M.T. Ortega Hernández-Agero, O.M. Palomino Ruiz-Poveda, M.P. Gómez-Serranillos Cuadrado and M.E. Carretero Accame*
Introduction
Melissa offi cinalis L., from the Lamiaceae family, is one of the vegetal species traditionally used in Europe for the treatment of nervousness, anxiety and sleep disorders, as well as for gastrointestinal complaints; it is usually known as melisa, toronjil or hierba limonera (Spanish), lemon balm (English), mélisse (French), melissa (Italian), melissenblätter or zitronenkraut (German), cidreira (Portuguese), citroenmelisse (Dutch) or φύλλο μελίσσης (Greek).
This species originated in southern Europe, but nowadays is naturalized around the world, from North America to New Zealand. Lemon balm occurs naturally in sandy and scrubby areas but has also been found on damp wasteland, at elevations ranging from sea level to the mountains, including most of the Iberian peninsula regions.
M. offi cinalis is an erect perennial aromatic herb which grows up to 70–150 cm tall with straight hairy stalks. The leaves grow in opposite pairs of toothed, smooth and ovate leaves growing on square, branching stems. They have a gentle lemon scent, close to mint, with 3–9 x 1.8–5 cm size and occasionally up to 15 x 8 cm. Flowers are two-lipped, grow in whorled clusters, and may be pale yellow, white or pinkish.
The fruit is a tiny nutlet (1.6–2 x 0.8–1 mm), with a dark brown colour.
The herbal drug consists of the dried leaf of Melissa offi cinalis L. According to the European Pharmacopoeia monograph (2013), it contains a minimum of 1.0% of rosmarinic acid (C18H16O8; Mr 360.3), calculated with reference to the dried drug. Other international organizations such as the European Scientifi c Cooperative on Phytotherapy (ESCOP), European Medicines Agency (EMA) and Commission E have also published a monograph for this species.
Department of Pharmacology, School of Pharmacy, Universidad Complutense de Madrid. Pza Ramón y Cajal s/n, 28040 Madrid, Spain.
Email: meca@farm.ucm.es
* Corresponding author
Melissa leaves chemical composition includes one essential oil which is mainly made up by sesquiterpenes (beta-caryophyllen, germacren-D) and especially monoterpenes (aldehydes: citral A and B, known as geranial and neral, respectively; citronellal; alcohols: linalol; esters: geranil acetate; hydrocarbon derivatives: beta-pinene); other groups have also been identifi ed, including triterpenes (ursolic and oleanolic acids), phenolic acids derived from hydroxycinnamic acid (rosmarinic, caffeic, chlorogenic), fl avonoids (quercitroside, apigenin derivatives, luteolin, quercetin and kaempferol) and tannins (Fig. 3.1).
The herbal drug has been used since ancient times. Greek and Roman civilizations used lemon balm preparations for oral and topical use, i.e., for wound healing and insect bites. Also the Persian medical doctor Avicenna recommended its topical use in wounds, ulcers and scabies. Later on, during the 15th century, Paracelsus used lemon balm for “all complaints supposed to proceed from a disordered state of the nervous system”. Traditional use also assigned benefi cial effects on the memory, as well as digestive, analgesic and sedative properties for the relief of insomnia, fever, common cold, etc. Ayurvedic traditional medicine used lemon balm for the treatment of dyspepsia associated with depression or anxiety.
Pharmacological and clinical studies have shown its antioxidant, sedative, anxiolytic, digestive, spasmolytic, hypoglycaemic, anti-infl ammatory, hepatoprotective and antimicrobial (antibacterial, antifungal and antiviral) activities. Recent research studies point the possible protective role of lemon balm in Alzheimer’s disease.
A summary of the scientifi c studies performed with M. offi cinalis L. leaves is shown in Tables 3.1 and 3.2 (pharmacological in vitro and in vivo studies, respectively).
Figure 3.1. Main chemical components of Melissa offi cinalis L. leaf.
Central nervous system activity
Several in vitro and in vivo pharmacological studies on the activity of lemon balm on CNS have been performed. Some of them intended to elucidate its mechanism of action. This research line was completed with some clinical trials to prove its effi cacy on human being.
