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May 7, 2009 - Vıctor López Æ Sara Martın Æ Maria Pilar Gómez-Serranillos Æ. Maria Emilia Carretero Æ Anna K. Jäger Æ. Maria Isabel Calvo. Accepted: 18 ...
Neurochem Res (2009) 34:1955–1961 DOI 10.1007/s11064-009-9981-0

ORIGINAL PAPER

Neuroprotective and Neurological Properties of Melissa officinalis Vı´ctor Lo´pez Æ Sara Martı´n Æ Maria Pilar Go´mez-Serranillos Æ Maria Emilia Carretero Æ Anna K. Ja¨ger Æ Maria Isabel Calvo

Accepted: 18 April 2009 / Published online: 7 May 2009 Ó Springer Science+Business Media, LLC 2009

Abstract Melissa officinalis has traditionally been used due to its effects on nervous system. Both methanolic and aqueous extracts were tested for protective effects on the PC12 cell line, free radical scavenging properties and neurological activities (inhibition of MAO-A and acetylcholinesterase enzymes and affinity to the GABAA-benzodiazepine receptor). The results suggest that the plant has a significant (P \ 0.05) protective effect on hydrogen peroxide induced toxicity in PC12 cells. The radical scavenging properties were also investigated in cells and in cell free systems, where this plant was shown to be a good free radical scavenger. The MAO-A bioassay was also performed to detect possible antidepressant activities demonstrating that both extracts inhibited this enzyme, which has a key role in neurotransmitters metabolism. However, no activity was detected in the acetylcholinesterase and GABA assays. In general, the methanolic extract was more effective than the aqueous. Keywords Melissa officinalis  Traditional medicine  Neuroprotective  PC12  Antioxidant  MAO

V. Lo´pez (&)  M. I. Calvo Department of Pharmacy and Pharmaceutical Technology, School of Pharmacy, University of Navarra, Irunlarrea sn, 31080 Pamplona, Spain e-mail: [email protected] V. Lo´pez  S. Martı´n  M. P. Go´mez-Serranillos  M. E. Carretero Department of Pharmacology, School of Pharmacy, Complutense University of Madrid, 28040 Madrid, Spain V. Lo´pez  A. K. Ja¨ger Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen O, Denmark

Introduction The leaves of Melissa officinalis L. (Lamiaceae), also known as lemon balm, are used in traditional medicine to prepare a tea for its nerve calming and spasmolytic effects [1–4] although there are a great variety of phytopharmaceutical preparations containing this plant or its extracts. Furthermore, this plant is used by food industry to flavour different products due to its particular taste. The number of people suffering from neurological disorders has lately increased worldwide, specially in the developed countries [5, 6]. Between them, neurodegenerative diseases (Parkinson, Alzheimer) as well as psychiatric ones (anxiety and depression) are the most common [7]. Oxidative stress is directly implicated in neurodegenerative diseases. Reactive oxygen species are a class of highly reactive molecules derived from oxygen and generated by metabolic processes and some external factors. An excessive production of ROS may cause DNA mutation and protein and lipid oxidation leading to cellular senescence and neuronal death [8]. Natural antioxidants from plants are well known to protect the human organism from free radicals, preventing from certain diseases. In this sense, plants are a source of compounds with antioxidant activity and Melissa officinalis might have some protective effects. Neuroprotection was studied using an in vitro cellular model with the PC12 (rat pheochromocytoma) cell line, which retains features of dopaminergic neurons and is a common model used in neurosciences [9, 10]. Antioxidant capacities were carried out in cell free systems with different free radicals. Neurological activities were investigated because lemon balm is mainly used for its calming effects: monoamine oxidase (MAO) and acetylcholinesterase (AChE) inhibition and affinity to the GABAA-benzodiazepine receptor.

