Morphophysiological and phytochemical properties of peppermint ecotypes: a comprehensive study in 11 provinces of Iran

doi:10.21203/rs.3.rs-4972754/v1

Download PDF

Research Article

Morphophysiological and phytochemical properties of peppermint ecotypes: a comprehensive study in 11 provinces of Iran

https://doi.org/10.21203/rs.3.rs-4972754/v1

This work is licensed under a CC BY 4.0 License

Version 1

posted

You are reading this latest preprint version

In order to evaluate the quantity and quality of 11 peppermint ecotypes, rhizomes were collected from the producing provinces and tested at the Research Institute of Forests and Rangelands in 2021 and 2022. The results showed that there was a statistically significant difference, in years in the morphological traits, shoot yield, the percentage of leaves and shoot essential oil, the yield of shoot essential oil, flavonoid, chlorophyll a, b, all essential oil compounds, total nitrogen, potassium, calcium, magnesium, iron, manganese, cadmium and lead absorption. Mean comparison of years showed that the highest shoot yield (5656.88 kg/ha), leaf essential oil percentage (2.48 percent), shoot essential oil percentage (1.87 percent) was obtained in the second year. The highest shoot yield with 6559 kg/ha and shoot essential oil percentage with 2.2% was obtained from Markazi province. The most menthone with 23.7% belonged to Mazandaran. The highest amount of menthol was in Kermanshah, with 59%. The result of cluster analysis showed that 11 accessions were placed in three main clusters. The results show the difference in the quantity and quality, absorption of macro, micro and heavy metal elements of different ecotypes and it is better to do preliminary tests and choose the appropriate ecotypes for specific uses (food, medicine, cosmetics and hygiene) before planting peppermint.

medicinal plants

peppermint

menthol

menthone

cadmium

cluster analysis

The value of the global trade in medicinal plants in 2019 was more than 60 billion dollars (Khan et al., 2019) and is expanding, so that it is estimated to reach more than 5 trillion dollars in 2050 (Purohit & Vyas, 2004). Medicinal and aromatic plants, in addition to being important in medical use, are widely used in many food, cosmetic, health and spice industries (Roodbari et al., 2013; Jesus, 2000; Simões et al., 2000). Almost 2000 tons of essential oils of mints are produced in the world every year, which ranks second after citrus species (Mucciarelli et al., 2001). The main producing countries are Bulgaria, Italy, China, and the United States, which account for about 90% of peppermint production in the world (Dwivedi et al., 2004).

Peppermint with the scientific name Mentha piperita L. belongs to the Lamiacea family, it is a hybrid species obtained from the crossing of Mentha aquatica and Mentha spicata species (Foster, 1996). Chromosomally, 2n = 144 has been reported (Lutkov et al., 1966; Bugaenko and Rezinkova, 1980). Peppermint is a perennial plant (Mamadalieva et al., 2017). It is used in traditional medicine in different countries (Prakash et al., 2016), It has biological effects such as anti-viral, anti-inflammatory, anti-cancer, anti-diabetic, anti-pain, anti-bacterial (Lorenzi and Matos, 2002), anti-fungal, anti-asthma, anti-headache (Parv Nayak et al., 2020) and anti mold (Sharifi-Rad et al., 2018).

The effect of its essential oil on Gram-negative and Gram-positive bacteria has been confirmed (Pattnaik et al., 1996; Moleyar and Narasimham, 1992). It is used in the treatment of colds and flu (Tetik et al., 2013). Also, the antioxidant (Park et al., 2012), antimicrobial, and detoxifying properties of mints have been reported (Gulluce et al., 2007). According to the emphasis of the World Health Organization (WHO), plants with medicinal and therapeutic functions should be tested to confirm the absence of heavy metals (WHO, 2007). Non-essential metals such as lead (Pb), cadmium (Cd), mercury (Hg) and arsenic (As) with unknown function are toxic to plants even in low amounts (Shahid et al., 2017). Reports indicate that heavy metal stress causes a significant increase in secondary metabolites in medicinal plants (Sinha and Saxena, 2006; Tirillini et al., 2006). New findings show that the production of secondary metabolites increases after exposure to heavy metal stress (Asgari Lajayer et al., 2017).

It seems that choosing ecotypes with low cadmium absorption capacity may be a way to limit the presence of this toxic metal in plant products. The ability to absorb cadmium can be tested by some cadmium-accumulating species (in experiments with artificially contaminated materials) (Khalili et al. 2022; Lorenzi and Matos, 2002; Mamadalieva et al., 2017; Prakash et al., 2016; Zakikhani et al. 2012). Morphological traits and shoot yield of 8 populations of 4 mint species were investigated: Mentha longifolia var. amphilema from Qazvin and Ardabil provinces, M. spicata from Tehran and Yazd provinces, M. piperita from Tehran and Ardabil provinces, M. aquatic from Gilan and Ardabil provinces (Sharifi-Rad et al., 2018). The results showed that between different species in terms of plant height, number of lateral stems, leaf length, leaf width, stem diameter, flower yield, flower essential oil percentage and yield, leaf yield, leaf essential oil percentage and yield, shoot yield and shoot essential oil yield, there was a statistical difference. The highest leaf yield with 914 kg/ha and leaf width with 2.13 cm belonged to M. piperita from Tehran province. The results of investigating the relationship between essential oil yield and some agricultural traits of two Nepeta ecotypes showed that there was a significant positive correlation between essential oil yield and flower and leaf yield, percentage of flower and leaf essential oil, and leaf essential oil yield had the highest direct and positive effect (0.97) on the yield of total essential oil (Pattnaik et al., 1996).