Melissa Offi cinalis Updated Overview 59
Neuroprotection Antioxidant
Extract/ Active principle Assay Model Activity Reference
Hydroalcoholic extract TBARS DPPH
- Antioxidant Pereira et al. 2009
Aqueous extracts/
Poliphenols
ABTS - Antioxidant Ivanova et al. 2005
Aqueous extract Methanolic extract
MAO-A Inhibitory activity
Acetylcholinesterase inhibitory activity ABTS
Superoxide Radical Scavenging A Inhibition on Xanthine Oxidase
PC12 cells Inhibition MAO-A
No activity on acetylcholinesterase Antioxidant
López et al. 2009
Hydroalcoholic extract Inhibition of lipid peroxidation Enzyme peroxidation system Antioxidant Hohmann et al. 1999 Ethanolic extract
(acidic/non acidic fraction)
Aβ-induced cytotoxicity Lipid peroxidation Glutation peroxidase activity
PC12 cells Anticholinesterase activity
Antioxidant
Sepand et al. 2013
Ethanolic extract CNS cholinergic receptor binding activity Human cerebral cortical cell membranes Displacement 3H-(N)-nicotine and 3H-(N)-scopolamine from muscarinic and nicotinic receptor
Kennedy et al. 2003
Ethanol-water extract TEAC - Antioxidant Lahucky et al. 2010
Ethanol extract Cholinergic receptor binding activity Displacement nicotinic and
muscarinic receptors
Wake et al. 2000
Hydroalcoholic extract DPPH - Antioxidant Lamaison et al. 1991
Extracts containing rosmarinic acid
Inhibition amylase activity Porcine pancreatic cells Antioxidant McCue and Shetty
2004
Polar extract DPPH/ TLC assay - Antioxidant López et al. 2007
Supercritical extract EDTA-mediated oxidation of linoleic acid - Antioxidant Marongiu et al. 2004
Balm oil Hypoxic-ischemic injury Primary neuronal culture LDH
Caspase-3
Bayat et al. 2012
Antitumoral
Ethanolic extract Aqueous extract Rosmarinic acid
MTT/Neutral Red (NR) assays ABTS
DPPH
Human colon cancer cell (HCT-116) Cytotoxicity Encalada et al. 2011
Aqueous extract Caspase 7 Apoptosis
Human cancer cells lines: MCF-7, MDA-MB-468, MDA-MB-231 cells
Saraydin et al. 2012
Essential oil DPPH/MTT assay Human cancer cell lines: A549,
MCF-7; Caco-2, HL-60; K562 Mouse cell line: B16F10
Antitumoral De Sousa et al. 2004
Table 3.1. contd....
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Antimicrobial/Antiviral/Antifungal
Extract/ Active principle Assay Model Activity Reference
Aqueous extract Herpes simplex virus HSV-1; HSV-2 Plaque reduction assay
RC-37 cells Antiviral Nolkemper et al.
2006
Aqueous Extract HIV-1 MT-4 cells Antiviral Yamasaki et al. 1998
Aqueous extract Caffeic, p-coumaric and rosmarinic acid
Plaque reduction assay Expression of viral protein ICP0
RC-37 cells
Herpes simplex virus type 1(HSV-1)
Antiviral Astani et al. 2012
Aqueous, methanolic and ethyl acetate extracts
Rhizopus growth Rhizopus stolonifer Antifungal Lopez et al. 2007
Apigenine-7-O-glucoside from MO
Rhesus rotavirus-(RRV) MA-104 cells Antiviral activity Knipping et al. 2012
Lemon balm oil Candida albicans growth Gram-positive/ gram-negative strains Anticandida Hancianu et al. 2008
Lemon balm oil Herpes simplex virus type 1 (HSV-1)
HSV-2
HSV-1 and HSV-2 Antiviral Schnitzler et al. 2008
Central Nervous System
Aqueous extract Inhibition of GABA-T activity Rat brain homogenate Anxiolytic Awad et al. 2007
Methanol extract Inhibition of GABA transaminase Rat brain homogenate Anxiolytic Awad et al. 2009
Aqueous extract Methanolic extract
GABAA-receptor Assay Cerebral cortex No activity López et al. 2009
Essential oil Inhibition of GABA receptor channel Inhibition of inhibitory and excitatory transmission
Radioligand binding Electrophysiological study
Sedative Abuhamdah et al.
