The volatile oil extracted from the leaves of Melissa officinalis is important due to its pharmacological effects, and is obtained in small quantities, unlike other plants in the Lamiaceae family. The major and minor components of the essential oil of the dried leaves of Melissa officinalis are volatile compounds that are found in different concentrations (Table 1).

2.2. Triterpenes

Triterpenes are non-volatile components extracted from plants, and are one of the largest classes of natural plant products, with more than twenty thousand different triterpenes (Table 2) [23,24]^[22][23]. Some triterpenes contain a different number of sulfate groups bound to sugar or glucones ^[10]. ^30]. Phenolic acids and flavonoids contain chemical structural elements that are responsible for the antioxidant process, and their antioxidant activities have been well established biochemically. Phenolic acids are important bioactive constituents of Melissa officinalis, among which are rosmarinic, caffeic, chlorogenic and ferulic acids. Following the phenolic profiles of the three types of extracts from the study performed by Ghiulai et al. ^[4], it was observed that rosmarinic acid was obtained in the highest amount (86,637.60 μg/g). The extraction with methanol, by sonication, favored the extraction of all of the phenolic compounds sought; thus, it can be observed that, depending on the extraction technique, some compounds can be in high concentrations and others cannot be detected.

2.4. Other Components

Melissa officinalis is a source of active biocompounds such as volatile compounds, triterpenes and polyphenolic compounds, but, in addition to these, it also contains other important biological active agents. Ashori et al. [35]^[31] conducted a study on the chemical composition of the stalk of Melissa officinalis. The content of extractive agents, lignin, polysaccharides and ash was determined, with the observation that the results showed a high content of alpha-cellulose and a low content of lignin. Komes et al. [36]^[32] reported the content of tannins, phenolic acids and flavonoids in non-hydrolyzed and hydrolyzed extracts from various plants, including Melissa officinalis. Dias et al. [37]^[33] performed a comparative study between two commercial samples, an in vitro culture sample and a normal culture sample. After the analysis of all of the samples, they observed the highest levels of proteins (8 g/100 g dw) and ash (12 g/100 g dw) in the in vitro cultured lemon balm content; the highest levels of carbohydrates were found in the granulate commercial sample (85 g/100 g dw), and a bag of commercial lemon balm had the highest energetic value (377 kcal/100 g dw) due to its higher fat content (3 g/100 g dw).

Overall, Melissa officinalis has a complex chemical composition, with many active biocompounds, which differ depending on the way in which the extraction is performed and the part of the plant that is subjected to extraction.

3. Pharmacological Studies

According to many biological studies, plant extracts and volatile oils are known for many beneficial activities for the human body. Melissa officinalis is considered to be a medicinal plant due to the numerous pharmacological effects associated with its chemical composition (Table 4).

Table 4.

Pharmacological effects reported from

Melissa officinalis

extracts.

Effect	Model	Dosage or Concentration	Tested Systems	Results	Type of Extract	Reference

Antiproliferative

in vitro

20, 100, 250 μg/mL

Breast cancer cells MDA-MB- 231 and healthy HaCat cells

Inhibitory effect on migration and proliferation of both types of cells

ethanolic extract

[38]

[34]

Oleanolic acid

915.03–6151.67

aerial parts

[4]

Caftaric acid

1.85–344.34

Dried leaves

in vitro

50%

Human Colon Cancer Cell Line (HCT-116)

[4]

The 50 % ethanolic extract showed significant differences after 72 h of treatment, reducing cell proliferation to values close to 40%

ethanolic and aqueous extracts

[39]

[35]

Ursolic acid

3577.00–11,234.97

aerial parts

Antitumor

in vitro

Different concentration

[4]

Human tumor cell lines: MCF-7,

AGS and NCI-H460

Obtained revealed that the ethanolic extract presented the highest cell growth inhibitory potential in all the human tumor cell lines tested

ethanolic, methanolic, hydro-methanolic,

23-Sulfate ester of niga-ichigoside F1

n.a.

leaves and stems

[25]

[24]

]

3β,16β,23-Trihydroxy-13,28-epoxyurs-11-ene-3-O-β-D-glucopyranoside

n.a.

dried leaves and stems

[24]

[23]

3,23-Disulfate ester of 2α,3β,19α,23-tetrahydroxyurs-12-en-28-oicacid

n.a.

dried leaves and stems

[24]