By examining the relationships between the traits of two populations of Mentha spicata with path and principle component analysis, the results showed that between different years and populations in the traits of plant height, leaf length and width, stem diameter, number of lateral stems, flower yield, percentage and yield of flower essential oil there was a statistically significant difference in leaf yield, percentage and yield of leaf essential oil and the yield of shoot essential oil. Also, the result of step-by-step analysis showed that more than 96% of the changes belonged to the characteristics of leaf essential oil yield, flower essential oil yield, number of lateral stems, flower essential oil percentage and stem diameter. The result of path analysis showed that the yield of leaf essential oil had the highest direct and positive effect (0.76) on the yield of total essential oil (Dwivedi et al., 2004). It was observed that the Sahne region with the highest percentage of essential oil is the most suitable area for the cultivation of this plant in terms of the quantity of essential oil, but in terms of quality, Kermanshah region has the highest amount of menthol. On the other hand, Gilanegharb region with the least essential oil had the most menthon in essential oil, which indicates that the climatic conditions are hot compared to other places. In another research on peppermint, it was observed that the highest morphological traits, essential oil yield, percentage of menthone and menthofuran were obtained in normal irrigation of 100% FC, and the highest percentage of menthol was obtained from irrigation of 70% FC (Foster, 1996). Flavonoids are a group of secondary metabolites that are involved in many plant biological activities, including plant defense activities (Lutkov et al., 1996).

In a 2020 research on peppermint, the number of side stems 10.10–30.20 per plant, height 59.6 cm, maximum shoot yield 3548 kg/ha, essential oil percentage 2.7%, essential oil yield 66 kg/ha, the amount of menthol 31.82–37.87%, menthone 23.85–30.90%, absorbed nitrogen 2.03–4.37%, phosphorus 1.50-0.223%, potassium 2.10–2.68%., iron 2.95 mg/g dry matter, zinc 0.86 mg/g dry matter, manganese 0.48 mg/g dry matter were reported (Bugaenko and Rezinkova, 1980). In another study, the yield of peppermint under different treatments was up to 8.56 tons, essential oil percentage 0.61–1.12%, essential oil yield 46.9–72.1 kg per hectare, menthol percentage 25.7–29.8%, the percentage of menthone was 14.2–20.8% and menthofuran was reported as 3.5–11.7% (Mamadalieva et al., 2017).

This research was conducted in order to evaluate the range of changes in different morphological and physiological traits, yield of shoot and essential oil, as well as the composition of essential oil, the amount of absorption of heavy metals and other macro and micro elements in 11 ecotypes of peppermint plants during two years in field conditions.

In order to evaluate the quantity and quality of 11 ecotypes of peppermint (Mentha piperita L.), mint rhizomes were collected from the provinces producing this plant (Table 1).

Table 1

The information and geographical location of the collecting place of ecotypes
altitude	Longitude and latitude	city	province
936m	34°38′24″N, 50°52′35″E	Qom	Qom	1
1850m	48°31′00″N, 34°48′00″E	Hamedan	Hamedan	2
1340m	37^o75'89''N, 45^o97'66''E	Khosrowshahr	East Azerbaijan	3
160m	54°26′00″N, 36°50′30″E	Gorgan	Golestan	4
185m	53°03′30″N, 36°34′00″E	Sari	Mazandaran	5
1350m	34^o32'77''N, 47^o07'78''	Kermanshah	Kermanshah	6
1755m	49°41′30″N, 34°05′30″E	Arak	Markazi	7
20m	37°16′51″N, 49°34′59″E	Rasht	Gilan	8
1320m	35^o46'21''N, 50^o46'07''E	Karaj	Alborz	9
1755m	30^o17'30''N, 57^o05'00''E	Kerman	Kerman	10
1767m	36^o51'10''N, 48^o78'57''E	Zanjan	Zanjan	11

The collected ecotypes were cultivated in the Alborz Research Station (soil information and climatic conditions have shown in Tables 2 & 3), Research Institute of Forests and rangelands, in the years 2021–2022 in the form of random complete blocks with 3 replications.

Table 2

Soil information of the test station
Absorbable phosphorus (g/100g)	Absorbable potassium (g/100g)	Total nitrogen (g/100g)	Organic matter (g/100g)	Organic carbon (g/100g)	TNV- total neutralized value (%)	Clay (%)	Silt (%)	Sand (%)	The electrical conductivity	Saturated soil reaction	Soil texture
9.3	176	0.08	1.54	0.88	11.2	26	29	45	0.7	7.5	Sand-Clay
*21.13 (kg/ha)	*40 (kg/ha)	*18.18(kg/ha)
*The amount of nitrogen, phosphorus and potassium in the root development depth (40 cm) per hectare was calculated based on the ƿ=m/v. (ƿ=Baulk Density (1.76 g/cm³), m = Mass, v = Volume)

Table 3

Climatic information of the test station
Year	Air Tem. Max (^OC)	Tem. Min (^OC)	Tem. Mean (^OC)	Relative Humidity (%). Max.	Relative Humidity (%). Min.	Relative Humidity (%). Mean.
2021	33	17	25.3	57.7	14.8	32.8
2022	31.8	16.3	24	60.6	15.9	34.3
Mean	32.4	16.65	24.65	59.15	15.35	33.55

The dimensions of the plots were 2 x 3 meters. The size of the rhizomes was 10 cm, the distance between the rhizomes was 20 x 30 cm. The distance between plots was 1.5 meters and the distance between repetitions was 2.5 meters. Harvesting was done at the full flowering stage (70% flowering). Every year, two harvests were done. Morphological traits, percentage of essential oils and accumulation of elements are presented based on the average of 2 harvests. Morphological traits including plant height, leaf length and width, number of leaves on one stem, number of lateral stems, number of inflorescences, length of main inflorescence and diameter of main inflorescence were measured. Flowering shoots were harvested from 5 cm above the soil, then transferred to the shade and dried with air flow at a temperature of 20–25°C and a relative humidity of about 60%.

The yield of flowering shoot, leaf, stem and flower were measured separately and calculated as yield per hectare. The essential oil of the leaf and flower shoot was extracted and calculated by Clevenger and water distillation method in 2 hours. Then, the percentage of essential oil of leaves and flowering shoots and the yield of essential oil of flowering shoots per hectare were calculated. The essential oils were stored in the refrigerator. The percentage of essential oil compounds was determined by GC and their identification was done using GC/MS. Plants were sampled in the flowering stage and their chlorophyll a, b, total, carotenoid, phenol and flavonoid were measured. Phosphorus and potassium accumulation were measured using a film photometer and nitrogen by the Kjeldahl method. Calcium, magnesium, iron, manganese, zinc, copper, cadmium, lead, chromium and nickel were measured in aerial parts by atomic absorption device.

