CULTIVATION OF PEPPERMINT (MENTHA PIPERITA RUBESCENS) USING WATER TREATED WITH LOW-PRESSURE, LOW-TEMPERATURE GLOW PLASMA OF LOW FREQUENCY
DOI:10.30825/5.EJPAU.76.2018.21.3

Elżbieta Pisulewska¹, Wojciech Ciesielski², Monika Jackowska³, Maciej Gąstoł³, Zdzisław Oszczęda⁴, Piotr Tomasik^4
1 Cracow College of Health Promotion, Cracow, Poland
² Institute of Chemistry, Environmental Protection and Biotechnology, Jan Długosz Academy, Częstochowa, Poland
³ Department of Fruit Growing and Bee-keeping, Cracow University of Agriculture, Poland
⁴ Nantes Nanotechnological Systems, Bolesławiec, Poland

ABSTRACT

Watering peppermint (Mentha piperita rubescens)cultivationwith water exposed to low-pressure, low-temperature glow plasma of low frequency (PW) for 150 days in a greenhouse stimulated the growth of leaves and stems by 24 and 5%, respectively. Application  of PW changed composition of extracts without any negative effect on their bactericidal properties. PW increased the content of chlorophylls, carotenoids and ascorbic acid, decreased the concentration of the Mn(II), Ni(II), Ca and Mg ions in leaves and stems, decreased the concentration of the Pb(II) and Fe(III) ions in leaves and increased their concentration in stems and had no influence of the concentration of the Zn,  Cu(II),  Cd, Cr(III) and Co(II) ions in both parts of the plant. Simultaneously, PW increased the level of the Na⁺ ions and had no influence on the level of the K⁺ ions in the leaves and stems.  PW considerably reduced the accumulation of sulfates, chlorides and nitrites, slightly reduced the level of phosphates and elevated the level of nitrate

Key words: bioaccumulation, essential oil, plant growth stimulation, plazmed water.

INTRODUCTION

Recently, Bialopiotrowicz et al. [3] reported the structure and selected physicochemical properties of water exposed to low-pressure and low-temperature glow plasma of low frequency (LPGP). The Raman spectra of that water were identical to the spectra of water subjected to a static magnetic field of induction ≈0.5 T.

It was shown that the magnetically treated water considerably stimulated the reproduction and pathogenicity of entomopathogenic fungi [13]. Thus, the effect of water treated with LPGP was tested [14]  with success on the same fungi, showing also that LPGP treated tap water performed better than distilled water. In this paper the effect of watering plants with LPGP treated water, called here plazmed water (PW) is presented. Since in herbs, apart from the stimulation of their growth, the resulting composition and yield of essential oils is also crucial the attention was paid to peppermint (Mentha piperita rubescens) growth and essential oil production.   

For its fragrance, biological and pharmacological properties of various species of peppermint are commonly used in many branches of industry. Recently, peppermints are considered as potential food preservatives, stabilizers and food quality improvers [1, 6–9, 12, 15, 17].

MATERIALS

Peppermint
Underground stolons of the plant placed into the experimental pots were  purchased in 2016 from the Napieralski Enterprise in Lodz, Poland.

Water
Distilled water or tap water from Boleslawiec with total hardness 129 mg/dm³ CaCO3, pH 7.1, conductivity 334 mS/cm, Fe < 50 mg/dm³, Mn < 5 mg/dm³ and dissolved oxygen 6.93 mg/dm³ were used.

Microorganisms
Escherichia  coli code PCM 2857 and Staphylococcus aureus code PCN 2602 were purchased in 2017  from Polish Collection of Microorganisms (Ludwik Hirszfeld Institute of  Immunology and Experimental Therapy  in Wroclaw, Poland).

