Phytochemical Profiling,Antioxidant,and Antifungal Activities of Ethyl Acetate and Chloroformic Extracts from Three Mentha Species

Abstract

Background

Phytopathogenic fungi remain the main infectious agents in plants, causing severe damage to the environment and human health. Thus, to reduce the usage of synthetically derived fungicides and perform agricultural crop production, the search for new control strategies including plant extracts constitutes an eco-friendly and safe alternative.

Objectives

This study aimed to quantify the phytochemical constituents of the three plant (Mentha pulegium L., Mentha spicata L., and Mentha longifolia L.) extracts and to screen their phytochemical composition including total phenolic (TPC), flavonoids (TFC) and condensed tannins contents (TCTC), and to evaluate their antioxidant activities. The efficacy of all mint extracts will be investigated against phytopathogenic fungal species.

Materials and Methods

The three plant extracts were screened to assess their total phenolic, flavonoids, and condensed tannin contents using spectrophotometric assays. The antioxidant activities include 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric ion reducing antioxidant power (FRAP), and β-carotene assays. The antifungal activities were investigated on phytopathogenic species including Botrytis cinerea, Fusarium culmorum, Fusarium oxysporum, Aspergillus niger, Aspergillus flavus, and Trichoderma sp.

Results

Quantitative analyses of phytochemical constituents of Mentha genus extracts revealed that both ethyl acetate (EtAc) and chloroformic (Chl) extracts are a rich source of phenols, flavonoids, and condensed tannins. Ethyl acetate extract of M. longifolia (EtAc L) displayed the highest content of phenols (69.9 ± 1.35 mg GAE/g DW) and flavonoids (53.26 ± 2.11 mg CE/g DW), while M. pulegium ethyl acetate extract (EtAc P) has the highest condensed tannins content (2.13 ± 0.4 mg CE/g DW). Moreover, the tested extracts exhibited potent antioxidant activities at low concentrations for EtAc L, followed by M. spicata (EtAc S), and EtAc P (IC₅₀ = 35.76 ± 1.32 µg/mL for scavenging DPPH free radicals; EC₅₀ 527.96 ± 5.45 µg/mL for FRAP, and IC₅₀ = 106.3 ± 3.75 µg/mL for β-carotene bleaching test). Finally, all tested extracts were able to inhibit the growth of several phytopathogenic micro-organisms on both agar and broth media.

Conclusion

The Mentha extracts derived from the three mint species (i.e., L, P, and S) could be used for their antifungal activities to provide sustainable crop pest management.

Keywords

Crops phytopathogens plant extracts chemical composition antioxidants antifungal activities

Introduction

The agricultural sector in the Hail region is one of the distinct sectors that demonstrate the efforts made to achieve comprehensive and balanced development in line with the Kingdom’s 2030 vision. Despite that, plant fungal pathogens are responsible for most of the diseases occurring in agriculture (Elahi et al., 2019; Sellar et al., 2020). To overcome this, synthetic fungicides are the most common way to prevent a fungal infection, but they will face major limitations with several significant concerns inducing excessive yield or quality loss and are rendered unfit for human consumption (Da et al., 2016; Elshafie et al., 2017; Usta, 2013). Due to their overuse and misuse in agriculture, synthetic pesticides remain very restricted and discouraged, causing the development of pesticide-resistant pests as well as chronic human ailments due to either consumption or exposure (Damalas & Koutroubas, 2019; Kumari et al., 2014). Therefore, the need to search for alternative control strategies that are affordable and environmentally safe, such as medicinal plants, especially plant extracts as control agents to prevent damage from pests and pathogens is of great interest, and researchers have been focused on understanding their effectiveness and potential to be another alternative solution (Enis et al., 2019; Muhammad et al., 2019). Traditional medicine provides an important healthcare service and can be used as an alternate therapy. Medicinal plants and their bioactive compounds have been used in traditional medicine for the treatment of various diseases (Felhi et al., 2017; Hajlaoui et al., 2019). Their application has been explored in the control of phytopathogenic organisms (Chandel & Kumar, 2017; Karabuyuk et al., 2018). The importance of plant-based pesticides is linked to their efficacy, biodegradability, safety, and availability of source materials (Neeraji et al., 2017). Plant extracts and their phytochemical are gaining popularity as they do not represent a health risk or pollute the environment (Alminderej et al., 2021).

Mentha (family Lamiaceae), also known as the “Mint” of the Lamiaceae family, encompasses several aromatic species, which are distributed worldwide (Lawerence, 2006). The Mentha genus was subdivided into five sections including Audibertia, Eriodontes, Mentha, Preslia, and Pulegium (Tucher, 2007). It consists of 20 worldwide-spread species, with the most important being L, P, S, Mentha piperita, Mentha gracilis, Mentha rotundifolia, and Mentha suaveolens (Amalich et al., 2016; Gonzalez-Tejero et al., 2008). Mentha species are well known for their distinct aroma and commercial value, commonly used in traditional food flavoring or as medicine for the treatment of cold, fever, and digestive and cardiovascular disorders as well as against flatulence, nausea, ulcerative colitis, anorexia, bronchitis, and liver diseases (Abdelli et al., 2016; Hajlaoui et al., 2010; Kumar & Patra, 2012; Kumar et al., 2011; Mahboubi, 2017; Moricia et al., 2016; Murad et al., 2016; Shaikh et al., 2014). It was a source of a number of biological activities such as anti-oxidant, anti-microbial, anti-tumor, anti-cancer, anti-viral, anti-allergic, anti-inflammatory, anti-hypertensive, and urease inhibitory (Abdelli et al., 2016; Mahboubi, 2017). The traditional pharmacological attributes of Mentha herbs value owing to its aromatic species and can be linked to the occurrence of bioactive phytochemicals such as phenolic compounds, tannins, terpenes, terpenoids, quinones, coumarins, flavonoids, alkaloids, sterols, and saponins (El-Gharbaoui et al., 2017; Malika et al., 2019).

This study aimed to curb the usage of synthetically derived fungicides by determining the anti-fungal activities of L, P, and S aerial part extracts against Botrytis cinerea, Fusarium culmorum, Fusarium oxysporum, Aspergillus niger, Aspergillus flavus, and Trichoderma sp. The obtained results will be correlated with the phytochemistry of each extract.

