Mentha spicata Linn., commonly known as spearmint, belongs to the Lamiaceae family [1]. The genus Mentha consists of more than 25 species, among which the monoterpenes such as menthol, menthone, carvone, and pulegone are well known. Monoterpenes are widely used by food and pharmaceutical industries as a flavour or in fragrance formulations. They are widely grown in temperate areas of the world, particularly in Europe, North America, North Africa, and Asia, although presently they are cultivated in regions all over the world.

The Lamiaceae family is a rich source of polyphenolic compounds and may therefore possess strong antioxidant properties [2]. Mints are herbaceous plants with high contents of essential oils. Due to their levels of secondary metabolites, they are commonly used as pharmaceuticals, fragrances, flavours, and foods [3]. The pharmacological activity of Mentha species, peppermint in particular, has been investigated extensively. Peppermint is used for gastrointestinal disorders (atony of the stomach, flatulence) and nervous system disorders (nervousness, insomnia, nervous sickness). Its antibacterial, antiviral, fungicidal, anti-inflammatory, choleretic, cholekinetic, and astringent activities have also been described [4]. Recent work examined the antioxidant activities of different Mentha species. Phenolic compounds play the most important role in the antioxidant properties of mint. The major phenolic constituents are caffeic acid derivatives and flavonoids (). This study is the first to report the larvicidal, nematicidal, antifeedant, and antifungal activities of the root extract of M. spicata.

Research on herbs has recently become more pressing because of their potential antifungal activity. Here, we estimated the total phenolic and flavonoid contents of dried M. spicata root using various solvent extractions (hexane, chloroform, ethyl acetate, and water) and screened for larvicidal, nematicidal, antifeedant, and antifungal activities.

All materials and reagents were purchased from Sigma Aldrich (Mississauga ON Canada) unless otherwise stated. (Chloroform, Ethyl acetate, Sodium carbonate, Aluminium chloride, Sodium nitrate, Catechin, Gallic acid)

Plant material

The fresh plant material used in this study, and fresh plant was collected (Juan 28, 2015) from Tiruchirappalli distric, Tamil Nadu, The plant was morphologically identified and authenticated by Anis Ahamed (One of the authors of this paper – Lecture in Botany department) the Botanist of Department of Botany, King Saud University (KSU), Riyadh, Saudi Arabia.

Extraction of solvent fractions

The shade-dried root powder of M. spicata (100 g) was immersed in 500 mL of double distilled (DW) water at 85 °C for 3 h (water extraction). The mixture was constantly stirred at every 24 hours interval of 72 h then the final filtrate volume found to be 1.2 L. Each extraction was performed three times. After filtration, the extracts was then concentrated under reduced pressure at 40 ^oC in a vacuum rotary evaporator. Afterwards, the concentrates were dried and weighed to determine total extractable compounds. Finally, the extracts were stored in a refrigerator at 4 °C until used. The aqueous layer further fractionated with chloroform (CHCl₃), ethyl acetate other solvent fractions were collected and concentrated with vacuum rotary evaporator.

Identification of total phenolic content

The total phenolic content of the extracts was determined by using the FC (Folin–Ciocalteu) method [5]. Briefly, 10 mg of each extract was dissolved in 1 mL of DW and different concentrations of gallic acid (0 - 1 mg/mL) were prepared in ethanol. 40 µL of each solution of Mentha spicata root extract or a standard reagent was mixed with 20 µL of 1 N FC reagent. Next, the mixture was incubated for 3 – 5 min at room temperature, followed by addition of 20 % sodium carbonate solution and incubated at RT for 30 min. Finally, the absorbance was measured at 700 nm wavelength, with a UV–visible spectrophotometer. Total phenolic content was determined from the standard calibration curve. Results were expressed as percentage of gallic acid equivalents (GAE) per 100 g dry mass.

Identification of total flavonoid content

Total flavonoid content was determined by aluminium chloride (AlCl₃) colorimetric assay [5]. The concentrations of the Mentha spicata root extract and standard (catechins) solutions were 10 mg/mL and 25, 20, 15, 10 and 5 µg/mL, respectively. The concentration (25 µL) of each sample or standard reagent was mixed with 125 µL of DW, followed by addition of 8 µL of 5 % sodium nitrate solution. The mixture was incubated at RT for 5 min. Aluminium chloride (10 %) solution was mixed with 15 µL of the above mixture. Finally, the absorbance was measured at 517 nm. Total flavonoid content was expressed a percentage of the catechins equivalent (CE) per 100 g dry mass obtained from the standard calibration curve.