[23]

Chlorogenic acid

0.62–75.529

Dried leaves

[4,29]

[4][28]

hydro-ethanolic and aqueous extracts)

[

40

]

[

36

]

Ferulic acid

1.03–45.489

Dried leaves

[4,29]

[4][28]

Antioxidant

in vitro

Different concentration

Encephalic tissue from male Wistar rats

Effective agent in the prevention of various neurological diseases associated with oxidative stress

ethanolic, methanolic and aqueous extracts

[41]

[37

[15]

Gentisic acid

[16]

10.40–60.48

[17][18]

Dried leaves

[

4

]

]

in vitro

1, 2.5, 5 and 10 mg/mL

DPPH radical scavenging activity assay, β-carotene bleaching test and ABTS assay

Good antioxidant activity

essential oil

[42]

Geranyl acetate

0.5–19.3

[12,13,14,17]

[12][13][14][17]

[

38

]

p-Coumaric acid

1.06–20.72

Dried leaves

[4]

Antiangiogenic

in vitro, in ovo

50 μg/mL

Two breast cancer cell lines, MCF-7 And MDA-MB-231

Highest cell inhibitory activity was exhibited by the 96% ethanolic extract

ethanolic extracts and methanolic extracts

[4]

Neral (citral B)

3,23-Disulfate ester of 2α,3β,19α,23-tetrahydroxyurs-12-en-28-oicacid 28-O-β-D-glucopyranoside4.28–35.02

[12,,18

n.a.

]

[

13,

12]

dried leaves and stems

[24]

[23]

Cardioprotective

13.37 ± 2.84

Aerialparts

in vivo

[30

[

14,

13

15,16,

]

[29]

][14][15][16]

17

[17

25, 50 and 100 mg/kg b.w. *

(4.23/8.46/16.91 mg/kg b.w. *)

][18]

Rats

Antioxidant and cardio-protective effects against arrhythmias induced by ischemia and ischemia-reperfusion

ethanolic leaf extract

[

43

]

[39]

α-Cadinol

5.64

[14]

3,23-Disulfate ester of2α,3β,23,29-tetrahydroxyolean-12-en-28-oicacid

n.a.

dried leaves and stems

[24]

[23]

Rosmarinic acid

3515.60–86,637.60

Dried leaves

[4]

Antinociceptive Antihyperglycemic

in vivo

0.01, 0.02 and 0.04 mg/day

(0.0063/0.0126/0.0252 mg/kg b.w. *)

Male adult Wistar rats

Long-term oral administration of essential oil (at an effective dose of 0.04 mg/day) can suppress chemical hyperalgesia in diabetic rats

essential oil

[44]

[40]

α-Copaene

0.1–7.02

[12

3,23-disulfate ester of 3β-23,29-trihydroxyolean-12-en-28-oic acid

,15,16]

[12]

n.a.[15

dried leaves and stems

[

6914.1 ± 779

Aerial parts

[30]

[29

]

24]

[23]

Anxiolytic Antidepressant

[

16]

in vivo

50, 75 and 150 mg/kg b.w./day *

(3.91/5.86/11.72 mg/kg b.w. *)

Albino BALB/c male mice

Hydro-alcoholic extract (75 and 150 mg/kg) significantly reversed anxiety- and depressive-like behaviors

hydro-alcoholic extract

[45]

[41]

β-Caryophyllene

1.3–29.14

[14,15,16,17]

[14]

3,23-Disulfate ester of 2α,3β-23,29-tetrahydroxyolean-12-ene-28-oicacid

[15]

n.a.

[16]

dried leaves and stems

Neuroprotective

in vitro

[17]

5, 10, 50, 100, 500 μg/mL

[

24

]

[23]

]

Flavonoids

Apigenin

0.66–84.53

Dried leaves

Cortical neuronal Culture system

[4,29]

[4][28]

Protective effects on neurons in the brain

balm oil

[

Minority components (<5%)

46

23-sulfate ester of 2α,3β,19 α,23-tetrahydroxyurs-12-en-28-oic acid

n.a.

fresh leaves and stems

[26]

[25]

]

[

42

(2E)-Nonen-1-al

23-sulfate ester of 2α,3β,19 α,23-tetrahydroxyurs-12-en-28-oic acid 28-O-β- 0.2

D-glucopyranoside

n.a.[12

fresh leaves and stems

[26]

[25]

]

]

(E)-Nerolidol

0.2

Melissioside A[12]

n.a.

leaves and stems

[

25]

[24]

(E)-α-Bergamotene

1.24

[14]

Melissioside B

n.a.

leaves and stems

[25]

[24]

Melissioside C

n.a.

leaves and stems

[25]

[24]

n.a. = not available; * expressed on a dry weight basis.

