly, one-fifth of the methanol/water extracts was stirredin hydrochloric acid (2.5 N, 5 ml) at 80 °C for 90 min. In allcases, samples were filtered through disposable MinisartSRP4 (Sartorius Stedim Biotech GmbH, Goettingn, Germany)filters in PTFE (0.45 μm), and injected (5 μl) into the HPLCsystem.

2.5. HPLC-DAD and HPLC-MS analysis

The HPLC system consisted of an Agilent Technologies(Palo Alto, CA) HP-1100 series, made of an autosampler anda binary solvent pump, with a diode array detector (DAD).LC separation was performed on a reversed phase columnPurosfer© C18 (150×4.6 mm i.d., 5 μm) from Merck KGaA(Darmstadt, Germany) thermostatted at 40 °C, using mobilephase A (water with 1% v/v formic acid) and mobile phase B(acetonitrile) in a gradient program with a flow of 1.0 ml/min: 0–20min: 10% B; 20–45 min: 90% B. The volume of a sin-gle injection was 5 μl. After each injection, column was equili-brated for 15 min. For quantitative analysis, the wavelengthwith the highest intensity was used (278 nm). Furthermore,UV/Vis spectra between 235 and 600 nm were recorded toverify the peak identity of coumarin and the peak purity. Thesame chromatographic conditions reported above were usedfor HPLC-MS (single quadrupole) analysis, performed by aHewlett Packard (Palo Alto, CA, USA) HP-1090 Series II, madeof an autosampler and a binary solvent pump, with a massspectrometer detector (MSD) equipped with an ESI interfacein negative ionization (NI) mode. The optimized parameterswere: drying temperature, 325 °C; nebulizer gas (nitrogen)pressure, 60 psi; drying gas (nitrogen) flow rate, 13 l/min;temperature, 350 °C; and capillary voltage, 3500 V. Quantita-tive data were collected using Chemstation for LC 3D systemssoftware rev. B.01.03[204].

2.6. Method validation

An eight point calibration curve was constructed by ana-lyzing various amounts (2, 10, 50, 100, 500, 1000, 2000 and3000 mg/l) of coumarin (n=3 each). It showed a good line-arity in the whole range of the tested concentrations with aregression coefficient (R2) greater than 0.9999. The limits ofdetection (LOD) and quantification (LOQ) were 0.17 mg/land 0.56 mg/l, respectively.

Among several procedures adopted for the extraction ofcoumarin from plant material, we chose that appearing thebest, i.e. using methanol (80%) with magnetic stirring for30 min at room temperature [3]. This procedure is currentlyincluded in the draft of an official German standard methodfor the determination of coumarin in foods. To evaluatewhether the extraction procedure was sufficient for quantify-ing coumarin in bastard balm leaves, known amounts of thecoumarin standard were added to fresh and dry samples attwo fortification levels (200 and 2000 mg/kg), and the recov-ery was calculated. It was found to be 104 and 90% for freshsamples, and 96 and 93% for dry samples at the two fortifica-tion levels, respectively, with a coefficients of variation (CV%)below 7% (n=3) in all cases. The CV values for intra-assayprecisions (n=4) ranged from 6.7 to 16.2% and from 0.3 to2.9% for total coumarin in fresh and dry samples, respectively.

These data indicated a good repeatability, since they arebelow the recommended limits [20].

