Chemical Composition and Biological Activities of Mentha Species

4.1. Essential oils

Essential oils are natural and volatile secondary metabolites characterized by a strong odor and a complex composition. They are usually obtained by steam or hydro-distillation from various aromatic plants, generally localized in temperate to warm countries like Mediterranean and tropical countries where they represent an important part of the traditional pharmacopoeia [34].

Several species of Menthaare cultivated for the production of essential oil. Indeed, mint oils are among the most important essential oils produced in the world, and their values are exceeding 400 million of US dollar/year. For instance, M. canadensisL. produces corn mint oil which represents the most important source of (–) menthol; M. piperitaL. produces peppermint oil, constituted of menthol, menthone, and menthyl acetate as main components; M. spicatassp., M. viridis(native spearmint), and M. gracilis(scotch spearmint) produce mostly carvone-rich oils, although different compositions have been reported; M. citratais a source of linalool and linalyl acetate; M. pulegiumproduces the so-called pennyroyal oil, which is a pulegone-rich oil; the composition of M. aquaticaoils is dominated by menthofuran [21]; M. haplocalyxcould be classified into six chemotypes, including linalool, pulegone, menthone, carvone, menthol, and piperitenone oxide [35].

Peppermint leaves typically contain 1.2–3.9% (v/w) of essential oil, with more than 300 identified compounds. The terpenic class is the most represented, comprising about 52% of monoterpenes and 9% of sesquiterpenes, whereas other groups, such as aldehydes (9%), aromatic hydrocarbons (9%), miscellaneous (8%), lactones (7%), and alcohols (6%), have been shown to be present in a smaller proportion. Among monoterpenes, menthol is the major constituent (35–60%), followed by menthone (2–44%), menthyl acetate (0.7–23%), 1,8-cineole (eucalyptol) (1–13%), menthofuran (0.3–14%), isomenthone (2–5%), neomenthol (3–4%), and limonene (0.1–6%), whereas β-caryophyllene is the main sesquiterpene (1.6–1.8%) [36]. Most of peppermint oil medicinal properties are ascribed to menthol, their major active component, while esters, such as menthyl acetate, provide the familiar minty taste and associated aroma [4].

Table 2 presents published compositions of some widespread mint essential oils with a more limited commercial interest, including M. pulegium, the source of the essential oil “pennyroyal” rich in pulegone; M. spicata, dominated by carvone; and M. rotundifoliaand longifoliaof varied composition.

4.2. Phenolic compounds

Phenolic compounds, secondary metabolites ubiquitously distributed in plants, include a large group of biologically active compounds, with over 8000 molecules, either small or large and complex molecules, presenting at least one aromatic ring with one or more hydroxyl groups attached. These compounds often appear in their natural sources as esters and glycosides [94].

Species of the genus Menthahave been reported to contain a range of components, including cinnamic acids and aglycon, glycoside, and/or acylated flavonoids [95]. Triantaphyllou et al. [96] reported that water extracts from Menthacontain esters of phenolic acids and flavonoid derivatives and glycosidic flavonoids hydroxylated in position 3 or 5.

Regarding phenolic acids, the genus Menthais particularly rich in caffeic acid and its derivatives, chlorogenic and rosmarinic acid [24, 25, 36, 94, 95, 97–99], the latter accounting for 60–80% of total phenolic compounds. In addition, seven salvianolic acids have been described in Menthaplants, such as salvianolic acid H/I, salvianolic acid E, salvianolic acid B, and isosalvianolic acid A (caffeate trimers) [30].

Menthaplants are rich in flavonoids, particularly in flavones and flavanones. Luteolin and its derivatives are the main flavones described in Menthaspecies [30]. The components eriocitrin, luteolin-7-O-glucoside, naringenin-7-O-glucoside, isorhoifolin, eriodictyol, luteolin, and apigenin were identified in aqueous extracts from Menthaspecies, hybrids, varieties, and cultivars [95]. Besides, Areias et al. [97] have reported the main component in aqueous Menthaextracts to be the glycoside eriocitrin.