Materials and Methods

Plant Materials and Culture Conditions

Young seedlings (15 days) of three mint species M. longifolia(L), M. pulegium (P), and M. spicata (S) were cultivated in the ground under greenhouse for five months until stage flowering.

Preparation of the Extracts

Aerial part extracts were prepared following the same protocol as done by Kais et al. (2022).

Determination of Total Phenolic, Flavonoid Contents, and Condensed Tannins

The total phenolic content of the two species of mint was determined using the same previous protocol with slight modification (Aouadi et al., 2021; Hajlaoui et al., 2022).

Antioxidant Activities

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activity, ferric ion reducing antioxidant power (FRAP), and β-carotene bleaching test were estimated (Bakari et al., 2017; Bakari et al., 2018; Patro et al., 2005).

Anti-fungal Activity

Micro-organisms

The used micro-organisms included the following six fungal strains: Botrytis cinerea, F. culmorum, F. oxysporum, Aspergillus. niger, A. flavus, and Trichoderma sp. These fungal strains are provided by the Laboratory of Phytopathology of the INRAT of Tunis under the direction of Prof. Mohammed Rabeh Hajlaoui.

Disc-diffusion Assay and Inhibition Test of the Growth Fungus on Liquid Medium

Antifungal activity testing was performed by using the same protocol described by Hajlaoui et al., (2009) with light modifications.

Statistical Analysis

All the experiments were conducted in triplicate, and the average values were calculated using the SPSS 26.0 statistics package for Windows. The mean differences were calculated using Duncan’s multiple-range tests for means with a 95% confidence limit (p ≤ 0.05).

Results and Discussion

Quantitative Analyses of Phytochemical Constituents of Mentha Genus Extracts

The results of the quantitative phytochemical screening of EtAc L EtAc S, and EtAc P are summarized in Table 1. Accordingly, the tested extracts showed a considerable quantity of phenolic compounds with the highest content of TPC, mainly TFC in the EtAc then in chloroform for the three plants (Table 1).

Table 1.

TPC (mg GAE/g DW), TFC (mg CE/g DW), and TCTC (mg CE/g DW) in Different Extracts in the Aerial Part Extract of Three Mint Species.

Extracts		M. longifolia	M. spicata	M. pulegium
EtAc	TPC	69.9 ± 1.35^aA	65.06 ± 1.10^aA	46.36 ± 1.3^bA
	TFC	53.26 ± 2.11^aA	40.83 ± 1.25^bA	33 ± 1.11^cA
	TCTC	1.43 ± 0.12^cA	1.81 ± 0.25^bA	2.13 ± 0.4^aA
Chl	TPC	54.13 ± 1.36^aB	52.1 ± 1.22^aB	36.13 ± 1.00^bB
	TFC	36.1 ± 1.85^aB	32.6 ± 1.10^bB	25.56 ± 1.12^cB
	TCTC	0.76 ± 0.05^bB	1.06 ± 0.25^aB	0.56 ± 0.05^cB

Note: Each data point represents mean ± SD of three independent replicates. ^a,b,cdifferent letters within the same rows differ significantly (p < 0.05). ^A,Bdifferent letters within the same columns differ significantly (p < 0.05).

Abbreviations: TPC, total phenolic content; TFC, total flavonoid content; TCTC, total condensed tannin content; GAE, gallic acid equivalent/g dry extract; CE, catechine equivalent/g dry extract.

Overall, our results showed high and not significantly different (p > 0.05) total polyphenolic content found in M. longifolia (69.9±1.35 mg GAE/g DW) and M. spicata(65.06±1.10 mg GAE/g DW), while M. pulegium extract displayed significantly (p < 0.05) lower TPC (46.36±1.3 mg GAE/g DW). Similarly, significantly different TFC (p < 0.05) was observed in three plants with the strongest level was observed in M. longifolia (53.26±2.11 mg CE/g DW), followed by M. spicata (40.83±1.25 mg CE/g DW) and M. pulegium (33 ±1.11 mg CE/g DW), respectively. Lower significantly (p < 0.05) different TCTC was obtained for the three Mentha species. In fact, M. pulegium extract exhibited the highest level. In chloroformic extracts, also strong and not significantly different (p > 0.05) TPC are shown in M. longifolia (54.13±1.36 mg GAE/g DW) and M. spicata extracts (52.1±1.22 mg GAE/g DW), however the lowest level (36.13±1.00mg GAE/g DW) was recorded for M. pulegium extract, which was significantly (p < 0.05) different.

Likewise, the Mentha species had significantly (p < 0.05) different TFC and TCTC with the highest levels were recorded specifically to M. longifolia (36.1±1.85 mg CE/g DW), and M. spicata extracts (1.06±0.25 mg CE/g DW), respectively. Previous studies have reported that Mentha genus members are a rich source of phenolic compounds (mainly caffeic acid and its derivatives, chlorogenic acid, and rosmarinic acid), flavonoids, terpenoids, steroids, ceramides, and tannins with different pharmacological activities (Soleimani et al., 2022; Parham et al., 2020; Kumar et al. 2010; Eftekhari et al., 2021). In fact, it has been reported that methanolic extract from M. longifolia collected from Saudi Arabia have a very high phenol (157.99 ± 12.3 mg GAE/g DW) and flavonoid content and (16.96 ± 1.48 mg RTE/g DW) (Osman, 2013). Similarly, the methanolic extract from M. spicata, M. longifolia, and M. pulegium are a rich source of polyphenols, flavonoids, and tannins. The highest polyphenols were recorded in the methanolic extract from M. longifolia (89.16±0.96 mg GAE/g DW), followed by M. pulegium (62.06±2.31 mg GAE/g DW), and M. spicata (39.8±1.21 mg GAE/g DW). Similarly, the highest yield of flavonoids was recorded in the methanolic extract from M. longifolia (63.93±1.68 mg GAE/g DW), followed by M. pulegium (51.66±2.12 mg GAE/g DW), and M. spicata (32.83±1.25 mg GAE/g DW) (Hajlaoui et al., 2015).

