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Polyphenolic Composition andAntioxidative Properties of Lemon Balm(Melissa officinalis L.) Extract Affectedby Different Brewing ProcessesAleksandra Sentkowskaa, Magdalena Biesagaa & Krystyna Pyrzynskaa

a University of Warsaw, Department of Chemistry, Warsaw, PolandAccepted author version posted online: 31 Dec 2014.Publishedonline: 31 Dec 2015.

To cite this article: Aleksandra Sentkowska, Magdalena Biesaga & Krystyna Pyrzynska (2015)Polyphenolic Composition and Antioxidative Properties of Lemon Balm (Melissa officinalis L.) ExtractAffected by Different Brewing Processes, International Journal of Food Properties, 18:9, 2009-2014,DOI: 10.1080/10942912.2014.960932

To link to this article: http://dx.doi.org/10.1080/10942912.2014.960932

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International Journal of Food Properties, 18:2009–2014, 2015Copyright © Taylor & Francis Group, LLCISSN: 1094-2912 print/1532-2386 onlineDOI: 10.1080/10942912.2014.960932

Polyphenolic Composition and Antioxidative Properties ofLemon Balm (Melissa officinalis L.) Extract Affected by

Different Brewing Processes

Aleksandra Sentkowska, Magdalena Biesaga, and Krystyna Pyrzynska

University of Warsaw, Department of Chemistry, Warsaw, Poland

Polyphenolic composition of lemon balm (Mellisa officinalis L.) aqueous extracts prepared as infusionand decoction were evaluated by high performance liquid chromatography/tandem mass spectrometry.All the samples showed a similar phenolic profile but presenting differences in the quantities found ofeach compound. Higher content was found in plant infusion extract compared to decoction process.During the prolonged time of extractions the contents of some compounds were changed. Rutin andcaffeic acid contents were decreased in the infusion mode, while increased concentration was observedduring decoction process. The antioxidant activities measured by Folin-Ciocalteu and CUOPRAC assayswere higher for the extract prepared in decoction mode (1245 mg/g GAE and 4.91 mmol/g TRE,respectively) in comparison to infusion process (267 mg/g GAE and 2.62 mmol/g TRE).

Keywords: Lemon balm, Melissa officinalis L., Polyphenols, Antioxidant activity, Extraction conditions.

INTRODUCTION

Lemon balm (Melissa officinalis L.), a perennial herb belonging to Lamiaceae family, is cultivated inmany countries for its carminative and antispasmodic properties. It is used for treatment of headache,rheumatism, indigestion, and hypersensitivities.[1] Natural essential oils (mixtures of fragrant chem-ical) obtained from various parts of plant are efficient active antimicrobial agents[2] and due to itscitrus fragrance is used in the cosmetic industry and perfumery. All of these properties of lemonbalm have been related to the high levels of polyphenolic compounds.[3−5]

Antioxidative properties of polyphenolic compounds are manifested particularly by their abilitiesto inhibit free radical generation, scavenge free radicals, and chelate transition metal ions, whichare catalysts of free radical reactions. Polyphenols also prevent free radical generation by inhibitingactivity of existing enzymes participating in their generation or by increasing the activity of enzymeswith antioxidative properties.[6] The antioxidant capacities of many polyphenolic compounds aremuch stronger than those of vitamin C and E.[7]

There is an increasing interest in the antioxidant effects of natural compounds from medicinalplants and pharmaceutical products. Herbal therapies are perceived by many as natural, safer, gentler,

Received 12 May 2014; accepted 29 August 2014.Address correspondence to Krystyna Pyrzynska, Department of Chemistry, University of Warsaw, Pasteura 093, Warsaw

02-093, Poland. E-mail: kryspyrz@chem.uw.edu.pl

2009

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and lower in cost than synthetic pharmaceuticals. Several different species and aromatic herbs havebeen investigated for their chemical composition and antioxidant activity.[8−10]

Infusion and decoction are the most consumed preparation of Melissa officinalis. During theseprocesses, hydrophilic compounds will be released, including flavonoids and phenolic acids. Theobjective of this study was to compare the content of some phenolic compounds in aqueous extractsof lemon balm prepared as infusion and decoction. These processes were examined using differentbrewing time. Additionally, antioxidant properties of the prepared extracts were studied by Folin-Ciocalteu (FC) and cupric reducing ability (CUPRAC) assays.