2.7. Extraction and MS/MS analysis of the o-coumaric acidglucoside precursor

There is evidence that o-coumaric acid glucoside is presentas a glycosidically-bound precursor of coumarin in someplants [8]. To investigate its presence in bastard balm leaves,the methanol/water extract was purified as follows: part ofthe dried extract was suspended in water, washed twicewith n-hexane and ethyl acetate, and extracted twice withwater-saturated n-butanol. The butanolic fraction was thenevaporated to dryness, dissolved in methanol/water 50:50(100 μg/ml), filtered through 0.45 μm disposable Minisart SRP4filters, and used for the characterization with FIA-ESI-IT massspectrometry by an Agilent (Santa Clara, CA, USA) MSD Trap SLequipped with an ESI source operating in negative ionizationmode. The operating ESI conditions were: drying gas tempera-ture, 350 °C; nebulizer gas (nitrogen) pressure, 30 psi; dryinggas (nitrogen) flow rate, 8 l/min; and capillary voltage, 3500 V.The mass spectrometer was used in the Smart Tune mode ofthe using Daltonic Esquire Control software, while infusing theextracted solution via a syringe pump at a flow rate of 5 μl/min.The acquisition was in full scan and in Manual MS mode; nega-tive ions were detected using the normal resolution and thetrap parameters were set in Ion Charge Control (ICC) usingrolling averaging set at 5 with a target of 30,000, and maximumaccumulation time of 300 ms at m/z range from 50 to 1500.

2.8. Statistical analysis

One-way ANOVA, performed by SPSS (v. 13.0) softwarepackage for Windows (SPSS Inc., Chicago, IL, USA), was used toestimate overall significance. A probability level of 5% (pb0.05)was considered significant.

3. Results and discussion

3.1. Identification of coumarin and its glucosylated precursor

Plants have been reported to liberate coumarin from itsprecursor by enzyme action after disrupture of cells [21] oc-curring during harvesting and processing. In the applied pro-cedure this happened especially after nitrogen treatment anddisruption during plant processing.

Fig. 1 reports the HPLC-DAD chromatograms of freshleaves before (a) and after (b) acidic hydrolysis. It can beseen as coumarin (r. t. 17.0 min, identified by comparisonwith authentic coumarin) and its precursor, o-coumaric acidglucoside (r. t. 7.3 min), were efficiently separated within17 min. In particular, the peak with retention time of7.3 min showed a UV spectrum similar to that of coumarin,although there is a difference in the absorbance range be-tween 310 and 320 nm (Fig. 2a). Moreover, HPLC-MS analy-sis showed that the mass spectrum referred to the peakwith retention time of 7.3 min (Fig. 2b), presents two princi-pal signals at m/z 325.1 and 651.2, that correspond to the [M-H]− and [2 M-H]− ions. At the beginning of work, these sig-nals have been hypothesized to be typical of o-coumaricacid glucoside.

Fig. 1. HPLC-DAD chromatograms of bastard balm samples (collection of 14June): a) fresh leaves; b) fresh leaves after acidic hydrolysis.

Fig. 2. a) UV spectra of coumarin and its glucosidic precursor; b) mass spec-trum of glucosidic precursor of coumarin in negative ionization mode usingsingle quadrupole.

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To confirm the identity of this molecule, the glucosidic frac-tion was purified according to the procedure reported above,and analyzed by ESI-MSn obtaining the spectra shown inFig. 3. The MS2 of m/z 325 [M-H]− gave two peaks at 163 and119m/z, corresponding to the successive loss of one glucoseand one CO2 units, respectively. The MS3 of m/z 163 gave apeak at 119m/z confirming the above fragmentation pattern.On this basis and according to the literature [6], the compoundwas therefore identified as o-coumaric acid glucoside. In thepresent work, the isomeric form in which the precursor occursin bastard balm leaves was not investigated.

3.2. Total coumarin content in fresh and dry bastard balm leavesand changes during the phenological cycle

M. melissophyllum has been included in the “EFSA Com-pendium of botanicals that have been reported to containtoxic substances” [19] for the occurrence of coumarin. How-ever, no information is available regarding the levels of thismolecule, as well as the plant part with the highest concen-tration. On the other hand, seasonal changes and environ-mental conditions may affect the production of secondarymetabolites in various parts of the plant. Therefore, besidesthe first quantification of coumarin levels in bastard balmleaves, in the present study the changes in content duringthe phenological cycle of the plant, as well as those occurringduring processing of plant material, have been reported(Table 2).

The analytical data obtained as reported in the experi-mental section were corrected for the moisture content andwere expressed on a dry weight basis. Liberation of coumarinwas initiated during sample manipulation, by nitrogen treat-ment and homogenization of fresh leaves and by shredding ofdry leaves. Consequently, cells were mechanically destroyed,enabling the enzymes such as glucosidase to interact with the

substrate, and leading to the production of coumarin fromglucosidic precursor. In addition, in order to quantify thetotal coumarin occurring in the plant matrix, the glucosidicprecursor was completely hydrolysed, as described above.