Moreover, it has been demonstrated that Mentha genus is a source of diverse bioactive molecules with important biological activities (Hajlaoui et al., 2009; Mikaili et al., 2013; Hajlaoui et al., 2015; Pereira et al., 2016; Parham et al., 2020; Eftekhari et al., 2021). In fact, Hajlaoui and colleagues reported the chemical composition of the methanolic extract of the same studies Mentha species by using RP-HPLC technique. These authors reported the identification of chlorogenic acid (6.88%), trans-cinnamic acid (10.32%), myricetin (10.93%), and morin (20.87%) in M. spicata. While, myricetin (8.36%), chlorogenic acid (14.9%), and trans-2-dihydroxycinnamic acid (59.73%) were the main compounds identified in M. pulegium . In addition, trans-2-dihydroxycinnamic acid (4.48%), apigenin (8.23%), ferulic acid (12.37%), and chlorogenic acid (23.31%) were dominant in the methanolic extract from M. longifolia (Hajlaoui et al., 2015). It has been also demonstrated that Mentha species contain other compounds like trace elements, triacylglycerol, fatty acids, sugar, saponins, alkaloids, anthraquinones, and quinines (Padmini et al., 2010; Kaizil et al., 2010; Perez et al., 2014).

It has been well documented that the obtained compounds are the main compounds identified in M. pulegium (Goudjil et al., 2015; Fancello et al., 2017; Marwa et al., 2017). Other phenolic compounds are reported in M. spicata including rosmarinic acid, salvianolic acid, hydroxybenzoic acid, caffeoylquinic acid, hydroxycinnamic acid, flavanones, and flavones (Derwich et al., 2009; Riahi et al. 2013; Singh et al., 2015; Bahadori et al., 2018). Prasterone acetate and sclareol were also reported in M. longifolia (Shelepova et al., 2014).

Antioxidant Activity Evaluation

The antioxidant power of the investigated extracts for the three species plants was assessed through three assays including DPPH, FRAP, and β-carotenes. The results shown in Table 2 revealed that EtAc exhibited better antioxidant potency then chloroform extract in three assays. Indeed, in EtAc, M. longilfolia (IC₅₀ = 35.76±1.32 mg/mL), and M. spicata (IC₅₀ = 38.16±1.25 mg/mL) exhibited high and not significantly different (p > 0.05) DPPH scavenging ability when compared to the standard, BHT (IC₅₀ = 11.56±0.35 mg/mL). Poor FRAP activity was found for the three studied species; however moderate antioxidant activity was obtained with b-carotenes test for the three plants met the following order: M. longilfolia = M. spicata > M. pulegium.

In contrast, chloroformic extract from M. longilfolia and M. spicta exhibited moderate and not significant DPPH scavenging capacity in comparison to the standard both, however, in FRAP and b-carotenes assays, the three species recorded a significantly (p < 0.05) weaker activity. This result correlates well with our evaluation of the polyphenolic compounds and flavonoids of the two extracts studied, because high level of phenolics compounds were found in the extract whose constituents possessed high potentials for DPPH radicals scavenging activity.

Table 2.

DPPH Test (IC₅₀; µg/mL), Reducing Power (EC₅₀; µg/mL) and β-Carotene (IC₅₀; µg/mL) of Mentha spp. Aerial Part Extracts, and Authentic Standards (BHT, BHA, and Ascorbic Acid) (µg/mL).

Extracts	Tests	M. longilfolia	M. spicata	M. pulegium	BHT	BHA	Vitamin C
EtAc	DPPH	35.76 ± 1.32^b	38.16 ± 1.25^b	81.4 ± 1.82^a	11.56 ± 0.35^c	–	–
	FRAP	527.96 ± 5.45^b	528.26 ± 2.89^b	713.76 ± 4.27^a	–	–	36 ± 1^c
	β-carotene	106.3 ± 3.75^b	110.6 ± 4.27^b	153.46 ± 3.99^a	75 ± 1^c	48 ± 1^d	–
Chl	DPPH	123.86 ± 3.35^b	118.63 ± 1.18^c	199.4 ± 1.96^a	11.56 ± 0.35^d	–	–
	FRAP	703.93 ± 3.52^b	696.92 ± 1.67^c	920.06 ± 2.15^a	–	–	36 ± 1^d
	β-carotene	229 ± 6.55^b	213.06 ± 2.92^c	446.26 ± 8.10^a	75 ± 1^d	48 ± 1^e	–

Note: Means (three replicates) followed by at least one same letter are not significantly different at p < 0.05. ^a,b,c,d,eMeans followed by the same letters are not significantly different at p = 0.05 based on Duncan’s multiple range test.

Abbreviations: DPPH, 1,1-diphenyl-2-picrylhydrazyl.

Previous reports have discussed the antioxidant activities of Mentha species, and mainly their essential oil obtained by hydrodistillation (Hajlaoui et al., 2015; Bahadori et al., 2018; El Hassani, 2020; Tafrihi et al., 2021). It has been reported the antioxidant activity of M. piperita organic (Petroleum ether, Chl, EtAc, ethanol) and aqueous extracts. In fact, the antioxidant capacity (%) ranged from 69.8±5.2 (%) for the aqueous extract to 91.2±5.6 (%) for the Chl. Similarly, the highest DPPH free radical scavenging activity (%) was about 91.8±5.8 (%) for the Chl extract followed by the EtAc extract (84.9±4.2%), and the ethanolic extract (84.9±4.2%) (Singh et al., 2015).

Similar results have been reported previously showing the high antioxidant activities of M. longifolia, M. pulegium, and M. spicata (methanolic extract) by using DPPH test, O2.- scavenging assay, iron chelating/reducing assays, and b-carotene system. The lowest IC₅₀ concentration (DPPH and b-carotene) was recorded for M. longifolia methanolic extract (68.96±3.9 µg /mL and 23.06±2.8 µg /mL, respectively) (Hajlaoui et al., 2015). It has been demonstrated that Phenolic acids (caffeic acids and rosmarinic), flavones (luteolin derivatives), ascorbic acid, and flavanones (eriocitrin derivatives) are known to show promising antioxidant capacities, whereas vitamin antioxidants such as carotenoids exhibit weak radical scavenging traits (Eftekhari et al., 2021).