MATERIALS AND METHODS

Reagents and Apparatus

The commercial standards of flavonoids and polyphenolic acids, FC’s reagent as well as the restof the chemicals were purchased from Sigma (Steinheim, Germany). Acetonitrile were of HPLCgradient grade from Merck (Darmstadt, Germany). Ultrapure water from Milli-Q system (Millipore,Bedford, MA, USA) with a conductivity of 18 MQ was used in all experiments.

Chromatographic analysis was performed with the Shimadzu LC system with 3200 QTRAP Massspectrometer (Applied Biosystem/MDS SCIEX). A mass spectrometry (MS) system was equippedwith electrospray ionization source (ESI). ESI in negative mode conditions were used. For eachcompound the optimum conditions of selected reaction mode (SRM) were determined in infusionmode. Continuous mass spectra were obtained by scanning m/z from 50 to 650.[11]

Compounds were separated on SeQuantTM ZIC-HILIC column (100 × 2.1 mm, 3.5 µm) fromMerck (Darmstadt, Germany) at 30◦C. Ten mM ammonium acetate (pH 7) as eluent A andacetonitrile as eluent B were used. The mobile phase was delivered at 0.2 mL/min in gradient mode:0–4 min 98%B, 6–7 min 90% B, 8–8.4 min 80% B, 8.4–12 min 50% B, and 13–20 min 98% B. Theanalytes were identified by comparing retention time and m/z values obtained by MS and MS2 withthe mass spectra.[11] Quantification of compounds was done from the calibration curves obtained inSRM mode.

Brewing Processes

Lemon balm tea bags were purchased from a local market. In order to simulate household brewingconditions, the samples were prepared using an aqueous extraction procedure. Infusion was carriedout by pouring 2 g of dried plant in 50 mL of freshly boiled distilled water and allowed steep forappropriate interval time at room temperature. For decoction, 50 mL of distilled water was added to2 g of a herb and the liquid was heated on the heating plate under the cover and boiled for appropriateinterval time. The extracts were then filtered and analyzed. For each experiment the brewing processwas conducted separately from three bags.

FC Assay

The FC assay was conducted according to Singleton et al.[12] with a slight modification. Briefly,1 mL of extract was introduced into test tubes followed by 0.1 mL of FC’s reagent and 0.9 mL ofwater. The tubes were allowed to stand for 5 min. Then, 1 mL of Na2CO3 (7%, w/v) and 0.4 mL ofwater were added and 10 more min was allowed for stabilization of the formed color. The absorbanceagainst a reagent blank was measured at 765 nm. The data were expressed as gallic acid equivalent(GAE) in mg per gram of dry matter.

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LEMON BALM (MELISSA OFFICINALIS L.) EXTRACT 2011

CUPRAC

For CUPRAC method, the assay described by Apak et al. was adopted.[13] One mL of CuCl2 solution(1.0 × 10−2 M) was mixed with 1 mL of neocuproine solution (7.5 × 10−3 M) and 1 mL of 1 MNH4AC buffer (pH 7), followed by mixing 0.5 mL of extract and 0.6 mL of water. The reagentswere then incubated in a water bath at 50◦C for 20 min. Absorbance against the reagent blank wasmeasured at 450 nm. The antioxidant activity of the extracts were expressed as trolox equivalent(mmol TR/g of dry matter).

Statistical Analysis

The results were expressed as mean ± standard deviation at least three independent determinations.Statistical analysis was conducted using the software package STATISTICA 8.0 for Windows fromStatsoft. A p-value of less than 0.05 was considered statistically different.

RESULTS AND DISCUSSION

HPLC Analysis

The data from the HPLC analysis of the aqueous lemon balm extracts prepared as infusion anddecoction is presented in Table 1, while the chromatogram of this herb infusion is shown in Fig. 1.Rosmarinic acid was the major phenolic compound present in the plant extract, which is in accor-dance with the previous studies.[5,14] From the flavonoid family, high content of rutin was found aswell as traces of quercetrin and myricetin. The presence of rutin in the aqueous extract of lemon balmwas not reported, although, its aglycones quercetin was found in the hydrolyzed extract.[15] Borroset al.[5] found only one flavonoid, luteolin-3-O-glucoronide, in cultivated, in vitro cultured andcommercial samples of Melissa officinalis. Some hydroxycinnamic acids (caffeic acid, chlorogenicacid, and p-coumaric acid) and hydroxybenzoic acid derivatives (gallic acid and p-HBA) were also