Gas chromatography (GC) and gas chromatography–mass spectrometry (GC–MS)

Specifications of Gas Chromatograph (GC) device

Ultra-fast gas chromatograph (GC-FID) with Thermo-UFM model made in Italy, equipped with FID detector and data processor with chrom-card 2006 software was used. Semi-polar DB-5 column (length 10m, inner diameter 0.1mm and thickness of stationary phase layer equal to 0.4 microns) was used. The injection chamber temperature was set to 280°C and also the detector temperature was set to 280°C. The thermal programming of the column consisted of increasing the temperature from 60 to 285°C at an increasing rate of 40°C/min and then holding it at 285°C for 3min. The carrier gas used was helium at a flow rate of 0.5ml/min.

Specifications of GC/MS device

Analysis of essential oils was carried out with Agilent 7890A gas chromatograph device connected to Agilent 5975C quadrupole mass spectrometer (made in USA), equipped with DB-5 column (length 30m, internal diameter 0.25mm and the thickness of the static phase layer of 0.25microns). The thermal programming of the column includes increasing the temperature from 60 to 220°C with an increase rate of 3°C per minute and then increasing to 260°C with an increase rate of 20°C per minute and finally keeping 5 minutes at this temperature. The injection chamber temperature is set at 260°C and the transfer line temperature is set at 280°C. The carrier gas was helium, which moves along the column at a speed of 30.6cm/s. The scan time was equal to one second, the ionization energy was 70 electron volts and the scan of the mass region was from 30 to 340a.m.u.

Statistical analysis

Data were analyzed using SPSS software (version 18) and the means were compared using LSD test at the level of P ≥ 0.05.

The results of composite variance analysis of morphological traits, aerial organs yield and essential oil of different ecotypes of peppermint showed that between the years of experiment in the traits of inflorescence length, number of branches, leaf width, number of inflorescences, stem yield, inflorescence yield, shoot yield, percentage of leaf and shoot essential oil, was a statistically significant difference (Table 4).

Statistically significant differences at 1% level were observed between different ecotypes in all morphological traits, yield of different organs, percentage and yield of essential oil. The interaction effect of year in accessions showed a statistical difference in the stem diameter and leaf yield at the level of 1%. The results of composite variance analysis of the physiological traits of different peppermint ecotypes showed that there was a statistical difference of 1% in the amount of flavonoid, chlorophyll a and b between the years of the experiment (Table 5).

A statistical difference was observed at the level of 1% between different ecotypes in phenol, flavonoid, chlorophyll a, chlorophyll b, total chlorophyll and carotenoid. The interaction effect of year on ecotype was not significant on any of the traits (Table 5).

The results of composite variance analysis of essential oil compounds of different ecotypes of peppermint showed that between the two years of the experiment, there is a statistical difference in all essential oil compounds at the 1% level (Table 6). A statistical difference of 1% was observed between the accessions.

The results of the interaction effect of year on ecotypes on β-pinene, 3-octanal, limonene, 1,8-cineole, cis-sabinene hydrate, menthone, iso-menthone, menthofurane, γ-terpineol, menthol, iso-menthol, α-terpineol , pulegone, neo-menthyl acetate, menthyl acetate showed a statistical difference at the level of 1%.

Results of composite variance analysis of elements absorbed by different ecotypes of peppermint showed that there was a statistical difference of 1% in the amount of absorbed total nitrogen, potassium, calcium, magnesium, iron, manganese, cadmium and lead between the years of the experiment (Table 7).

A statistical difference of 1% was observed between the ecotypes in all macro elements (total nitrogen, phosphorus, potassium, calcium and magnesium), micro elements (manganese, zinc and copper) and heavy metals chromium and nickel.

Mean comparison of two years of traits showed that inflorescence length (5.8 cm), leaf width (1.84 cm), flavonoid (16.01 mg/g dry matter) and chlorophyll a (0.0004 μg/ml/g fresh matter) were the most in the first year (Table 8).

The number of branches (14 in per stem), the number of inflorescences (10.18 in per plant), stem yield (2545.2 kg/ha), inflorescence yield (588.4 kg/ha), shoot yield (5656.88) kg/ha), the percentage of leaf essential oil (2.48%), the percentage of shoot essential oil (1.87%) and the amount of chlorophyll b (0.00016 micrograms/ml/gram of fresh material) were higher in the second year compared to the first year (Table) 8).

Mean comparison of two years showed that menthofurane (3.81%), α-terpineol (0.46%), neo-menthyl acetate (0.6%) and menthyl acetate (9.77%) in the second year was more than the first year.

Total nitrogen (12.4 g/kg), potassium (10.33 g/kg), calcium (19.12 g/kg), magnesium (3.25 g/kg), iron (45.63 mg/g), Manganese (22.16 mg/g), zinc (1.44 mg/g), cadmium (0.19 mg/g) and lead (0.13 mg/g) in the first year were higher than the second year (Table 9).

The results of mean comparison of different peppermint ecotypes in two years (Table 10) showed that the highest plant height with 71.33 cm belonged to Markazi province. The longest inflorescence length (9.08 cm) was the Zanjan variety. Golestan (20.83 numbers per plant) and Zanjan (20.16 numbers per plant) ecotypes had the highest number of branches. The longest leaf length (5.31 cm) belonged to the Markazi. The highest number of flowers with 13.66 belonged to the Gorgan ecotype, although it was statistically in the same group as the Kerman and Zanjan. The highest stem yield with 3116 kg per hectare belonged to Gilan ecotype. The highest flower yield was observed in the Golestan ecotype with 980 kg/ha. The highest shoot yield with 6559 kg/ha was observed in the Markazi Province. The highest percentage of leaf essential oil with 2.95% belonged to East Azerbaijan. The Markazi ecotype had the highest percentage of shoot essential oil (2.2%). Maximum phenol was owned by Kerman ecotype. Alborz province extract had the lowest flavonoid and the highest amount of chlorophyll a and total chlorophyll. The highest amount of carotenoid belonged to the Qom province.

The mean comparison of the essential oil composition of different extracts of peppermint (Table 11) showed that the highest amount of limonene with 7.58% belonged to Kerman. Hamedan and Golestan had the highest amount of 1,8-cineole. The most menthone with 23.7% belonged to Mazandaran. The most iso-menthone with 17.59% and menthofurane with 5.5% belonged to Qom. The highest amount of menthol was in Kermanshah with 59%. The maximum menthyl acetate with 12.84% belonged to a Golestan.