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Depression is a common form of mental illness. The WHO estimates that at least 3% of the world’s population is suffering from depression [11]. The sympthoms of depression can be alleviated by increasing the synaptic availability of monoamines. Such increase can be brought about by decreasing the metabolism of these neurotransmitters by inhibition of oxidative enzymes as for example MAO-A. Alzheimer’s disease is the most common form of dementia and the current treatments include acetylcholinesterase inhibitors such as galantamine. Binding to the GABA-benzodiazepine receptor was also studied because affinity to this site produces a sedative and anticonvulsive effect. Studies on neuroprotective and neurological activities of this plant may demonstrate the effects of Melissa officinalis on the central nervous system as well as to elucidate the mechanisms involved in the activity.

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until dryness in a rotatory evaporator and the aqueous extracts were lyophilized. The dry extracts were kept in glass vials at -40°C prior to biological assays. Only the methanolic and aqueous extracts were used in these assays. Extracts were dissolved in PBS and filtered before use. Protective Effect Against H2O2-induced Toxicity in PC12 Cells Cell Culture Rat pheochromocytoma cells (PC12) were obtained from the American Type Culture Collection. Cells were grown in DMEM supplemented with 10% heat-inactivated horse serum, 5% heat-inactivated fetal bovine serum, penicillin (10 U/ml), streptomycin (10 lg/ml) and 0.2 mM sodium pyruvate. Cultures were incubated in the presence of 5% CO2 at 37°C and 100% relative humidified atmosphere.

Experimental Procedure MTT Assay Chemicals The cytotoxicity detection kit was purchased from Roche (Indianapolis, USA). Dulbecco’s Modified Eagles Medium (DMEM), penicillin–streptomycin, fetal bovine serum (FBS), horse serum (HS), sodium pyruvate and Dulbecco’s phosphate buffered saline (PBS) were obtained from Gibco (Barcelona, Spain). 2,2-azino-bis(3-ethylbenzothiazoline6-sulfonic acid) diammonium salt (ABTS), xanthine, xanthine oxidase from buttermilk, nitroblue tetrazolium salt chloride (NBT), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), acetylthiocholineiodide (ATCI), 5,50 -dithiobis[2-nitrobenzoic acid] (DNTB) and some others were purchased from Sigma-Aldrich (Spain). Caffeic acid was obtained from Extrasynthe`se (Genay, France). 3H-Ro 15-1788 (flumazenil) was purchased from Perkin Elmer Life Sciences. Plant Material The plant was collected in Pamplona (province of Navarra, Spain) in June 2004 and was authenticated by Silvia Akerreta and Rita Yolanda Cavero (Department of Plant Biology, University of Navarra). Voucher specimens (18687) have been deposited in the PAMP Herbarium of the University of Navarra. Preparation of Extracts The powder of dried aerial parts of the plants (5 g) was successively extracted three times with 200 ml of dichloromethane, ethyl acetate, methanol and water after maceration at 4°C for 24 h. Solvents were removed under vacuum

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The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is a yellow tetrazolium salt that is converted into a purple compound (formazan) in viable cells by mitochondrial enzymes [12]. PC12 cells were seeded in 96-multiwell plates at 2 9 104 cells/well. After 48 h, plant extracts were added to the wells at different concentrations and incubated for 24 h. Cells were treated with PBS containing H2O2 250 lM for 30 min. After 24 h, the medium was removed and MTT (0.3 mg/ml final concentration in the wells) was added for 1 h incubation at 37°C. Cell survival (%) was measured in terms of absorbance at 550 nm in a microplate reader (Bio-Tek, USA) comparing treated (hydrogen peroxide or plant extract plus hydrogen peroxide) with untreated cells. LDH Assay The protective effect of the extracts in the PC12 cell line was also studied by the determination of lactate dehydrogenase (LDH) into the incubation medium using a commercial kit from Roche. LDH is a cytosolic enzyme released into the medium when the integrity of the cell membrane deteriorates suffering from necrotic cell death [13]. Cells were seeded in 96-multiwell plates at 2 9 103 cells/well and treated as in the MTT assay. The assay was carried out following the instructions of the manufacturer. Background interferences were deducted by calculating the LDH activity of the medium. Spontaneous release of LDH was also calculated and subtracted by measuring LDH activity of untreated cells. Total intracellular LDH was measured in cell lysates obtained by treatment with the manufacturer lysis solution. Percentage of cell death (LDH