Anti-fungal Activity Evaluation

Plant pathogens such as fungi are responsible for various plant diseases resulting in severe damage to plants with considerable loss of crop yields in agricultural industries. The screening of the antifungal potential of plant extracts toward some phytopathogenic fungi has been studied. As shown in Table 3, both ethyl and chloroform extracts significantly (p < 0.05) inhibit the growth of all tested fungi in a different manner with potent to moderate activity, and globally, EtAc extract exhibited better antifungal inhibitory effect than chloroform extract in the three plant species (Table 4).

Table 3.

Zones of Growth Inhibition (mm) Showing Antifungal Activity for Three Mint Species (L, S, and P) Extracts.

Inhibition zone in diameter (mm ± SD) around the discs
	M. longifolia		M. spicata		M. pulegium		Anti-fungal
Fungal species	EtAc	Chl	EtAc	Chl	EtAc	Chl	Amp B
Botrytis cinerea	11 ± 0^cB	8 ± 0^eC	11.5 ± 0.5^cB	8.33 ± 0.57^cC	8 ± 0^dC	7 ± 0^cC	15.67 ± 0.58^eA
Fusarium culmorum	14 ± 1^aB	12 ± 0^cC	14 ± 0^aB	11.33±0.57^aC	10.66 ± 0.57^bCD	8 ± 0^bE	18.33 ± 0.58^dA
Fusarium oxysporum	14 ± 1^aB	12.5 ± 0.5^cC	13.33 ± 0.57^aB	10.33±0.57^abD	9.66 ± 0.57^cE	9.33 ± 0.57^aE	18 ± 0^dA
Trichoderma sp.	12.33 ± 6.35^bD	14.33 ± 0.57^aB	13.33 ± 1.52^aC	11 ± 1^aE	12 ± 0^aD	8 ± 0^bF	28 ± 1^aA
Aspergillus niger	14 ± 1^aB	13.33 ± 0.57^bB	13 ± 0^abB	11 ± 0^aC	12.16 ± 1.04^aC	8 ± 0^bD	24 ± 1^bA
Aspergillus flavus	12.33 ± 0.57^bB	11.33 ± 0.57^dC	11 ± 1^cC	10 ± 0.57^bCD	10.33±0.57^bCD	7.33 ± 028^cE	22.67 ± 0.58^cA

Note: Inhibition zone in diameter (mm) around the discs (6mm) impregnated with 100 µg/disc of extract; Amp B = Amphotericin B (20 µg/disc) was used as positive reference standard antifungal discs; ^{a,b,c,d,e,A,B,C,D,E,F}Means followed by the same letters are not significantly different at p = 0.05 based on Duncan’s multiple range test. Small letters are used to compare means between strains for each extract, while capital letters are used to compare means between extracts and amphotericin B for each strain.

Table 4.

Percentage of Inhibition (%I) of the Growth of Fungus Species Cultivated on Liquid Medium with Various Concentrations of Three Mint Extracts (100 µg/mL YNB broth), Compared to Positive Standard Drug (Amphotericin B, 10 µg/mL YNB broth).

	M. longifolia		M. spicata		M. pulegium		Amp B
Fungal species	EtAc	Chl	EtAc	Chl	EtAc	Chl
Botrytis cinerea	52.5 ± 0.91^d	45.5±1.05^e	50.3±2.15^e	48 ± 1c	39.42 ± 1.23e	36 ± 1b	96.27 ± 1.25b
Fusarium culmorum	70.8 ± 1.57^a	64 ± 1^a	70.21 ± 1.2^a	55.16 ± 1.24^a	44.48 ± 0.73^c	37.0 ± 1.0^b	100 ± 0^a
Fusarium oxysporum	72.41 ± 0.53^a	63.35 ± 1.26^a	70.10 ± 1.38^a	52.00 ± 1.00^b	46.07 ± 1.64^b	40.35 ± 2.05^a	94.19 ± 1.30^d
Trichoderma sp.	52.34 ± 0.90^d	48.080.89^d	55.98 ± 1.51^d	45.71 ± 1.09^d	41.31 ± 1.22^d	35.56 ± 0.87^bc	98.84 ± 0.34^c
Aspergillus niger	68.15 ± 1.11^ab	55.53 ± 1.32^b	63.53 ± 0.8^b	49.32 ± 0.98c	52.38 ± 1.14a	32.22 ± 1.14^e	94.43 ± 1.13^d
Aspergillus flavus	62.41 ± 1.27^c	51.50 ± 0.7^c	58.6 ± 1.00^c	40.90 ± 0.31^e	53.55 ± 0.62^a	34.12 ± 1.49^d	93.47 ± 0.95^d

Note: ^a,b,c,d,eMeans followed by the same letters are not significantly different at p = 0.05 based on Duncan’s multiple range test.

EtAc L had good antifungal activity against the three most sensitive test organisms B. cinerea (IZ = 11±0 mm, %I = 52.5±0.9), F. culmorum (IZ = 14±1 mm, %I = 70.8±1.57) and F. oxysporum (IZ= 14±1 mm, %I = 72.41±0.53). EtAc S displayed also good activity towards the same strains, B. cinerea (IZ = 11.5±0.5 mm, %I =50.3±2.15), F. culmorum (IZ = 14±0 mm, %I = 70.21±1.2) and F. oxysporum (IZ= 13.33±0.57 mm, %I = 70.10±1.38). besides all chloroform extracts revealed significantly weaker (p < 0.05) antifungal inhibitory effect over all strains for the three investigated plants.

Previous reports have discussed the antimicrobial activities of Mentha essential oil and extracts (Sokovic et al., 2009, Hussain et al., 2010a; Hussain et al., 2010b; Džamić et al., 2010; Hajlaoui et al., 2015). In fact, Sokovic and colleagues reported that essential oils from M. piperita (Menthol-chemotype) and M. spicata (Carvone-chemotype) exhibited antifungal against the mostly cited strains. In another studies, Hussain and colleagues (Hussain et al., 2010a; Hussain et al., 2010b) reported the antifungal activities of M. piperita, M. arvensis, M. spicata, and M. longifolia essential oils and their main compounds (Menthol, menthone, piperitenone oxide, and carvone) against A. niger, A. flavus, Alternaria solani, F. solani, Rhizopus solani, Alternaria alternata, Botryodiplodia theobromae, Mucor mucedo, and Rhizopusspp. with high growth inhibition zones, and low minimum inhibitory concentrations (especially for M. arvensis mint species).