Mean comparing the elements absorbed by different ecotypes (Table 12) showed that the maximum total nitrogen absorbed with 16.28 g/kg belonged to Kermanshah accession. The maximum calcium and magnesium with 33.16 and 7.3 g/kg, respectively, were related to Hamadan. The most manganese and zinc were observed with 28.2 and 2.39 mg/g respectively in Zanjan.

The mean comparison of interaction effect of year * ecotype on different traits of peppermint (Table 13) showed that the largest stem diameter with 1.2 cm was related to Kerman ecotype in 2021. Hamedan ecotype had the highest leaf yield with 3386.7 and 3333.3 kg per hectare in 2022 and 2021, respectively. The maximum production of β-pinene with 1.89% was achieved in Alborz province in the second year.

Examining the correlation results (Table 14) showed that plant height has a significant positive correlation with leaf length and width, inflorescence diameter, stem yield, leaf yield, chlorophyll content and it had a significant negative correlation with the branches number, stem diameter, inflorescence number, inflorescence yield and flavonoids. Stem yield showed significant positive correlation with plant height, inflorescence length, leaf width, and significant negative correlation with stem diameter and number of inflorescences. Leaf yield showed a significant positive correlation with plant height, leaf length and width and a significant negative correlation with stem diameter and number of inflorescences. Inflorescence yield had a significant positive correlation with the number of branches, stem diameter, number of inflorescences and a significant negative correlation with plant height, leaf width and carotenoid.

A positive correlation was observed between the shoot yield with inflorescence length, number of branches, leaf width, stem and leaf yield, inflorescence yield, and a significant negative correlation was observed with stem diameter and carotenoid. A significant positive correlation was observed between phenol with flavonoid and carotenoid, and a significant negative correlation was observed with total chlorophyll. There was a significant negative correlation between flavonoid and chlorophyll a, b and total. A significant positive correlation was observed between carotenoid and chlorophyll b.

The correlation results of essential oil compounds (Table 15) showed that menthone had a significant positive correlation with menthofurane, γ-terpineol, α-terpineol and a significant negative correlation with α-pinene, sabinene, iso-menthone, pulegone, neo-menthyl acetate and menthyl acetate. Menthol had a significant positive correlation with γ-terpineol and α-terpineol, and also had a significant negative correlation with iso-menthone and neo-menthyl acetate.

Correlation results of absorbed elements (Table 16) showed that nitrogen had a significant negative correlation with phosphorus, magnesium, copper, chromium and nickel. Potassium had a significant positive correlation with iron, manganese, and copper and a significant negative correlation with cadmium. Calcium showed a significant positive correlation with magnesium, copper, cadmium and lead. Magnesium had a significant positive correlation with copper, cadmium, lead, chromium and nickel. Also, magnesium showed a significant negative correlation with zinc. Iron showed a significant positive relationship with manganese. Manganese had a significant positive correlation with zinc and copper. Zinc showed a significant negative correlation with lead. Cadmium showed a significant positive correlation with lead, chromium and nickel. Lead had a significant positive relationship with chromium and nickel, and a significant positive correlation was observed between chromium and nickel.

Examining the amount of non-similar essential oil compounds of different ecotype of peppermint in 2021, showed that among the non-similar essential oil compounds of the extracts (Table 17), p-cymene of Gilan with 8.3%, iso-menthyl acetate of Mazandaran with 2.29%, E-caryophllene of Kermanshah (2.05%) and Arak (2.67%) were significant.

The average composition of different essential oils of different ecotypes in 2022 showed that p-cymene was the highest in Gilan with 11.6%. E-caryophllene from Qom with 2.21%, and Arak with 3.31%, Jermacrene D from Arak with 2.59% were significant and the amount of other compounds was very low (Table 18).

The result of cluster analysis (Dendogram 1) showed that 11 ecotypes were placed in three main clusters. Ecotype 1 (Qom), 9 (Alborz) and 10 (Kerman) were placed in one cluster. Ecotypes 2 (Hamadan) and 7 (Arak) are in a separate cluster, as well as ecotypes 3 (Ardebil), 8 (Gilan), 4 (Gorgan), 11 (Zanjan), 5 (Mazandaran) and 6 (Kermanshah) are in a separate cluster.

The existence of a significant difference between the years of the experiment and different ecotypes (Tables 4, 5 and 6) indicates the appropriate choice of the research topic and selected traits for evaluation. Absence of significant difference between the interaction of year and ecotype in important traits, indicating the proximity of environmental factors, especially the average temperature and relative humidity in the two years of the experiment (Table 3) and the lower impact of environmental factors than genetic factors in the control and incidence of some traits And also the suitability in the process of changes of traits among different ecotypes in two years of testing. Also, the existence of a significant difference in the morphological traits and the yield of different organs between the years of the experiment can be caused by the age of the plants, their complete establishment, the start of vegetative growth as soon as the weather conditions become favorable in the second year (increasing the length of the growth period) and as a result, increasing the number of components performance such as the number of lateral stems and the number of inflorescences, etc. (Table 8).

The existence of a significant difference between the years of the experiment in the morphological traits of mint types has also been reported in previous researches (Abaszadeh, 2014; Abaszadeh et al., 2014). The higher of some morphological traits such as plant height, leaf length and width (Table 5) in the first year can be due to the suitable growth conditions of the plant, especially the ground is not firm due to the substrate preparation operation and the lack of compression of the roots and the availability of elements at the disposal of the plant. So that the high amount of most elements absorbed in the first year (Table 9), the high amount of chlorophyll a and the total and low percentage of essential oil (Table 9) confirm this issue. The high amount of carotenoid can be caused by the high transpiration of the plant due to the high surface of the leaf and the low volume of sukers compared to the second year.

The high level of heavy metal elements in the first year indicates the contamination of rhizomes transferred from different provinces, and its decrease in the second year confirms the removal of part of the heavy metals in the rhizomes and its reduction compared to the first year, so peppermint can be considered as one introduced plants that absorb and transfer heavy metals and thus clean up polluted lands (Grejtovsky and Piri, 2000; Chizzola, 2005; Grejtovsky et al., 2006; Salamon et l., 2007).

