Medicinal Uses, Phytochemistry, Pharmacology, and Toxicology of Mentha spicata

Mentha spicata, also called Mentha viridis, is a medicinal plant of the Lamiaceae family characterized by its potency to synthesize and secret secondary metabolites, essentially essential oils. Different populations use the aerial parts of this plant for tea preparation, and this tisane has shown several effects, according to ethnopharmacological surveys carried out in different areas around the world. These effects are attributed to different compounds of M. spicata, in which their biological effects were recently proved experimentally. Pharmacological properties of M. spicata extracts and essential oils were investigated for different health benefits such as antioxidant, anticancer, antiparasitic, antimicrobial, and antidiabetic effects. In vitro and in vivo studies showed positives effects that could be certainly related to different bioactive compounds identified in M. spicata. Indeed, volatile compounds seem to be efficient in inhibiting different microbial agents such as bacteria, fungi, and parasites through several mechanisms. Moreover, M. spicata exhibited, according to some studies, promising antioxidant, antidiabetic, anti-inflammatory, and anticancer effects, which show its potential to be used as a source for identifying natural drugs against cellular oxidative stress and its related diseases. Importantly, toxicological investigations of M. spicata show the safety of this species at different doses and several periods of use which justify its use in traditional medicines as tisane with tea. Here, we report, explore, and highlight the data published on M. spicata concerning its botanical description and geographical distribution, its phytochemical compounds, its pharmacological properties, and its toxicological investigations of M. spicata.


Introduction
e use of M. spicata is importantly characterized in several populations, including Moroccan population, which has used the aerial parts (with tea) of this plant since time against several diseases including diabetes, digestive and respiratory disorders, throat ailments, and skin disease [1,2].
Certainly, M. spicata contains molecules biologically active having biological effects, and effective spectroscopic analysis of extracts and essential oils of M. spicata using GC-MS, HPLC, HPLC-MS, and RMN revealed the presence of several phytochemical bioactive compounds belonging to different classes of secondary metabolites in particularly the classes of flavonoids, phenolic acids, and terpenes [3,4]. Indeed, the distribution of these chemical compounds between different plant parts and collection regions is variable, which explains different traditional uses (with efficacy) of this species according to each region. In addition, the extraction of these chemical compounds depends on used methods and therefore can justify the difference in traditional applications according to used methods of pharmaceutical formulations preparation.
In vitro and in vivo experimental explorations showed that M. spicata extracts and essential oils exhibit remarkable biological activities, including antimicrobial, antiparasitic, antidiabetic, anti-inflammatory, and anticancer effects. Indeed, different organic extracts (rich in bioactive compounds) revealed important antifungal activity by their potency to inhibit the growth of some strains involved in human infections such as Aspergillus niger, Candida albicans, Cryptococcus neoformans, and Microsporum audouinii [5]. Moreover, M. spicata showed antibacterial properties against various bacterial strains, either clinical or reference [6,7]. It was also revealed that M. spicata extracts target some human complex diseases, including chronic inflammatory diseases, diabetes, and cancers. Plant extracts inhibit or activate targets and/or pathways involved in these pathologies, including membrane receptors, signaling pathways, and molecular targets [8,9].
To the best of our knowledge, despite numerous investigations that have been carried out until now showing remarkable results, there are now literature reviews exploring M. spicata as a source of potential lead compounds. erefore, this review aims to explore, discuss, and highlight all data concerning M. spicata and give suggestions about its exploitation as a source for developing bioactive compounds in the pharmaceutical and cosmetic fields. It is an aromatic plant that belongs to the genus Mentha, family Lamiaceae, subfamily Nepetoideae, placed in Magnoliopsida class, and belongs to order Lamiales. e genus Mentha, one of the most important members of the Lamiaceae family, is represented by 19 species and 13 natural hybrids, and Lamiaceae family consists of over 7000 species and around 260 genera of trees and shrubs [10]. e spearmint, M. spicata, is a hybrid of M. longifolia and M. rotundifolia.

Results and Discussion
is species is widely grown in Europe, North America, and Asia, but nowadays cultivated throughout all regions of the world [11].
M. spicata L. (spearmint) is a creeping rhizomatous, glabrous, and perennial herb with a strong aromatic odor, growing up to 30-100 cm tall with variably hairless to hairy stems and foliage, and a wide spreading fleshy underground rhizome [12]. e leaves are ovate to lancolate, 5-9 cm long and 1.5-3 cm broad, with a serrated margin. Spearmint produces flowers in slender spikes, each flower pink or white, and 2.5-3 mm long and broad. e stem is square-shaped, a trademark of the mint family of herbs [13]. M. spicata L is well adapted to climatic conditions in tropical and subtropical areas. It can be cultivated in wide range of soils and found in back gardens of homesteads [14].