In an older study, external lipophilic methylated flavonoids have been extracted from dried leaves of Mentha aquatica, M. spicata, M. x piperita, and M. citrata. Twenty flavonoids have been identified. 5,6-Dihydroxy-7,8,3′,4′-tetramethoxyflavone was identified as major flavonoid of M. spicataand M. x piperitaand 5-hydroxy-6,7,8,4′-tetramethoxyflavone (gardenin B) as a major compound of M. citrataand M. aquatica[100].

The phenolic composition of other species of different origins is summarized in Table 3.

4.3. Other compounds

Various other classes of compounds have been characterized and quantified in the mints. M. spicataand M. piperitacontain different trace elements [46, 131]. Maffei and Scannerini [132] studied the variability of the triacylglycerol, diacylglycerol, and free fatty acids in some Menthaspecies. They found a high level of C₁₈:3 only in the leaves of certain species (M. longifolia, M. crispa, and M. sachalinensis). Among the major components found in peppermint leaves are fatty acids such as linoleic, linolenic, and palmitic acid [98]. In addition, recent studies identified two new ceramides from the methanolic extract of M. longifolia, longifoamides A and B [10].

Triterpenoids and steroids were also isolated from mints. So, two triterpenoids ursolic acid and uvaol and three steroids stigmast-5-en-3-β-yl formate, stigmast-5-en-3-one, and β-sitosterol were isolated from the aerial parts of M. longifoliasubsp. noeana[128].

On the other hand, different pigments were identified and quantified in Menthaspecies. The analysis of M. spicatarevealed the presence of xanthophylls (neoxanthin, violaxanthin, and lutein, zeaxanthin), carotenes (α-carotene) [133], and chlorophylls (chlorophylls a and b) [134, 135]. Carotenoids (lutein and β-carotene isomers) were determined in dry peppermint tea, but only lutein was found in infusion [36]. Among vitamins, α-tocopherols and ascorbic acid were present in mints [36, 98, 135].

Mint was also reported to contain sugars, saponins, alkaloids, anthraquinones, and quinines [136], but these absolutely surprising HPTLC-based phytochemical data as well as the identity/purity of investigated samples should be thoroughly verified.

5.1. Antioxidant activity

Various types of compounds from aromatic and medicinal plants are receiving particular attention due to their radical scavenging properties. Reactive oxygen species (ROS) are chemical species formed in the body during metabolism that are highly reactive and may have one or more unpaired electrons. Oxidative stress, i.e., an imbalance between ROS and antioxidant defenses, has deleterious effects, such as the peroxidation of membrane lipids and the attack on biomolecules (proteins, membrane enzymes, carbohydrates, and DNA) [137].

Various Menthaspecies and their extracts or essential oils have been shown to possess antioxidant activity [30]. Phenolic acids (e.g., rosmarinic and caffeic acids), flavones (e.g., luteolin derivatives), and flavanones (e.g., eriocitrin derivatives) are possibly the major antioxidants. Vitamin antioxidants (e.g., ascorbic acid and carotenoids) are minor contributors to the overall antioxidant potential. In essential oils, unsaturated terpenes having a cyclohexadiene structure (e.g., terpinene) and minor cyclic oxygenated terpenes (e.g., thymol) may contribute to antioxidant potential, while acyclic unsaturated oxygenated monoterpenes (e.g., linalool) may act as pro-oxidants [36].

Menthaextracts are widely known to act as free radical scavengers in vitro. The acetonic extract and essential oil of peppermint act as scavengers of hydroxyl radical (•OH) [25, 138], the hydroalcoholic extract of M. piperita[139] and peppermint essential oil [140] as scavengers of nitric oxide (•NO), and the ethanolic and water extracts of M. pulegium[141] as scavengers of hydrogen peroxide (H₂O₂). Besides, different fractions of the ethanol extract of M. spicata[142]; the ethanolic extracts from M. spicata, M. pulegium, and M. rotundifolia[99]; the methanolic extract of M. pulegium[68, 143] and M. longifolia[68] were shown to quench superoxide (O₂•⁻) radicals.