Determination of larvicidal activity

The larvicidal activity was assessed by the procedure of the WHO (1996) with some modifications and as per the method [6]. Mentha spicata root extract was tested against the urban mosquito larvae (Culex quinquefasiatus) using standard bio assay protocol. Eggs of C. quinquefasiatus were obtained from drainage system.  Eggs were placed in clean water and kept at room temperature for hatching. Larval development was monitored for seven days. The second stage larvae were collected at the tip of a pasture pipette and placed in cotton bud to remove excess water and transferred to the test vial. The larval mortality was observed using various concentrations of Mentha spicata root extract (10, 20, 30 and 40 mg/mL).

Assessment of nematicidal activity

Nematicidal activity was evaluated using juvenile nematodes of Meloidogyne javanica [7]. Assay system was prepared with 2 mL Milli Q water containing different concentrations of Mentha spicata root extract (10, 20, 30 and 40 mg / mL) in glass tubes. The different concentrations of solutions are prepared treated and control nematodes were held under the same conditions as used for colony maintenance. Total 25 juveniles of M. javanica were transferred in test tube, positive control (sample) and negative control (only solvent). Mortality was observed under a zoom stereomicroscope after 24 h of exposure.

Evaluation of antifeedant activity

Fingerlings (1.5 - 2.0 cm) of marine acclimated Oreochromis mossambicus were used for evaluating the ichthyotoxic potential [8]. Total 25 fingerlings fish were introduced in experimental and control glass bowls containing 1,000 mL seawater and chosen concentrations of Mentha spicata root extract. Immediate reflex changes and mortality were observed continuously for six hours at 1 h interval for the next 12 h. After 24 h of exposure, the number dead and live fish were counted.

In vitro antifungal screening

Mentha spicata root extract were evaluated for their in vitro antifungal activity against Aspergillus niger, Candida albicans, Cryptococcus neoformans (recultured) and Microsporum audouinii using an disc diffusion method [9,10] with Sabouraud’s dextrose agar (Hi-Media).

Determination of the minimal inhibitory concentration (MIC):  Test sample was concentrated at two fold dilutions method (356, 128, 64, 32…., 0.5 µg/mL, etc). The microorganism suspensions at 10⁶ cfu/mL (colony forming unit/mL) concentrations were incubated to the corresponding wells. The test sample solution (0.01 mL per disk) was added to sterile disks to prepare disks of potency with different concentration. The disks were stored at 4 °C until use.

The plates were incubated at 36 ºC at 48 h and observe for inhibition zones and then note the MIC. The minimum inhibitory concentration (MIC) values were determined as the lowest concentration that completely inhibited visible growth of the microorganism.

Statistical analysis

All experiments were performed in triplicate and all data were expressed at least 3 independent evaluations and the standard deviations (SD) were also calculated using Microsoft Excel 2007 software (Microsoft, Redmond, WA, USA).

Total phenolic content

The reagent, Folin-Ciocalteu, was used to determine the total polyphenol content in root extracts. Folin-Ciocalteu reagent consists of a yellow acidic solution containing complex polymeric ions, which are formed from phosphomolybdic and phosphotungstic heteropoly acids [11]. The total phenolic content in the various organic and water extracts was determined and expressed as milligrams of gallic acid equivalent (GAE) per gram of dry plant. The total phenolic content from the different solvents ranged from 0.3 ± 0.43 to 83.4 ± 0.89 mg GAE/g. The water extract exhibited the highest phenolic content, whereas the lowest content was from the n-propanol extract. The total phenolic contents from each of the different extracts are presented in .

Total flavonoid content

Flavonoids are the most common and widely distributed group of plant phenolic compounds, ever-present in fruits and vegetables. The total flavonoid values of the extracts ranged from 0.4 ± 0.12 to 91.9 ± 0.14 mg catechin equivalent (CE)/100 g. The highest value was observed in the water extract, whereas the lowest values were observed in the toluene, n-butanol, n-propanol, and isopropanol extracts. Total flavonoid content analysis is shown in .

Biological profile

shows the total biological screening outline of M. spicata root extract compounds. The water extract exhibited the highest levels of larvicidal (LD₅₀ = 11.77 µg/mL), nematicidal (LD₅₀ = 11.78 µg/mL), antifeedant (LD₅₀ ≥ 40 µg/mL), and antifungal activities (28 mm at minimum inhibitory concentration [MIC]: 16 μg/mL) against Microsporum audouinii.

M. spicata root extract was screened for larvicidal activity against second instar larvae for 24 h at room temperature. Water extract, at 30 µg/mL, induced 100 % mortality while hexane extract exhibited low toxicity compared with the other solvent extracts. The extracts induced 50 % mortality of second instar larvae.