In Morocco, Mentha viridis is a medicinal plant most used in the treatment of throat ailments. e use of this plant to treat throat ailments has been demonstrated by Orch et al. [20], who reported the use of aerial parts' infusion of Mentha viridis in Moroccan oriental folklore. e leaves of M. viridis are also administered as a decoction to treat diabetes in the Al Haouz-Rhamna region (Morocco) [1]. Idm' hand et al. [17] showed that the leaves and stems of M. viridis are also used as a decoction and infusion to treat diabetes; on the other hand, El-hilaly et al. [16] showed that these parts were used to treat headache and tiredness. e leaves and flowers of M. viridis have also been widely used to treat asthma,

Phytochemical Compounds.
Extracts and essential oils extracted from M. spicata (viridis) are considered as valuable source phytochemicals, including natural phenolics and EO S . ese volatile compounds are complex mixtures of substances that have been found to create different chemotypes distinguished based on the dominant compound in the essential oil, which depends on the plant species, and within the same variety, the essential oil composition can vary according to the geographical region [24]. In terms of phytochemical content, terpenes and terpenoids are the major components of EOs obtained from aerial parts of M. spicata. us, more detailed discussion regarding chemical aspects of EOs of these species is described (Table 2). Previous studies reported the existence of different chemotypes in the chemical composition of M. spicata, naturally grown as cultivated, around the world, and the essential oil mainly composed of carvone, carvacrol, trans-carveol, piperitone oxide, limonene, 1,8-cinéole, camphene, p-cymene, dihydrocarvone, pulegone, β-caryophyllene, germacrene D, menthone, α-pinene, and linalool [3,5,26,27]; whereas, carvone is mentioned as the absolute predominant constituent of M. spicata oil as well as monoterpenes including linalool, piperitone, piperitone oxide, menthone, isomenthone, and pulegone ( Figure 1 and Table 2). e composition of M. spicata EO S from Morocco is relatively stable and has strong homogeneity [7,40,53,56]. No significant difference between samples was observed; whatever the locality (region), the main essential oil compounds are carvone and trans-carveol, showing variation in a narrow range of 29-47.3% and 14-20%, respectively [34,46,47,51,52]. Various chemotypes of M. spicata were also identified for plants cultivated in Italy and Turkey. In plants from Italy, carvone (39.13-59.26%) was detected as the main compound [29], while for the species from Turkey, piperitenone oxide (25.84%), pulegone (24.72%), cis-piperitenone oxide (12.55%), and limonene 1.59% were the principal constituents of the EO S [31]. It is worth noting that chemotype carvone represented the most variation, 79.70% in spearmint M. spicata EO S [24]. Other examples of M. spicata producing EOs with high piperitone oxide content (above 70%) are samples from India [36]. As established in the literature, such compound is one of the most abundant components of M. spicata EO, which offers spearmint its unique smooth characteristic scent [57], and it also varies according to the spearmint oil grown in different countries. Similarly, EOs from Cyprus is reported to possess a higher carvone content (69.23-74.27%) [55].
However, four chemotypes of M. spicata were found in Brazil, characterized by the dominant occurrence of carvone which vary from 39.42% to 72.28% and piperitone presented high level 81.18% [7,56], Although carvone was constantly present as a chief component among spearmint species, there was one landrace with linalool content up to 58.51%. Since all the studies were carried out in the same environmental conditions, this variation may be triggered by their different genetic backgrounds, having evolved due to complex geographic-environmental differences across Brazil. Interestingly, in most M. spicata EOs, carvone is the major constituent, notably found in quantities above 50% in EOs extracted from plants cultivated in Hungary, Iran, Bangladesh, Serbia, Czech Republic, and Pakistan [3,5,40,46,47,52,53].
Furthermore, the occurrence of huge chemical variations among Mentha accessions collected from diverse countries seems to be due to the divergent climatological and geographical conditions; existing variations in oil content and composition may be attributed to factors related to ecotype and the environment including temperature, relative humidity, irradiance, and photoperiod [34]. Additionally, the reported yields of carvone for M. spicata range from 39.21% to 75.53%, being the highest value found for plants cultivated in Tunisia [51].
As given in Table 2, plants cultivated in several states in Iran usually produce EOs with high (>50%) 1,8-cineole content [39]. Similarly, M. spicata populations in China also show certain stability in essential oils, with carvone chemotype affording high yield 46.7-65.4% above, while dihydrocarveol acetate (0.2-7%) observed in Chinese spearmint is the only oxygenated sesquiterpenes [46]. Also, a large chemical variability is observed among M. spicata essential oil extracted by different methods. Such variation can be attributed to several factors, including genetic, environmental, and their interaction effects, such as plant part, harvest time, extraction method, ecotype, and geographic origin (climate, edaphic, elevation, and topography) [4].