METHODS

Peppermint plantation
The monofactorial experiment was carried out in the 2016/2017 break in a greenhouse at the University of Agriculture in Cracow  The greenhouse was set for 23°C  and automatic additional 16h illumination. Experiments were arranged on 24 November 2016 in six parallel series each containing 20 pots of 10 cm in diameter, filled with a universal horticulture soil for flowers purchased from Biovita, Krzeszowice at Cracow, Poland. In order to eliminate the parietal effect, all edges of each collection of the pots were surrounded with double row of pots with peppermint. Each row contained 10 pots. The watering was carried out always at 9:30 a.m. Three of six series of the pots were watered always at 9:30 a.m. with tap water and they were considered as control. The other three another series were watered with plasma treated water (PW). In total the watering was carried out 38 times consuming 395 dm³ of both tap water and PW.  When the plants reached 5–8 cm in height they were transferred into pots of 15 cm in diameter and watering was continued in the same regime and with the same volume of water. The experiment terminated on 24th April 2017 when the plants were collected and separated into leaves and stems. The plant was then dried at 105°C for 4 hours to determine dry mass of the crops.

Separation of essential oils
Sample of the plant dried at 35°C to constant weight (1 g) was steam distilled in a Deryng apparatus with a closed water circulation. The collected oil was  transferred to a closed vial then analyzed chromatographically.

Treating water with LPGP (PW)
Either distilled or tap water (1000 cm³) was placed in the chamber of the reactor [20] and exposed to plasma for 30 min. Plasma of 38°C was generated at 5x10^-3 mbar, 600 V, 50 mA and 280 GHz frequency. The produced water was stored at ambient temperature in 100 mL closed Teflon containers.

Gas chromatographic analyses
An Agilent 7890A gas chromatograph (Agilent Technologies, Inc., Santa Clara, Cf. USA) was equipped with a Supelcowax-10 30 cm x 0.32 mm x 0.25 mm column. An injector was maintained at 270°C. Initial temperature of 40°C was maintained for 1 min, then rose to 220°C with a rate of 4°C/min. Helium (0.5 cm³/min) was used as the gas. Analyzed sample mass ranged from 33 to 333Da. Temperature of the ion generator was maintained at 220°C. SPMR injections were performed in a splitless manner.   

Analyses for cations
Samples were mineralized in a microwave oven (MarsXpress CEM company). Samples (0.5 g) were digested with nitric acid 65% analytical grade (10 cm³). Determination of metals content was performed with atomic absorption spectrometry with electrothermal device (AA  Varian 240 instrument). A palladium standard solution (1000 mg/dm³ ) was used as a modifier.

Anion analyses with ion chromatography
A DX500 micropore (2 mm) ion chromatograph with a CD20 conductivity detector and GP40 gradient pump (Dionex, California) was used for ion separation and detection. Commercially available Ionpac CG12A guard and CS12A analytical columns (Dionex, California) with  carboxylic-phosphonic acid functional groups were used for cation analysis. Ionpac AG14 guard and AS14 analytical columns (Dionex, California) with quaternary ammonium functional groups were used for anion separation. Eluents were stored in vessels pressurized at 8 p.s.i. using high purity argon (BOC gases), and flow-rates were maintained at 0.45 ml/min for anions and 0.40 cm³/min for cations using a GP40 gradient pump (Dionex, California). Samples were loaded from an AS40 automated sampler (Dionex, California).

Determination of chlorophylls and carotenoids
Leaves of peppermint (200 mg) were homogenized for 2 min in a cooled mortar then homogenized for further an additional 2 min with the acetone/ammonia (0.05 mol/dm³) 8/2 blend (5 cm³) cooled to 0–5°C.  The extraction was continued for 2 more min. by addition of a subsequent 5 cm³ of extracting acetone/ammonia blend.  The resulting suspension of well disintegrated sample was transferred into 25 cm³ measuring cylinder, the mortar was washed with extracting blend (10 cm³) and the wash was combined with the extract. The extract was then centrifuged for 10 min at 5000 rpm, and decanted. The volume of the extract was increased to 25 cm³ by adding the extracting blend. The experiments were run in triplicates.   