The larvicidal activities of various plant components, natural products, and secondary metabolites of plant origin have been evaluated against mosquitoes, with varying results [15].

M. spicata essential oil exhibits a significant toxic effect against early third-stage larvae of C. quinquefasciatus, with LC₅₀ values of 62.62 μg/mL [16]. The water extract of the Mentha spicata root showed high levels of larvicidal activity. Toluene extract was less active than the other solvent extracts in larvicidal screening, reaching 38 % mortality at 40 μg/mL, with an LD₅₀ value of > 100.37 μg/mL. The chloroform extract was slightly more active compared with the hexane and ethyl acetate extracts, reaching 100 % mortality at 40 μg/mL, with an LD₅₀ of 15.78 μg/mL. The water extract was highly active compared with the other solvents, with an LD₅₀ of 11.77 μg/mL. The remarkably toxic effects exhibited by the M. spicata root extracts against the third and fourth instar larvae of C. quinquefasiatus indicate its potential use as a natural larvicide.

The water extract at 30 µg/mL induced 100 % mortality, whereas the toluene, n-butanol, and isopropanol extracts exhibited lower toxicity. Chloroform and ethyl acetate extracts showed even lower toxicity. 

Many investigations have addressed the effects of root solvent extracts of various plants against several plant parasitic nematode species. Aqueous root extract samples of the marigold Tagetes erecta, the dusty miller Cineraria maritime, and the common rue Ruta graveolens exhibit high nematicidal activity against Heterodera schachtii and against the root knot nematodes Meloidogyne arenaria, Meloidogyne hapla, Meloidogyne incognita, and Meloidogyne javanica [17].  However, the root extract of M. spicata exhibited greater nematicidal activity than that of its leaf extracts against the juvenile nematodes of M. javanica. Previous studies reported an LC₅₀ value of 0.293 mg/mL for 100–200 mg/kg for M. spicata and M. javanica [18]. The hexane extract was found to be less active than the water solvent extracts in nematicidal screening, with 55 % mortality induced by the 40 μg/mL concentration and an LD₅₀ of 36.45 μg/mL. Chloroform and ethyl acetate extracts were equally active compared with the water extract, while the water extract was highly active compared with the other solvents, with an LD₅₀ of 11.78 μg/mL.

Antifeedant activity was measured to evaluate the ichthyotoxicity profile of M. spicata root extracts of various solvents. Water extract exhibited low toxicity with 48 % mortality induced at 6 h at a concentration of 40 µg/mL while the other solvent extracts were highly toxic, with 100 % mortality induced at 3 to 5 h at the same extract concentration.

Feeding deterrent activity of plant extracts and plant products against P. xylostella has been reported by many investigators [19]. Koul et al and Chen et al stated that Aglaia elaeagnoidea extract induced maximum antifeedant and larvicidal activities against Helicoverpa armigera and Pieris rapae [20].

Ling et al reported that an ethanol extract of Mimordica charantia had both feeding deterrent and larvicidal activities against P. xylostella [21]. Using the same method to screen marine acclimated Oreochromis mossambicus, we evaluated the ichthyotoxic potential of M. spicata root extracts and found that the toluene extract was less active than were the other solvent extracts evaluated.

The water extract was highly active against M. audouinii assay (MIC: 16 μg/mL) and also showed a significant response against the fungal species, Aspergillus niger, Candida albicans, and Cryptococcus neoformans.

There is great demand for more efficient and safer treatments. Medicinal herbs could provide an alternative source for the treatment of dermatophytosis. Herbal medicines are generally safer and free from side effects. Moreover, the effectiveness of many traditional medicines is now accepted [22]. Some Indian plants have gained reputation for their treatment of fungal infections [23], but no systematic study has been conducted to investigate their efficacy. Few studies on M. spicata extracts have comprehensively investigated their antifungal activity against various microorganisms. One study, however, showed that the crude extracts (MIC: 0.75 mg/mL) are highly active against C. albicans compared with water extracts (MIC: 3.5 mg/mL) [24]. The present study showed that while the hexane and chloroform extracts did not show significant activity against the fungal strains evaluated, the water extract of the M. spicata root (MIC: 16 μg/mL) exhibits significant fungicidal activity against M. audouinii.

The findings of this study demonstrate that the M. spicata root water extract exhibits higher phenolic and flavonoid contents and it has higher larvicidal and nematicidal activity but not antifeedant activity compared with the other extracts evaluated.  It also showed high antifungal activity against M. audouinii. Further investigation will be required to determine the active constituents of the extract as well as the mechanism of action to develop potent larvicidal, nematicidal, and antifungal agents.