The levels of total coumarin in bastard balm samples var-ied between 4548 mg/kg and 14,392 mg/kg in fresh samples,and between 3091 mg/kg and 11,125 mg/kg in dry samples(Table 2). There was a similar pattern in coumarin levels dur-ing the phenological cycle both in fresh and dry material. Themaximum levels (14,392 mg/kg and 11,125 mg/kg in freshand dry leaves, respectively) were observed at the beginningof cycle (3 May) when the plant shows a high growth rate(renewal after winter dormant). As a consequence, also thewater content of the plant leaves resulted the maximum(above 80%), allowing cell vacuoles to contain higher amountsof the hydrophilic coumarin precursor. This might explain therole of coumarin as phytoalexin: since the plant at the begin-ning of the cycle is quite susceptible to wounds or attacks byother organisms, it produces coumarin as an antipathogenic

Fig. 3. a) Fragmentation pattern proposed for the glucosidic precursor of coumarin; b), c) and d) mass spectra (MS, MS2 and MS3) obtained in the ion trap.

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compound. Furthermore, the primary site of synthesis of cou-marin is suggested to be the young, actively growing leaves.The above values resulted among the highest detected inplantmatrices so far, but substantially lower than the publishedhigh contents, ranging between 16,000 and 25,000 mg/kg,detected in cassia cinnamon [7]. The total coumarin content de-creased at early stages of flowering (18May) and raised again in

Table 2Endogenous levels of free and total coumarin (mg/kg dry weight) in Bastardbalm leaves (subsp. melissophyllum) during the phenological cycle.a

Date ofcollection

Fresh leaves Dry leaves

Free Total Free Total

3 May 7076 14,392 2537 11,12518 May 3815 6635 1168 536330 May 5380 8861 1330 610214 June 6181 9742 2216 629728 June 5536 7984 1816 564512 July 2617 4548 691 30912 August 2843 4588 363 3143Mean 4779 8107 1446 5824

a Data are means of four determinations.

the fruiting (14 June) when it reached the concentration of9742mg/kg and 6297 mg/kg in fresh and dry leaves, respec-tively. After this stage, the coumarin content decreased, andreached the lowest values (4588mg/kg and 3143 mg/kg infresh and dry leaves, respectively) at the later stages of cycle(2 August) when the plant lost significant amount of water(water content about 60%). The differences observed betweenfresh and dry samples (pb0.05) for each collection can beexplained by the evaporation of coumarin (from 19% to 35%depending on the different collection periods) liberated fromits precursor during the drying process. At this regard, dryingis reported to inducesù the formation of coumarin on the sur-face of the plant leaves, as a response to traumatic injury duringthe wilting process [22]. Therefore, the total coumarin contentresulted significantly (p≤0.05) higher in fresh samples.

Comparing the levels of coumarin in bastard balm leavesobtained with and without acidic treatment, we can noticethe following points: 1) as can be seen in the chromatogramreported in Fig. 1b, the boundprecursor is hydrolysed effective-ly by acidic treatment; 2) there is a significant increase in totalcoumarin content both in fresh (pb0.05) and dry samples(pb0.001) after treatment with hydrochloric acid. This can be

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due to the high amounts of the bound precursor (mean of 40and 80% in fresh and dry samples, respectively) found in bas-tard balm leaves where it is stored in the vacuole of cells. Con-sequently, the content of coumarin after acidic treatmentshowed a mean value from ~2- to 4-fold higher than in freshand dry leaves without hydrolysis, respectively (Table 2).These results are in contrast to those reported for cassia andCeylon cinnamon, and green tea leaves [4,6], where it wasfound that coumarin mostly occurred in its free form. On theother hand, our results are in accordance with those observedin woodruff (Asperula odorata), where only a minor part ofthe total coumarin occurred in its free form, being 88% presentas bound precursor [4]; and 3) after the drying process, freecoumarin decreased under a factor of about 3 orders of magni-tude (Table 2), suggesting that this process influenced the con-tent of coumarin in the final product.