The most common triterpenes in Melissa officinalis extracts are ursolic acid and oleanolic acid. In the studies of Ghiulai et al. ^[4] and Ibarra et al. [27]^[26], ursolic acid and oleanolic acid were extracted together with polyphenolic compounds in order to show the biological properties of the plant. Using different extraction conditions, different extracts can be obtained with different compositions, and thus different activities. The first extraction was obtained by maceration for 9 days in 70% ethanol, the second was obtained by maceration in 96% ethanol for 24 h under continuous stirring, and the third was extracted by sonication in 80% methanol for one hour. In the three extractions, the largest amount of ursolic acid was obtained, followed by oleanolic acid and small amounts of betulinic acid. Comparing the values of the obtained concentrations, the extraction in methanol with sonication is the most favorable, obtaining the best results: ursolic acid (11,234.97 μg/g), oleanolic acid (6151.67 μg/g) and betulinic acid (169.88 μg/g).

2.3. Polyphenolic Compounds

Polyphenolic compounds are a group of secondary metabolites which includes flavonoids (e.g., anthocyanins, flavones, isoflavones) and phenolic acids, which have many biological properties (Table 3) [28]^[27].

Table 3. Major polyphenolic compounds from Melissa officinalis extracts.

Group Name	Compound Name	Content *, μg/g
41.71 ± 20.6
Aerial parts
[
30
]
[
29
]
Cynaroside	408.13 ± 30.0
in vivo	50, 100, 200 and 400 mg/kg b.w. *	(8.35/16.71/33.41/66.83 mg/kg		b.w. *)	Male rats	Treatment with 100 mg/kg of oil attenuated the increased caspase-3 like protease activity significantly	balm oil	[46]	[42]	Aerial parts	[30]	[29]
(E)-β-Ionone	0.9
GABA-T inhibitor	in vitro	0–4 mg/mL	Rat brain	Phytochemical characterization of the crude extract determined rosmarinic acid as 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	methanol extract	[12]
[	47	]	[	43	]	Daidzein	51.25 ± 8.07	Aerial parts	[30]	[29]
Anti-kinetoplastidae	in vitro	31.25, 62.5, 125, 250 μg/mL	T. cruzi	,	L. brasiliensi	,	L. infantum	A potential source of natural product featuring anti-	Leishmania	and anti-	Trypanosoma	activity	ethanol extract	[48]	[44]	Hyperoside	3.30–16.240	(E)-β-Ocimene
Analgesic	Dried leaves	in vivo	5, 10, 20 mg/kg b.w. *	(0.87/1.73/3.46 mg/kg		b.w. *)	[4]	0.1–0.5
Male Wistar rats	Intrathecal administration could significantly improve hot-water and formalin-induced pain in male Wistar rats	hydro-alcoholic extract	[	49	]	[45]	Isoquercetin	6.82–162.40	Dried leaves	[4]
Hypnotic	in vivo	200, 400 and 800 mg/kg b.w. *	(14.81/29.61/59.23 mg/kg		b.w. *)	Male Swiss mice	Extracts may be useful for insomnia	hydro-alcoholic extracts	[50]	[46]	[12,13]	Kaempherol	[12]	21.84	[13]
