Biochemical responses and volatile compounds in a peppermint chemotype grown in a controlled environment

doi:10.21203/rs.3.rs-2391173/v1

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Biochemical responses and volatile compounds in a peppermint chemotype grown in a controlled environment

https://doi.org/10.21203/rs.3.rs-2391173/v1

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Peppermint is a medicinal plant with great economic importance for its protective effects against biotic and abiotic factors. This study aimed to assess the vegetative growth, biochemistry and volatile production of peppermint plants under elicitation. Nodal segments were inoculated in flasks containing Murashige and Skoog medium with one of four treatments (50 µM salicylic acid, 200 mg L^− 1 chitosan, 25 µM copper sulphate, and control) and maintained for 90 days in a controlled environment. Copper treatment increased shoot growth by 43% and 68% compared with salicylic acid and chitosan, respectively. Furthermore, copper elicitation reduced the oxidation rate to only 13% and produced plants with better architecture. Salicylic acid and chitosan treatments increased the total phenolic content by 38% and 40%, respectively, compared with the control. The ferric reducing assay showed that salicylic acid and chitosan treatments increased the plant’s antioxidant activity by 82% and 96%, respectively, compared with the control. However, β-carotene, flavonoids and anthocyanins decreased with these treatments. Phenylalanine ammonia-lyase activity increased by 63% and 54% in shoots elicited with salicylic acid and chitosan, respectively. Elicitor treatment increased the number of volatile compounds detected (control = 29, salicylic acid = 32, copper = 37, chitosan = 38). Elicitation promoted significant changes in plant metabolism and chemical composition, evidenced mainly by differences in the levels of 2-hydroxy-3-(3-methyl-2-butenyl)-3-cyclopenten-1-one, d-limonene, eucalyptol, caryophyllene and l-alanine ethylamide. Monoterpene hydrocarbons were the major class in control shoots, whereas oxygenated monoterpenes were the major compounds in elicited shoots.

Mentha × piperita L.

Oxygenated monoterpenes

Secondary metabolite

Tissue culture

Medicinal plants are excellent producers of secondary compounds, a considerable fraction of which is found in essential oils (D'Amelia et al. 2021). Such compounds have a wide range of applications in health products and nutraceutical foods (Juárez et al. 2017). Changes in consumption patterns in recent years have motivated the industry to develop raw materials and technologies with reduced environmental impact (Fejér et al. 2018). Studies have indicated a growing demand for essential oils from medicinal plants; however, this has not been met by a parallel increase in supply (Singh et al. 2020).

Peppermint (Mentha × piperita L.) is a plant species belonging to the family Lamiaceae and native to Europe (Balakrishnan 2015). Peppermint essential oil is rich in compounds widely used in the pharmaceutical, cosmetic and food industries (Balakrishnan 2015). Recent research has demonstrated its pesticidal and biocidal action, suggesting its great potential for use in agriculture as an alternative to chemical pesticides (Jamiołkowska 2020). Essential oils from species of the genus Mentha L. are the second most valued by the market, after those of citrus plants (Barbieri and Borsotto 2018). According to Yu et al. (2018), the genus Mentha has seven chemotypes and shows great variations in chemical composition between species and cultivars owing to the high rates of interspecific hybridisation. The peppermint chemotype analysed in the current study is characterised by high concentrations of 2-hydroxy-3-(3-methyl-2-butenyl)-3-cyclopenten-1-one and l-alanine ethylamide.

According to Cardoso et al. (2019), a key strategy to induce and standardise the production of specific chemical compounds in plants is elicitation. Elicitors are substances that modulate plant defence genes and promote resistance to biotic and abiotic factors. This technology, when applied in combination with biotechnological methods such as tissue culture, has shown promise in the production of several plant species (Meena et al. 2022). In an in vitro environment, it is much easier to control and predict the production of specific compound classes, which can be extracted from calli, cell cultures and leaves (Fejér et al. 2018). It must be noted, however, that plants require an adequate culture medium and balanced concentrations of elicitors to fully develop under in vitro conditions, as any excess or deficiency may lead to toxic effects and physiological disorders (Montejo et al. 2021), resulting in oxidised, hyperhydric or abnormal shoots (Trettel et al. 2020). Therefore, it is of paramount importance to monitor the occurrence of these conditions during culture (Cardoso et al. 2019).

Among the abiotic elicitors, plant hormones and metal ions such as salicylic acid, chitosan and copper sulphate deserve special mention for their easy application, low cost and sustainability (Fejér et al. 2018; Siddaiah et al. 2018). Salicylic acid is a hormonal elicitor with multiple functions that result in increased defence against pathogens and enhanced growth (Gorni and Pacheco 2016). Chitosan is a polysaccharide produced by the deacetylation of chitin and is found in the exoskeleton of crustaceans (Malerba and Cerana 2016). Copper sulphate is a salt of mineral origin (chalcanthite) that serves as a source of copper, a cofactor of several enzymes in plant metabolism (Trettel et al. 2017).

These elicitors have been reported to be efficient in stimulating biochemical pathways related to biomass accumulation, essential oil production and chemical standardisation in the family Lamiaceae (Trettel et al. 2017; Choudhary et al. 2021; Nowak et al. 2021). For instance, Pedroso et al. (2019) reported a 16.56-fold increase in rutin production in Hyptis marrubioides Epl. grown in vitro in the presence of salicylic acid (30 µM). Another study found increased levels of phenolic acids, such as gallic acid, caffeic acid, benzoic acid and rosmarinic acid, in Salvia leriifolia Benth. treated with chitosan (50 mg L^− 1) (Jami et al. 2018).

Studies on the elicitation of peppermint have mainly focused on the effects of salicylic acid and chitosan on the chemical composition, oil yield and biocidal activity of plants grown under in vivo, greenhouse or field conditions (Chrysargyris et al. 2021). Studies on compound production using tissue culture are incipient and often inconclusive, with a lack of information on antioxidant activity, enzyme activity or primary metabolite production. Given that plant product quality is determined by physiological and biochemical processes, it is of great interest to producers to understand how elicitation interferes with the metabolism of peppermint physiology and biochemistry in a controlled environment (Salimgandomi and Shabrangi 2016). The types and concentrations of elicitors, together with temperature and photoperiod conditions, should be carefully adjusted to achieve good biomass yields and physiological parameters (Cardoso et al. 2019). In addition to plant growth, metabolic pathways of interest should be stimulated by elicitation, such as terpene biosynthesis and antioxidant compound production (Salimgandomi and Shabrangi 2016).