According to the results of Tables 10 and 12, it does not seem that the presence of heavy metals in the peppermint plant causes a decrease in yield and an increase in the percentage of essential oil, and the results of this part of the research with the results of (Shahid et al., 2017; Sinha and Saxena, 2006; Tirillini et al., 2006; Asgari Lajayer et al., 2017) showed a mismatch.

The absence of significant differences in most of the essential oil compounds in the two years of the experiment (Table 6) can indicate the greater effect of the genetic factor in the process of producing and converting the essential oil in the peppermint and also the closeness of the climatic parameters in the two years of the experiment (Table 3).

Also, the presence of high menthol compared to menthone (Table 11) among the essential oil compounds can be caused by the environmental temperature conditions, because it was observed in the research that in mild and cool climates, the amount of menthol production was higher than in hot areas (Telci et al., 2010).

It was observed that there was a statistically significant difference between different ecotypes in morphological traits, essential oil compounds, physiological traits and macro, micro and heavy metal elements (Tables 4, 5 and 6) and the results related to morphological traits, yield of different organs, percentage And the yield of the essential oil is also similar to the reports (Abaszadeh, 2014; Abaszadeh et al., 2014).

Examining the mean of different ecotypes of peppermint in two years (Table 10) showed that in terms of plant height, the ecotype of Markazi Province was higher than previous reports (Abaszadeh et al., 2014; Ostadi et al., 2020). The number of branches was in the range reported by Ostadi et al. (2020). In terms of the shoot yield, the production rate of the examined ecotypes is higher than the ecotypes reported in Iran by Ostadi et al. (2020) and Abbaszadeh et al. (2014) and it is lower than the amount reported by Zheljazkov et al. (2009).

The main components of essential oils of plants dried in the shade that has been reported are menthol (C10H20O) (Gavahian et al., 2015), menthone (C10H18O) (Stanfill et al., 2003), polgon (C10H16O) (Stanfill et al., 2003) and methyl acetate (Croteau et al., 2005), in our research also, menthone, iso-menthone, menthofurane, γ-terpineol, menthol, pulegone and menthyl acetate formed the main components of essential oil.

Although in terms of the percentage of compounds mentioned in this research, the percentage of menthone was lower than the values reported by Zheljazkov et al. (2009) and menthol was higher in all accessions, so it can be concluded that under suitable growth conditions for the aerial organs that result in having access to a lot of light, a lot of water (Khorasaninejad et al., 2011) and a lot of nutrition (Ostadi et al., 2020), the percentage of menthol is reduced and vice versa in optimal growth conditions for light and temperature, (Duriyaprapan et al., 1986; Salehi etal., 2018), enough water, enough food (Rios-Estepa et al., 2008), in addition to the proper growth of aerial organs, the percentage of essential oil and the percentage of menthol will increase.

Considering that peppermint essential oil has a global standard under ISO 856 (Anynames, 2006) and according to the European Scientific Cooperative on Phytotherapy standard, the amount of menthol is the main criterion in determining the quality of peppermint (Kumar et al., 2004), therefore, in choosing optimal growth conditions and accessions, menthol attribute will be the main indicator in terms of percentage and yield per hectare. Considering this issue, it can be said that East Azerbaijan ecotype with more than 42% of menthol (Table 11), high shoot yield, high percentage and yield of essential oil (Table 10) is one of the suitable ecotypes for agricultural and breeding. Also, Arak ecotype was ranked first among accessions with the highest percentage (2.2%) and essential oil yield (144 kg per hectare), and in terms of menthol percentage, it was 20% less than Kermanshah ecotype.

It was observed that the maximum total nitrogen absorbed with 16.28 g/kg belonged to the Kermanshah (Table 12), so it can be concluded that taking into account the yield of the shoot and root of the plant in each year, the need of this plant for nitrogen element is about It is 100 kg per hectare. Considering that phosphorus is one of the essential elements for plant growth and development and constitutes about 2% of the dry matter of plants (Toth et al., 2014) and this element is involved in biochemical processes, energy production and signal transmission in plant cells (Azziz et al., 2012; Tak et al., 2012), so evaluating its absorption rate is of great importance.

It was observed that the maximum absorption of phosphorus was 3.73 g/kg in Qom (Table 12). It has been reported that the increase of phosphorus increased the shoot yield, parasimen and polgon compounds of Mentha arvensis L, and on the other hand, it decreased the percentage of menthol (Souza et al., 2014). Phosphorus deficiency decreased plant growth and yield (Marschner, 1985). Potassium is another macro element that plays a high physiological and biochemical role in plant cells in regulating cell osmosis and enzyme activity (Marschner, 1995). Also, potassium plays a role in the regulation of p-transferase, acetate-kinase, and acetyl-thiokinase enzymes, it is involved in increasing the synthesis of Rubisco (ribulose-bis-p-carboxylase) and increasing ATPase activity (Marschner, 1995; Mengel and Kirkby, 1987). The results of our investigation showed that the maximum absorption of potassium was 11.33 g/kg in the Kerman extension, taking into account shoot yield in this extension, its absorption rate was about 54 kg per hectare.

In the research, it was reported that the increase in potassium decreased the absorption of magnesium, iron, boron and molybdenum, and on the contrary, it increased the absorption of zinc, copper and manganese (Wilkinson, 1994). According to Table 12, in most of the accessions, the need for calcium was slightly more than nitrogen or equal to it, and the reason for its increase in plants can be attributed to the role of this element in strengthening the cell wall and the plant's erectness, as well as its immobility and the need Young organs are related to its absorption through the roots. Also, in Hamedan, the amount of magnesium absorption was 7.3 g/kg, which, taking into account the shoot yield, the plant's requirement per hectare is more than 50 kg. In the Italian Pharmacopoeia, the permissible limits for lead and cadmium are 3 and 0.5 mg/kg, respectively (Kosalec et al., 2009). Therefore, according to the results of Table 9, the examined ecotypes are within the permissible limits in terms of cadmium and lead (Kabata-Pendias, 2000). Therefore, it can be concluded that the examined ecotypes have suitable conditions in terms of micro metals, and the lower content of some heavy metals such as lead, cadmium, and zinc in the second year can be due to absorption in the first year and the reduction of its amount in the soil in the second year. The increase in leaf yield in the second year (Table 13) can be caused by the increase in the number of secondary stems.