Dried leaves	[	4	]
Antidiabetic	in vivo	0.0125 mg/d	db/db mice	Anti-hyperglycaemic agent	essential oil	[51]	[47]	(E-E)-Geranyl linalool	1.59	Luteolin	0.81–26.32	Dried leaves	[4]
in vivo	0.4%, 0.8% (	w	/	w	)	[14]
Otsuka Long-Evans Tokushima fatty rats	An effective therapeutic strategy to treat human obesity and type 2 diabetes	herbal extract	[	52	]	[48]	(Z)-β-Ocimene	0.1	Myricetin	[15]
3.45–17.92	Dried leaves	[	4]
Anti-Alzheimer	in vitro	8.8 mg/mL	GSK-3Β, CK-1δ, and BACE-1	Best activity for ck-1δ inhibitory activity with maximum inhibitory concentration values at half (IC50) below 250 μg/mL	methanol extract	[53]	[49]	1,2-Benzenedicarboxilic acid, butyl 2-methylopropyl ester	0.6	[	Quercetin	13]
153.46	Dried leaves	[	29]	[28]	1,8-Dehydro-cineol	0.1	[13]
14-Hydroxy-9-epi-(E)-caryophyllene	0.2	[13]
1-Octen-3-ol	0.2–0.3	[12,13,15]	[12][13][15]
3,5-Heptadienal,2-ethylidene-6-methyl	0.4	[13]
3-Octanone	0.2	[17]
6-Methyl-5-hepten-2-ol	0.2–1.7	[12,13,15]	[12][13][15]
Benzene acetaldehyde	0.3	[12]
Camphene	0.38–1.38	[14,16]	[14][16]
Camphor	0.1–0.4	[13,15]	[13][15]
Carvacrol	0.3–1	[12,13]	[12][13]
Caryophyllenol	0.5–2.23	[14]
cis-2H-3a-Methyl-octahydro-Inden-2-one	4.7	[17]
Cis-Chrysanthenol	0.7–1.7	[12,13,15]	[12][13][15]
Cis-Rose oxide	0.1–0.2	[12,15]	[12][15]
Citronellol	0.4–1.88	[12,	[12	14]	][14]
Citronellyl acetate	0.1	[12]
Dihydrocitronellol acetate	0.3	[15]
Geraniol	0.6–0.7	[12,	[	13,	12	15]	][13][15]
Germacrene D	0.2–2.0	[12,13,14]	[12][13][14]
Humulene epoxide II	0.2–1.29	[13,14]	[13][14]
iso Aromadendren epoxide	0.46	[14]
Isogeranial	1.4–2.0	[13]
Isomenthol	2.4	[15]
Linalool	0.3–0.5	[12
Antispasmodic	ex vivo	1, 5, 10, 25, and 50 mg/mL	Different segments of the gastrointestinal tract of mice	Site- and dose-dependent effects on the contractile activity of the gastrointestinal tract	hydro-ethanolic leaf extract	[54]	[50]	Quercetrol	5.72–33.60	Dried leaves	,15]	[12][15]
[	4
Antiviral	]	Linalool + trans-Sabinene hydrate	0.5–0.8	[13]
Menthol
in vitro	1.5–150 μg/mL	RC-37 cells	High virucidal activity against	0.3	[15]
Methyl citronellate	0.5–2.78	[12,13,16]	[12][13][16]
Methyl eugenol	0.1	[12]
Methyl geranate	0.2–0.4	[12,13,17]	[12][13][17]
Myrcene	0.1–0.3	[13,15]	[13][15]
n-Eicosane	0.6	[15]
Nerol	0.2	[15]
Neryl acetate	0.1	[12]
n-Heneicosane	0.4	[15]
	n-Nonanal	0.1–0.4	[12,15]	[12][15]
para-Mentha-1(7),8-diene	0.1	[13]
p-Cymene	0.1	[12,13]	[12][13]
Phytol	3.64	[14]
Rosefuran epoxide	0.6–0.7	[13]
Sabinene	0.4	[13]
Thymol	0.1–3.1	[12,13,14]	[12][13][14]
t-Muurolol	0.59	[14]
trans-Limonene oxide	0.6	[13]
trans-para-Mentha-1(7),8-dien-2-ol	2.3	[17]
HSV-1	aqueous extract	[	55	]	[	51]	Rutin	8.11–1462.99	Dried leaves	Trans-Rose oxide
Antifungal	in vitro	[	4,29]	[4][28]	0.1

* expressed on a dry weight basis.