Menthaplants have also been reported for antioxidant activities in several functional tests. The DPPH test, a test widely used to measure the ability to donate hydrogen atoms [41], was applied to measure the antioxidant capacities of Menthaspecies extracted by different solvent systems; these include the ethanol extracts of M. longifolia, M. piperita[144], M. pulegium[73, 99, 141, 144], M. spicata, and M. rotundifolia[96, 144]; the methanol extracts from M. pulegium[68, 69, 143, 145], M. longifolia[68, 93], M. aquatica, M. arvensis, M. piperita, M. rotundifolia, and M. villosa[145]; the water extracts from M. pulegium[69, 73, 141]; and the acetonic extracts from peppermint [25] and M. spicata[146]. DPPH was also used to evaluate the antioxidant activity of the essential oils from M. aquatica[92], M. longifolia[6, 68, 92, 93], M. spicata[6, 46, 51], M. pulegium[68, 69, 72, 73], M. rotundifolia[89, 147], and M. piperita[46, 92, 138, 140].

Other tests are less used in literature to evaluate the antioxidant potential/radical scavenger capacity of Menthaspecies polar extracts and essential oils (Table 4).

The most studied species are M. spicata, M. piperita, M. longifolia, M. pulegium, M. rotundifolia, M. arvensis, and M. aquatica. M. piperitaand M. spicataextracts showed good antioxidant activities in several in vitro assay systems compared to other species [95, 99, 144, 149]. The antioxidant compounds present in these extracts act as hydrogen- or electron-donating agents and/or metal chelators. Moreover, as expected from their composition, the polar extracts of Menthaspecies showed much better activity than the essential oils [6, 41, 69, 93].

5.2. Antimicrobial activity

The antibacterial and antifungal activities of Menthaspecies have been studied on various bacteria and fungi [30]. These studies indicate that essential oils are more efficient antifungals and antibacterials compared to the polar extracts [6, 68, 73]. Menthaessential oils showed remarkable antimicrobial activity against bacteria and other microorganisms, such as yeasts and periodontopathogens [4], mainly due to the presence of oxygenated monoterpenes in their chemical compositions [22]. Bactericidal and bacteriostatic activities are observed in the 1/1 to 1/1000 (V/V) and 1–5 mg/mL concentration ranges, respectively.

Thus, M. rotundifoliaoils showed effect against Bacillus subtilis, B. cereus, Escherichia coli, Proteus mirabilis, Salmonella typhimurium, and Staphylococcus aureus[21, 88, 89, 152]. The pulegone-rich essential oil of M. suaveolensefficiently inhibited all the microorganisms (20 stains) tested by Oumzil et al. [85]. Furthermore, according to Brahmi et al. [71], M. rotundifoliaessential oils exhibited stronger antimicrobial effect than M. pulegiumoils against all the microorganisms studied (three Gram⁺, three Gram⁻, two fungal, and one yeast). Nevertheless, M. pulegiumoil showed good antimicrobial activity against 11 bacteria (3 Gram⁺ and 8 Gram⁻) and 2 yeasts [72].

M. pulegiumpresents an appreciable activity toward all microorganisms (five Gram⁺, five Gram⁻, and six fungal strains) tested by Hadjlaoui et al. [68] and Streptococcus pyogenes[47]. Similarly, they showed the best bacteriostatic and bactericidal effect compared to tested medicinal and aromatic plants from other genera [70]. Besides, the essential oil of the flowering aerial parts of M. pulegiumshowed a significant activity against microorganisms especially Gram-positive bacteria [19].

The essential oil of M. spicatahas an appreciable activity against Streptococcus pyogenes[47], E. coli, S. aureus, S. pyogenes[46], and C. albicans[46, 51]. Oils of Mentha longifoliashowed strong antimicrobial activity against all 16 microorganisms tested by Hadjlaoui et al. [68] and against Escherichia coli, Shigella sonnei, and Micrococcus flavus.These bacteria were also inhibited by the essential oils from M. aquaticaand M. piperita[92]. Of the Menthaessential oils tested by Hussain et al., the oil from M. arvensisshowed relatively higher antimicrobial activity [45]; the essential oils of Mentha officinalistotally inhibited E. coli, Bacillus aureus, Streptococcus lactis, and S. aureus[153].

Besides, the essential oils from Menthaspp. have been considered a safe ingredient for the development of antibiofilm agents that could find a role in the pharmaceutical industry [4].

