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Effect of Ozone Treatment on the Contents of Selected Bioactive Phytochemicals in Leaves of Alligator Plant Kalanchoe daigremontiana

Effect of Ozone Treatment on the Contents of Selected Bioactive Phytochemicals in Leaves of... applied sciences Article Effect of Ozone Treatment on the Contents of Selected Bioactive Phytochemicals in Leaves of Alligator Plant Kalanchoe daigremontiana 1 , 2 1 2 Natalia Matłok * , Tomasz Piechowiak , Miłosz Zardzewiały and Maciej Balawejder Department of Food and Agriculture Production Engineering, University of Rzeszow, St. Zelwerowicza 4, 35-601 Rzeszow, Poland Department of Chemistry and Food Toxicology, University of Rzeszow, St. Cwiklinskiej ´ 1a, 35-601 Rzeszow, Poland * Correspondence: nmatlok@ur.edu.pl Abstract: The study investigated the effect of ozone treatment applied to Kalanchoe daigremontiana plants on the contents of selected phytochemicals and on markers of oxidative stress in the leaves. For this purpose, alligator plants were exposed to the gaseous ozone applied at a rate of 5 and 10 ppm for 1, 5, and 10 min. Subsequently, tests were performed to assess the ozone-treated plants for the ability to produce reactive oxygen species (ROS), and for the activation of superoxide dismutase (SOD) and catalase (CAT), i.e., enzymes responsible for the decomposition of ROS. Measurements were also carried out to determine antioxidant potential, total contents of polyphenols, and vitamin C in plants as well as their mechanical properties. The findings show that the use of controlled conditions of ozone treatment (10 ppm; 1 min) resulted in increased contents of selected bioactive compounds (enhancement of total polyphenols 79%, enhancement of antioxidant potential ABTS 55.6% and DPPH 65.8%) in the ozone-treated raw material, with no phytotoxic effects of the process Citation: Matłok, N.; Piechowiak, T.; observed. It was shown that a short duration of ozone treatment is related to the increased activity of Zardzewiały, M.; Balawejder, M. SOD (max 44%) and CAT (max 18.8%), which contributes to the lower production of ROS in cells of Effect of Ozone Treatment on the Kalanchoe daigremontiana. Contents of Selected Bioactive Phytochemicals in Leaves of Alligator Plant Kalanchoe Keywords: Kalanchoe daigremontiana; ozone; antioxidant activity; polyphenols; mechanical properties; daigremontiana. Appl. Sci. 2022, 12, ROS generation; SOD activity; COT activity 8934. https://doi.org/10.3390/ app12188934 Academic Editor: Monica Gallo 1. Introduction Received: 8 August 2022 The genus Kalanchoe (Crassulaceae) comprises succulent plants [1,2] native to subtropi- Accepted: 2 September 2022 cal and tropical regions of Asia, Africa, and America, as well as Australia and Madagascar. Published: 6 September 2022 Most commonly grown in Europe, K. daigremontiana is an ornamental house plant [3,4], Publisher’s Note: MDPI stays neutral well known for its aesthetic attributes. Regarding its characteristics, the plant has a single with regard to jurisdictional claims in straight stalk, up to 100 cm in height, with fleshy, spiky lanceolate-shaped leaves and published maps and institutional affil- purple to brown spots on the underside [5]. iations. The multi-coloured leaves of Kalanchoe daigremontiana are a good source of phenolic compounds, bufadienolides and vitamins, including ascorbic acid, vitamins B1, B2, and B3, vitamin E, and macronutrients such as calcium, potassium, and sodium [6]. Moreover, alligator plants also contain organic acids, such as chlorogenic, ferulic, gallic, caffeic, p- Copyright: © 2022 by the authors. coumaric, protocatechuic, syringic, b- and g-resorcylic, and malic acids [7]. Owing to the Licensee MDPI, Basel, Switzerland. ample contents of bioactive compounds, Kalanchoe plants are used in herbal medicine. This article is an open access article Traditional medicine mainly makes use of juice made from the leaves, applied to promote distributed under the terms and the healing of wounds and skin damage [8]. Nowadays, extracts obtained from plants in conditions of the Creative Commons the genus Kalanchoe are used in the treatment of a number of conditions, including asthma, Attribution (CC BY) license (https:// gastric ulcers, diarrhoea, and diabetes. creativecommons.org/licenses/by/ 4.0/). Appl. Sci. 2022, 12, 8934. https://doi.org/10.3390/app12188934 https://www.mdpi.com/journal/applsci Appl. Sci. 2022, 12, 8934 2 of 11 Studies investigating the biological activity of isolates acquired from Kalanchoe daigre- montiana showed their antiviral [9], antioxidant, cytotoxic and insecticidal properties [10]. Moreover, the juice obtained from Kalanchoe plants was found to have anti-inflammatory and antiseptic activity [1,11,12]. The medicinal properties of isolates acquired from these plants are linked to the high contents of bioactive compounds with antioxidant activity; these include flavonols, phenolic acid glycosides [13], and quercetin [14]. Research focusing on the modification of plants rich in bioactive compounds has shown that the controlled exposure of such plants to stressors induces a defence re- sponse, [15] which, under appropriate conditions, leads to the increased production of compounds classified as small-molecule antioxidants, and to the activation of selected groups of enzymes. These stressors include abiotic elicitors such as ozone, which by its activity induces changes in the chemical composition of plant material [16]. Exposure to ozone induces oxidative stress in plants [17]. It has been demonstrated that, by inducing a partial reduction in molecular oxygen (O ), this factor is involved in the production of reac- tive oxygen species (ROS), such as hydrogen peroxide H O , hydroxyl radical OH , singlet 2 2 oxygen O , and superoxide anion radical O [18]. Notably, however, severe oxidative 2 2 stress results in the excessive production of reactive oxygen species, and in a disturbed antioxidant balance, which adversely affects redox homeostasis in the organism [19]. How- ever, by adequately selecting the parameters of ozone treatment, it is possible to control the severity of the stress [15]. Correctly conducted ozone treatment leads to increased contents of antioxidants representing various groups of chemical compounds [15]. This happens owing to the activation of an antioxidant defence mechanism, which makes it possible to effectively and quickly remove reactive oxygen species [20]. The system successfully removes oxygen free radicals generated by the ozone from cell organelles [21]. For this purpose, cells use enzymatic systems directly scavenging ROS, i.e., glutathione peroxidases (SOD), catalases (CAT), peroxidases (POX), and Halliwell–Asada cycle enzymes [22], as well as non-enzymatic antioxidants such as glutathione (GSH), vitamins C and E, as well as cysteine, homoglutathione, carotenoids, polyphenols, and mannitol. Under controlled stress, it is possible to increase the contents of these factors in plant material subjected to ozone treatment. It has also been shown that ozonation may affect mechanical properties of plant material [23]. The study aims to define advantageous conditions for ozone treatment to be applied to alligator plant Kalanchoe daigremontiana, making it possible to expose the plant to controlled oxidative stress in order to directly modify the contents of bioactive compounds and the activity of selected enzymes, as well as to affect the mechanical properties of the leaves. It is hypothesised that gaseous ozone applied in correctly defined conditions stimulates Kalanchoe daigremontiana plants to increase the production of small-molecule antioxidants and to change the mechanical properties of leaves so that they are more manageable for technological processes, making it possible to isolate bioactive substances from these plants. 2. Materials and Methods 2.1. Plant Materials The material used in the experiment comprised Kalanchoe daigremontiana plants com- mercially grown in pots. Dry matter content in the leaves of alligator plant amounted to 92  3%. 2.2. Determination of Antioxidant Activity The antioxidant activity of Kalanchoe daigremontiana leaves was assayed using ABTS (2,2 -Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) described by R. Re et al. [24] and DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical described by Brand-Williams et al. [25] and Matłok et al. [26]. The results are expressed with Trolox equivalent (mg) per 1 kg of plant. Appl. Sci. 2022, 12, 8934 3 of 11 2.3. Total Phenolic Content Assay The level of total phenolic content was measured following the method presented by Matłok et al. [27]. The results are expressed as gallic acid equivalent (mg) per 1 g of leaf dry matter. 