The purpose of this study was to assess the effects of elicitation with chitosan, salicylic acid and copper sulphate on biomass production, volatile composition, antioxidant potential and enzyme activity in peppermint (Mentha × piperita L.) plants cultured in vitro.

Plant material and study site

Seeds of peppermint were obtained commercially from Isla Sementes^®, Porto Alegre, RS, Brazil. The experiment was conducted at the Plant Tissue Culture Laboratory of Paranaense University (UNIPAR), Umuarama, PR, Brazil. Analytical standards were purchased from Sigma–Aldrich^®, St. Louis, MO, USA.

In vitro elicitation

Peppermint seeds were disinfected for 1 min in 70% alcohol and for 10 min in 2.5% sodium hypochlorite in a laminar flow hood, according to the protocol of Trettel et al. (2018). Seeds were then inoculated in Murashige and Skoog (MS) basal medium (Murashige and Skoog 1962) supplemented with 30 g L^− 1 sucrose and 6.5 g L^− 1 agar. Before use in the experiment, the medium was adjusted to pH 5.8 and autoclaved at 121°C for 20 min. A total of 150 flasks were prepared, each with a total volume of 350 mL and containing 50 mL of culture medium and 3 peppermint seeds. Flasks were kept in a growth room at 25 ± 2°C and a light intensity of 72.02 µmol m^− 2 s^− 1 under a 24 h light photoperiod provided by light emitter diode (LED) lamps (model T8, 10 W) (Trettel et al. 2018). After 70 days of culture, shoots were used as explant donors.

At the end of the culture period, new 350 mL flasks were prepared by adding 50 mL of culture medium and one of the following four treatments: 50 µM salicylic acid, 200 mg L^− 1 chitosan (degree of deacetylation of 75–85%), 25 µM copper sulphate (CuSO₄) or an untreated control. Elicitor concentrations were based on previous studies (Trettel et al. 2018; Miclea et al. 2020). A nodal segment (1 cm) with two buds was then inoculated in each flask (Fig. 1a). Each treatment comprised 250 flasks, totalling 1000 plants. Shortly after preparation, samples were stored in a growth chamber for 90 days under the same conditions described above.

Growth Monitoring

Shoots and oxidised plants were counted after 30, 60 and 90 days of inoculation of the nodal segments. The fresh and dry weights (g) of the shoots and roots were determined after 90 days of inoculation. For dry weight determination, samples were oven-dried at 60°C for 3 days and then weighed on a precision digital scale. Shoot and primary root lengths (cm) were measured after 90 days of incubation using a calliper (Fig. 1c,d).

Determination Of Volatile Compounds

Volatile compounds were characterised by headspace gas chromatography/mass spectrometry. First, 2 g of fresh peppermint leaves were harvested after 90 days of inoculation, added to 20 mL flasks, and incubated for 30 min at 120°C under intermittent stirring at 550 rpm (1 min on, followed by 10 s off). The temperature of the injection syringe was set at 130°C. Volatile compounds were determined using a 7890B Agilent gas chromatograph coupled to a 5977A Agilent mass spectrometer equipped with an HP-5MS UI capillary column (30 m × 0.25 mm × 0.25 µm). The oven temperature was set at 60°C for 1 min, ramped to 250°C at 4°C min^− 1, held for 2 min, ramped to 300°C at 15°C min^− 1, and held for 1 min. Helium was used as carrier gas at a flow rate of 1 mL min^− 1 and a constant pressure of 8,230 psi. The injector, transfer line, ionization source and quadrupole temperatures were 250, 280, 230 and 150°C, respectively. The mass spectral detection system was operated in scan mode in the range of 40–550 m/z. Individual compounds were identified by comparison of their mass spectra with the Wiley spectral database (http://www.sisweb.com/software/ms/wiley.htm) and by comparison of their retention indices with those described by Adams (2017).

Determination Of Antioxidant Activity

Antioxidant, reducing sugar, flavonoid, anthocyanin and enzyme activity analyses were performed using 96-well flat-bottom ELISA microplates. The absorbance was measured using a spectrophotometer (UV-Vis SpectraMax Plus^®) equipped with SoftMax Pro 6.5.1 software. Three biological repeats were analysed in triplicate. Results are presented in graphs, as mean and standard deviation (n = 3) per treatment.

The antioxidant activity was analysed in 100 mg samples of fresh leaves collected at the end of the experiment (90 days after inoculation). Leaves were ground in liquid nitrogen and maintained at − 80°C until analysis. Four methods were used to assess antioxidant activity, namely total phenolic quantification, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH^•) assay, β-carotene/linoleic acid co-oxidation, and ferric reducing antioxidant power (FRAP) assay. All procedures were performed in the absence of light. Extracts were prepared by mixing ground leaves in 50% methanol and 70% acetone, except for the β-carotene method, whose procedures are detailed below. After preparation, samples were kept at rest for 2 h and centrifuged at 8,000 × g. The supernatant was collected and used as the crude extract (Waterhouse 2002).

Total phenolics were determined using 10% Folin–Ciocalteu solution and 4% sodium carbonate (Larrauri et al. 1997). Readings were taken at 750 nm. Results are expressed in milligrams of gallic acid equivalents (GAE) per 100 g of sample. Quantification was performed against a standard curve of gallic acid (0, 10, 20, 30, 40 and 50 mg mL^− 1), given by the equation y = 0.2708 − 0.0994x, with R² = 0.99.

The DPPH^• assay, which is based on the loss of absorbance of a DPPH^• solution (60 µM), was performed according to Rufino et al. (2009). Readings (515 nm) were taken every 30 min until stabilisation. Results are expressed as the free radical scavenging (FRS) percentage, calculated using Eq. (1):

$$\text{F}\text{R}\text{S} = ({A}_{\text{c}}-{A}_{\text{s}})÷{A}_{\text{c}}\times 100$$

where A_c is the absorbance of the control and A_s is the absorbance of the sample.