Pharmacological
Properties of M. spicata. M. spicata essential oils and extracts exhibit different biological and pharmacological properties ( Figure 6). ese properties will be discussed in the following sections.

Antifungal Activity.
Several studies investigated the antifungal activity of Mentha spicata extracts using different parts of the plant and different methods such as the disc diffusion method, microdilution method, agar well diffusion method, spots method, and microdilution broth susceptibility assay [5,11,62,63]. Table 3 provides all studies that examined the antifungal potential of M. spicata extracts, showing the type of extract, plant part used, used method, tested strains, and key results. Using the disc diffusion method, Alaklabi et al. [62] assessed the antifungal activity of hexane, chloroform, ethyl acetate, methanol, ethanol, toluene, n-butanol, n-propanol, isopropanol, and water extracts from the root of M. spicata against Aspergillus niger, Candida albicans, Cryptococcus neoformans, and Microsporum audouinii. Water extract showed the highest activity against M. audouinii (MIC: 16 μg/mL). It revealed a remarkable antifungal response against other fungal species, A. niger (MIC � 32 μg/mL), C. albicans (MIC � 64 μg/mL), and C. neoformans (MIC � 32 μg/mL). Hexane, chloroform, and ethyl acetate extracts exhibited high antifungal activity against M. audouinii with a MIC equal to 32 μg/mL, 64 μg/ mL, and 32 μg/mL, respectively. In contrast, the same extracts did not show a significant effect against the other fungal strains tested. Moreover, C. albicans was significantly inhibited by toluene and n-butanol extracts (MIC � 64 μg/mL), whereas the fungal activity of A. niger was highly reduced by using methanol and ethanol extracts (MIC � 64 μg/mL). Using the same method to screen the antifungal activity of M. spicata root extracts, isopropanol Evidence-Based Complementary and Alternative Medicine extract was found to be less active for the four fungal strains evaluated [62].
To investigate the antifungal properties of essential oil isolated from the aerial parts of M. spicata cultivated in the Algerian Saharan Atlas, the results published by Bardaweel et al. [48] showed a lower activity of essential oil of M. spicata against Candida glabrata (MIC � 256 μg/mL) by employing the microdilution method. Nevertheless, in the Turkish study conducted by Bayan et al. [64], the volatile oil from M. spicata extracted of aerial parts exhibited a strong fungitoxicity effect with 100% of inhibition of mycelium growth in F. oxysporum f.sp. radicis-lycopersici (FORL), Verticillium dahliae Kleb (V. dahliae), Alternaria solani (A. solani), and Rhizoctonia solani J.G. Kühn. (R. solani) at a dose of 12 μL petri −1 by using the agar well diffusion method.
In another study from Pakistan, Hussain et al. [5] evaluated the antifungal activity of essential oil of spearmint (Mentha spicata L.) isolated from dried aerial parts against five fungal strains. e results showed that Aspergillus niger was the most responsive fungal species presenting the largest zone of inhibition (26.9   Fusarium solani (V � 25.2 ± 1.0 mm and MIC � 0.09 ± 0.00 μg/mL). However, B. theobromae was observed to be the most resistant fungus with the smallest inhibition zone (23.0 mm) and a MIC value equal to 0.11 mg/ mL by using microdilution broth susceptibility assay.
Additionally, Kedia et al. [66] tested the antifungal potency of essential oil of spearmint against 19 food-deteriorating molds using the poisoned food assay. e fundings showed that the oil of M. spicata has a notable potential to inhibit the fungal growth of all fungi species, causing 100% of mycelial inhibition at 1.0 μL ml −1 excluding Aspergillus luchuensis and Aspergillus terreus, where the percentage of mycelial inhibition was 91.72 ± 0.36% and 75.67 ± 0.74%, respectively. e results of testing the nature toxicity of the oil from M. spicata revealed that spearmint essential oil possessed a fungicidal effect in Cladosporium cladosporioides, Mycelia sterilia, Alternaria alternata, and Curvularia lunata at 1.0 μL mL −1 . In their study, Liu et al. [11] investigated the biological properties of the essential oil isolated from aerial parts of M. spicata from China. Using the disc diffusion method, the results of this study showed quite strong antifungal potency against A. niger with an MIC value of 6.25 μg/mL and an MBC value of 12.50 μg/mL. Compared to a study carried out by Şarer et al. [67] from eastern Turkey, the oil of M. spicata subsp. spicata exhibited high antifungal activity against Candida albicans and Candida tropicalis with an MIC value less than 3.19 μg/mL.