Few studies have reported the antifungal activities of mint extracts. In fact, Džamić et al., (2010) reported the antifungal activities of M. longifolia methanolic extract tested against P. ochrochloron, Cladosporium fulvum, and Cladosporium cladosporioides were observed to be the most sensitive to M. longifolia extract (Džamić et al., 2010). It has been demonstrated that crude extracts from M. piperita collected from Sultanate of Oman by using different solvents including hexane, EtAc, Chl, and butanol were active against Staphylococcus aureus, Escherichia coli and Xanthomonas campestris bacteria at low concentrations ranging from 0.335 mg/mL to 2.5 mg/mL.

Conclusion

In this study, the phytochemical composition of the ethyacetate and chloroformic extrcats from three Mentha species including M. longifolia, M. spicata, and M. pulegium was assessed. Overall results showed that both EtAc and Chl exhibited an important antioxidant and antifungal potential due to their richness in phenolics, flavonoids, and condensed tannins. Further analyses are necessary in order to study the chemical composition of the tested extract and to elucidate the possible mechanism of action of the identified molecules.

Footnotes

Abbreviations

CE/g DW: Catechine equivalent/g dry; Chl: Chloroformic extract; DPPH: 1,1-diphenyl-2-picrylhydrazyl; EtAc L: M. longifolia ethyl acetate extract; EtAc P: M. pulegium ethyl acetate extract; EtAc S: M. spicata ethyl acetate extract ; pEtAc: Ethyl acetate extract; FRAP: Ferric ion reducing antioxidant power; GAE/g DW: Gallic acid equivalent/g dry extract; IC₅₀: Inhibition concentration of 50%; TCTC: Total condensed tannin content; TFC: Total flavonoid content; TPC: Total phenolic content; SD: Standard deviation; ANOVA: Analysis of variance; SPSS: Statistical Package for the Social Sciences.

Acknowledgments

The authors would like to express gratitude to the Sheikh Ali Al-Jumaiah Scientific Research Chair for Sustainable Development in Agricultural Communities at the University of Ha’il, Saudi Arabia, for their generous funding support through project number SCR-22 088.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This research has been funded by Sheikh Ali Al-Jumaiah Scientific Research Chair for Sustainable Development in Agricultural Communities at the University of Ha’il-Saudi Arabia through project number SCR-22 088.

Statement of Informed Consent and Ethical Approval

Necessary ethical clearances and informed consent were received and obtained, respectively, before initiating the study from all participants.

ORCID iDs

Mohd Saeed

Snoussi Mejdi

References

Abdelli

, Moghrani

, Aboun

, & Maachi

(2016). Algerian Mentha pulegium L. leaves essential oil: Chemical composition, antimicrobial, insecticidal and antioxidant activities. Industrial Crops and Products , 94, 197–205. https://doi.org/10.1016/j.indcrop.2016.08.042

Alminderej

, Bakari

, Almundarij

T. I.

, Snoussi

, Aouadi

, & Kadri

(2021). Antimicrobial and wound healing potential of a new chemotype from Pipercubeba L. essential oil and in silico study on S. aureus tyrosyl-tRNA synthetase protein. Plants , 10(2), 205. https://doi.org/10.3390/plants10020205

Amalich

S. H.

, Zerkani

, Cherrat

N. K.

, Mezrioui

N. E.

, Hassani

, & Pages

J. M.

(2016). Essential oils from Moroccan plants as potential chemosensitizers restoring antibiotic activity in resistant Gram-negative bacteria. International Journal of Antimicrobial Agents , 38(4), 325–330. https://doi.org/10.1016/j.ijantimicag.2011.05.005

Aouadi

, Hajlaoui

, Arraouadi

, Ghannay

, Snoussi

, & Kadri

(2022). Phytochemical profiling, antimicrobial and α-glucosidase inhibitory potential of phenolic-enriched extracts of the aerial parts from Echium humile Desf.: In vitro combined with in silico approach. Plants , 11(9), 1131. https://doi.org/10.3390/plants11091131

Aouadi

, Hajlaoui

, Arraouadi

, Ghannay

, Snoussi

, & Kadri

(2021). HPLC/MS phytochemical profiling with antioxidant activities of Echium humile Desf. extracts: ADMET prediction and computational study targeting human peroxiredoxin 5 receptor. Agronomy , 11(11), 2165. https://doi.org/10.3390/agronomy11112165

Arshad

, Jeelani

S. M.

, Salim

, & Hussain

B. D.

(2019). Multiple magnet ingestion leading to bowel perforation: A relatively sinister foreign body. Cureus , 11(10), e5866. https://doi.org/10.7759/cureus.5866

Bahadori

M. B.

, Zengin

, Bahadori

, Dinparast

, & Movahhedin

(2018). Phenolic composition and functional properties of wild mint (Mentha longifolia var. calliantha (Stapf) Briq.). International Journal of Food Properties , 21(1), 183–193. https://doi.org/10.1080/10942912.2018.1440238

Bakari

, Daoud

, Felhi

, Smaoui

, Gharsallah

, & Kadri

(2017). Proximate analysis, mineral composition, phytochemical contents, antioxidant and antimicrobial activities and GC-MS investigation of various solvent extracts of cactus cladode. Food Science and Technology (Campinas) , 27, 286–293. https://doi.org/10.1590/1678-457X.20116

Bakari

, Hajlaoui

, Daoud

, Mighri

, Ross-garcia

J. M.

, Gharsallah

, & Kadri

(2018). Phytochemicals, antioxidant and antimicrobial potentials and LC-MS analysis of hydroalcoholic extracts of leaves and flowers of Erodium glaucophyllum collected from Tunisian Sahara. Food Science and Technology , 38(2), 310–317. https://doi.org/10.1590/fst.04517

10.

Bilal Goud

, Ladjel

, Eddine Ben

, Zighmi

, & Hamada

(2015). Chemical composition, antibacterial and antioxidant activities of the essential oil extracted from the Mentha piperita of Southern Algeria. Research Journal of Phytochemistry , 9(2), 79–87. https://doi.org/10.3923/rjphyto.2015.79.87

11.