2.4. Total Ascorbic Acid Assay The content of ascorbic acid in the tissue was determined using 2,6-Dichloropheno- lindophenol titration, in line with the protocol proposed by Piechowiak et al. [15]. See Supplementary Materials: S1. 2.5. ROS The level of reactive oxygen species in plant tissues was measured using a fluorimetric 0 0 method and 2 ,7 -dichlorodihydrofluorescein diacetate. The results are expressed as an in- crease in fluorescence during 1 min in 1 g of tissue [15,28]. See Supplementary Materials: S2. 2.6. SOD and CAT The activity of superoxide dismutase was assessed using a calorimetric method, mak- ing it possible to determine the degree of inhibition of adrenalin auto-oxidation by SOD present in the plant extract. A unit of SOD activity was defined as the amount of enzyme inhibiting adrenaline oxidation by 50%. The activity of catalase was assessed in accordance with the methodology presented by Piechowiak et al. [29,30], which involved a colorimetric estimation of H O residue in the enzyme mixture comprising catalase from plant extract. A 2 2 unit of CAT activity is defined as the amount of the enzyme which results in neutralisation of 1 mmol H O during 1 min. See Supplementary Materials: S3. 2 2 2.7. Mechanical Properties The mechanical parameters of Kalanchoe daigremontiana leaves were assessed by per- forming an epidermis and flesh puncture test using a Zwick/Roell 2000 testing machine, as described by Gorzelany et al. [31]. 2.8. Statistical Analysis Multi-dimensional ANOVA analysis of variance of results were conducted at the significance level = 0.05, and utilized STATISTICA 13.1 software (TIBCO Software Inc., Hillview Avenue, Palo Alto, CA, USA). The mean values calculated from the three indepen- dent replications were analyzed statistically by comparing the results between the variants of the experiment. 3. Results and Discussion 3.1. Antioxidant Activity and Total Phenolic Content Ozone treatment applied to Kalanchoe daigremontiana plants affected the contents of bioactive compounds. An increase in the contents of bioactive compounds, measured one day after the treatment, was observed in the case of each dose of ozone applied. The findings show that samples treated with ozone at a rate to 10 ppm for 1 min had the highest antioxidant potentials, assayed using ABTS (2,2 -Azino-bis-(3-ethylbenzothiazoline- 6-sulfonic acid) and DPPH (2,2-Diphenyl-1-picrylhydrazyl) (Figure 1). An assessment based on the ABTS method showed an increase of 54% compared to the antioxidant potential of the control samples (untreated alligator plants). A similar increase of 65% was identified in a DPPH assay. This change most possibly was an effect of oxidative burst in the apoplastic space of cells in the ozone-treated plants. This led to a greater activation of the mechanisms involved in the biosynthesis of low-molecular-weight antioxidants easily reacting with free radicals [32]. Increased antioxidant potential in plant materials exposed to gaseous ozone was also observed by other researchers [15]. Importantly, once a certain dose of ozone applied to plant materials is exceeded, the gas starts to induce catabolism which eventually leads to a significant decrease in antioxidant potential. In the case of alligator plants, this Appl. Sci. 2022, 12, x FOR PEER REVIEW 4 of 12 to gaseous ozone was also observed by other researchers [15]. Importantly, once a certain dose of ozone applied to plant materials is exceeded, the gas starts to induce catabolism which eventually leads to a significant decrease in antioxidant potential. In the case of alligator plants, this effect was also observed when a dose of 10 ppm was applied; when the duration of the ozone treatment exceeded 1 min, antioxidant potential was considera- bly lower, and in samples treated for 10 min its value was similar to that in the control samples (untreated). On the other hand, when ozone was applied at a rate of 5 ppm, no Appl. Sci. 2022, 12, 8934 4 of 11 significant