The β-carotene/linoleic acid co-oxidation assay was performed according to Mattos et al. (2009). Samples were mixed with ethanol, stirred for 1 h in a shaker, incubated for 20 min in an ultrasonic bath, and centrifuged at 8,000 × g to collect the supernatant. This procedure was repeated twice. The β-carotene/linoleic acid emulsion was prepared by mixing 20 µL of linoleic acid, 265 µL of Tween 40, 25 µL of β-carotene solution (20 mg mL^− 1) and 0.5 mL of chloroform. Two readings were taken at 470 nm, one at time zero and the other after 120 min of reaction. Results were calculated using Eq. (2):

$$\text{A}\text{A} = [1-({A}_{\text{i}\text{c}}-{A}_{\text{f}\text{c}}) / ({A}_{\text{i}\text{s}}-{A}_{\text{f}\text{s}}\left)\right]\times 100$$

where AA is the antioxidant activity (%), A_ic is the initial absorbance of the control, A_fc is the final absorbance of the control, A_is is the initial absorbance of the sample, and A_fs is the final absorbance of the sample.

For the FRAP assay, the FRAP reagent was prepared by mixing sodium acetate buffer (0.3 M, pH 3.6), iron chloride (20 mM), and 2,3,5-triphenyltetrazolium chloride (10 mM) (Rufino et al. 2006). Readings were taken at 595 nm. Results are expressed in milligrams of Trolox equivalents (TE) per 100 g of sample, determined against a standard curve (0, 160, 320, 480, 640, 800, 1600 µmol L^− 1 Trolox), given by the equation y = 0.3904x − 0.0548, with R² = 0.9962 (Rodríguez-Bonilla et al. 2017).

Determination Of Yellow Flavonoids And Anthocyanins

For the determination of yellow flavonoids and anthocyanins, 100 mg of fresh leaves were mixed with an ethanol–HCl solution (1.5 M) and centrifuged at 8,000 × g for 10 min, according to Francis (1982). The supernatant was collected and used as the crude extract. Reactions were read at 535 nm for anthocyanin quantification and 374 nm for flavonoid quantification. The results are expressed in mg 100 g^− 1 sample, calculated using a dilution factor of 500 (Eqs. 3 and 4):

$$\text{A}\text{n}\text{t}\text{h}\text{o}\text{c}\text{y}\text{a}\text{n}\text{i}\text{n}\text{s}= {(\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}}_{535} \times \text{D}\text{i}\text{l}\text{u}\text{t}\text{i}\text{o}\text{n} \text{f}\text{a}\text{c}\text{t}\text{o}\text{r})÷98.2$$

$$\text{F}\text{l}\text{a}\text{v}\text{o}\text{n}\text{o}\text{i}\text{d}\text{s}= {(\text{A}\text{b}\text{s}\text{o}\text{r}\text{b}\text{a}\text{n}\text{c}\text{e}}_{374} \times \text{D}\text{i}\text{l}\text{u}\text{t}\text{i}\text{o}\text{n} \text{f}\text{a}\text{c}\text{t}\text{o}\text{r})÷76.6$$

Determination Of Reducing Sugars

The extract was prepared using 100 mg of fresh leaves and 80% ethanol, according to Santos et al. (2017). The material was incubated at 75°C for 10 min and centrifuged at 8,000 × g for 15 min. Subsequently, reducing sugars were measured by the method proposed by Maldonade et al. (2013), based on the reaction with 3,5-dinitrosalicylic acid. Readings were taken at 490 nm. Results are expressed in milligrams of reducing sugars per gram of sample. Quantification was performed using a standard curve of glucose (0, 0.2, 0.4, 0.8, 1.0, 1.6 and 2.0 g L^− 1) (y = 0.672x − 0.4675, R² = 0.97).

Determination Of Phenylalanine Ammonia-lyase (Pal) Activity

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.24) activity was measured using 100 mg of fresh leaves in phosphate buffer pH 6.0, according to Redman et al. (1999) and Umesha (2006). The reaction medium was prepared using 100 µL of 0.5 M Tris-HCl buffer (pH 8.0) and 100 µL of 6 µM l-phenylalanine. The calibration curve (y = 0.0397x + 0.0506, R² = 0.97) was constructed using six concentrations of trans-cinnamic acid (0, 3.32, 6.65, 10.0, 13.32 and 20.0 µL mL^− 1). Absorbance readings were taken at 490 nm. Results are expressed as mg trans-cinnamic acid g^− 1 sample.

Statistical analysis

Data on the number of shoots and the number of oxidised shoots at 30, 60 and 90 days of in vitro culture were converted to percentages and plotted as graphs using Microsoft Excel. Data on the chemical composition and chemical classes were subjected to principal component analysis (PCA) using the Kaiser criterion using Statistica 7 software (StatSoft 2020).

The experiment had a completely randomised design. Plant growth, biochemical activity, antioxidant potential and enzyme activity data were analysed for normality by the Shapiro–Wilk test (p < 0.05) and subjected to analysis of variance by the F-test (p < 0.05). Means were compared by Tukey's test and Skott-knott test (p < 0.05) using Sisvar^® 5.6 software (Ferreira 2011).

Elicitor effects on plant growth

Elicitors induced varied responses throughout the 90 days of culture (Fig. 1cd). The mean number of shoots ranged from 1.0 to 3.0 in the first 30 days (Fig. 2a). Most plants had only one shoot or required more than 60 days to produce additional shoots, as observed in control nodal segments and those treated with CuSO₄. Plants cultured in media containing chitosan and salicylic acid had similar shoot numbers (1.0–1.5). CuSO₄ treatment increased the shoot number by 68% compared with chitosan treatment and by 43% compared with salicylic acid treatment at the end of the 90-day period (Fig. 2a).

Shoot oxidation was observed throughout the culture period, evidenced by the presence of dark necrotic tissues. The intensity of this phenomenon was variable, sometimes manifesting only in some parts of the plant and other times affecting the plant as a whole (Fig. 1b). In the first 30 days, oxidation was observed in all treatments. CuSO₄-treated plants had the lowest occurrence of oxidised shoots, corresponding to 13% at 90 days of cultivation. In the same period (90 days), the prevalence of oxidised plants in the control and chitosan groups was 39% and 12% higher, respectively, than in the CuSO₄ group (Fig. 2b). Explants grown in medium containing salicylic acid had an oxidation rate of 14% at 30 days, 19% at 60 days, and 38% at 90 days (Fig. 2b).