The absorbance (A) of resulting extract was taken at 470, 647 and 664 nm. The content of chlorophylls a and b in mg/g was estimated from the formulas (1) and (2), respectively.  

chl.a = 25a/m

(1)

 where  a = 11.78 A664 – 2.29 A647

chl.b = 25b/m

(2)

where  b = 20.05 A647 – 4.77 A664

The content of carotenoids (b-carotene and xanthophyll) was calculated from formulas (3).

car.=  25c//229m

(3)

where  c = 1000 A470 – 3.27a – 104b

In Eqs. (1)–(3) m denotes the weight [mg] of the fresh plant material.

Determination of ascorbic acid
Sample of the dried plant (1 g) was disintegrated in a mortar 50 cm³ distilled water and 5 cm³ of 0.1M aqueous solution of potato starch added. This solution was titrated with a iodine solution following paper [2]. The estimations were triplicated.

Test for bactericidal properties of essential oils and extracts 
The Blanc method [4] was followed. Filter paper circles, f = 6 mm, were soaked for 1–2 min in  essential oil or extract (10 μl) and immediately transferred on inoculated plate which was then incubated for 24 h at 37 °C. Extract was prepared on 30 min. grounding of the plant material in a mortar (20 g) 96% ethanol (100 cm³) added  The diameter of the  area of suppressed growth was measured with a caliper. The experiments were run in triplicates. The results were statistically elaborated with the Excel 97 software. The tests were run in triplicates.

RESULTS AND DISCUSSION

One may see from Table 1 that watering peppermint with PW [3] only slightly stimulates the growth of the plant expressed in terms of the yield of dry plant. In case of leaves there was the 24% increase in the dried material whereas in case of stems that increase reached only 5%. The comparison of the crop yield expressed in grams  for plants watered with water and PW showed that there was reduced demand for watering when PW was used. The same volume of PW provided more dry crop than did tap water. The yield of essential oil from the plants watered with tap water and PW were practically identical.

Table 1. Crops of peppermint watered with non-treated water and PW

Cropsa [g]
Fresh						Dried
Total	Tap water		Total	PW		Total	Tap water		Total	PW
	Leaves	Stems		Leaves	Stems		Leaves	Stems		Leaves	Stems
1301.6	720.4	581.2	1199.2	589.2	609.3	594.8	269.4	325.4	678.4	334.6	341.8
a Total results from 60 pots.

The chromatograms showed that the stimulation of the growth of peppermint with PW was accompanied by essential changes in the composition of extracts from leaves (Tab. 2) and stems (Tab. 3).

Table 2. Retention time, area and height of major (≥ 100 pA x s) peaks in chromatograms of extracts of leaves of peppermint watered with PW and tap water

Tap water (control)				PW
Retention time [min]	Areaa [pA x s]	Heighta [pA]	Area [%]	Retention time [min]	Areaa [pA x s]	Heighta [pA]	Area [%]
2.09	100.81	55.34	0.41	2.08	232.96	106.85	1.53
3.07	137.66	55.90	0.56	3.06	104.70	39.82	0.69
3.30	177.12	141.59	1.53	3.30	284.50	104.94	1.87
3.50	186.12	235.86	1.20	3.50	667.38	252.36	4.39
3.99	120.22	18.50	0.49	3.99	104.13	23.33	0.69
11.05	15300.0	2165.47	62.24	11.01	7212.31	1139.22	47.46
12.01	1158.47	195.47	4.21	12.02	921.75	153.11	0.07
14.55	550.34	81.83	2.24	14.57	315.08	46.13	2.07
16.12	557.07	39.34	2.26	16.14	210.37	19.77	1.38
				16.28	214.07	22.27	1.41
17.55	4602.51	492.03	18.71	17.56	3082.05	320.00	20.28
19.75	321.36	60.18	1.31	19.77	263.02	49.40	1.73
				43.83	151.11	8.54	0.99
				44.59	204.12	4.97	1.34
a In relative units