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release) was calculated comparing LDH activity of treatments with total intracellular LDH. Measurement of Intracellular Reactive Oxygen Species Formation This assay was performed as previously described with some modifications [14]. Cells were seeded in 96-multiwell plates at 2 9 104 cells/well. After 48 h, cells were incubated with 20 ,70 -dichlorofluorescein diacetate (DCFH/DA) for 30 min in a final concentration of 10 lM. DCFH-DA solution was removed and cells were co-incubated with different extract doses and 250 lM H2O2. The fluorescence intensity was measured at 485 nm excitation and 530 nm emission for 2 h. Antioxidant Activity in Cell Free Systems ABTS Assay The free radical scavenging activity was measured as previously described [15] but adapted to 96-well microplates. About 200 ll of the ABTS solution was added to 20 ll of plant extracts at different concentrations. Absorbance was read after 6 min at 734 nm and percentage of inhibition was calculated with the following equation: (Abscontrol-Absample/Abscontrol) 9 100. The radical scaveging capacity (RSC) of the extracts is expressed as IC50, defined as the concentration of extract that scavenge 50% of free radicals. IC50 values were calculated by a non-linear regression with a one phase exponential association equation using GraphPad Prism version 4.0. Superoxide Radical Scavenging Activity Superoxide radicals were generated by the xanthine/xanthine oxidase (X/XO) system following a described procedure [16] with some modifications. The reaction mixture contained 75 ll NBT (50 lM), 75 ll xanthine (145 lM), 75 ll plant extract (different concentrations) and 75 ll xanthine oxidase (0.29 U/ml). The reaction was initiated by the addition of the enzyme and the mixture was incubated for 2 min at 37°C. Antioxidant activity was determined by monitoring the effect of the extracts on the reduction of NBT to the blue chromogen formazan by O2- at 560 nm: % RSC = (Abscontrol-Absample/Abscontrol) 9 100. IC50 values were calculated by a non-linear regression using GraphPad Prism version 4.0. Inhibition on Xanthine Oxidase The effect of the extracts on xantine oxidase was also evaluated at the same concentrations by measuring the

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formation of uric acid from xanthine in order to detect possible enzyme inhibition [17]. The reaction mixture contained the same proportion of components as described above except NBT: 75 ll phosphate buffer, 75 ll xanthine (145 lM), 75 ll plant extract (different concentrations) and 75 ll xanthine oxidase (0.29 U/ml). Absorbance was read at 295 nm in this case after 2 min. Neurological Activities MAO-A Inhibitory Activity The bioassay was performed in a 96-well microplate [18]. Each well contained fifty microliters plant extract (or DMSO as blank), 50 ll chromogenic solution (0.8 mM vanillic acid, 417 mM 4-aminoantipyrine and 4 U/ml horseradish peroxidase in potassium phosphate buffer pH 7.6), 100 ll 3 mM tyramine and 50 ll 8 U/ml MAO-A. Absobance was read at 490 nm every 5 min for 30 min. Background interferences were deducted as the same way described above but without MAO enzyme. Data were analyzed using GraFit 5 to obtain IC50. Clorgyline was used as a reference MAO-A inhibitor. Acetylcholinesterase Inhibitory Activity The activity was measured using a 96-microplate reader based on Ellman’s method [19]. Each well contained 25 ll of 15 mM ATCI in Millipore water, 125 ll of 3 mM DTNB in buffer C (50 mM Tris–HCl, pH 8, 0.1 M NaCl, 0.02 M MgCl2 6 H2O), 50 ll buffer B (50 mM Tris–HCl, pH 8, 0.1% Bovine Serum), 25 ll plant extract in buffer A (50 mM Tris–HCl, pH 8). The absorbance was read five times every 13 s for five times at 405 nm. Then, 25 ll 0.22 U/ml AChE were added and the absorbance was measured again eight times every 13 s at 405 nm. Galantamine was used as control substance. GABAA-receptor Assay Membrane Preparation The preparation was performed using cerebral cortex of four rats as described by Risa et al. [20]. Flumazenil Binding Assay The membrane preparation was washed with ice-cold buffer (50 mM Tris–citrate pH 7.1). The suspension was centrifuged at 0–4°C for 10 min at 27,000g. The pellet was resuspended in Tris– citrate buffer (2 mg original tissue per ml) and used for the assay. 25 ll of 3H-Ro 15-1788 (flumazenil) was added to 25 ll of test solutions (1, 0.1, 0.01 mg/ml) and 500 ll of membrane preparation. Total and unspecific binding was measured using buffer or diazepam (1 lM assay final