Bnina

E. B.

, Hajlaoui

, Chaieb

, Daami-Remadi

, Said

M. B.

, & Jannet

H. B.

(2019). Chemical composition, antimicrobial and insecticidal activities of the Tunisian Citrus aurantium essential oils. Czech Journal of Food Sciences , 37(2), 81–92. https://doi.org/10.17221/202/2017-CJFS

12.

Chandel

, & Kumar

(2017). Effect of plant extracts as prestorage seed treatment on storage fungi, germination percentage and seedling vigour of pea (Pisum sativum). Indian Journal of Agricultural Sciences , 87(11), 1476–1481. https://doi.org/10.56093/ijas.v87i11.75703

13.

, Nishiyama

, Tie

, Hein

K.Z.

, Yamamoto

, & Morita

(2019). Antifungal activity and mechanism of action of Ou-gon (Scutellaria root extract) components against pathogenic fungi. Scientific Reports , 9(1683), 43–55. https://doi.org/10.1038/s41598-019-38916w

14.

Damalas

C. A.

, & Koutroubas

S. D.

(2016). Farmers’ exposure to pesticides: Toxicity types and ways of prevention. Toxics , 4(1), 1–10. https://doi.org/10.3390/toxics4010001

15.

Derwich

, Benziane

, Boukir

, & Benaabidate

(2009). GC-MS analysis of the leaf essential oil of Mentha rotundifolia, a traditional herbal medicine in Morocco. Chemistry Bulletin “Politehnica”, 54, 85–88.

16.

Džamić

, Soković

, Ristić

, Novaković

, Grujić

, Tešević

, & Marin

(2010). Antifungal and antioxidant activity of Mentha longifolia (L.) Hudson (Lamiaceae) essential oil. Botanica Serbica , 34(1), 57–61.

17.

Eftekhari

, Khusro

, Ahmadian

, Dizaj

S. M.

, Hasanzadeh

, & Cucchiarini

(2021). Phytochemical and nutra-pharmaceutical attributes of Mentha spp.: A comprehensive review. Arabian Journal of Chemistry , 14(5), 103106. https://doi.org/10.1016/j.arabjc.2021.103106

18.

El Hassani

F. Z.

(2020). Characterization, activities, and ethnobotanical uses of Mentha species in Morocco. Heliyon , 6(11), e05480. https://doi.org/10.1016/j.heliyon.2020.e05480

19.

Elahi

, Weijun

, Zhang

, & Nazeer

(2019). Agricultural intensification and damages to human health in relation to agrochemicals: Application of artificial intelligence. Land Use Policy , 83, 461–474. https://doi.org/10.1016/j.landusepol.2019.02.023

20.

El-Gharbaoui

A. G.

, Beníteza

, Gonz Molero-Mesa

, & Merzouki

(2017). Comparison of Lamiaceae medicinal uses in eastern Morocco and eastern Andalusia and in Ibn al-Baytar’s Compendium of Simple Medicaments (13th century CE). Journal of Ethnopharmacology , 202, 208–224. https://doi.org/10.1016/j.jep.2017.03.014

21.

Elshafie

H. S.

, Shimaa

, Bufo

S. A.

, & Camele

(2017). An attempt of biocontrol the tomato-wilt disease caused by Verticillium dahlia using Burkholderia gladioli pv. agaricicola and its bioactive secondary metabolites. International Journal of Plant Biology , 8, 57–60. https://doi.org/10.4081/pb2017.7263

22.

Fancello

, Zara

, Petretto

G. L.

, Chessa

, Addis

, Rourke

J. P.

, & Pintore

(2017). Essential oils from three species of Mentha harvested in Sardinia: Chemical characterization and evaluation of their biological activity. International Journal of Food Properties , 20, 1–18.

23.

Felhi

, Daoud

, Hajlaoui

, Mnafgui

, Gharsallah

, & Kadri

(2017). Solvent extraction effects on phytochemical constituents profiles, antioxidant and antimicrobial activities and functional group analysis of Ecballium elaterium seeds and peels fruits. Food Science and Technology , 37(3), 483–492. https://doi.org/10.1590/1678-457X.23516

24.

Figueroa Pérez

M. G.

, Rocha-Guzmán

N. E.

, Mercado-Silva

, Loarca-Piña

, & Reynoso-Camacho

(2014). Effect of chemical elicitors on peppermint (Mentha piperita) plants and their impact on the metabolite profile and antioxidant capacity of resulting infusions. Food Chemistry , 156, 273–278. https://doi.org/10.1016/j.foodchem.2014.01.101

25.

González-Tejero

M. R.

, Casares-Porcel

, Sánchez-Rojas

C. P.

, Ramiro-Gutiérrez

J. M.

, Molero-Mesa

, Pieroni

, Giusti

M. E.

, Censorii

, de Pasquale

, Della

, Paraskeva-Hadijchambi

, Hadjichambis

, Houmani

, El-Demerdash

, El-Zayat

, Hmamouchi

, & Eljohrig

(2008). Medicinal plants in the Mediterranean area: Synthesis of the results of the project Rubia. Journal of Ethnopharmacology , 116(2), 341–357. https://doi.org/10.1016/j.jep.2007.11.045

26.

Hafedh

, Fethi

B. A.

, Mejdi

, Emira

, & Amina

(2010). Effect of Mentha longifolia L. ssp longifolia essential oil on the morphology of four pathogenic bacteria visualized by atomic force microscopy. African Journal of Microbiology Research , 4(11), 1122–1127.

27.

Hajlaoui

, Arraouadi

, Mighri

, Chaaibia

, Gharsallah

, Ros

, Nieto

, & Kadri

A. A.

(2019). Phytochemical constituents and antioxidant activity of Oudneya Africana L. leaves extracts: Evaluation effects on fatty acids and proteins oxidation of beef burger during refrigerated storage. Antioxidants , 8(10), 442. https://doi.org/10.3390/antiox8100442

28.

Hajlaoui

, Arraouadi

, Mighri

, Ghannay

, Aouadi

, Adnan

, Elasbali

A. M.

, Noumi

, Snoussi

, & Kadri

(2022). HPLC-MS profiling, antioxidant, antimicrobial, antidiabetic, and cytotoxicity activities of Arthrocnemum indicum (Willd.) Moq. extracts. Plants , 11(2), 232. https://doi.org/10.3390/plants11020232

29.