Peppermint plants showed different growth and biomass accumulation patterns when exposed to different elicitors (Table 1). Shoots were larger (7.95 cm) when grown in the presence of CuSO₄, which promoted a 19.49% increase in shoot length in relation to the control and a 71.69% increase in relation to the chitosan group (lowest shoot growth) (Fig. 1a,b) (Table 1). None of the elicitor-treated shoots had larger roots than control shoots (10.60 cm). In the CuSO₄ treatment, the root length (7.95 cm) was 30.18% shorter than in the control.

Table 1

Shoot length (SL), root length (RL), shoot fresh weight (SFW), shoot dry weight (SDW), root fresh weight (RFW), and root dry weight (RDW) of *Mentha* × *piperita* shoot cultured in vitro with different elicitors
Treatments	SL (cm)	RL (cm)	SFW (g)
Control	6.400 ± 2.04^b*	10.600 ± 3.50^a	0.1196 ± 0.12^ab
SA 50 µM	2.625 ± 0.99^c	2.025 ± 1.12^c	0.0599 ± 0.02^b
Chi 200 mg L^− 1	2.250 ± 0.79^c	1.275 ± 0.68^c	0.1318 ± 0.07^a
CuSO₄ 25 µM	7.950 ± 2.74^a	7.400 ± 1.90^b	0.0910 ± 0.03^ab
Treatments	SDW (g)	RFW (g)	RDW (g)
Control	0.0381 ± 0,01^a	0.0520 ± 0.02^b	0.0213 ± 0,01^a
SA 50 µM	0.0239 ± 0,02^b	0.0304 ± 0.02^b	0.0083 ± 0.01^b
Chi 200 mg L^− 1	0.0249 ± 0,01^b	0.0306 ± 0.02^b	0.0083 ± 0.01^b
CuSO₄ 25 µM	0.0358 ± 0,02^ab	0.1644 ± 0.18^a	0.0178 ± 0.02^ab
* Means followed by the same letter in a column are not significantly different from each other by Tukey's test (p < 0.05)
Control = without elicitor; Chi = Chitosan; CuSO_{4 =} copper sulfate; SA = acid salicylic

The highest weights for fresh shoots were observed in control, chitosan and CuSO₄ treated plants, and the lowest biomass accumulation (0.0599 g) was recorded in shoots treated with salicylic acid (Fig. 1e). Shoot dry weights were highest in control shoots, with the other treatments did not differ significantly from each other (Table 1).

Elicitation with CuSO₄ increased the fresh weight of the roots. Responses to the other elicitors were similar, without significant differences between groups. On a dry basis, the highest root weights were observed in the control and CuSO₄ groups; the responses to chitosan and salicylic acid did not differ significantly (Table 1).

Volatile compounds in elicited peppermint shoots

PCA explained 93.73% of the variance in compounds and 96.53% of the variance in chemical classes (Fig. 3a,3b). 2-Hydroxy-3-(3-methyl-2-butenyl)-3-cyclopenten-1-one was identified in all treatments, and the highest concentrations were detected in shoots elicited with salicylic acid, CuSO₄ and chitosan, in that order. d-Limonene was the second most frequent compound, found in control and chitosan treatments. Eucalyptol was detected in shoots grown with salicylic acid and CuSO₄, and l-β-pinene and l-alanine ethylamide were detected in all treatments (Fig. 3a). PCA of the chemical classes showed that the control and chitosan treatments had a predominance of monoterpene hydrocarbons, whereas there was a predominance of oxygenated monoterpenes in salicylic acid- and CuSO₄-treated shoots (Fig. 3b).

Table 2 lists all volatile compounds found in peppermint shoots grown in medium with and without elicitors. In the control treatment, 29 volatile compounds were identified, the major compound being d-limonene (31.28%), which was also found in chitosan-elicited shoots. Monoterpene hydrocarbons were the major class in the control group (54.11%) (Fig. 3c). Plants grown in salicylic acid medium were found to contain 32 compounds, the major compound being 2-hydroxy-3-(3-methyl-2-butenyl)-3-cyclopenten-1-one (40.41%), followed by eucalyptol (17.49%). The major classes were oxygenated monoterpenes (63.99%) and sesquiterpene hydrocarbons (11.85%) (Fig. 3c). Chitosan-elicited peppermint shoots had 38 compounds, with 2-hydroxy-3-(3-methyl-2-butenyl)-3-cyclopenten-1-one (25.39%), d-limonene (18.59%) and l-β-pinene (11.87%) being the major compounds (Table 2). The predominant classes were monoterpene hydrocarbons (35.95%) and oxygenated monoterpenes (32.24%) (Fig. 3c). Shoots elicited with CuSO₄ were found to contain 37 compounds, the major compounds being 2-hydroxy-3-(3-methyl-2-butenyl)-3-cyclopenten-1-one (32.53%), eucalyptol (15.21%) and l-β-pinene (12.49%). Oxygenated monoterpenes were the major class (Fig. 3c).