TABLE 1The content of polyphenols in the extracts of lemon balm

Infusion time, min Decoction, time, min

10 15 20 10 15 20

Myricetin 0.05 ± 0.02a 0.07 ± 0.03a 0.43 ± 0.02b 0.05 ± 0.02a 0.09 ± 0.03b 0.16 ± 0.05c

Quercetrin 0.03 ± 0.01a 0.05 ± 0.02a 0.05 ± 0.03a 0.01 ± 0.007a 0.02 ± 0.01a 0.03 ± 0.01a

Rutin 6.10 ± 0.43a 6.67 ± 0.41b 4.46 ± 0.31c 1.03 ± 0.02a 2.81 ± 0.04b 4.59 ± 0.38c

p-HBA 1.76 ± 0.05a 1.25 ± 0.05b 1.90 ± 0.11c 0.87 ± 0.07a 1.41 ± 0.06b 1.28 ± 0.04c

Gallic acid 0.46 ± 0.04a 0.55 ± 0.04b 0.51 ± 0.03a 0.02 ± 0.01a 0.03 ± 0.02a 0.07 ± 0.02b

Caffeic acid 3.93 ± 0.14a 3.10 ± 0.11b 3.38 ± 0.15c 3.75 ± 0.19a 3.77 ± 0.17a 4.61 ± 0.25b

p-Coumaric acid 0.50 ± 0.06a 0.52 ± 0.02a 0.48 ± 0.02a 0.19 ± 0.02a 0.32 ± 0.03b 0.36 ± 0.04b

Chlorogenic acid 0.24 ± 0.02a 0.27 ± 0.01a 0.28 ± 0.01a 0.09 ± 0.02a 0.14 ± 0.03b 0.17 ± 0.04b

Rosmarinic acid 62.8 ± 3.41a 67.8 ± 3.56a 72.3 ± 4.09b 58.6 ± 2.93a 84.4 ± 4.15b 68.3 ± 3.12c

Antioxidant activityFC assay (mg GAE/g) 267 ± 7.0a 525 ± 3.6b 708 ± 5.6c 1245 ± 5.5a 1399 ± 5.0b 1408 ± 6.1b

CUPRAC (mmol TR/g) 2.62 ± 0.03a 2.31 ± 0.07b 2.37 ± 0.04b 4.91 ± 0.05a 5.09 ± 0.04b 4.79 ± 0.06c

Values (mg/L) are expresses as mean ± sd (n =3);nd: not detected;In each row different letters mean significant differences (p < 0.05).

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FIGURE 1 Total ion chromatogram (TIC) in SRM mode of lemon balm water extract and extracted ion chro-matogram of polyphenolic compounds. Column: SeQuantTM ZIC-HILIC (100 × 2.1 mm, 3.5 µm). Eluent:CH3COONH4/acetonitrile.

found. From the quantified phenolic acids, caffeic, and coumaric acids have already been reportedin the lemon balm extracts.[13,5,14] The differences in chemical composition may be due to a vari-ety of reason ranging from climate and geography to difference in the specificity of the extractionprocedures.[3]

The amounts of the phenolic compounds found in water extracts of lemon balm varied among thedifferent preparation processes. Generally, higher content of determined polyphenolic compoundswas found in plant infusion compared to decoction process. The same was observed in chamomile(Matricaria recutita L.) extracts.[16] During the prolonged time of extraction the contents of somepolyphenolic compounds were changed. Rutin and caffeic acid contents were decreased in the infu-sion mode, while increase of their concentration was observed during decoction process. Myricetincontent was increasing in both brewing processes. Polyphenols are present in the plant matrix atdifferent sites, some of which can be readily extracted, while others are tightly bound. Therefore,the longer time of extraction and increase in temperature may have influenced the release of thetightly bound compounds and improves the efficiency of extraction. However, degradation of sometermolable compounds also could occur as a result of polyphenol oxidase activity.[17]

Antioxidant Activity

For the evaluation of antioxidant properties of studied lemon balm extracts FC and CUPRAC assayswere used as no single assay accurately reflects the mechanism of action of all radial sources or allantioxidants in a complex sample.[18] These assays used different chromogenic redox reagents as