In Table 14, a significant positive correlation was observed between the shoot yield with length of inflorescence, number of branches, leaf width, yield of stem and leaf, yield of inflorescence, therefore, the increase of any of the above traits as well as any factor that causes their increase can cause Increase yield. The results of this part of the research with the observations of Abbaszadeh (2014) and Abbaszadeh et al. (2014) was consistent. The existence of a positive correlation between phenol with flavonoid and carotenoid is due to the fact that these compounds generally increase their production in unfavorable environmental conditions and decrease in favorable plant growth conditions, as well as the existence of a negative correlation between flavonoid and chlorophyll a, b and total The confirmation is that in unfavorable growth conditions, the amount of chlorophyll is reduced and the production of phenolic compounds is increased, and this result can be used in the production of effective substances in medicinal plants. The absence of a significant correlation between menthone and menthol (Table 15) indicates a very important and important point in choosing the best ecotypes as well as correction methods in peppermint. The table of correlation results of elements (Table 16) can be used well in managing the consumption of fertilizers and choosing the type of fertilizer, as well as in choosing the land where the plant is grown. The presence of a negative relationship between zinc and lead or the presence of a positive relationship between cadmium and lead, chromium and nickel indicates the absorption of these elements in the plant, and it is possible to prevent the absorption of others by adding some of them to the soil.

The result of the cluster analysis (Dendogram 1) showed that the 11 investigated ecotypes have high diversity, also it seems that the weather conditions were effective in the emergence and transfer of traits and their placement in different clusters .For example, the ecotypes of the relatively dry provinces of Qom, Alborz and Kerman were placed in one group, or the neighboring and similar climatic provinces of Hamedan and Arak in another group, as well as the moderately cold and humid provinces such as Ardabil, Gilan, Gorgan, Mazandaran, Zanjan and Kermanshah were placed in a separate group. Therefore, by using cluster results and all morphological, physiological, essential oil compounds and elements results, it is possible to select the appropriate ecotypes for various agricultural purposes, industrial medicine (use of essential oil) or direct consumption.

In general, it was observed that the differences between the two years were caused by soil factors, the age of the plants, the increase in the length of the growth period, and the increase in yield components in the second year compared to the first year. It was found that some morphological traits such as plant height, leaf length and width in the first year were higher due to the suitable growth conditions of the plant, especially the ground was not firm due to the substrate preparation operation and the lack of compression of the roots and the availability of elements at the disposal of the plant.

The high level of heavy metal elements in the first year indicated the contamination of rhizomes transferred from different provinces, and its decrease in the second year confirmed the removal of part of the heavy metals in the rhizomes and its reduction compared to the first year. Also, peppermint was introduced as one of the plants that absorbs and transports heavy metals and thus cleans polluted lands. The absence of significant differences in most of the essential oil compounds in the two years of the experiment indicates the greater effect of the genetic factor in the process of the production and transformation of the essential oil in the peppermint plant, and also the closeness of the climatic parameters in the two years of the experiment. Also, the presence of high menthol compared to menthone among the essential oil compounds was detected due to environmental temperature conditions.

It was observed that there was a statistically significant difference in morphological traits, essential oil compounds, physiological traits and macro, micro and heavy metal elements among different accessions. The main components of the essential oil were menthone, iso-menthone, menthofurane, γ-terpineol, menthol, pulegone and menthyl acetate, and the extracts studied had a much higher percentage of menthol than other countries. Therefore, in the selection of optimal growth conditions and accessions, menthol attribute was reported as the main indicator in terms of percentage and yield per hectare. Considering the large difference in shoot yields, the percentage and yield of essential oil, the amount of macro elements and heavy metals, a preliminary study before the development of ecotypes cultivation and the selection of the desired ecotypes for the aforementioned traits in order to be optimally used in the food, pharmaceutical, cosmetic, and health industries It was recognized as very important and necessary. The fertilizer requirement of this plant for macro and micro fertilizers was presented practically, and each of the producers can have a good estimate of the amount of fertilizer needed to be added to the farm by having the soil analysis in hand. So that the nitrogen requirement of this plant is about 100 kg per hectare in a year, its phosphorus requirement is about 20 kg. Due to the non-correlation of the main compounds of menthol and menthone, it is possible to manage according to crop and species in choosing the type of treatments, excisions and also correction methods to produce plants with the desired essential oil composition. It also seems that weather conditions have been effective in the emergence and transmission of traits and their placement in different clusters. For example, the relatively dry provinces of Qom, Alborz and Kerman were placed in one group, or the neighboring and similar climatic provinces of Hamedan and Arak were in another group, as well as cold and humid temperate provinces such as Ardabil, Gilan, Gorgan, Mazandaran, Zanjan and Kermanshah were placed in a separate group. Therefore, by using cluster results and all morphological, physiological results, essential oil compounds and elements, it is possible to select the appropriate ecotypes for various agricultural purposes, industrial medicine (use of essential oil) or direct consumption.

Conflicts of Interest:

We declare that this manuscript is original, has not been published and is not under consideration for publication elsewhere and there are no conflicts of interest to disclose.

Author Contribution

MRS collected the data (field and lab works), data analysis and writing the manuscript. BA performed the project administration, investigation, conceptualization, formal analysis and methodology. MRA field works and critical reviewing of the manuscript. DH contributed in lab works and phytochemical analysis. MNl contributed in field works. All authors reviewed the manuscript.

Acknowledgement

The authors would like to acknowledge all technicians who actively supported the field and lab works from the Islamic Azad University of Karaj Branch and the Research Institute of Forests and Rangelands.

Data Availability Statement:

The authors confirm that most of the data supporting the findings of this study are available within the article. If raw data or other information is needed, it can be accessed by requesting the corresponding author [B. A].