Regarding testing the potential antimicrobial effects of M. spicata, [45] investigated the essential oil extracted from air-dried leaves of Algerian spearmint against Candida albicans (ATCC 1024) strain and two Aspergillus species (flavus NRRL 391 and niger 2CA 936). Using the spots       On the other hand, Ojewumi et al. [63] demonstrated the antimicrobial role of the leaf oil extract of M. spicata from Nigeria by using two types of petroleum ether and hexane extract. ey found that the hexane extract showed higher activity against Aspergillus niger (V � 26 mm) followed by Saccharomyces cerevisiae (V � 25). In addition, they observed that petroleum ether extract showed potent activity against Aspergillus niger (V � 27 mm) followed by S. cerevisiae (V � 27). erefore, it was noted that the effectiveness of the two extracts was significantly comparable as the inhibitory zone values are very similar. Furthermore, the ethanolic extract exhibited 100% of inhibition against Fusarium oxysporum f.sp. lentis in the investigation performed by Singh et al. [68] that aimed to study the antifungal activity of M. spicata. e results found were supported by the study conducted in Sudan by Sulieman et al. [69]; they indicated that spearmint oil leaves have demonstrated potent activity against Aspergillus niger (ATCC 9763) with an inhibition zone equal to 19 mm at a high concentration (20%) and (15 mm) at low concentration (5%). In addition, the oil of M. spicata exhibited considerable inhibition capacity against C. albicans with an inhibition zone diameter of 18 mm at higher concentration (20%) and 14 mm at lower concentration (5%). Similarly, the concentration of 100 mg/mL was able to inhibit C. albicans with a diameter of growth inhibition zone reached 16 mm using the agar diffusion method [71].
Zaidi et al. [70] evaluated the antifungal efficiency of oil leaves from M. spicata against four fungal species including A. niger and Aspergillus spp., C. albicans, and Rhizopus nigricans, using the agar well diffusion method. e results showed that Mentha spicata oil exhibited an excellent potential against fungal strains tested but with differing sensitivity. A. niger showed a strong inhibition zone of 15.7 ± 0.09 mm compared to C. albicans, which possessed an inhibition zone of 11.8 ± 0.10 mm. However, M. spicata oil was not able to inhibit the growth of R. nigricans strain. e oil also exhibited an antifungal effect against Aspergillus spp. (13 ± 0.13 mm). In another study, using the agar well diffusion method, essential oil isolated from spearmint was observed to act as a stronger bioactive source against fungal species with a different zone of inhibition. Indeed, inhibition zone diameters for Aspergillus ochraceus (NRRL 3174) (V � 43 mm) and Mucor ramamnianus (ATCC 9314) (V � 40 mm) were higher than inhibition zone diameters for S. cerevisiae (ATCC 4226 A) (V � 25 mm) and C. albicans IPA 200 (V � 21 mm) [72].

Antibacterial Activity.
For over 60 years, antimicrobial agents have been used to treat infections in humans, animals, and plants. Currently, they are among the most widely used therapeutic agents in human and veterinary medicine [73]. At the start of antibiotic therapy, as resistant strains were low and highly effective antimicrobial agents of different classes were detected, antimicrobial resistance was not considered a major problem. is has forced sensitive bacteria living in close contact with antimicrobial producers to develop mechanisms to bypass the inhibitory effects of antimicrobial agents (Table 4). In the context of this study, several in vitro studies have determined the antibacterial activity of M. spicata essential oils and solvent extracts against various bacterial strains, either clinical or reference, using the agar diffusion methods (disks or well) and the agar and broth dilution methods [5,74,89]. In most of these studies, qualitative inhibition was determined by the dilution method, which is used to assess minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values [5,74,89]. Indeed, the increased selective pressure imposed by the widespread use of antimicrobial agents has clearly accelerated the development and spread of bacterial resistance to antimicrobial agents [5,74,89]. ese observations underscore the enormous flexibility of bacteria to resist less favorable environmental conditions by constantly developing new survival strategies. Table 5 provides investigations interested in the antiparasitic effect of spearmint [90,91]. Zandi-Sohani and Ramezani [90] investigated the antiparasitic effect of essential oil isolated from spearmint leaves collected from southwestern Iran against Tetranychus turkestani. ey discovered that the essential oil of spearmint exhibited acaricidal potential and can be employed to protect against Tetranychus turkestani, which showed to cause 100% adult mortality at a concentration of 20 μL/L. e lethal concentration values (LC 50 and LC 95 ) for essential oil spearmint were estimated to be 15.3 μL.L −1 and 23.4 μL.L −1 , respectively. However, the study conducted by Koumad and Berkani [91] demonstrated that spearmint leaves revealed the lowest acaricidal activity against Varroa destructor by smoke. Results showed that spearmint killed 26.20% of Varroa destructor and reduced the infestation rate by 2.35%. e mortality rate was estimated at 30.65%, and infestation rate was 13.18%.