2008 by Taylor & Francis Group, LLC
Melissa Offi cinalis Updated Overview 61
Central Nervous System
Extract/ Active principle Assay Model Activity Reference
Ethanolic/aqueous extract Sleep induced by pentobarbital Staircase test
Two compartment test
Mice Sedative Soulimani et al. 1991
Ethanolic extract EPM
Forced swimming test Open fi eld test
Rats(male and female) Anxiolytic Antidepressant Taiwo et al. 2012
Ethanolic extract EPM Mice Anxiolytic Ibarra et al. 2010
Ethanolic/aqueous extract Hippocampal dentate gyrus functionality Mice Decrease corsticoesterone levels Increase GABA
Yoo et al. 2011
Rosmaric acid EPM Rats Anxiolytic Pereira et al. 2005
Antioxidant
Essential oil Trasient Hippocampal ischemia Rats Antioxidant Bayat et al. 2012
Aqueous extract Mn Oxidative model Mice Antioxidant Martins et al. 2012
Ethanolic/aqueous extract TBARS Pigs Lipidic peroxidation inhibition Lahucky et al. 2010
Antiinfl ammatory/ analgesic
Ethanolic extract Writhing test
Formalin Test
Mice Analgesic Guginski et al. 2009
Ethanolic/aqueous extract Writhing test Mice Analgesic Soulimani et al. 1991
Essential oil Paw oedema (carrageenan)
Traumatism
Rat Antiinfl ammatory Bounihi et al. 2013
Rosmarinic acid Paw oedema (CVF) Rats Antiinfl ammatory Englberger et al. 1988
Antitumoral
Aqueous extract Mammary tumors (DMBA) Rats Antitumoural Saradyn et al. 2012
Others
Aqueous extract Induced gastric ulcer Rat Cytoprotective Khayyal et al.
2001 Ethanolic extract Fat rich diet
Type 2 DM model
Obese mice Glycemia and triglyceridemia reduction
Weidner et al. 2013
Essential oil Fat rich diet
Type 2 DM model
Mice Glycemia and triglyceridemia
reduction
Chung et al. 2010
Essential oil APOE2 (R158C) transgenic mice Mice Decrease TG Jun et al. 2012
Aqueous extract Fat rich diet Rats Cholesterol, total lipids, ALT, AST,
ALP reduction Hepatoprotective
Bolkent et al. 2005
EPM: elevated plus-maze by Taylor & Francis Group, LLC
In vitro studies
Methanolic and aqueous extracts were tested in relation to their affi nity to the GABA A-benzodiazepine Receptor, but no activity was detected (López et al. 2009).
Chemically-validated essential oil derived from Melissa offi cinalis elicited a signifi cant dose-dependent reduction in both inhibitory and excitatory transmission, with a net depressant effect on neurotransmission, in contrast to the classical GABA-A antagonist picrotoxinin (Abuhamdah et al. 2008).
The aqueous extract of Melissa offi cinalis (lemon balm) exhibited the greatest inhibition of GABA transaminase (GABA-T) activity (IC50 = 0.35 mg/mL) in vitro rat brain homogenate assays (Awad et al.
2009). The same authors found that the methanol extract of lemon balm was a potent in vitro inhibitor of rat brain GABA-T, an enzyme target in the therapy of anxiety, epilepsy and related neurological disorders.
Rosmarinic acid and the triterpenoids, ursolic acid and oleanolic acid, were the main compounds of the extract and thus were supposed to be responsible for the sedative effect. Phytochemical characterization of the crude extract determined that rosmarinic acid was the major compound responsible for activity (40% inhibition at 100 μg/mL) since it represented approximately 1.5% of the dry mass of the leaves.
Synergistic effects may also play an important role (Awad et al. 2007).
In vivo studies
Sedative properties of the hydroalcoholic extract of M. offi cinalis leaf were proved in different animal models. In mice, intraperitoneal administration of low doses exerted a sedative and sleep inductive effect, as it improved the sleep induced by an infra-hypnotic dose of pentobarbital (Soulimani et al. 1991).
The anxiolytic and antidepressant effects have been tested in male and female rats. Ethanolic extract was capable of reducing anxiety in stressed rats, although the response was dependent on the treatment duration. The administration of 30, 100 or 300 mg/kg/day of the ethanolic extract for 10 days showed a percentage of open arm entries and open arm times for both male and female rats which was signifi cantly higher than the control group alone, with similar values to diazepan group (1 mg/kg), when tested in the elevated plus-maze (EPM) test. With respect to the antidepressant activity which was tested by the forced swimming test, immobility duration was signifi cantly lower in rats treated with the plant extract when compared to control-animals. Also in this assay, a signifi cant infl uence of the animal genera and treatment duration in the effi cacy was observed: a single administration was ineffective; the lowest extract dose was not active in male rats in subacute treatment. On the contrary, no effect on locomotor activity was observed in the open fi eld test. Effects obtained with fl uoxetine (10 mg/kg) were superior to lemon balm extract (Taiwo et al. 2012).