Abaszadeh B, Rezaiee MB, Paknejad P (2011) Evaluation relationship between essential oil yield and some agriculture characters by using of path analysis of two ecotypes of Mentha longifolia (L.) Huds. Var. amphilema L. Iranian Journal of Medicinal and Aromatic Plants (IJMAPR) 2011. 27:1:36–46.
Abaszadeh B, Rezaiee MB, Layegh Haghighi M (2014) The study of morphological characteristics and composition of the essential oil of two ecotypes of Mentha aquatica L. J Med Plants 11:8:249–257.
Abbaszadeh B (2014) Determining the relation of traits in two Mentha spitaca L. Populations from Iran, by path and principal component analysis. J Essent Oil-Bear 17(1). 42–48.
Anynames (2006) ISO 856, Oil of peppermint (Mentha piperita L.) was prepared by Technical Committee ISO/TC 54, Essential oils.
Asgari Lajayer H, Savaghebi GH, Hadian J, Hatami M, Pezhmanmehr M (2018) Comparison of copper and zinc effects on growth, micro-and macronutrients status and essential oil constituents in pennyroyal (Mentha pulegium L.). Braz J Bot. 40 (2): 379–388.
Azziz G, Bajsa N, Haghjou T, Taulé C, Valverde A, Igual J, et al (2012) Abundance, diversity and prospecting of culturable phosphate solubilizing bacteria on soils under crop–pasture rotations in a no-tillage regime in Uruguay. Appl Soil Ecol 61: 320–326.
Bahraminejad S, Asenntorfer RE, Riley IT, Zwer P, Schultz CJ., Schmidt O (2008) Genetic variation of flavonoid defense compound concentration in oat (Avena sativa L.) entries and testing of their biological activity. Australasian Plant Breeding Conference, Christchurch, New Zealand. 18 – 21, pp: 1127–1132.
Bugaenko LA, Rezinkova SA (1980) Cytological stud of interspecific mint hybrids. Tsitologiya igenetika. 14 (2), 23–6.
Chizzola R (2005) Cadmium and micronutrient accumulation in yarrow. Phyton. 45: 159–171.
Croteau RB, Davis EM, Ringer KL, Wildung MR (2005) Menthol biosynthesis and molecular genetics. Naturwissenschaften. 92(12): 562–577.
Duriyaprapan S, Britten E, Basford K (1986) The effect of temperature on growth, oil yield and oil quality of japanese mint. Ann Bot. 58: 729–736.
Dwivedi S, Khan M, Srivastava SK, Syamasunnder KV, Srivastava A (2004) Essential oil composition of different accessions of Mentha piperita L. grown on the northern plains of India Flavour Frag J 19: 437–440.
Foster, S. Peppermint. Menta pipeeita.American Botanical council – Botantical Series 1996. 306:3.
Gavahian M, Farahnaky A, Farhoosh R, Javidnia K, Shahidi F (2015) Extraction of essential oils from Mentha piperita using advanced techniques: Microwave versus ohmic assisted hydrodistillation. Food Bioprod Process. 94: 50–58.
Grejtovsky A, Markusova K, Eliasova A (2006) The response of chamomile (Matricaria chamomilla L.) plants to soil zinc supply. Plant Soil Environ. 52: 1–7.
Grejtovsky A, Piri R (2000) Effect of high cadmium concentrations in soil on growth, uptake of nutrients and some heavy metals of Chamomilla recutita (L.) Rauschert. J Appl Bot 74: 169–174.
Gulluce M, Shain F, Sokmen M, Ozer H, Daferera D, Sokmen A, Polissiou M, Adiguzel A, Ozcan H (2007) Antimicrobial and antioxidant properties of the essential oils and methanol extract from Mentha longifolia L. spp. longifolia. Food Chem. 103: 1449–1456.
Jesus DL (2000) Effect of artificial polyploidy in transformed roots of Artemisia annua L. A thesis in Biotechnology,of Woreester polytechnic Institute, for the Degree of Master of Science in Biotechnology, 111p.
Kabata-Pendias A (2000) Trace Elements in Soils and Plants, Third ed. CRC Press, Boca Raton, pp. 432.
Khalili F, Aghayari F, Ardakani MR (2020) Effect of alternate furrow irrigation on maize productivity in interaction with different irrigation regimes and biochar amendment. Commun Soil Sci Plant Anal 51: 757–768.
Khan MSA, Ahmad I (2019) Chap. 1, 3–13. Herbal Medicine: Current Trends and Future Prospects in Advancements in Herbal Products as Novel Drug Leads; Khan, M.S.A., Ahmad, I., Chattopadhyay, D., Eds.; Academic Press: Cambridge, MA, USA.
Khorasaninejad S, Mousavi A, Soltanloo H, Hemmati K, Khalighi A (2011) The effect of drought stress on growth parameters, essential oil yield and constituent of Peppermint (Mentha piperita L.). J Med Plant Res. 5(22): 5360–5365.
Kosalec I, Cvek J, Tomić S (2009) Contaminants of medicinal plants herbs and herbal products. Arh Hig Rada Toksikol. 350–380.
Kumar A, Samarth RM, Yasmeen S (2004) Anticancer and radio protective potentials of Mentha piperita L. Bio Factors. 22: 87–91.
Lorenzi H, Matos FJA (2002) Plantas medicinais do Brasil: nativas e exóticas. Instituto Plantarum,Nova Odessa.
Lutkov AN, Butgakov SV, Bejaeva RG (1966) Allopolyploidy and inter specific hybridization of peppermints (Mentha piperita L.) Exp Polyploidy Plant Breed. 172 – 85.
Mamadalieva NZ, Akramov DK, Ovidi E, Tiezzi A, Nahar L, Azimova SS, Sarker SD (2017) Aromatic medicinal plants of the Lamiaceae family from Uzbekistan: Ethnopharmacology, essential oils composition, and biological activities. Medicines 4, 8.
Marschner H (1995) Mineral Nutrition of Higher Plants (2"J i:dn). Academic Press, Harcoun Press and Co., London. 889.
Mengel K, Kirkby EA (1987) Principles of plant nutrition. 4th Ed. International Potash, Institute Switzerland.
Moleyar V, Narasimham P (1992) Antibacterial activity of essential oilcomponents. Int J of Food Microbiology. 16:337–342.
Mucciarelli M, Camusso W, Bertea CM, Bossi S, Maffei M (2001) Effect of (+)-pulegone and other oil components of Mentha × piperita on cucumber respiration. Phytoche. 57: 91–98.