3.3. Coumarin as chemotaxonomic marker

As reported above, bastard balm occurs in Italy with twosubspecies: the subsp. melissophyllum, occurring in the North-Western and central Italy; and the subsp. albida occurring insouthern Italy and Islands. So far, the classification of these sub-species was exclusively morphology-based, being the densityof glandular trichomes, the dimension of the leaves and thenumber of teeth on each side used as the main discriminantcharacters.

Previously, by using SPME coupled to GC-FID and GC-MS,we found that headspace levels of coumarin in the subsp. albidawere significantly lower than in the subsp.melissophyllum [23],suggesting that this molecule can be used as a chemotaxonom-ic marker.

Therefore, another aim of this work was to discriminatethe two subspecies by detecting significantly different con-centrations of this marker compound in the plant matrix,hence to support the botanical classification.

The total coumarin content detected in dry leaves of thesubsp. albida were 19mg/kg in the population from Calabria,26 mg/kg in the population from Basilicata and 35 mg/kg inthe population from Sicily, thus far lower (about 220-fold)than the mean content detected in dry leaves of the subsp.melissophyllum (5824mg/kg) (pb0.001). These results are inagreement with the chemical differences previously reportedat headspace level between the two subspecies [23], supportingthe use of coumarin as a chemotaxonomic marker for bastardbalm. Thus, the subsp. albida resulted to have concentrationsof coumarin in line with those reported for Ceylon cinnamon(Cinnamomum zeylanicum) bark essential oil (40 mg/kg) andblue-white clover seeds (Trigonella caerulea) (37 mg/kg) [4].Therefore, this subspecies should be preferred to the subsp.melissophyllum in the use as flavoring in food products andbeverages.

4. Conclusions

The present work reports the first quantification of cou-marin in bastard balm (M. melissophyllum). In this way, theEFSA ‘Compendium of botanicals that have been reported tocontain toxic, addictive, psychotropic or other substances ofconcern’ can be updated. A simple, fast and low-cost HPLCmethod for determination of endogenous coumarin levels

has been applied. It resulted suitable for a rapid screeningof numerous plant samples to be analyzed.

This plant (notably the subsp.melissophyllum) was found tocontain among the highest levels of coumarin detected so far.Hence, if bastard balm is regarded as a foodstuff, the coumarinconcentration in the leaves is much higher than the maximumlevel permitted posing health risk to humans. However, theplant was traditionally used under decoction or infusion as di-gestive, hence it might be treated as a beverage. Assuming amean content of coumarin of 6 g/kg, as detected in our study,only 750 mg of dried leaves in infusion with 500 ml water areneeded to equal themaximum limits of coumarin in beverages(2 mg/l). On the other hand, about 1 g of dried leaves, if eaten,are enough to reach a daily coumarin intake corresponding tothe TDI.

The changes in the content of coumarin during the pheno-logical cycle were investigated highlighting the early stagesof the cycle and the fruiting time as the most appropriate pe-riods for the production of the molecule in the plant. Thus,coumarin levels can vary widely (up to more than 3-fold) be-tween different sample harvesting times.

The precursor molecule, o-coumaric acid glucoside, wasdetected in bastard balm leaves. The results indicated that cou-marin occurred asmuch as in its bound and free form, and thatdrying processing was found to influence the content of cou-marin in the final product leading to a loss of the volatile inits free form due to evaporation.

Coumarin turns out as amolecule of chemotaxonomic value,to be successfully used in the support of the taxonomy of bas-tard balm since its amount clearly differentiated the subsp.melissophyllum with respect to the subsp. albida. Thus, thiswork represents the first phytochemical approach using HPLCmethod as an effective tool to distinguish the two chemotypesof bastard balm, giving a rapid and unequivocal discriminationusing coumarin as marker compound.

Finally, it is recommended that traditional consumers useprudentially bastard balm leaves with low coumarin levels,corresponding to dry material collected at the latter stagesof the plant cycle and belonging to the subsp. albida.

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