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concentration). After incubation for 40 min in an ice bath, 5 ml of ice-cold buffer was added to the samples and poured onto Adventic glass fibre filters (GC-50) under vacuum, and inmediatly washed with another 5 ml of icecold buffer. The amount of radioactivity of the filters was measured by conventional liquid scintillation counting using Ultimo Gold XR as scintillation fluid. Clonazepam was used as positive control. Statistical Analysis Data are means ± SD of at least three independent experiments. One-way ANOVA followed by Dunnett’s multiple comparison test was used to compare control and treated groups in cells experiments. IC50 values were analyzed by t-test.

Table 2 Protective effect of Melissa officinalis extracts on hydrogen peroxide induced toxicity in PC12 cells by the LDH release assay Methanolic extract

Aqueous extract

56.0 ± 5.1

56.0 ± 5.1

20 lg/ml 40 lg/ml

54.7 ± 6.8 41.9 ± 6.2

61.9 ± 6.1 47.6 ± 3.2

60 lg/ml

38.6 ± 6.4*

42.7 ± 4.8

80 lg/ml

29.7 ± 9.8*

62.7 ± 6.7

Control (250 lM H2O2) Treated (Melisa officinalis ? H2O2)

Results are expressed as % of cell death in terms of LDH release (mean ± SD) of three experiments performed in triplicates. Concentrations 20–80 lg/ml refer to the dose of extracts in the wells * P \ 0.05 versus control (250 lM H2O2)

200 180

Hydrogen Peroxide 250µM

160

20 µg/ml

Effect Against H2O2-induced Toxicity in PC12 Cells The effect of the extracts in PC12 cells was determined using the MTT, LDH and intracellular ROS formation assays. Pretreatments with the methanolic extract at concentrations of 60 and 80 lg/ml protected PC12 cells against hydrogen peroxide toxicity by the MTT assay. Neuroprotection was also observed by the LDH test at the same doses (Tables 1, 2). Aqueous extract obtained from Melissa officinalis did not protect cells by either MTT or LDH assays. For the study of the preventive effect on ROS formation, cells were co-incubated with extracts and 250 lM H2O2. Both aqueous and methanolic extracts reduced the percentage of ROS formation for the 2 h of the experiment (Figs. 1, 2).