The antibacterial or antifungal activity of Menthaplant polar extracts have been studied to a much lesser extent; bactericidal and bacteriostatic activities are observed in the 2–4 mg/mL and 100–250 μg/mL concentration ranges, respectively, and at 6 μg/disk. The extracts were shown to possess antibacterial and antifungal activity [30]. Methanolic extracts of M. viridisand M. pulegiumshowed slight antimicrobial capacity against S. enteritidisand E. coli, respectively [104]; infusions of M. piperitaand M. spicatawere active on Vibrio parahaemolyticus[154]. Fractions from M. spicataethanol extract showed effective antibacterial activity against Escherichia coli, Salmonella paratyphi, Shigella boydii, Staphylococcus aureus, and Vibrio cholerae[142]. Peppermint tea extracts were active against Chlamydia pneumoniae[24].

5.3. Insecticidal activity

Mint is also known to exhibit insecticidal activity against a wide variety of insects. Menthahas been used as insecticides mainly in the form of essential oils [155]. M. spicata, M. pulegium, and M. rotundifoliaoils demonstrated insecticidal properties against adults of Rhyzopertha dominica, in contact and fumigation bioassays and repellency [51, 71]. M. pulegiumand M. rotundifoliaoils were also very toxic in the first 24 h in a contact toxicity bioassay against the same pest [156].

M. arvensisoil was toxic against Sitophilus oryzae(LC₅₀, 45.5 μL/L) [157, 158]. Similarly, the essential oil of M. microphyllagave remarkable activity against this insect (LC₅₀, 0.2 μL/L) in fumigation bioassays and in contact bioassays (24 h; LC₅₀, 0.01 mg/cm²) [159], and the ethanolic extract of M. longifoliawas also efficient against it (24.2% repellency) [160]. Additionally, M. pulegiumoil was toxic against Sitophilus granarius(contact LD₅₀, 9.1 μL/mL) [72], and M. longifoliaessential oil has 100% repellence against Sitophilus zeamais[22].

Varma and Dubey [158] reported complete inhibition of Tribolium castaneum, through the treatment of wheat samples with M. arvensisessential oil. The essential oil of M. microphyllagave remarkable activity against adults of this insect (LC₅₀, 4.5 μL/L) in fumigation bioassays [159]. Furthermore, the insecticidal properties of M. longifoliaessential oil against this pest have been attributed to piperitenone oxide (LC₅₀, 9.95 mg/L) [22]. In another study, Lee et al. [157] observed that M. piperita(LD₅₀, 25.8 μL/L) was a slightly better fumigant than M. spicata(LD₅₀, 33.1 μL/L) against T. castaneum. Besides, in both contact and fumigation assays, the M. rotundifoliaoil samples rich in pulegone and menthone, compared to other chemotypes, exhibited superior insecticidal activity against the adults of the same insect [161].

Menthaessential oils and polar extracts showed also insecticidal properties toward other insect species. The ethanolic extract of M. longifoliawas efficient against third- and fourth-instar larvae of Culex pipiens(LC₅₀-26.8 ppm). M. arvensisoil efficiently repelled (85%) Callosobruchus chinensis[160]. Feeding on M. longifoliacaused death in Chrysolina herbacea[22]. M. pulegiumL. oil also caused 100% mortality of Mayetiola destructor[162]. Studies have shown that essential oils of spearmint were effective against Lycoriella ingenuaat 20 × 10⁻³ mg/mL [10]; fumigation allowed controlling all stages of Callosobruchus maculatus; and the egg stage was the most susceptible stage [163]. Also, compared to M. pulegium, a M. suaveolenshydrosol showed higher insecticidal activity toward an insect pest of citrus, Toxoptera aurantii[164].

5.4. Cytotoxicity

Several studies have indicated that Menthaplants contain constituents with cytotoxic properties that may find use in developing anticancer agents. For example, M. arvensis, M. longifolia, M. spicata, and M. viridismethanolic and aqueous extracts showed antiproliferative effect against various cancer cell lines in vitro at a concentration of 100 μg/mL [165]. Similarly, in Yi and Wetzstein [166] study, spearmint and peppermint methanolic extracts significantly inhibited SW-480 colon cancer cell growth (IC_50s: 143.6 ± 25.6 μg/mL for spearmint and 92.3 ± 17.8 μg/mL for peppermint). The cytotoxic effect of the essential oil of M. pulegiumon ovarian adenocarcinoma (SK-OV-3), human malignant cervix carcinoma (HeLa), and human lung carcinoma (A549) cell lines has been shown by other investigators (IC₅₀s ranging from 14.10 to 59.10 μg/mL) [167]. In an in vitro screening for the tumoricidal properties of international medicinal herbs, M. spicataand M. piperitaexhibited extremely weak tumoricidal effects (LC₅₀ > 5.0 mg/mL), while M. pulegiumshowed a weak activity (LC₅₀, 1.2–2.5 mg/mL) [168].