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LEMON BALM (MELISSA OFFICINALIS L.) EXTRACT 2013

0

200

400

600

800

1000

1200

FC assay

mg

GA

E/g

infusion decoction

Lemon balm St. John` wort Lemon balm St. John` wort0

1

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5

CUPRAC

mm

ol T

R/g

infusion decoction

FIGURE 2 Comparison of antioxidant activity of lemon balm and St. John’s wort water extracts using FC andCUPRAC assays. Extraction time 10 min.

well as different conditions for measurement. FC assay has been used for many years by the foodand agricultural industries to determine the phenolic content of plant products. However, FC reagentreacts with other antioxidants besides phenols and possible contributiors include proteins, carbo-hydrates, amino acids, thiols, vitamins, amines, aldehydes, and ketones.[19] The chemistry behindthis assay counts on the transfer of electrons in alkaline medium from phenolic compounds andother reducing species to molybdenum, forming blue complexes that can be monitored spectropho-tometrically. CUPRAC method is based on reduction of Cu(II) to Cu(I) at neutral pH by reductants(antioxidants) present in a sample utilizing the copper(II)-neocuproine reagent as the chromogenicoxidant.[13]

The antioxidant capacities of lemon balm extracts using both reagents are presented in Table 1.The values in FC assay increased with prolonged time of brewing, particularly for infusion mode.However, during decoction process more compounds with antioxidant (reductative) properties, arereleased and the highest value for so-called total phenols was obtained for this brewing mode.Some organic acids (oxalic or ascorbic acid) released from the plant during brewing, may con-tribute to the antioxidant activity of the samples studied herein. As literature data for lemon balminfusion in FC assay are expressed in different units – as 8240 mg GAE/100 g,[20] 959.5 mgGAE/mL,[14] or 268.0 mg GAE/g (dry weight) extract,[3] thus, it is difficult to compare them.The values for antioxidant activity in CUPRAC method decreased slightly when the extraction timewas increased. Apak et al.[13] reported CUPRAC value for Melissa officinalis infusion as 0.99 mmolTR/g. No values were found for antioxidant activity of this herb prepared in decoction mode.

The obtained value for the antioxidant capacities of lemon balm using both FC and CUPRACassays were compared with the aqueous extracts of St. John’s wort (Hypericum perforatum), popularmedicinal plant, using the same procedures (Fig. 2). In FC assay very similar results were obtainedfor both plants in decoction mode, while the infusion of St. John’s wort exhibited higher value.Using the CUPRAC method the values for lemon balm extracts were higher despite of kind ofbrewing process. Similar results were reported by Kratachanova et al.,[20] who compared the resultsfor these two plants using FC and ORAC assays.

CONCLUSIONS

The present study showed that the aqueous extracts of lemon balm can be a source of phenolic com-pounds such as rutin, quercetrin, and myricetin from the flavonoid family as well as some phenolic

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acids, known by their bioactive effects. A higher content of these compounds was found in plantinfusion extract compared to decoction process. However, higher antioxidant activity was obtainedfor the extract prepared in decoction mode.

REFERENCES

1. Petersen, M.; Simmonds, M.S.J. Molecules of interest: Rosmarinic acid. Phytochemistry 2003, 62, 121–125.2. Allahverdiyev, A.; Duran, N.; Ozguven, M.; Koltas, S. Antiviral activity of the volatile oils of Melissa officinalis L.

against Herpes simplex virus type-2. Phytomedcine 2004, 11, 657–661.3. Dastmalchi, K.; Dorman, H.J.D.; Oinonen, P.P.; Darwis, Y.; Laakso, I.; Hiltumen, R. Chemical composition and in vitro

antioxidative activity of a lemon balm (Melissa officinalis L.) extract. LWT–Food Science and Technology 2008, 41,391–400.

4. Dias, M.I.; Barros, L.; Sousa, M.J.; Ferreira, I.C.F. Systematic comparison of nutraceuticals and antioxidant potentialof cultivated, in vitro cultured, and commercial Melissa officinalis samples. Food and Chemical Toxicology 2012, 50,1866–1873.