% ROS

Results

40 µg/ml

140

60 µg/ml

120

80 µg/ml

100

100 µg/ml

80

120 µg/ml

60 0

15

30

45

60

90

120

Time (min)

Fig. 1 Protective effect of methanolic extract of M. officinalis on ROS intracellular production after exposition to 250 lM H2O2. Results are means of three experiments, each one performed in eight replicates. Error bars not shown as SD was less than 10% 200 Hydrogen Peroxide 250µM

180

20 µg/ml 160

% ROS

40 µg/ml

Table 1 Protective effect of Melissa officinalis extracts on hydrogen peroxide induced toxicity in PC12 cells by the MTT assay

140 60 µg/ml 120 80 µg/ml 100 100 µg/ml 80

Methanolic extract

Aqueous extract

50.2 ± 3.8

50.2 ± 3.8

20 lg/ml

43.8 ± 5.1

42.1 ± 3.9

40 lg/ml

51.5 ± 4.6

44.6 ± 2.5

60 lg/ml

61.4 ± 4.0*

50.4 ± 6.1

80 lg/ml

64.4 ± 7.3*

49.0 ± 5.2

Control (250 lM H2O2) Treated (Melisa officinalis ? H2O2)

Results are expressed as % of cell survival (mean ± SD) of three experiments performed in eight replicates. Concentrations 20–80 lg/ml refer to the dose of extracts in the wells * P \ 0.05 versus control (250 lM H2O2)

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120 µg/ml 60 0

15

30

45

60

90

120

Time (min)

Fig. 2 Protective effect of aqueous extract of M. offficinalis on ROS intracellular production after exposition to 250 lM H2O2. Results are means of three experiments, each one performed in eight replicates. Error bars not shown as SD was less than 10%

Antioxidant Activity in Cell Free Systems Methanolic and aqueous extracts possessed high antioxidant properties in the cell free system experimental models.

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Table 3 Antioxidant activity of Melissa officinalis in cell free systems IC50 (lg/ml) ABTS

Superoxide radical

Xanthine oxidase 500.2 ± 47.1

Methanolic extract

12.7 ± 0.4**

5.9 ± 0.9*

Aqueous extract

17.1 ± 0.9

7.8 ± 0.8

2.2 ± 0.4

1.4 ± 0.2

Caffeic acid Allopurinol

n.d. 5.5 ± 0.3

Results are presented as IC50 values (mean ± SD) obtained from non linear regression of three experiments performed in triplicates. IC50 is defined as the concentration of extract that inhibits 50% of the free radical or the enzyme * P \ 0.05; ** P \ 0.01 versus aqueous extract

However, this activity was not superior to the control substances used in the assays (Table 3). Neurological Activities Results on the MAO-A/AChE inhibition and binding the GABAA-benzodiazepine receptor are shown on Table 4. Both the methanolic and aqueous extracts inhibited MAO-A, the methanolic extract being the most effective (Fig. 3) whereas inhibition of AChE was not detected. Clorgyline was used a a selective MAO-A inhibitor, resulting twenty times more potent than the methanolic extract. Neither methanolic nor aqueous extracts exerted affinity to the GABAA receptor as flumazenil was not displaced from binding the receptor.

Discussion Oxidative stress produces cell death and reactive oxygen species, including hydrogen peroxide, is involved in neurotoxic events related to some neurodegenerative processes

such as Alzheimer’s disease. It is known than lemon balm extracts contain phenolic compounds such as flavonoids and phenolic acids [21] that may scavenge free radicals. The antioxidant activity of those extracts have previously been established in different experimental models [21–25] but the protective effects in a PC12 cell line have never been reported. PC12 cells are a useful model in neuroscience due to its phenotypic characteristics with sympathetic neurons. Therefore, a protective activity in these cells may mean that lemon balm might have a protective effect in neurons. In this study, exposition of cells to 250 lM H2O2 reduced cell survival around 50% and pretreatments with methanolic extracts increased the survival in 15%. The co-treatment of PC12 cells with extract (methanolic and aqueous) and 250 lM H2O2 produced a significant reduction in ROS intracellular formation. Therefore these results suggest that Melissa officinalis exert protective activities in the PC12 cell line and might protect neurons from oxidative stress. Both extracts exhibited a dose dependent antioxidant activity in the cell free systems. In all cases, the methanolic extract was more effective than the aqueous, with a lower IC50. Three different methodologies were performed to evaluate the antioxidant activity. The ABTS method is fast and easy but the ABTS radical is not involved in human biology; another method using the superoxide radical, which is a physiological radical, was then performed. The superoxide radical was generated by the X/XO system [26] and both extracts showed superoxide radical scavenging activities. Taking into account that the scavenging effect on superoxide radical might be due to an inhibition of the enzyme, the effect of the extracts on XO was checked out. The results show that only the methanolic extract of Melissa officinalis had a weak inhibition on XO, with a IC50 value around 500 lg/ml, a hundred times lower effect than the xanthine oxidase inhibitor allopurinol. The aqueous extract acted as a superoxide scavenger but no inhibition on XO was detected. The results on antioxidant activity were in part expected as this species is known to