Table 3. Retention time, area and height of major (≥ 100 pA x s) peaks in chromatograms of extracts of stems of peppermint watered with tap water and PW

Tap water				PW
Retention time [min]	Areaa [pA x s]	Heighta [pA]	Area [%]	Retention time [min]	Areaa [pA x s]	Heighta [pA]	Area [%]
				3.53	223.79	67.25	4.74
				10.99	2037.39	126.38	43.19
				12.04	178.02	29.01	3.77
				14.59	109.19	16.02	2.31
				17.56	856.25	87.97	18.15
37.75	1330.70	10.06	4.94
39.48	1011.62	14.83	3.75
40.34	1216.61	20.30	4.51
42.56	3235.82	34.43	12.00
43.58	2207.22	41.83	8.19
44.68	3797.60	53.62	14.09	44.68	299.10	5.32	6.34
45.32	3076.40	54.44	11.41
46/15	1295.19	54.02	4.81
46.54	2860.17	54.49	6.90
47.27	2194.19	55.76	8.14
48.01	2755.88	54.98	10.22
48.45	146.76	53.60	2.77
49.14	2018.56	54.26	7.49
a In relative units

Table 2 collects data solely for the peaks areas of whose were higher from 100 pA x s. One could see that watering peppermint with PW resulted in a higher intensity of the peaks of the retention time of 2.08, 3.50 and 3.99 min. In contrast to that the peaks of the retention time of 3.07 min and from 17.55 min were considerably higher in the extracts from control plants, that is, those watered with tap, non-plazmed water. Particularly, the peak belonging to menthol, the major peak in chromatogram [1] at 11.05 min faced tremendous decrease in the extract from plant watered with PW. Instead, in that extract components with the retention time of 16.28 , 43.83 and 44.59 appeared solely in the extract from the plant watered with PW. 

Extracts from stems of the plant watered with PW contained numerous components of lower retention time from 3.53 to 17.56 min. Among them the peak at the retention time of 10.99 min likely belonged to menthol. The extracts were also rich in the components of the retention time from 37.75 to 49.14 min. Except relatively small amount of the component of the retention time of 44.68 min they were absent in the extracts from plant watered with tap water. 

The characterization of the components will be presented in a subsequent paper.

Essential oil from the plant watered with PW exhibited higher bactericidal properties against Escherichia coli and Staphylococcus aureus thanthe oil from control plant. The oil from stems was slightly more bactericidal than the oil from the leaves and these from PW watered plants were slightly more bactericidal. Always these oils performed better for S. aureus. Extracts were less bactericidal than the oil regardless their origin (Tab. 4).

Table 4. Diameters of the areas of growth of microorganisms [mm]

Microorganisms	Leaves				Stems
	Water		PW		Water		PW
	Oil	Extract	Oil	Extract	Oil	Extract	Oil	Extract
Escherichia coli	8±1	3±1	10±1	3±1	6±1	2±1	7±1	2±1
Staphylococcus aureus	6±1	3±1	7±1	3±1	3±1	1±1	4±1	1±1

Watering with PW influenced accumulation of metal ions in the plant (Tab. 5). It decreased the concentration of the Mn(II) and Ni(II) ions in leaves and stems, decreased the concentration of the Pb(II) and Fe(III) ions in leaves and increased their concentration in stems and had no influence of the concentration of the Zn, Cu(II), Cd, Cr(III) and Co(II) ions in both parts of the plant. Simultaneously, PW increased the level of the Na+ ions, had no influence on the level of the K+ ions and decreased the level of the Ca2+ and Mg2+ ions in leaves and stems. 