Ostadi A, Javanmard A, Amani Machiani M, Morshedloo MR, Nouraein M, Rasouli F, Maggi F (2020) Effect of different fertilizer sources and harvesting time on the growth characteristics, nutrient uptakes, essential oil productivity and composition of Mentha piperita L. Ind Crops Prod. 148 112290.
Park KJ, Vohnikova Z, Reis Brod FP (2002) Evaluation of drying parameters and desorption isotherms of garden mint leaves (Mentha crispa L.). J Food Eng. 51:193–199.
Parv Nayak, Tankesh Kumar, Gupta AK, Joshi NU (2020) Peppermint a medicinal herb and treasure of health. J Pharmacogn Phytochem. 9(3): 1519–1528.
Pattnaik S, Subramanyam VR, Kole C (1996) Antibacterial and antifungal activity of ten essential oils in vitro. Microbios. 86:237–46.
Prakash O, Chandra M, Pant AK, Rawat DS (2016) Mint (Mentha spicata L.) Oils; Elsevier: Amsterdam, The Netherlands.
Purohit SS, Vyas SP (2004) Marketing of medicinal and aromatic plants in Rajasthan. In Marketing of Medicinal and Aromatic Plants in Rajasthan, National Consultative Workshop on Medicinal Plants; GBPUAT: Pantnagar, India.
Rios-Estepa R, Turner GW, Lee JM, Croteau RB, Lange BM (2008) A systems biology approach identifies the biochemical mechanisms regulating monoterpenoid essential oil composition in peppermint. Proceedings of the National Academy of Sciences.105(8): 2818–2823.
Roodbari N, Roodbari S, Ganjali A, Ansarifar M (2013) The effect of salinity stress on growth parameters and essential oil percentage of peppermint (Mentha piperita L.). Int.J Basic Appl Sci.1: 294–299.
Salamon I, Labun P, Skoula M, Fabian M (2007) Cadmium, lead and nickel accumulation in chamomile plants grown on heavy metal enriched soil. Acta Hort. 749: 231–236.
Salehi B, Stojanovi´c-Radi´C, Mateji´C, Sharopov J (2018) Plants of Genus Mentha: From Farm to Food Factory. Plants. 7: 70.
Shahid M, Dumat C, Khalid S, Schreck E, Xiong T, Niazi NK (2017) Foliar heavy metal uptake, toxicity and detoxification in plants: a comparison of foliar and root metal uptake. J Hazard Mater. 325: 36–58.
Sharifi-Rad J, Tayeboon GS, Niknam F, Sharifi-Rad M, Mohajeri M, Salehi B, Iriti M (2018) Veronica persica poir. Extract-antibacterial, antifungal and scolicidal activities, and inhibitory potential on acetylcholinesterase, tyrosinase, lipoxygenase and xanthine oxidase. Cell Mol Biol (Noisy-le-Grand France). 64: 50–56.
Simões CMO, Spitzer V, Óleos V (2000) In - Farmacognosia da planta ao medicamento, C. M. O. Simões et al. 2 ed. Universidade Federal do Rio Grande do Sul, Porto Alegre and Universidade Federal de Santa Catarina, Florianópolis. pp. 394–412.
Sinha S, Saxena R (2006) Effect of iron on lipid peroxidation, and enzymatic and nonenzymatic antioxidants and bacoside-A content in medicinal plant Bacopa monnieri L. Chemosphere. 62 (8): 1340–1350.
Souza MAA, Araujo OJL, Brito DMC, Fernandes MS, Castro RN, Souza SR (2014) Chemical composition of the essential oil and nitrogen metabolism of menthol mint under different phosphorus levels. Am J Plant Sci. 5: 2312–2322.
Stanfill SB, Calafat AM, Brown CR, Polzin GM, Chiang JM, Watson CH, et al (2003) Concentrations of nine alkenyl benzenes, coumarin, piperonal and pulegone in Indian bidi cigarette tobacco. Food Chem Toxicol. 41:303–17.
Tak HI, Ahmad F, Babalola OO, Inam A (2012) Growth, photosynthesis and yield of chickpea as influenced by urban wastewater and different levels of phosphorus. Int J Plant Res. 2: 6–13.
Telci I, Demirtas I, Bayram E, Arabac O, Kacar O (2010) Environmental variation on aroma components of pulegone/piperitone rich spearmint (Mentha spicata L.). Ind Crops Prod. 32: 588–592.
Tetik F, Civelek S, Cakilcioglu U (2013) Traditional uses of some medicinal plants in Malatya (Turkey). J Ethnopharmacol. 146: 331–346.
Tirillini B, Ricci A, Pintore G, Chessa M, Sighinolfi S (2006) Induction of hypericins in Hypericum perforatum L. in response to chromium. Fitoterapia. 77 (3): 164–170.
Toth G, Guicharnaud RA, Toth B, Hermann T (2014) Phosphorus levels in croplands of the European Union with implications for P fertilizer use. Eur J Agron. 55, 42–52.
Wilkinson RE (1994) Plant: environment interactions. Marcel Dekker, New York.
World Health Organization (2007) WHO Guidelines for assessing quality of herbal medicines with reference to contaminants and residues. WHO Publishers: Geneva.
Zakikhani H, Ardakani MR, Rejali F, Gholamhoseini M, Khodaei Joghan A, Dolatabadian A (2012) Influence of diazotrophic bacteria on antioxidant enzymes and some biochemical characteristics of soybean subjected to water stress. J Integr Agric 11(11): 1828–1835
Zheljazkov VD, Cerven V, Cantrel CL, Ebelhar WM, Horgan T (2009) Effect of nitrogen, location, and harvesting stage on peppermint productivity, oil Content, and oil Composition. Hortscience. 44(5):1267–1270. 2009.

Tables 1 to 18 are available in the Supplementary Files section.

No competing interests reported.

Tables.docx

Download PDF

Version 1

posted

You are reading this latest preprint version

Morphophysiological and phytochemical properties of peppermint ecotypes: a comprehensive study in 11 provinces of Iran

Status:

Version 1

Abstract

Introduction

Materials and Methods

Statistical analysis

Results

Discussion

Conclusions

Declarations

Conflicts of Interest:

Author Contribution

Acknowledgement

Data Availability Statement:

References

Tables

Additional Declarations

Supplementary Files

Status:

Version 1