Table 4 Neurological activities of Melissa officinalis MAO-A

AChE assay

GABAA assay (% Flumazenil binding)

IC50 (lg/ml)

IC50 (lg/ml)

10 mg/ml

1 mg/ml

0.1 mg/ml

Metanolic extract

19.3 ± 2.3**

na

78.7 ± 2.3

94.7 ± 16.1

84.7 ± 16.1

Aqueous extract

48.2 ± 5.8

na

94.8 ± 9.0

94.3 ± 4.4

95.2 ± 11.3

Clorgyline

0.1 ± 0.01

Galantamine

19.9 ± 4.8

Results expressed as IC50 for MAO-A and AChE assays. Results for GABAA assay are expressed as % of flumazenil binding (IC50 for clonazepam is 0.002 nm). In all cases, data (mean ± SD) were obtained from three experiments performed in triplicates na no activity ** P \ 0.01 versus aqueous extract

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Neurochem Res (2009) 34:1955–1961 80

80

60

60

% MAO-A inhibition

% MAO-A inhibition

1960

40

20

40

20

0

0 10-3

10-2

10-3

[Methanolic extract]

10-2

[Aqueous extract ]

Fig. 3 MAO-A inhibitory effect of lemon balm extracts. Extracts were assayed at concentrations of 3.75, 7.5, 15, 30 and 60 lg/ml. Data are means ± SD of three independent experiments performed in triplicates

contain caffeic acid derivatives and flavonoids, which have previously shown free radical scavenging properties [27]. As far as neurological activities are concerned only inhibition of MAO-A was detected. It is the first time this activity is reported for Melissa officinalis. MAO-A is an enzyme distributed in the central nervous system and has a key role in catecholamine metabolism. The inhibition of this enzyme is related to alleviation of depression symptoms [7] so this could be one of the mechanisms involved in its central nervous system effects as in many cases the antidepressant activity is related to an anxyolitic effect. Though lemon balm is usually used for its calming effect, methanolic and aqueous extracts did not bind to the GABAA receptor as previous studies demonstrated [28]. On the contary, Melissa officinalis from Lebanon showed some kind of activity in the GABA-receptor assay [29]. Some previous reports also show the lack of activity of the plant in the acetylcholinesterase assay [30]. Despite of the unclear results about the methanolic and aqueous extracts in the GABA assays, some authors found the affinity to the GABA-receptor in the essential oil obtained from lemon balm [31], which may be in major part, the responsible for the anxyolitic effect. Lemon balm contains some flavonoids such as quercitrin as well as apigenin and luteolin derivatives that might inhibit monoamine oxidases as previous studies demonstrated that a number of flavonoids possesed MAO-inhibitory activity [32–34]. In conclusion, Melissa officinalis has shown protective effects in the PC12 cell line, free radical scavenging properties and MAO-A inhibitory effects, the methanolic extract being the most effective. These results suggest the potential use of this plant or its constituents for central

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nervous system disorders and as a neuroprotective agent to prevent diseases in which oxidative stress is involved. Acknowledegments University of Navarra Foundation and Alumni Navarrensis Association are thanked for finantial support and fellowships.

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