One marketed ethanolic extract (30%) of M. offi cinalis leaf (Cyracos®), standardized to a rosmarinic acid content superior to 7% and hydroxycinnamic acid derivatives superior to 15%, when administered orally to mice (120, 240 and 360 mg/kg) for 15 days signifi cantly reduced anxiety-like reactivity dose- dependently in an elevated plus maze task. These doses did not alter those tests evaluating exploratory or circadian activities (Ibarra et al. 2010).
Administration of an infusion containing Melissa and Passifl ora caerulea decreased immobilization—
stress in mice. A signifi cant decrease in plasma levels of corticosterone was observed in the treated group when compared to the control group (Feliú-Hemmelmann et al. 2013). The administration of a freeze-dried aqueous ethanolic (20%) extract of lemon balm leaf (50–200 mg/kg/day for three weeks) to 12 months old mice induced a dose-dependent increase in cellular proliferation, neuroblast differentiation and integration into granule cells in the hippocampal dentate gyrus; this structure may have a functional role in stress and depression. The observed activity was related to the decrease in corticosterone levels in serum and the GABA increase in that brain structure (Yoo et al. 2011).
Only a few studies have been performed with the isolated active principles in order to determine which of them are responsible for the pharmacological effects obtained with M. offi cinalis leaf. Rosmarinic acid exerts anxiolytic activity; intraperitoneal administration of low doses to rats increased exploratory conduct on elevated plus-maze test, without affecting locomotor activity and short or long term-memory (Pereira et al. 2005).
Interestingly, intraperitoneal administration of the essential oil did not induce a sedative effect, while oral administration (3.16 mg/kg or superior) exerted a sedative and narcotic effect (ESCOP 2003).
Clinical trials
Several clinical trials proved that lemon balm improves cognitive behaviour and mood, while decreasing induced stress and exerting anxiolytic properties after a single dose treatment. Doses of 600 mg/day of extract increased calm, decreased vigilance and improved the quality of attention as proved by a randomized, placebo-controlled, double-blind, balanced-crossover study including 20 healthy subjects treated with daily single doses of 300, 600 or 900 mg of a standardized, commercial extract of M. offi cinalis (30:70 methanol/
water) (Kennedy et al. 2002). The same research group studied the effect on mood and cognitive performance of the same herbal preparation at 300 and 600 mg on 18 healthy subjects. Participants underwent light psychological stress that was induced in the laboratory one hour after treatment. No adverse events were recorded and all the participants completed the study. Doses of 600 mg improved the subjective effects of laboratory-induced stress (Kennedy et al. 2004).
A prospective, open-label study lasting 15 days included 20 volunteers aged 18 to 70 years with mild to moderate anxiety and sleep disorders. A clear improvement in anxiety symptoms were observed after treatment with 600 mg/day of M. offi cinalis extract (standardized hydroalcoholic leaf extract at 30%, > 7% rosmarinic acid and > 15% hydroxycinnamic acid derivatives) (Cases et al. 2011).
As a general conclusion, lemon balm extract properties were attributed to the presence of some polyphenolic compounds, mainly rosmarinic acid, fl avonoids, monoterpene glycosides and the triterpenoids ursolic and oleanoic acids. Again, the synergistic effect of the extract was superior to the addition of the activity of the isolated compounds.
M. offi cinalis essential oil seems to be more effi cient in patients suffering from dementia; aromatherapy proved to be effective in 71 patients with severe dementia. For this double-blind, placebo-controlled study, patients received local administration of the essential oil combined in a lotion on the face and forearms.
An improvement in the nervousness status and quality of life of patients was observed after four weeks’
treatment when compared to the placebo group (less socially unassertive and more participative in different activities). Safety profi le was positive, as no signifi cant adverse events were recorded (Ballard et al. 2002).
Oral administration of lemon balm was also tested in Alzheimer’s disease patients. One randomized, double blind, placebo-controlled trial evaluated the effi cacy and safety of an alcoholic extract (45% V/V) standardized to 500 μg citral/ml for a four month period; a daily dose of 60 drops was administered to 42 patients of both sexes (21 received lemon balm extract; 21 received a placebo) aged between 65 and 80 years with light to moderate Alzheimer’s disease. Thirty fi ve patients completed the study and showed a signifi cant benefi cial effect on cognitive function. Related to the adverse events, no signifi cant differences were observed between placebo and treated groups, except for the agitation frequency (not considered as an adverse event) which was superior in the placebo group. These results show a benefi cial effect of M. offi cinalis treatment (Akhondzadeh et al. 2003).
The essential oil obtained by the leaves distillation showed inhibitory activity on cholinesterase.