The cytotoxicity of essential oils from four Menthaspecies (M. arvensis, M. piperita, M. longifolia, and M. spicata) was tested on breast cancer (MCF-7) and prostate cancer (LNCaP) cell lines using the MTT assay. The tested Menthaessential oils showed prominent cytotoxic activity against both cancer cell lines (IC₅₀s ranging from 43.5 ± 2.1–95.7 ± 4.5 μg/mL) [45].

In another study, aqueous extract of M. spicatasignificantly reduced the proliferation of Wehi-164 and U937 cells dose and time dependently (LD₅₀s ranging from 4.63 to 5.97 mg/mL) [169]. Jain et al. [170] examined the possible molecular mechanisms underlying the cytotoxicity and anticarcinogenic potential of Mentha piperitaleaf extracts on six human cancer (HeLa, MCF-7, Jurkat, T24, HT-29, MIA PaCa-2). The chloroform and ethyl acetate extracts showed significant dose- and time-dependent anticarcinogenic activity, leading to G1 cell cycle arrest and mitochondrial-mediated apoptosis, perturbation of oxidative balance, upregulation of Bax gene, elevated expression of p53 and p21 in the treated cells, and acquisition of senescence phenotype (effective doses ranging from 10 × (10 μg/μL) to 100 × (100 μg/μL)).

Lv et al. [25] also evaluated the antiproliferative activity of a peppermint extract against the human tumor cell line HT-29 (effective doses 250 and 500 μg/mL). Similarly, the cytotoxic effect of Mentha piperitaessential oil was assessed against four human cancer cells. It was found to be significantly active against human lung carcinoma SPC-A1, human leukemia K562, and human gastric cancer SGC-7901 cells, with IC₅₀ values of 10.9, 16.2, and 38.8 μg/mL, respectively [138].

M. longifoliamethanolic extract and M. piperitaethanolic extract presented a cytotoxic activity, respectively, against human breast cancer (IC₅₀ = 191.2 μg/mL) [171] and human laryngeal epidermoid carcinoma (IC₅₀ = 94 μg/mL) [172]. Besides, peppermint extract showed cytotoxicity against four human tumor cell lines (MCF-7, NCI-H460, HeLa, and HepG2; IC₅₀s ranging from 98 ± 9 to 226 ± 11 μg/mL) [94].

5.5. Anti-inflammatory properties

Menthaextracts contain numerous constituents which could have anti-inflammatory effects. In vitro, the anti-inflammatory activity of the M. piperitaessential oil has been determined by 5-lipoxygenase (5-LOX) inhibition assay (IC₅₀s ranging from 0.03 ± 0.01–0.08 ± 0.01 μg/mL) [140]. It could also effectively inhibit nitric oxide (^•NO) and prostaglandin E2 (PGE2) production in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages [138]. Lv et al. [25] using J774A.1 mouse macrophage cells showed that peppermint extracts were efficient in inhibiting IL-1 and COX-2 expression and have inhibitory effect on IL-6 and MCP-1 (IC₅₀s ranging from 50 to 100 μg/mL).

In vivo, pretreatment of albino mice and female Wistar rats with M. suaveolensmethanol extract induced an anti-inflammatory effect [173]. The anti-inflammatory effects of aqueous, chloroform, ethyl acetate, and hexane extracts of M. spicataethyl acetate and aqueous fractions were both effective in reducing the chronic and acute inflammation of Wistar albinorats [11]. In addition, edema reduction was also observed by topic use of M. aquaticaL. alcohol extract on Male CD-1 mice [174]. The M. piperitaessential oil exhibited potent anti-inflammatory activities in a croton oil-induced mouse ear edema model. The oil reduced the edematous response by 5.77, 7.37, and 30.24% at the dose of 200, 400, and 800 μg, respectively [138].