Table 5. Concentration of selected metal ions in leaves and stems of peppermint watered with PW and with untreated water (control)

Sample

Metal cation concentration [mg/g d.m]

Medium

Zn

Mn(II)

Cu(II)

Pb(II)

Ni(II)

Cd

Fe(III)

Cr(III)

Co(II)

Na

K

Ca

Mg

Leaves

Water

1.204 ±0.009

4.584 ±0.011

0.015 ±0.004

1.247 ±0.009

0.879 ±0.007

0.041 ±0.005

25.324 ±0.017

0.020 ±0.003

0.006 ±0.002

0.57 ±0.15

27.04 ±1.07

8.04 ±1.29

2.28 ±0.46

PW

1.208 ±0.004

4.311 ±0.008

0.016 ±0.005

1.113 ±0.007

0.776 ±0.004

0.031 ±0.007

24.431 ±0.005

0.017 ±0.004

0.008 ±0.003

0.90 ±0.06

27.11 ±0.38

2.49 ±0.16

1.01 ±0.03

Stems

Water

1.380 ±0.008

4.496 ±0.008

0.002 ±0.0007

1.064 ±0.004

0.706 ±0.004

0.033 ±0.004

22.952 ±0.008

0.016 ±0.004

0.006 ±0.001

0.57 ±0.07

25.80 ±1.15

7.26 ±0.35

2.25 ±0.10

PW

1.386 ±0.005

4.317 ±0.006

0.001 ±0.0008

1.113 ±0.003

0.657 ±0.006

0.023 ±0.007

24.612 ±0.008

0.016 ±0.003

0.007 ±0.002

0.78 ±0.02

26.33 ±1.25

1.79 ±0.28

0.91 ±0.05

Watering peppermint with PW influenced also the anion uptake (Tab. 6).  PW the most considerably reduced the accumulation of sulfates in leaves and stems. Also, the accumulation of chlorides in both parts of the plant after watering with PW significantly declined. At the same time, after watering with PW, the level of phosphates was relatively slightly reduced whereas the level of nitrates tremendously increased.

Table 6. Accumulation of anions in peppermint watered with PW and plazma untreated water (control)

Sample	Medium		Cl-	NO2-	NO3-	SO42-	PO43-
Leaves	Water	mg/L	269.07 ±24.43	0.07 ±0.13	47.34 ±5.81	138.50 ±5.64	9.36 ±0.89
		mg/g	13.45 ±1.02	0.00 ±0.00	8.37 ±0.09	6.92 ±0.28	0.47 ±0.04
	PW	mg/L	198.64 ±16.24	0.00 ±0.00	500.62 ±48.32	45.01 ±2.32	5.56 ±0.58
		mg/g	9.39 ±0.81	0.00 ±0.00	25.03 ±2.42	2.25 ±0.12	0.28 ±0.03
Stems	Water	mg/L	239.63 ±13.83	0.23 ±0.02	113.77 ±5.24	127.86 ±4.47	9.47 ±1.02
		mg/g	11.98 ±0.69	0.01 ±0.00	5.69 ±0.26	6.39 ±0.22	0.47 ±0.05
	PW	mg/L	165.57 ±7.85	0.04 ±0.02	349.72 ±12.64	52.17 ±3.65	6.96 ±0.26
		mg/g	8.28 ±0.39	0.00 ±0.00	17.49 ±0.63	2.61 ±0.18	0.35 ±0.01

Watering with PW reduced the content of chlorophyll a but simultaneously, stimulated synthesis of chlorophyll b to a such extent that the total chlorophyll content increased. At the same time,  syntheses of carotenoids and ascorbic acid were also stimulated (Tab. 7).