Brahmi et al. [65] studied the impact of essential oil from M. spicata leaves against Rhyzopertha dominica. is study revealed that the essential oil from M. spicata leaf was effectively toxic against Rhyzopertha dominica adults. At a high concentration of 2 μL/mL, M. spicata oil showed high repellent activity against Rhyzopertha dominica (56.2% at 30 minutes), and the mortality rate was 43% after 96 hours of treatment. Furthermore, the toxicity contact assay showed that spearmint oil showed a low insecticidal effect with DL 50 equal to 6.1 μL/mL. In another study, Kedia et al. [66]

Whole plant
Staphylococcus aureus (MBLA) In an effort to identify biopesticides for granary weevil to avoid losses of crops caused by insects, Lamiri et al. [94] screened a variety of essential oils for their pesticide effects against Sitophilus granarius. ey discovered that essential oil of spearmint caused 80% and 43% mortality after 24 h and 48 h of exposure, respectively. ese findings indicate that the rate of adult mortality rises as the concentration of oil used in the test increases. e study by Papachristos and Stamopoulos [95] assessed the repellent effects of essential oil extracted from whole flowering plants of spearmint against Acanthoscelides obtectus. e results showed that this oil exhibited a highly toxic effect in both males and females with LC 50 values of 1.2 mL/L air for males and 4.4 mL/L air for females, where males are more affected than females. Also, the oil of spearmint exhibited the most repellent property against Acanthoscelides obtectus and appears to be more promising for potential use against this pest.
Abdel-Shafy and Soliman [96] in their research hypothesized that essential oil of spearmint (M. viridis) possesses the toxicity effect against embryonated eggs, larvae, and fed females of the cattle tick Boophilus annulatus (Acari: Ixodida: Amblyommidae) in Egypt. It was found that oil spearmint (M. viridis) was less toxic on embryonated eggs (LC 50 � 1.20%) as well as on unfed larvae (LC 50 � 0.90%) and fed females (LC 50 � 10.57%) than other oils tested, including peppermint (Mentha piperita), marjoram (Majorana hortensis), lavender (Lavandula officinalis), and sweet basil (Ocimum basilicum). Compared to the study performed by Derbalah and Ahmed [92], spearmint oil leaf was highly effective against Callosobruchus maculatus with an LC 50 value of 235 ppm. e results showed that oil spearmint could be used as a botanical product to control C. maculatus insect in cowpea seeds.   22 Evidence-Based Complementary and Alternative Medicine Pavela et al. [3] showed the effects of a variety of essential oils from the genus Mentha L., including M. spicata, against the larvae and adults of Culex quinquefasciatus Say (Diptera: Culicidae). eir finding indicates that the oil of M. spicata revealed lower larvicidal efficacy against C. quinquefasciatus compared to other oils tested. e lethal response of the oil towards the larvae for LC 50 was estimated as 92 mg/L and for LC 90 was estimated as 160 mg/L. Similarly, the study carried out by Govindarajan et al. [38] focused on the possible larvicidal properties of essential oil from M. spicata against three larvae species: A. stephensi, C. quinquefasciatus, and A. aegypti. After the exposure of treatment (24 h), the essential oil from M. spicata leaves showed a significant larvicidal effect against A. stephensi, C. quinquefasciatus, and A. aegypti, with LC 50  To test the application for alone or combined, three essential oils were isolated from three medicinal plant species belonging to the Mentha genus to manage the rice weevil Sitophilus oryzae (Curculionidae). e study conducted by Haouel-Hamdi et al. [93] showed that binary combined Tunisian spearmint oils from M. rotundifolia, M. viridis, and M. longifolia leaves have exerted an important anti-insecticide activity against Sitophilus oryzae. However, Mentha essential oils alone revealed the lowest repellent activity to S. oryzae adults. After 24 days of exposure, LC 50 and LC 95 values of fumigant toxicity of M. viridis essential oils alone was 100.16 μL/L air and 192.197 μL/L air, respectively, against S. oryzae adults. In addition, the LT 50 value was 45.52 h for M. viridis, and the percentage of mortality was 22% at a concentration of 71.43 μL/L air. Table 7 provides studies focused on the anti-inflammatory propriety of the M. spicata in different in vivo experiments [97,99]. Using the carrageen-induced paw edema method, Yousuf et al. [97] showed that methanol extract from the whole plant of M. spicata exhibited a strong anti-inflammatory activity which presenting at both doses 250 and 500 mg/kg of methanol extract a significant dose-dependent reduction of paw edema. Furthermore, the anti-inflammatory action of the extract remained significant until the 6 th hour of the test. In another study, Arumugam et al. [98] evaluated in vivo anti-inflammatory effect of different solvent fractions of the ethanolic extract of the dried leaves of M. spicata on rats with acute and chronic inflammation by using two experimental approaches, carrageenan and cotton pellet-induced inflammation models. e finding showed that ethyl acetate extract and aqueous fraction were potent in cotton pellet (chronic) induced inflammation where the rate of inflammation was reduced by 65% and 54%, respectively. However, inflammation was reduced with less effectiveness in hexane extract (0-20%) and aqueous fraction (7-11%); only the ethyl acetate fraction was found to be effective in carrageenan (acute) induced inflammation, while chloroform fraction has not been able to decrease inflammation. e study conducted by Jabbar and Kathem [99] evaluated the preventive effect of ethanolic extract of leaves from M. spicata on irinotecan-induced mucositis in mice. e results revealed that the ethanolic extract of M. spicata markedly reduced jejunal tissue IL-1β (3.47 ± 1.23 vs. 6.5 ± 0.36 ng/mL), and fecal β-glucuronidase activity (79.78 ± 10.7 vs. 120.6 ± 8.3 U) compared to no-treated mice. In addition, histological investigation of the jejunum section of the animal after administration of irinotecan and ethanolic extract of M. spicata showed enhancements in mucositis features.