Table 2

Volatile composition determined by headspace analysis of *Mentha* × *piperita* shoots cultured in vitro with different elicitors
Peak	I.M	Composition	Area (%)
Peak	I.M	Composition	Control	SA50 µM		Chi 200 mgL^-1	CuSO₄ 25 µM
1	a,b,c	1,4-Dioxane-2,6-dione	-	-	1.33			1.58
2	a,b,c	l-Alanine ethylamide, (S)-	29.93	6.37	7.85			8.41
3	a,b,c	α-Pinene, (D)-	5.38	2.63	4.02			4.08
4	a,b,c	L-β-Pinene	10.05	7.79	11.87			12.49
5	a,b,c	Terpinolen	0.96	0.22	0.16			0.47
6	a,b,c	Eucalyptol	-	17.49	-			15.21
7	a,b,c	D-Limonene	31.28	-	18.59			-
8	a,b,c	1,3,6-Octatriene, 3,7-dimethyl-, (Z)-	0.16	0.23	0.16			-
9	a,b,c	γ-Terpinene	5.82	0.79	0.40			0.80
10	a,b,c	p-Mentha-1,4(8)-diene	0.46	0.22	0.75			0.18
11	a,b,c	Linalool	-	0.11	-			0.32
12	a,b,c	Sabinol	-	-	0.11			0.11
13	a,b,c	3-Octanol, acetate	-	-	-			0.12
14	a,b,c	Cyclopropane, trimethyl(2-methyl-1-propenylidene)-	-	-	-			0.61
15	a,b,c	Cyclohexanone, 2-(1-methylethylidene)-	-	-	0.87			-
16	a,b,c	L-α-Terpineol	-	0.25	-			0.26
17	a,b,c	Ethanol, 2-(3,3-dimethylcyclohexylidene)-, (Z)-	-	-	0.17			-
18	a,b,c	Terpinen-4-ol	0.26	0.28	0.41			0.70
19	a,b,c	Benzenemethanol, α,α,4-trimethyl-	0.21	0.28	0.78			1.86
20	a,b,c	3-Cyclohexen-1-ol, 5-methylene-6-(1-methylethenyl)-, acetate	-	-	0.56			-
21	a,b,c	Myrtenal	-	0.68	-			-
22	a,b,c	Cyclohexanol, 2-methyl-5-(1-methylethenyl)-	-	-	0.12			-
23	a,b,c	2-Allyl-4-methylphenol	-	1.05	1.41			1.17
24	a,b,c	cis-3-Hexenyl isovalerate	-	-	0.18			-
25	a,b,c	L-perillaldehyde	-	-	-			0.14
26	a,b,c	Limonen-6-ol, pivalate	-	-	0.17			-
27	a,b,c	Piperiton epoxid	-	-	-			0.19
28	a,b,c	Humulene epoxide II	-	1.03	0.73			0.87
29	a,b,c	Dihydroedulan II	0.27	0.95	1.56			0.42
30	a,b,c	2-Heptanone, 6-(3-acetyl-2-methyl-1-cyclopropen-1-yl)-6-methyl-	-	-	-			0.79
31	a,b,c	Thymol	1.40	-	0.43			1.19
32	a,b,c	Ascaridole epoxide	0.42	-	-			-
33	a,b,c	Spiro[4.5]dec-6-en-8-one, 1,7-dimethyl-4-(1-methylethyl)-	-	-	0.28			-
34	a,b,c	Myrtenyl acetate	0.14	-	-			-
35	a,b,c	Phenol, 2,3,5,6-tetramethyl-	-	2.02	0.50			-
36	a,b,c	Leden	-	-	0.28			-
37	a,b,c	3-Cyclopenten-1-one, 2-hydroxy-3-(3-methyl-2-butenyl)-	2.58	40.41	25.39			32.53
38	a,b,c	β-Elemene	0.14	0.13	-			0.58
39	a,b,c	trans-Sesquisabinene hydrate	-	-	-			0.28
40	a,b,c	α-ylangene	-	0.19	0.13			-
41	a,b,c	Cinerolon	0.12	3.44	2.74			2.64
42	a,b,c	Guaia-1(10),11-diene	-	0.39	0.53			-
43	a,b,c	Caryophyllene	0.43	2.25	6.95			1.00
44	a,b,c	7-epi-cis-sesquisabinene hydrate	-	-	-			0.11
45	a,b,c	γ-Muurolene	0.12	-	-			0.15
46	a,b,c	δ-EIemene	-	-	-			0.27
47	a,b,c	β-copaene	0.16	-	-			-
48	a,b,c	Elixene	-	0.13	-			-
49	a,b,c	β-Cuvebene	1.46	-	-			0.18
50	a,b,c	β-Cubebene	-	3.52	4.21			3.58
51	a,b,c	cis-muurola-3,5-diene	0.92	-	-			-
52	a,b,c	(+)-epi-Bicyclosesquiphellandrene	1.06	3.85	4.54			1.67
53	a,b,c	Isocaryophillene	-	0.15	-			-
54	a,b,c	Humulene	-	-	0.34			-
55	a,b,c	β-Patchoulene	-	0.11	-			-
56	a,b,c	.alfa.-Copaene	0.16	-	-			-
57	a,b,c	γ-Gurjunene	-	-	0.22			0.29
58	a,b,c	γ-Elemene	-	0.22	-			-
59	a,b,c	Naphthalene, 1,2,3,4,4a,5,6,8a-octahydro-7-methyl-4-methylene-1-(1-methylethyl)-, (1α,4aβ,8aα)-	1.06	0.93	1.63			1.66
60	a,b,c	Benzene, 2-chloro-1-ethyl-5-methoxy-3-methyl-	-	-	0.41			-
61	a,b,c	Diethyl Phthalate	0.29	-	-			-
62	a,b,c	4-epi-cubedol	1.68	-	-			-
63	a,b,c	Cubenol	-	0.69	0.19			0.24
64	a,b,c	Hexanedioic acid, bis(2-ethylhexyl) ester	2.23	0.43	0.30			0.30
65	a,b,c	2,2,4a,6a,8a,9,12b,14a-Octamethyl-1,2,3,4,4a,5,6,6a,6b,7,8,8a,9,12,12a,12b,13,14,14a,14b-eicosahydropicene	0.10	-	-			-
	Total		99.13	98.69	100.61			97.10
Groups of compounds (%)
Monoterpene hydrocarbons			54.11	11.65	35.95			18.63
Oxygenated monoterpenes			5.00	63.99	32.24			56.43
Sesquiterpene hydrocarbons			1.80	5.16	4.11			4.13
Oxygenated sesquiterpenes			5.52	11.85	18.84			8.81
Others			32.82	9.48	12.21			11.74
^a Compounds are listed in order of elution on an HP-5MS capillary column
^b Compounds were identified by comparison of retention indices with relative retention indices found in the literature for HP-5MS capillary columns and/or comparison of mass spectra with literature data (Adams 2017)
^c Mass spectra compared with the NIST mass spectral library
-, not detected
Control = without elicitor; Chi = Chitosan; CuSO_{4 =} copper sulfate; SA = acid salicylic

Elicitor effects on antioxidant and enzyme activities

There was a 40% increase in total phenolic content (250 mg GAE 100 g^− 1) in plants grown in chitosan medium compared with the control, which had the lowest phenolic content, followed by salicylic acid and CuSO₄ elicited plants (Fig. 4a).