Table 7. Effect of watering peppermint with PW on the content of selected components in the plant leaves [mg/g d.m]

Medium	Chlorophyll			Carotenoids	Ascorbicacid
	a	b	total
Water	2.216 ±0.091	0.483 ±0.011	2.333 ±0.024	0.417 ±0.005	0.241 ±0.015
PW	1.948 ±0.038	0.640 ±0.021	2.788 ±0.055	0.463 ±0.003	0.348 ±0.024

Chlorophyll a synthesis is light stimulated whereas the synthesis of chlorophyll b proceeds in plants cultivated in the shadow [6]. The increase in the chlorophyll b in the peppermint watered with PW could be stimulated by the clathrates of singlet oxygen in PW quenching the near UV radiation at 195 and 230 nm [5].

Presented results show a positive role of PW [3]  in a slight enhancing the crop yield of peppermint cultivation. Although watering the plantations with PW reduced the content of menthol in the plant, the essential oil and extract from leaves and stems retains their bactericidal properties on the same level. Instead of components of essential oils content of which is reduced, watering with PW offers enhanced level of total chlorophyll, carotenoids and ascorbic acid. The watering with PW increased practical application of the plant stems which contained more essential oil including menthol than stems of the plant watered with tap water  (see Tab. 3).

In her studies Sitarska [22] checked the effect of watering with PW on dynamics of the growth of wheat. The watering had a little effect on the growth dynamics and the dry mass of the crops but clearly influenced the growth of the soil microorganisms depending on the soil fertility. The fertility provided more efficient stimulation of the microorganisms.

The present studies confirmed that watering plants with PW had a minor effect on the yield of its crop. Also the yield of the ethereal oil after watering with PW practically did not change. The change in the composition of the ethereal oil appeared the most beneficial effect of watering with PW. Menthol which is the principal component of the peppermint oil [1], especially when overdosed, shows several negative effects such as  abdominal pain, ataxia, atrial fibrillation, bradycardia, coma, dizziness, lethargy, nausea, skin rash, tremor, vomiting and vertigo [10, 16, 18, 19, 21, 24]. Also smoking common menthol containing cigarettes appeared hazardous [11, 23]. For that sake the finding that the watering peppermint with PW reduced the content of menthol and, simultaneously provided the oil of non-reduced bactericidal properties rationalized the use of PW in the peppermint cultivation. An increase in the yield of total chlorophyll and ascorbic acid also attracted attention.

CONCLUSIONS

Watering peppermint with water treated with low-pressure, low temperature glow plasma of low frequency (PW) stimulated the growth of the plant and reduced its demand for water. Simultaneously, the composition of essential oil and extracts from leaves and stems beneficially changed with no negative effect upon their bactericidal properties. Such watering increased the content of carotenoids, ascorbic acid and total chlorophyll with domination of chlorophyll b. Watering with PW influences accumulation of metal ions and their counterions in the plant.

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Accepted for print: 17.07.2018

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Elżbieta Pisulewska
Cracow College of Health Promotion, Cracow, Poland
Krowoderska 73
31-450 Kraków
Poland
email: elzbieta.pisulewska@gmail.com

Wojciech Ciesielski
Institute of Chemistry, Environmental Protection and Biotechnology, Jan Długosz Academy, Częstochowa, Poland
Armii Krajowej 21
42-200 Częstochowa
Poland
email: w.ciesielski@interia.pl

Monika Jackowska
Department of Fruit Growing and Bee-keeping, Cracow University of Agriculture, Poland
29 Listopada 48
31-425 Kraków
Poland
email: monikajackowska05@gmail.com

Maciej Gąstoł
Department of Fruit Growing and Bee-keeping, Cracow University of Agriculture, Poland
29 Listopada 48
31-425 Kraków
Poland
email: rogastol@ogr.ur.krakow.pl

Zdzisław Oszczęda
Nantes Nanotechnological Systems, Bolesławiec, Poland
Dolne Młyny 21
59-700 Bolesławiec
Poland
email: oszczeda@nantes.com.pl

Piotr Tomasik
Nantes Nanotechnological Systems, Bolesławiec, Poland
Dolne Młyny 21
59-700 Bolesławiec
Poland
email: rrtomasi@cyf-kr.edu.pl

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Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.

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