Anti-Inflammatory Activity.
3.5.6. Antidiabetic Activity. Diabetes mellitus is a metabolic disease that affects the endocrine system, often occurring when the pancreas does not secrete enough insulin or when the body cannot use this hormone effectively, resulting in chronic hyperglycemia with disruptions in protein, lipid, and carbohydrate metabolism.
In order to understand the mechanism of antidiabetic action of M. spicata better, several recent studies (in vivo and in vitro) performed in chronological order were discussed in this review [8,100,101] (Table 8).
Regarding in vivo studies, Al-Fartosi and collaborators evaluated this activity on male rats rendered diabetic by alloxan intraperitoneal injection (125 mg/kg b.w) and treated with phenolic compounds (200 mg/kg b.w) extracted from the leaves of this plant [100]. During 14 days of daily treatment, a decrease in the level of blood glucose, triglycerides, cholesterol, plasma LDL, and VLDL and a significant increase in plasma HDL levels were recorded. is work confirmed the potential of M. spicata in the management of diabetes and its complications. In 2017, two similar studies verified these findings on the same animal model. Indeed, the aqueous ethanolic extract (200 and 400 mg/kg b.w) [101] and the aqueous extract (300 mg/kg b.w) [13] of the leaves of this species presented the same results as the previous study. e following year, 40 streptozotocin-induced diabetic rats were treated for 4 weeks with butanol extract from M. spicata roots [8]. At the end of this period, the authors observed antidiabetic properties represented by a decrease in blood glucose level and an increase in bodyweight.
A very recent investigation tested this powder on two carbohydrate hydrolyzing enzymes, namely, α-amylase and α-glucosidase [86]. In fact, inhibiting these two enzymes prevents the digestion of carbohydrates, which is a promising strategy in the treatment of diabetes. e results of this study showed that the leaf essential oil of this herb at doses of 200 and 250 μL was able to inhibit α-amylase (IC 50 � 101.72 ± 1.86 μg/mL) and α-glucosidase (IC 50 � 86.93 ± 2.43 μg/mL), respectively.
From these studies, it can be inferred that M. spicata may be used as an antidiabetic agent; however, further investigations, as well as clinical trials, must be carried out to evaluate this benefit in humans.
Evidence-Based Complementary and Alternative Medicine 3.5.7. Antioxidant Activity. Oxidative stress corresponds to an attack on cells by free radicals, also called reactive oxygen species (ROS), produced continuously from oxygen in the cell, particularly in the mitochondrial respiratory chain. ROS are reactive and very toxic substances. Oxidative stress is caused by an imbalance between the production of prooxidant free radicals and antioxidants. Regarding M. spicata, many studies have evaluated its antioxidant activity either by measuring its effectiveness in scavenging free radicals or by directly assaying the products formed using photometric techniques [5,78,102] (Table 9). Indeed, Getahun et al. [78] obtained essential oils by hydrodistillation from M. spicata leaves to determine their radical scavenging potentials in vitro in DPPH and deoxyribose degradation assays. ese oils exhibited potent radical scavenging activities, with IC 50 values of 5.96 and 0.57 μL/ mL in the DPPH and deoxyribose degradation assays, respectively. In the same year, Nickavar et al. [102] found that the ethanolic extract of M. spicata aerial parts showed IC 50 values of 87.89 and 173.80 μg/mL by the DPPH • and ABTS •+ assays, respectively.
e following year, using the same methods, Mkaddem et al. [72] showed that the essential oil from the leaves of this plant has significant anti-free radical potential.