This activity may induce some improvement in symptoms from Alzheimer’s disease. One double-blind parallel-group placebo-controlled randomized trial performed with 114 patients was designed to compare the administration of lemon balm essential oil twice daily (massage over the hands and upper arms) with a standard treatment (donepezil). Results showed that the essential oil was not better than the placebo or donezepil for the relief of agitation in patients suffering from Alzheimer’s disease (Burns et al. 2011).
Antioxidant activity In vitro studies
Current lifestyle is causing the overproduction of free radicals and Reactive Oxygen Species (ROS) and decreasing the physiological antioxidant capacity. Consistent evidence demonstrated that hydrogen peroxide (H2O2), the major ROS in the human body, is involved in molecular mechanisms by which
oxidative stress causes cell death in several types of dementia including Alzheimer’s disease (Tabner et al.
2005). Phytochemical compounds with antioxidants properties are considered as a promising preventive or therapeutic strategy for reducing H2O2-mediated oxidative stress (Speciale et al. 2011). Extracts from plants of the Lamiaceae family have been described for antioxidant and antibacterial effects which are linked to their polyphenolic composition.
Medicinal plants constitute the main source of natural antioxidants which protect the human body from free radicals, prevent oxidative stress and associated diseases. For these reasons they play a very important role in health care. Plants are a source of compounds with antioxidant activity such as phenolic acids, fl avonoids, anthocyanins, tannins and carotenoids that may be used as pharmacologically active products.
Melissa offi cinalis exhibited high antioxidant potential, comparable to that of black and green tea (Ivanova et al. 2005). The aqueous-methanolic extract of lemon balm caused a considerable concentration- dependent inhibition of lipid peroxidation; the phenolics from the extract demonstrated antioxidant activity (Hohmann et al. 1999). Hydroalcoholic extracts from lemon balm have also shown signifi cant antioxidant activities, by free radical scavenger effect on DPPH (N,N-Diphenyl-N-(2,4,6-trinitrophenyl)-hydrazyl), partly in relation to their rosmarinic acid content (Lamaison et al. 1991, McCue and Shetty 2004, Pereira et al. 2009).
Methanolic and aqueous extracts from M. offi cinalis were tested for protective effects on the PC12 cell line, free radical scavenging properties and neurological activities such as inhibition of monoaminooxidase A (MAO-A) and acetylcholinesterase enzymes, as well as the affi nity to GABAA-benzodiazepine receptors.
Results suggested a protective effect on hydrogen peroxide induced toxicity in PC12 cells (López et al.
2009).
Bayat et al. (2012) examined the effect of Melissa offi cinalis on a model of neuronal death induced by hypoxia in neuronal culture. Cytotoxicity assays showed a signifi cant protection against hypoxia, which was confi rmed by a conventional staining. Melissa treatment decreased caspase3 activity and also inhibited malondialdehyde (MDA) level, as a marker of lipid peroxidation.
In vivo studies
Related to the antioxidant and neuroprotective properties, the effect of lemon balm essential oil was evaluated by a transient hippocampal ischemia induced in male rats by carotid occlusion for 20 minutes.
After reperfusion, a recovery of the antioxidant ability was observed in those animals treated with lemon balm; also a decrease in MDA levels was measured. These results indicate that lemon balm could attenuate oxidative damage induced by ischemic brain injury. In addition the antioxidant activity studies, in vitro tests correlated this activity with a possible inhibitory effect on HIF-1alfa and activation of caspase3;
this mechanism minimized apoptosis induced by ischemia in neurons and astrocytes (Bayat et al. 2012).
Mice receiving an aqueous extract of Melissa offi cinalis for three months showed a decrease in oxidative cerebral damage induced by manganese, a metal which is capable of inducing neurodegenerative effects similar to Parkinson’s disease after long exposure (Martins et al. 2012).
The antioxidant effect was also tested in pork, although results were mild. Animals received different doses of one hydroalcoholic extract of lemon balm (100 or 20 mL) alone or combined with vitamin E for 30 days. After this period, samples of the muscle longissimus thoracis were taken and the ability to protect lipid peroxidation induced by Fe2+/ascorbate (TBARS) was measured. Results were signifi cantly better for those animals supplemented with vitamin E (Lahucky et al. 2010).
Clinical trials
Infusion from the leaves of lemon balm (1.5 g/100 mL), when taken twice daily for 30 days, improved the oxidative stress and protected DNA from oxidative injury. These conclusions were obtained by means of a clinical study including 55 volunteers who were exposed to low radiation levels during the workday (Zeraatpishe et al. 2011).