DPPH^• scavenging activity was highest in the control (66%) and chitosan (65%) groups. The lowest antioxidant activity was observed with the CuSO₄ treatment (18%) (Fig. 4b). The β-carotene/linoleic acid assay showed that the control had the highest antioxidant activity (15%), followed by CuSO₄ (8%). The salicylic acid and chitosan treatments did not differ from each other (p > 0.05) (Fig. 4c). FRAP activity was highest in salicylic acid-elicited shoots, followed by chitosan-treated shoots, with 643 and 557 mg TE 100 g^− 1, respectively, being 96% and 82% higher than the activity found in CuSO₄-elicited shoots (Fig. 4d).

The analysis of reducing sugars showed that the salicylic acid treatment differed from chitosan and CuSO₄ treatments but did not differ from the control (Fig. 5a). Peppermint shoots elicited with CuSO₄ had a 6% lower reducing sugars content than the control.

Elicited shoots had a lower flavonoid content than the control (17.33 mg 100 g^− 1): the levels were 8% lower in salicylic acid, 32% lower in chitosan, and 71% lower in CuSO₄ treatments (Fig. 5b). Plants elicited with salicylic acid did not differ from the control in anthocyanin content (1.63 and 1.80 mg 100 g^− 1, respectively). However, the chitosan and CuSO₄ treatments had 70% and 73% lower anthocyanin content than the control, respectively (Fig. 5c).

Salicylic acid-treated plants showed high PAL activity (1.65 mg trans-cinnamic acid g^− 1). This value was 63% higher than that found in shoots elicited with CuSO₄ (0.61 mg trans-cinnamic acid g^− 1) and chitosan (1.34 mg trans-cinnamic acid g^− 1). These treatments had 54.47% and 30.68% higher PAL content than the control (0.88 mg trans-cinnamic acid g^− 1), respectively (Fig. 5d).

Elicitor effects on peppermint growth

Copper is an essential micronutrient for plants (Shabbir et al. 2020) and was the elicitor that best promoted peppermint growth, resulting in a high number of shoots and a low number of oxidised plants. Peppermint grown in medium containing copper also exhibited better architecture. This micronutrient is a cofactor of important enzymes for plant metabolism, such as plastocyanin, which serves as an electron donor to P700 during photosynthesis, and cytochrome oxidase, which participates in respiration and protein synthesis. Copper also plays a role in the activation of important mechanisms for plant resistance to biotic factors (Ahmad et al. 2017).

Studies have associated such responses with an increase in lignin deposition, resulting in a greater number of leaves and shoots, better architecture and reduced oxidation (Bahabadi et al. 2014; Trettel et al. 2018). Bahabadi et al. (2014) and Trettel et al. (2018) argued that the phenylpropanoid route, which involves aromatic precursors such as trans-coumaric acid, is stimulated by biotic and abiotic elicitors, as these compounds are used for lignin synthesis. Together with cellulose, lignin provides rigidity to the cell wall, preventing oxidative damage and serving as a powerful physical barrier to microbial invasion. The phenylpropanoid pathway is one of the major routes to defence and lignification in species of the family Lamiaceae, being stimulated by elicitors such as jasmonic acid, methyl jasmonate and CuSO₄ (Krzyzanowska et al. 2012; Trettel et al. 2018; Figueroa-Pérez et al. 2019).

The role of copper in plant growth was observed in other medicinal plants, such as Ocimum basilicum L. cultured in vitro in medium containing 25 µM CuSO₄, where treated plants had more leaves, were more vigorous and showed a 17% reduction in shoot oxidation (Trettel et al. 2018). Callus cultures of O. basilicum had higher biomass accumulation when subjected to abiotic elicitation (10 mg L^− 1 copper), in addition to a higher production of secondary metabolites, such as antioxidants, flavonoids and phenolics (Nazir et al. 2021). CuSO₄ (2 mg L^− 1) positively influenced fresh and dry biomass weights, total phenolic accumulation, and antioxidant activity in Artemisia annua L. calli (Zarad et al. 2021). By contrast, Jesionek et al. (2018) did not obtain positive results in Rhododendron tomentosum treated with CuSO₄ (100 µM): there was a decrease in microshoot and essential oil yields. Copper elicitation may be toxic, depending on the concentration used, and plants might not respond to certain concentrations.

Salicylic acid and chitosan elicitation reduced peppermint growth compared with the control and CuSO₄ treatments. Salicylic acid is a phytohormone that acts in plant defence, having a great influence on the synthesis of secondary metabolites associated with stress (Kandoudi et al. 2022). Chitosan is a natural polysaccharide derived from chitin acetylation. Studies have shown that the degree of chitin acetylation affects the action of chitosan. Positive effects of chitosan and salicylic acid treatment on Mentha growth were reported by Abdou and Mohamed (2014) and Choudhary et al. (2021). As underscored by Silva et al. (2021), such responses are complex, as they vary depending on the plant genotype, degree of tissue maturation, development stage, elicitor chemical structure and elicitor concentration.

In Lavandula angustifolia Mill., for instance, 108 µM salicylic acid treatment reduced the number and length of shoots grown in vitro (Miclea et al. 2020). In Coleus aromaticus Benth., treatment with 7 µM salicylic acid + 1.0 mg L^− 1 benzylaminopurine resulted in a decrease in shoot numbers and plant regeneration (Govindaraju and Arulselvi 2018). Our results corroborate those of Oliveira et al. (2020), who observed a decrease in the fresh and dry weights of Mentha arvensis L. shoots and roots with increasing chitosan concentrations. Nowak et al. (2021) treated Melissa officinalis L. and O. basilicum with chitosan (100 mg L^− 1) and observed positive effects on growth and biochemical activity. In M. arvensis, the responses varied according to the plant tissue and organ: the dry weight of shoots was highest at a high chitosan concentration (0.75%), whereas the dry weight of roots decreased by 50% compared with the control at the same chitosan concentration (Silva et al. 2021).

Elicitor effects on volatile compounds and chemical classes

The genus Mentha comprises different chemotypes. A chemotype is a plant whose genetic constitution is directed toward the synthesis of one or more specific compounds, not necessarily having morphological differences, as described by Yu et al. (2018). These alterations arise mainly from interspecific hybridisation, which is very common in Mentha (Yu et al. 2018). Kokkini (1992) reported nine chemotypes in the genus Mentha. For instance, the major compound of Mentha piperita pallescens is menthol, that of Mentha spicata is carvone, and that of Mentha suaveolens is piperitenone oxide (Mogosan et al. 2017). The peppermint species studied here was found to be of the chemotype 2-hydroxy-3-(3-methyl-2-butenyl)-3-cyclopenten-1-one and l-alanine ethylamide. The species also produced l-β-pinene, eucalyptol and d-limonene, which are found in other species of the genus Mentha (Mogosan et al. 2017; Yu et al. 2018).