By respecting the chronology of the studies carried out over time, Ebrahimzadeh et al. [9] examined the antioxidant capacity of M. spicata aerial parts in vitro using eight assay systems. ey recorded the best activity with the DPPH test (IC 50 � 105.8 ± 3.98 μg/mL), followed by the assay of nitric oxide-scavenging activity (IC 50 � 210.6 ± 7.7 μg/mL) and scavenging of H 2 O 2 (IC 50 � 631.1 ± 26.0 μg/mL). In addition, good antioxidant activity has been demonstrated by Hussain   [11] (IC 50 � 72.07 ± 0.34 mg/mL), using DPPH free radicalscavenging ability. Moreover, the antioxidant power of M. spicata aerial parts has been tested by Benedec et al. [50] using only the DPPH radical scavenging assay, which showed a value of 18.34 ± 2.2% at the concentration of 0.4 mg/mL. A Tunisian research team also confirmed this when they recorded an important antiradical (IC 50 � 10 ± 0.24 μg/mL) and superoxide anion (IC 50 � 1.33 ± 0.10 μg/mL) scavenging ability [104]. Furthermore, according to Teixeira and collaborators, the essential oil of this plant was shown to be a potent antioxidant by exhibiting a dose-dependent antioxidant effect at the concentrations tested (25,50,100,150,200,250, 300, and 500 μg/mL), determined by the sequestration of the DPPH radical and by the β-carotene-linoleic acid method [44].
Using the same methods as previous studies, other more recent investigations have confirmed the important antioxidant activity of M. spicata, regardless of its harvest region or parts used (Table 9). e antioxidant activity of different parts of M. spicata is certainly attributed to its major compounds. Indeed, L-menthone (32.74%) and pulegone (26.67%) were the main volatiles of its essential oil, while apigenin (38.4 mg/100 g dry weight) was the main flavonoid in methanolic extracts [104]. ese molecules are renowned for their antioxidant potential [109].

Diuretic Activity.
e in vivo study performed by Aziz et al. [110] assessed the diuretic property of the aqueous methanol extract from aerial parts of spearmint in rat models. e treatment administered to experimental rats at dose 100 mg/kg revealed significant diuresis (3.74 ± 0.41 mL). e values obtained are more or less close to the reference standard (furosemide, 4.05 ± 0.34 mL) (p < 0.05). Also, the extract of spearmint significantly increased the excretion of potassium and sodium (p < 0.05), while a significant change in the pH has not been observed after administration of M. viridis extract.
3.5.9. Analgesic and Antipyretic Activities. For testing the analgesic and antipyretic effects of methanol extract from M. spicata, Yousuf et al. [97] in their study demonstrated that the methanol extract from the whole plant of M. spicata had markedly increased the reaction time of mice in a dosedependent manner by the hot-plate test (p < 0.001) proving its marked analgesic effect. In addition, using the acetic acidinduced writhing method, the methanol extract of M. spicata also exhibited a significant analgesic action. e inhibition at the dose of 500 mg/kg was estimated at 60.30%. On the other hand, using Brewer's yeast-induced pyrexia in rats, the methanol extract of M. spicata was revealed to exert a strong marked (p < 0.01) antipyretic activity at the dose of 500 mg/ kg at 3 h than at a dose of 100 mg/kg at 2 h.
3.5.10. Antihemolytic Activity. In order to investigate the biological functions of M. spicata, Ebrahimzadeh et al. [9] decided to study the antihemolytic effect of ethanol-water extract from aerial parts of M. spicata. e results showed that this extract possesses a weak inhibiting effect with an IC 50 � 1250.7 ± 46.1 μg/mL by H 2 O 2 -induced membrane damage and hemolysis.
3.5.11. Protective Effects. In their research, Saad et al. [111] were interested in studying the protective activity of M. spicata treatment against nicotine-induced oxidative damage in the liver and erythrocytes Wistar rats. e findings showed that aqueous extract from aerial parts of M. spicata exhibited a strong protective action. On the hematological parameters, it was found to restore to normal levels the levels of erythrocytes, haematocrit, hemoglobin, and white blood cells. However, on hepatic dysfunction parameters, the aqueous extract of spearmint significantly decreased ALT and ALP activities resulting in a decrease in liver toxicity. Furthermore, the aqueous extract of M. spicata to nicotine-treated rats provided a statistically significant (p ≤ 0.01) enhancement of antioxidant enzyme capacities, including CAT, SOD, and GPX activities, suggesting an improvement in antioxidant status. According to liver histological analysis, the treatment with the aqueous extract of M. spicata showed considerable recovery in the form of hepatic histoarchitecture. Similarly, Saad et al. [111] aimed to screen the in vivo and in vitro antioxidative effect of M. spicata extract against nicotine-induced oxidative injury in the kidney and brain of rats. e in vivo results obtained reported that Mentha extract significantly increased the bodyweight of rats as well as exhibited a significant increase in testis, brain, and accessory sex organ weights. In addition, treatment with the aqueous extract of M. spicata had a significant decrease in the MDA levels, but no significant changes in brain AChE were recorded. Also, M. spicata extract supplementation could restore the antioxidant enzymes activities to normal levels and participate to ameliorate cerebral cortex histological pictures and histological damages.