Hajlaoui

, Mighri

, Hamdaui

, & Mahjoub

Aouni.

(2015). Antioxidant activities and RP-HPLC identification of polyphenols in the methanolic extract of Mentha genus. Tunisian Journal of Medicinal Plants and Natural Products , 14, 1–11.

30.

Hajlaoui

, Trabelsi

, Noumi

, Snoussi

, Fallah

, Ksouri

, & Bakhrouf

(2009). Biological activities of the essential oils and methanol extract of tow cultivated mint species (Mentha longifolia and Mentha pulegium) used in the Tunisian folkloric medicine. World Journal of Microbiology and Biotechnology , 25(12), 2227–2238. https://doi.org/10.1007/s11274-009-0130-3

31.

Hussain

A. I.

, Anwar

, Nigam

P. S.

, Ashraf

, & Gilani

A. H.

(2010a). Seasonal variation in content, chemical composition and antimicrobial and cytotoxic activities of essential oils from four Mentha species. Journal of the Science of Food and Agriculture , 90(11), 1827–1836. https://doi.org/10.1002/jsfa.4021

32.

Hussain

A. I.

, Anwar

, Shahid

, Ashraf

, & Przybylski

(2010b). Chemical composition, and antioxidant and antimicrobial activities of essential oil of spearmint (Mentha spicata L.) from Pakistan. Journal of Essential Oil Research , 22(1), 78–84. https://doi.org/10.1080/10412905.2010.9700269

33.

Karabuyuk

, & Aysan

(2018). Aqueous plant extracts as seed treatments on tomato bacterial speck disease. Acta Horticulturae , (1207), 193–196. https://doi.org/10.17660/ActaHortic.2018.1207.25

34.

Kizil

, Hasimi

, Tolan

, Kilinç

, & Yüksel

(2010). Mineral content, essential oil components and biological activity of two Mentha species (M. piperita L., M. spicata L.). Turkish Journal of Field Crops , 15(2), 148–153.

35.

Kumar

, & Patra

N. K.

(2012). Inheritance pattern and genetics of yield and component traits in opium poppy (Papaver somniferum L.). Industrial Crops and Products , 36(1), 445–448. https://doi.org/10.1016/j.indcrop.2011.10.024

36.

Kumar

, Mishra

, Malik

, & Satya

(2011). Insecticidal properties of Mentha species: A review. Industrial Crops and Products , 34(1), 802–817. https://doi.org/10.1016/j.indcrop.2011.02.019

37.

Kumari

K. A.

, Kumar

K. N. R.

, & Rao

C. N.

(2014). Adverse effects of chemical fertilizers and pesticides on human health and environment. Proceedings of the National Seminar on Impact of Toxic Metals, Minerals and Solvents Leading to Environmental Pollution. Journal of Chemical and Pharmaceutical Research , 3, 150–151.

38.

Mahboubi

(2017). Mentha spicata as natural analgesia for treatment of pain in osteoarthritis patients. Complementary Therapies in Clinical Practice , 26, 1–4. https://doi.org/10.1016/j.ctcp.2016.11.001

39.

Malika Ait

S. B.

, Markouk

, & Mustapha

(2019). Moroccan medicinal plants as anti-infective and antioxidant agents. In Khan

M. S. A.

, Ahmad

, * Chattopadhyay

(Eds.), New look to phytomedicine: Advancements in herbal products as novel drug leads (pp. 91–142). https://doi.org/10.1016/B978-0-12-814619-4.00005-7

40.

Marwa

, Fikri-Benbrahim

, Ou-Yahia

, & Farah

(2017). African peppermint (Mentha piperita) from Morocco: Chemical composition and antimicrobial properties of essential oil. Journal of Advanced Pharmaceutical Technology and Research , 8(3), 86–90. https://doi.org/10.4103/japtr.JAPTR_11_17

41.

Mikaili

, Mojaverrostami

, Moloudizargari

, & Aghajanshakeri

(2013). Pharmacological and therapeutic effects of Mentha longifolia L. and its main constituent, menthol. Ancient Science of Life , 33(2), 131–138. https://doi.org/10.4103/0257-7941.139059

42.

Mint

L. B. M.

(2006). The genus Mentha . CRC Press.

43.

Morcia

, Tumino

, Ghizzoni

, & Terzi

(2016). Carvone (Mentha spicata L.) oil. In Essential oils in food preservation, flavor and safety (pp. 309–316). Elsevier.

44.

Murad

H. A.

, Abdallah

H. M.

, & Ali

S. S.

(2016). Mentha longifolia protects against acetic-acid induced colitis in rats. Journal of Ethnopharmacology , 190, 354–361. https://doi.org/10.1016/j.jep.2016.06.016

45.

Neeraj

G. S.

, Kumar

, Ram

, & Kumar

(2017). Evaluation of nematicidal activity of ethanolic extracts of medicinal plants to meloidogyne incognita (kofoid and white) Chitwood under lab conditions. International Journal of Pure and Applied Bioscience , 1, 827–831. http://doi.org/10.18782/2320-7051.2525

46.

Osman

I. H.

(2013). In vitro antioxidant activity of Mentha pulegium from Saudi Arabia. Bioscience Research , 10(1), 33–37.

47.

Padmini

, Valarmathi

, & Rani

(2010). Comparative analysis of chemical composition and antibacterial activities of Mentha spicata and Camellia sinensis. Asian Journal of Experimental Biological Sciences , 1(4), 772–781.

48.

Parham

, Kharazi

A. Z.

, Bakhsheshi-Rad

H. R.

, Nur

, Ismail

A. F.

, Sharif

, RamaKrishna

, & Berto

(2020). Antioxidant, antimicrobial and antiviral properties of herbal materials. Antioxidants , 9(12), 1309. https://doi.org/10.3390/antiox9121309

49.

Parham

, Kharazi

A. Z.

, Bakhsheshi-Rad

H. R.

, Nur

, Ismail

A. F.

, Sharif

, RamaKrishna

, & Berto

(2020). Antioxidant, antimicrobial and antiviral properties of herbal materials. Antioxidants , 9(12), 1309. https://doi.org/10.3390/antiox9121309

50.