Our results showed that elicitation led to changes in the profile of volatile compounds of the terpene fraction. There was a decrease in the l-alanine ethylamide content in shoots, as well as an increase in 2-hydroxy-3-(3-methyl-2-butenyl)-3-cyclopenten-1-one, particularly in plants exposed to salicylic acid and copper, compared with the control. Eucalyptol was detected in salicylic acid- and copper-elicited plants only. d-Limonene was found only in the control and chitosan-treated shoots. Monoterpene hydrocarbons were the predominant class in the control, whereas oxygenated monoterpenes were the major class in elicited plants.

Such an increase in the synthesis of oxygenated monoterpenes with elicitation suggests that, for certain species, these compounds may provide greater protection against biotic and abiotic stressors (Abd-ElGawad et al. 2020). The exposure of plants to essential oils or extracts rich in oxygenated monoterpenes improved the control of insects, weeds and fungi (Thakur et al. 2019). Oxygenated monoterpenes accounted for more than 70% of the chemical composition of Argemone ochroleuca, including trans-chrysanthenyl acetate (25.71%) and cadinene (11.70%). A. ochroleuca showed significant phytotoxic activity against Lactuca sativa and the harmful weed Peganum harmala (Abd-ElGawad et al. 2020). The fungus Fusarium oxysporum, known to cause tomato rot, was controlled with different concentrations of M. spicata and Mentha longifolia essential oils. Such control was attributed to the stimulation of transcription factors WRKY1, WRKY4, WRKY33 and WRKY53, and an increase in oxygenated monoterpenes (Soliman et al. 2022). Mentha rotundifolia L. showed 100% toxicity against the insect Sitophilus granarius, and 2-hydroxy-3-(3-methyl-2-butenyl)-3-cyclopenten-1-one (89.09%) was indicated as being responsible for this effect (Amina et al. 2018). Overall, the results demonstrate that oxygenated monoterpenes may have a greater action against stressors in this peppermint chemotype, given that their synthesis was stimulated by the tested elicitors.

Biochemical activities

Elicitors activate plant defence mechanisms via enzymatic or non-enzymatic pathways. Both have the function of combating and minimising the damage caused to plant cells by free radicals, which are generated from cellular respiration or stressors (Munteanu and Apetrei 2021). Secondary metabolites and vitamins play key roles in the non-enzymatic mechanism. Various studies have focused on the effects of elicitors on these compounds. Elicitation is used to stimulate the production of secondary metabolites, thereby influencing antioxidant capacity.

Several methods can be used to assess the capacity of plants to neutralise free radicals; however, the tests differ in their nature and may provide different results, precluding comparison (Munteanu and Apetrei 2021). By applying various antioxidant capacity tests, it is possible to gain a better understanding of the mechanisms most used by plants and the types of radicals generated (Alves et al. 2010). It should be noted that elicitation does not stimulate all antioxidant mechanisms at once, given that such activation has energy costs and is not always necessary; plants will try to neutralise the most harmful factors depending on the radical generated (Alves et al. 2010; Thakur et al. 2019; Munteanu and Apetrei 2021).

Here, we found a substantial increase in antioxidant activity by the total phenolic and FRAP methods, mainly in chitosan- and salicylic acid-elicited plants. There was also an increase in DPPH^• scavenging activity in the control. Copper elicitation, on the other hand, led to a decrease in antioxidant activity. The results demonstrated that elicitors induced the production of phenolic compounds, and this response was probably associated with an increase in free radical generation. DPPH^• is reduced by the donation of an electron or hydrogen atom. FRAP measures the tissue's ability to oxidize Fe³⁺ to Fe²⁺ (Munteanu and Apetrei 2021). The phenolic compounds rosmarinic acid, caffeic acid and chlorogenic acid, produced by Mentha species, are known to have such a capacity (Elansary et al. 2020; Jurić et al. 2021).

Our results corroborate those obtained for plants of the family Lamiaceae and other species. The treatment of Achillea millefolium L. with salicylic acid promoted an increase in essential oil and total phenolic contents, consequently improving the antioxidant power (Gorni et al. 2016). The elicitor did not influence growth or morphology but did enhance the biochemical properties of the plant. Kundu et al. (2018) found that salicylic acid at concentrations of 200 µg L^− 1 improved M. spicata growth and antioxidant activity. Salicylic acid at 10 µM enhanced total phenolic, flavonoid, chicoric acid and rosmarinic acid levels in calli of Thai basil L. (Nazir et al. 2021), while Nowak et al. (2021) reported that chitosan (200 mg L^− 1) promoted rosmarinic acid, anthocyanin and phenolic accumulation in M. officinalis.

The β-carotene/linoleic acid co-oxidation method revealed a decrease in antioxidant activity in plants elicited with salicylic acid and chitosan. This technique measures the amount of lipid peroxidation triggered by peroxides. β-Carotene, a precursor of vitamin A, has the capacity to neutralise free radicals generated by lipid peroxidation (Alves et al. 2010). Our findings suggest that this type of oxidation might not be the most deleterious to plant cells, in contrast to the effects of hydroxyl radicals (HO•), which are the most destructive reactive oxygen species (i.e., these species have the highest reduction potential, 2,310 mV) (Singh et al. 2015; Munteanu and Apetrei 2021). Phenolic acids, such as rosmarinic and caffeic acids, have a greater ability to neutralise this type of free radical. This hypothesis was proposed by Asghari et al. (2019), who demonstrated the ability of rosmarinic acid in Echium amoenum to neutralise radicals by the β-carotene/linoleic acid method. Such activity was accompanied by an increase in the DPPH^• and FRAP capacities. UV-B light and melatonin (elicitors) increased rosmarinic, caffeic and chicoric acid levels, as well as the antioxidant capacity, in calli of O. basilicum var. purpurascens (Nazir et al. 2021).