5. Barros, L.; Dueñas, M.; Dias, M.I.; Sousa, M.J.; Santos-Buelga, C.; Ferreira, I.C.F. Phenolic profiles of cultivated, invitro cultured, and commercial samples of Melissa officinalis L. infusions. Food Chemistry 2013, 136, 1–8.

6. Galleano, M.; Verstraeten, S.V.; Oteiza, P.I.; Fraga, C.G. Antioxidant actions of flavonoids: Thermodynamic and kineticanalysis. Archives of Biochemistry and Biophysics 2010, 501, 23–30.

7. Prochazková, D.; Bousová, I.; Wilhelmová, N. Antioxidant and prooxidant properties of flavonoids. Fitoteriapia 2011,82, 513–523.

8. Jaberian, H.; Piri, K.; Nazari, J. Phytochemical composition and in vitro antimicrobial and antioxidant activities of somemedicinal plants. Food Chemistry 2013, 136, 237–244.

9. Alinezhad, H.; Azimi, R.; Zare, M.; Ebrahimzadeh, M.A.; Eslami, S.; Nabavi, S.F.; Nabavi, S.M. Antioxidant andantihemolytic activities of ethanolic extract of flowers, leaves, and stems of hyssopus officinalis L. Var. angustifolius.International Journal of Food Properties 2013, 16, 1169–1178.

10. Nabavi, S.F.; Nabavi, S.M.; Ebrahimzadeh, M.A.; Eslami, B.; Jafari, N. In vitro antioxidant and antihemolytic activitiesof hydroalcoholic extracts of Allium scabriscapum Boiss. & Ky. aerial parts and bulbs. International Journal of FoodProperties 2013, 16, 713–722.

11. Sentkowska, A.; Biesaga, M.; Pyrzynska, K. Effects of the operation parameters on HILIC separation of flavonoids onzwitterionic column. Talanta 2013, 115, 284–290.

12. Singleton, V.L.; Orthofer, R.; Lamuela-Ravwntos, R.M. Analysis of total phenolics and other oxidation substrates andantioxidants by means of Folin-Ciocaltea reagent. Methods in Enzymology 1999, 299, 152–178.

13. Apak, R.; Güçlü, K.; Özyürek, M.; Karademir, S.E.; Erçag, E. The cupric reducing antioxidant capacity and polyphenoliccontent of some herbal teas. International Journal of Food Science and Nutrition 2006, 57, 292–304.

14. Fecka, I.; Turek, S. Determination of water-soluble polyphenolic compounds in commercial herbal teas from Lamiaceae:Peppermint, melisa, and sage. Journal of Agricultural and Food Chemistry 2007, 55, 10908–10917.

15. Komes, D.; Bešcak-Cvitanovic, A.; Horžic, D.; Rusak, G.; Likic, S.; Berendika, M. Phenolic composition andantioxidant properties of some traditionally used medicinal plants affected by the extraction time and hydrolysis.Phytochemical Analysis 2010, 22, 172–180.

16. Guimarães, R.; Barros, L.; Duenas, M.; Calhelha, R.; Carvallh, A.M.; Santos-Buelga, C.; Queiroz, M.J.; Ferreira, I.F.F.Infusion and decoction of wild German chamomile: Bioactivity and characterization of organic acids and phenoliccompounds. Food Chemistry 2010, 136, 947–954.

17. Marete, E.N.; Jacquier, J.C.; O’Riordan, D. Effects of extraction temperature on the phenolic and parthenolide contents,and colour of aqueous feverfew (Tanacetum parthenium) extracts. Food Chemistry 2009, 117, 226–231.

18. Magalhâes, L.M.; Segundo, M.A.; Reis, S.; Lim, J.L.F. Methodological aspects about in vitro evaluation of antioxidantproperties. Analytica Chimica Acta 2008, 613, 1–19.

19. Everette, J.D.; Bryant, Q.M.; Green, A.M.; Abbey, Y.A.; Wangila, G.W.; Walker, R.B. Through study of reactivity ofvarious compound classes toward the Folin-Ciocalteu reagent. Journal of Agricultural and Food Chemistry 2010, 58,8139–8144.

20. Kratchanova, M.; Denev, P.; Ciz, M.; Lojek, A.; Atanas Mihailov, A. Evaluation of antioxidant activity of medicinalplants containing polyphenol compounds. Comparison of two extraction systems. Acta Biochimica Polonica 2010, 57,229–234.

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