Larvicidal, nematicidal, antifeedant and antifungal, antioxidant activities of Mentha spicata (Lamiaceae) root extracts

Purpose: To evaluate the larvicidal, nematicidal, antifeedant, and antifungal effects of 10 solvent extracts of Mentha spicata root. Methods: Ten solvent extracts were investigated for their total flavonoid and phenolic content and screened for larvicidal, nematicidal, antifeedant, and antifungal activities. The total phenolic content of the extracts was determined using the Folin–Ciocalteu method, while total flavonoid content was determined by aluminium chloride (AlCl 3 ) colorimetric assay. Four solvents extracts were screened for antifungal activity against Aspergillus niger, Candida albicans, recultured Cryptococcus neoformans, and Microsporum audouinii using the agar diffusion method. The nematicidal activity of the compounds was evaluated against the juvenile Meloidogyne javanica organism, while larvicidal properties were evaluated against the urban mosquito Culex quinquefasciatus using a standard bioassay protocol. The antifeedant activity of marine acclimated Oreochromis mossambicus was used for evaluating ichthyotoxic potential. Results: The total flavonoid content in the extracts ranged from 18.5 to 83.4 mg/g, and the amount of free phenolic compounds ranged from 14.7 to 91.9 mg/g of extract powder. The water extract of these plants exhibited significant antioxidant activity and significant levels of phenolics and flavonoids. The water extract exhibited higher larvicidal (LD 50 = 11.77 µg/mL), nematicidal (LD 50 = 11.78 µg/mL), antifeedant (LD 50 > 40 µg/mL), and antifungal activities (minimum inhibitory concentration: 16 μg/mL) against M. audouinii compared with the other extracts. Conclusion: These results show that the water extract of Mentha spicata may be used as a potential natural alternative source of nutritional and pharmaceutical ingredients.

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, antiinflammatory, 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 ( Figure 1). 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.

EXPERIMENTAL
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 o C 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 (CHCl 3 ), 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 UVvisible 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 (AlCl 3 ) 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.

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 10 6 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 Table 1.

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, npropanol, and isopropanol extracts. Total flavonoid content analysis is shown in Table  1. Phenolics are secondary metabolites, which are major components of plants that scavenge free radicals and perform important biological activities [12,13]. Flavonoids are a group of polyphenolic compounds with various chemical structures and characteristics that exhibit numerous biological activities, including antiinflammatory, antiviral, and antibacterial activity,   in addition to their antioxidant properties [14]. The total phenolic content of the M. spicata root extracts was determined from a regression equation of the calibration curve (y = 0.5181x -0.5953) and expressed in GAE. Values were 83.4, 45.9, 32.0, 18.5, 12.8, 10.8, 0.8, 0.7, 0.6, and 0.3 mg GAE/100 g dry mass for water, ethyl acetate, chloroform, hexane, methanol, ethanol, n-butanol, toluene, isopropanol, and n-propanol extracts respectively. Total flavonoid content was determined from the regression equation of the calibration curve (y = 0.244x + 0.2849) and expressed in catechin equivalents (CE). The water, ethyl acetate, chloroform, hexane, methanol, ethanol, toluene, n-butanol, and isopropanol extracts of M. spicata root exhibited total flavonoid contents of 91.9, 44., 34.0, 14.7, 11.2, 10.1, 0.,0.6, 0.4, and 0.4 mg CE/100 g dry mass, respectively. The water extract contained greater phenolic and flavonoid contents than did any of the other solvent extracts.

DISCUSSION
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 LC 50 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 LD 50 value of > 100.37 μg/mL. The chloroform extract was slightly more active compared with   [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.

CONCLUSION
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.