In the current study, we also analysed the profile of phenolic compounds that act as pigments in plants (flavonoids and anthocyanins). Flavonoids comprise a large group of compounds with two aromatic rings connected by a three-carbon bridge, and anthocyanins are flavonoid glycosides that, in the major part, contain sugars in position three (Yonekura-Sakakibara et al. 2019). The level of total flavonoids increased in the following order of treatment: copper < chitosan < salicylic acid < control. The anthocyanin content was highest in the control and salicylic acid treatments, followed by copper and chitosan. The flavonoids apigenin 7-O-glucoside, apigenin, naringenin 7-rhamnoglucoside and hesperidin have been reported in peppermint (Elansary et al. 2020; Jurić et al. 2021). These compounds can protect plants against ultraviolet light, given their capacity to absorb short wavelengths compared with anthocyanins (Yonekura-Sakakibara et al. 2019).

The increase in flavonoid and anthocyanin content in the control and their reduction in elicited treatments suggest a more intense reaction of plants to the type of radicals generated from elicitation. As hypothesised above, such radicals are likely linked to phenolic acids, to the detriment of flavonoids. The results may also have been influenced by the time of analysis, given that, as argued by Jiao et al. (2018), the response of plants to elicitation varies as a function of time. Different results might be obtained depending on the time of elicitor application and the time of analysis. Our findings differ from those of Golkar et al. (2019), who observed an increase in flavonoid and anthocyanin contents in subcultures of Carthamus tinctorius L. calli exposed to 50 mg L^− 1 salicylic acid. A 7.08-fold increase in total flavonoid content was also observed in hairy root cultures of Isatis tinctoria L. treated with 150 and 200 mg L^− 1 chitosan; this response varied according to the time of analysis, with the highest means observed at 30 h after elicitor application. Following this period, there was a decrease in compound synthesis, up to 96 h, when there were no differences between treatments and the control (Jiao et al. 2018).

The analysis of reducing sugars showed a significant, albeit small, difference between treatments. Peppermint plants exposed to chitosan exhibited the highest reducing sugars content, followed by the control and salicylic acid-treated plants, which did not differ from each other. Copper elicitation resulted in the lowest reducing sugars content. Sugars are essential in that they provide carbon skeletons to molecules and are reduced in cellular respiration for ATP generation. Sugars may also serve as osmoprotectants, contributing to protein stabilisation, the formation of intracellular glasses, and the prevention of cellular collapse (Singh et al. 2015). Similar effects were observed in Perilla frutescens (L.) Britt.: treatment with 50 and 100 mg L^− 1 chitosan led to a 60 mg g^− 1 increase in reducing sugars (Salachna et al. 2022).

PAL is a key enzyme in compound synthesis, particularly that of phenolics. The enzyme converts phenylalanine into trans-cinnamic acid, which in turn is converted to p-coumaric acid, an important precursor of a variety of compounds (Radjabian et al. 2015). PAL activity was increased by salicylic acid and chitosan treatments, followed by the control and copper treatment. Plants exposed to salicylic acid and chitosan also had the highest total phenolic content and DPPH^• and FRAP antioxidant activities, suggesting that the elicitors stimulate these metabolic pathways. Radjabian et al. (2015) found that the exogenous application of salicylic acid (250 and 500 µM) to Salvia officinalis and Salvia virgata shoots led to the upregulation of PAL genes and an increase in rosmarinic acid synthesis. In cell suspensions of Zataria multiflora exposed to 40 mg L^− 1, there was an increase in PAL activity and phenolic content (Bavi et al. 2022). Meanwhile, Kamalipourazad et al. (2016) reported a reduction in Scrophularia striata Boiss. cell culture growth in response to chitosan (100 mg L^− 1); however, this concentration promoted an increase in total phenolic content and PAL activity on the fifth day after application.

Our results demonstrated that the tested elicitors not only altered peppermint growth but also promoted significant changes in the metabolism and chemical composition of plants grown in a controlled environment. Shoots exposed to 25 µM copper exhibited better growth and had l-β-pinene and eucalyptol as major compounds, but showed a reduced antioxidant activity. The growth of shoots was affected by exposure to 50 µM salicylic acid or 200 mg L^− 1 chitosan, but there was an increase in antioxidant activity. These shoots had 2-hydroxy-3-(3-methyl-2-butenyl)-3-cyclopenten-1-one, eucalyptol and d-limonene as major compounds. In the control, monoterpene hydrocarbons were the major chemical class, and l-alanine ethylamide and d-limonene were the major compounds.

Chi _ Chitosan; CuSO_{4_}Copper sulfate; GC/MS_ Gas chromatography/mass spectrometry; LED_ Light emitter diodes; MeJa_Methyl jasmonate; MS_ Medium Murashige and Skoog; NBI_ Nitrogen balance index; PCA_ Principal Component Analysis; RAI_ Relative anthocyanins index; RCI_ Relative chlorophyll index; ROS_ Reactive oxygen species; SA_ salicylic acid; SISVAR 5.6_ Statistical Analysis

Acknowledgments

We would like to thank the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) n° 001 and Universidade Paranaense (UNIPAR) for financial support.

Author Contributions

HM: project administration, validation, formal analysis, investigation, data curation and visualization, software, review and editing. LC and VM: writing of original draft, test performance and data collection, methodology, validation and resources. JG: chemical composition analysis. All authors read and approved the fnal manuscript.

Founding

Author L.C. has received research support from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) n° 001

Data availability

This manuscript has no associated data

Competing Interests

The authors declare that they have no conflict of interest.

Human and animal rights

This research did not involve experiments with human or animal participants.

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Biochemical responses and volatile compounds in a peppermint chemotype grown in a controlled environment

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Abstract

Figures

Introduction

Material And Methods

Plant material and study site

Growth Monitoring

Determination Of Volatile Compounds

Determination Of Antioxidant Activity

Determination Of Yellow Flavonoids And Anthocyanins

Determination Of Reducing Sugars

Determination Of Phenylalanine Ammonia-lyase (Pal) Activity

Statistical analysis

Results

Elicitor effects on plant growth

Volatile compounds in elicited peppermint shoots

Elicitor effects on antioxidant and enzyme activities

Discussion

Elicitor effects on peppermint growth

Elicitor effects on volatile compounds and chemical classes

Biochemical activities

Conclusions

Abreviations

Declarations

References

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