Patro

B. S.

, Bauri

A. K.

, Mishra

, & Chattopadhyay

(2005). Antioxidant activity of Myristica malabarica extracts and their constituents. Journal of Agricultural and Food Chemistry , 53(17), 6912–6918. https://doi.org/10.1021/jf050861x

51.

Pereira

, Pimenta

A. I.

, Calhelha

R. C.

, Antonio

A. L.

, Verde

S. C.

, Barros

, Santos-Buelga

, & Ferreira

I. C. F. R.

(2016). Effects of gamma irradiation on cytotoxicity and phenolic compounds of Thymus vulgaris L. and Mentha x piperita L. LWT – Food Science and Technology , 71, 370–377. https://doi.org/10.1016/j.lwt.2016.04.004

52.

Riahi

, Elferchichi

, Ghazghazi

, Jebali

, Ziadi

, Aouadhi

, Chograni

, Zaouali

, Zoghlami

, & Mliki

(2013). Phytochemistry, antioxidant and antimicrobial activities of the essential oils of Mentha rotundifolia L. in Tunisia. Industrial Crops and Products , 49, 883–889. https://doi.org/10.1016/j.indcrop.2013.06.032

53.

Sellare

, Meemken

E. M.

, & Qaim

(2020). Fairtrade, agrochemical input use, and effects on human health and the environment. Ecological Economics , 176, 2020. 106718. https://doi.org/10.1016/j.ecolecon.2020.106718

54.

Shaikh

, Yaacob

H. B.

, & Rahim

Z. H. A.

(2014). Prospective role in treatment of major illnesses and potential benefiits as a safe insecticide and natural food presertive of mint (Mentha spp.): A review. Asian Journal of Biomedical and Pharmaceutical Science , 4(35), 1–12.

55.

Shelepova

O. V.

, Voronkova

T. V.

, Kondrat’eva

V. V.

, Semenova

M. V.

, Bidyukova

G. F.

, & Olehnovich

L. S.

(2014). Phenotypic and phytochemical differences between Mentha arvensis L. and Mentha[YE1] canadiensis L. Biology Bulletin , 41(1), 19–23. https://doi.org/10.1134/S1062359014010099

56.

Singh

, Shushni

M. A. M.

, & Belkheir

(2015). Antibacterial and antioxidant activities of Mentha piperita L. Arabian Journal of Chemistry , 8(3), 322–328. https://doi.org/10.1016/j.arabjc.2011.01.019

57.

Soković

M. D.

, Vukojević

, Marin

P. D.

, Brkić

D. D.

, Vajs

, & van Griensven

L. J.

(2009). Chemical composition of essential oils of thymus and Mentha species and their antifungal activities. Molecules , 14(1), 238–249. https://doi.org/10.3390/molecules14010238

58.

Soleimani

, Arzani

, & Roberts

T. H.

(2022). Phenolic compounds and antimicrobial properties of mint and thyme. Journal of Herbal Medicine , 36, 100604. https://doi.org/10.1016/j.hermed.2022.100604

59.

Tafrihi

, Imran

, Tufail

, Gondal

T. A.

, Caruso

, Sharma

, Atanassova

, Atanassov

, Valere Tsouh Fokou

, & Pezzani

(2021). The wonderful activities of the genus Mentha: Not only antioxidant properties. Molecules , 26(4), 1118. https://doi.org/10.3390/molecules26041118

60.

Thawkar

B. S.

, Jawarkar

A. G.

, Kalamkar

P. V.

, Pawar

K. P.

, & Kale

M. K.

(2016). Phytochemical and pharmacological review of Mentha arvensis. International Journal of Green Pharmacy , 10, 71–76.

61.

Tucker

A. O.

(2007). Mentha: Economic uses. In Lawrence

B. M.

(Ed.), Mint: The genus Mentha (pp. 519–522). CRC Press, Taylor & Francis Group.

62.

Usta

(2013). Microorganisms in biological pest control-A review (bacterial toxin application and effect of environmental factors). Current Progress in Biological Research , 32, 137–144. https://doi.org/10.5772/55786

Phytochemical Profiling,Antioxidant,and Antifungal Activities of Ethyl Acetate and Chloroformic Extracts from Three Mentha Species

Abstract

Background

Objectives

Materials and Methods

Results

Conclusion

Keywords

Introduction

Materials and Methods

Plant Materials and Culture Conditions

Preparation of the Extracts

Determination of Total Phenolic, Flavonoid Contents, and Condensed Tannins

Antioxidant Activities

Anti-fungal Activity

Micro-organisms

Disc-diffusion Assay and Inhibition Test of the Growth Fungus on Liquid Medium

Statistical Analysis

Results and Discussion

Quantitative Analyses of Phytochemical Constituents of Mentha Genus Extracts

TPC (mg GAE/g DW), TFC (mg CE/g DW), and TCTC (mg CE/g DW) in Different Extracts in the Aerial Part Extract of Three Mint Species.

Antioxidant Activity Evaluation

DPPH Test (IC50; µg/mL), Reducing Power (EC50; µg/mL) and β-Carotene (IC50; µg/mL) of Mentha spp. Aerial Part Extracts, and Authentic Standards (BHT, BHA, and Ascorbic Acid) (µg/mL).

Anti-fungal Activity Evaluation

Zones of Growth Inhibition (mm) Showing Antifungal Activity for Three Mint Species (L, S, and P) Extracts.

Percentage of Inhibition (%I) of the Growth of Fungus Species Cultivated on Liquid Medium with Various Concentrations of Three Mint Extracts (100 µg/mL YNB broth), Compared to Positive Standard Drug (Amphotericin B, 10 µg/mL YNB broth).

Conclusion

Footnotes

Abbreviations

Acknowledgments

Declaration of Conflicting Interests

Funding

Statement of Informed Consent and Ethical Approval

ORCID iDs

References

DPPH Test (IC₅₀; µg/mL), Reducing Power (EC₅₀; µg/mL) and β-Carotene (IC₅₀; µg/mL) of Mentha spp. Aerial Part Extracts, and Authentic Standards (BHT, BHA, and Ascorbic Acid) (µg/mL).