Toxicity Investigations.
In pharmacology, the efficacy of a plant or a natural constituent is not sufficient to justify its therapeutic use. Indeed, each bioactive substance is likely to have deleterious effects for human health, at least in high doses and over long periods [112]. In addition to efficacy, the active dose must be free from any toxicity and demonstrate safety. erefore, in the therapeutic indication of any substance, it is imperative to define its risk-benefit ratio.
Despite the data paucity on its safety profile and given its wide use, the acute and subacute toxicities of M. spicata have been tested in four studies to optimize its use [66,113,114] (Table 10).
Initially, Yousuf et al. [97] orally administered single doses of 500, 1000, and 2000 mg/kg of whole plant methanolic extract to mice of both sexes. After 24 hours of observation, no mortality or signs of toxicity were noticed. One year later, aerial parts of the same extract at a dose of 5000 mg/kg of extract (the limit test dose according to OECD guidelines 425) showed similar results in female rats [113]. Indeed, during the 14 days of oral gavage, no mortality was recorded, considering the LD 50 to be greater than 5000 mg/kg. In addition, no changes in the behavior and the bodyweight of the animals were observed. At the end of the experiment and after sacrificing animals, there were no toxicologically significant biochemical and hematological changes compared to the control group. e histological evaluation did not reveal any morphological changes or gross lesions in the lung, kidney, liver, and heart tissues. ese results corroborate those obtained by Kedia et al. [66]. ey recorded low toxicity (LD 50 � 8342.33 μL/kg) of the essential oil of M. spicata aerial parts following oral administration of different doses (0.05-0.5 mL) to mice (Mus musculus L.).
In the same year, Mugisha and colleagues tested the acute and subacute toxicities of the leaves of this plant in Swiss mice and Wistar albino rats, respectively [114]. For acute toxicity, animals received intragastrically over 72 hours, doses of 10000, 12000, 14000, 16000, and 18000 g/kg b.w of the 70% ethanolic extract. erefore, a death rate of 100% was obtained at the highest dose with some signs of toxicity (convulsions, abdominal muscle contractions, and hyperurination) above 12000 mg/kg b.w. e LD 50 value was ethanol leaf extract (500, 1000, and 1500 mg/kg b.w) caused no mortality or signs of toxicity. However, it significantly increased the levels of mean corpuscular hemoglobin concentration, lymphocytes, blood cells count, and aspartate transferase and significantly reduced haematocrit. At the same time, serum urea and creatinine levels were not affected, confirmed by histopathological data.
From these toxicological investigations, it can be declared that M. spicata is an experimentally safe plant, thus justifying its use in treating numerous abnormalities. However, prolonged treatment in high doses can lead to specific problems. For this, other studies on this plant's chronic toxicity are necessary to complete its toxicological profile.

Conclusion and Perspectives
In this work, we reported the ethnobotanical, phytochemical, and pharmacological aspects of M. spicata (M. viridis). is medicinal plant is frequently used in traditional practices to treat certain diseases and showed interesting biological properties in various scientific investigations. Phytochemical studies of this species showed its richness in numerous bioactive compounds in particularly terpenoid components, exhibiting important biological effects. Pharmacological biology explorations demonstrated that extracts and essential oils of M. spicata showed different pharmacological properties such as antibacterial, antiparasitic activity, insecticidal, antiinflammatory, antidiabetic, antioxidant, diuretic, analgesic, antipyretic, antihemolytic, and protective activities. However, these effects were evaluated often using in vitro and in vivo approaches, and therefore, further investigations to validate these activities with determining mechanisms of their actions are needed. Toxicological investigation of M. spicata extracts was examined by some studies and showed a safety of this plant. However, clinical trials were not conducted, and there is an urgent need to perform such trials to promote the use of the plant especially after proving its excellent safety profile in the toxicological investigation. Indeed, bioactive compounds of M. spicata need further investigations concerning the pharmacodynamic and pharmacokinetic aspects to determine their bioavailability and their mechanisms of action of different targets.

Data Availability
e data used to support this study are included within the article. Methanol extract e animals treated with a single dose of 5000 mg/kg of M. spicata extract by oral gavage No mortality during the observation period [113] No toxicologically significant hematological and biochemical changes Any morphological changes in the heart, liver, kidney, and lung tissues of the rats LD 50  Conflicts of Interest e authors declare that they have no conflicts of interest.