Phenolic acids compose a large group of natural compounds, which exhibit a wide range of biological activities. Flavonoids are a class of polyphenolic compounds, classified according to their chemical structures into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanidins and chalcones [31]^[

15.5–2000 μg/mL

Human
Pathogenic fungi
Good antifungal activity
ethanol extracts
[
56
]
[
52
]
[
12
,
15
]
[
12
]
0.25–2 μL/mL	Phytopathogenic fungi in apples	essential oil	[57]	[53]
Antibacterial	in vitro	10 and 15 mg/mL	E. coli	,	L. monocytogenes	,	S. aureus	and	S. typhimurium	A significant antimicrobial effect	essential oil
[
15
]
[	42	]	[	38	]
Valencene
0.1
[
15
]
α-Humulene
0.2–2.6
[
12
,
13
,
15
,
16
]
[
12
]
[
13
]
[
15
]
[
16
]
α-Calacorene	0.76	[14]
α-Cubebene	0.42–1.23	[14]
β-Cubebene	0.1	[15]
β-Pinene oxide	1.1	[13]
β-sesquiphellandrene	0.97	[14]
γ-Cadinene	0.76–1.77	[14]
γ-Terpinene	0.3–0.5	[12,13]	[12][13]

Traces of components were not taken into account (contents below 0.05%).

Kowalski et al. ^[19] reported a 0.17% essential oil content, which was very low, unlike plants in the same family. The study by Seidler-Łożykowska et al. ^[16] showed that the essential oil content ranged from 0.08 to 0.20% due to fluctuations in weather conditions during the research years. For the variability of the essential oil content and its composition, Kittler et al. ^[20] studied a set of 28 accessions of lemon balm. They obtained an essential oil content that varied between 0.01 and 0.72%, and found out—based on statistical analysis on the composition of the essential oil—that there are two chemotypes of essential oil: chemotype citral and chemotype germacrene D. As it is known from the literature, there are two subspecies of the plant Melissa officinalis: officinalis and altissima. The difference between the two subspecies is given by the composition of the essential oil, such that the subspecies officinalis contains major amounts of citral and/or neral, but the subspecies altissima contains only traces. Basta et al. ^[21] reported, in a study on the composition of the essential oil of Melissa officinalis in Greece, the lack of the major constituents citral and citronellal. Souihi et al. ^[14] reported, in a comparative study between Melissa officinalis from Tunisia (from two different cities), Germany, and France, the lack of citral but the major presence of germacrene D. Some of the pharmacological activities may be connected with the polyphenolic compounds occurring in Melissa officinalis, which include phenolic acids and flavonoids [45,58]^[41][54]. Most studies have focused on Melissa officinalis leaf extracts, obtaining phenolic profiles correlated with antiproliferative [39]^[35], antiangiogenic ^[4], antiviral [55^[51][55],59], antioxidant [41^[37][38],42], anti-anxiety [45]^[41], antidepressant [45]^[41], anti-Alzheimer [53]^[49], neuroprotective [46]^[42], cardioprotective [43]^[39], antifungal [56,57]^[52][53] and antibacterial [42]^[38] effects. Moaca et al. [38]^[34] performed a comparative study between extractions from stems and leaves in order to evaluate the antioxidant activity, the total phenolic contents, and the cytotoxic and antiproliferative effects. In this study, a good antioxidant activity was observed to be correlated with the high total polyphenol content of the leaf ethanolic extract (32.76 mg gallic acid equivalents/g) as opposed to the seed ethanolic extract (8.4 mg gallic acid equivalents/g). The extracts obtained showed a different profile of cytotoxic effects on breast cancer cells, MDA-MB-231, but with significant antitumor activity for future in vitro studies. Ghiulai et al. ^[4] investigated the potential for angioprevention and chemoprevention in breast cancer from various extracts of Melissa officinalis. The antioxidant activity and in vitro effect on cell viability were evaluated on two breast cancer cell lines, MCF7 and MDA-MB-231. Based on the evaluation in ovo, using the chorioallantoic membrane test, it was found that 96% ethanolic extract is the strongest chemopreventive agent. Melissa officinalis and other plant extracts (Iberis amara, Silybum marianum and a mixture of Angelica archangelica and Carum carvi) were evaluated in association with STW5, a well-known fixed herbal multicomponent preparation recommended, at least, by the German treatment guideline for some gastrointestinal diseases under non-inflammatory and inflammatory conditions. It was found that only Melissa officinalis plant extract manifested a strong synergic effect when intestinal smooth muscle cells were considered [104]^[56]. The oral administration of a Melissa officinalis infusion can be also beneficial for the radiology staff, as the oxidative stress and DNA damage are reduced [105]^[57]. At present, only a few studies have been reported on the synergies between Melissa officinalis and other plants. Due to the volatile compounds in Melissa officinalis essential oil, the following effects have been reported: antifungal, antioxidant, antidiabetic, antibacterial and antimicrobial effects. These effects have also been reported in various extracts of Melissa officinalis.