effect of the treatment on the antioxidant potential of alligator plants was ob- served, irrespective of the duration of the treatment. The antioxidant potential of plant material is a combined effect produced by the ac- effect was also observed when a dose of 10 ppm was applied; when the duration of the tivity of a few groups of chemical compounds, most importantly including polyphenols. ozone treatment exceeded 1 min, antioxidant potential was considerably lower, and in In the case of alligator plants, there was a strong relationship between total polyphenol samples treated for 10 min its value was similar to that in the control samples (untreated). contents and total antioxidant potential (Figure 2). The highest polyphenol contents were On the other hand, when ozone was applied at a rate of 5 ppm, no significant effect of the observed in plants exposed to gaseous ozone at a rate of 10 ppm for 1 min. The value was treatment on the antioxidant potential of alligator plants was observed, irrespective of the 79% higher than the total polyphenol contents identified in the control plants (0 ppm, 0 duration of the treatment. min). Figure 1. Antioxidant activity ABTS (2,2′ -Azin 0 o-bis-(3-ethylbenzothiazoline-6-sulfonic acid) test (A) Figure 1. Antioxidant activity ABTS (2,2 -Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) test and DPPH (2,2-Diphenyl-1-picrylhydrazyl) test (B) in Kalanchoe daigremontiana leaves depending on (A) and DPPH (2,2-Diphenyl-1-picrylhydrazyl) test (B) in Kalanchoe daigremontiana leaves depending the duration of ozone treatment (n = 20): differences between the results for duration of ozone treat- on the duration of ozone treatment (n = 20): differences between the results for duration of ozone ment on specific days are marked with small letters, and differences between dose of ozone (ppm) treatment on specific days are marked with small letters, and differences between dose of ozone measurement are marked with capital letters; significance level is defined as p < 0.05. (ppm) measurement are marked with capital letters; significance level is defined as p < 0.05. The antioxidant potential of plant material is a combined effect produced by the activity of a few groups of chemical compounds, most importantly including polyphenols. In the case of alligator plants, there was a strong relationship between total polyphenol contents and total antioxidant potential (Figure 2). The highest polyphenol contents were observed in plants exposed to gaseous ozone at a rate of 10 ppm for 1 min. The value was 79% higher than the total polyphenol contents identified in the control plants (0 ppm, 0 min). 3.2. Ascorbic Acid Content The content of vitamin C in Kalanchoe daigremontiana significantly affects the quality of the raw herbal material. The concentration of ascorbic acid in plant material depends on the plant species, the conditions of cultivation, as well as the activity of abiotic factors occurring during plant growth. One of the abiotic factors affecting vitamin C content is gaseous ozone; applied in an appropriate way, it modifies the levels of this compound in the raw material [23]. Ozone treatment applied to alligator plants affected the content of vitamin C in the leaves (Figure 3). The value, however, was varied relative to the concentration of gaseous ozone applied and the duration of alligator plant exposure to its activity. Ozone treatment applied at a low rate of 5 ppm, irrespective of the process duration, contributed to a decrease in vitamin C content in the leaves. This decrease may have resulted from the activation of ascorbate oxidase, which is responsible for the degradation of ascorbic acid [33]. Appl. Sci. 2022, 12, 8934 5 of 11 Appl. Sci. 2022, 12, x FOR PEER REVIEW 5 of 12 5 ppm 10 ppm 966.27bB 699.55bA 650.74aA 648.55aA 623.04aA 579.10aA 538.48aA 538.48aA 0 1 5 10 Time of ozonation [min] Figure 2. Total polyphenolic content in Kalanchoe daigremontiana leaves depending on the duration Figure 2. Total polyphenolic content in Kalanchoe daigremontiana leaves depending on the duration of of ozone treatment (n = 20): differences between the results for duration of ozone treatment on spe- ozone treatment (n = 20): differences between the results for duration of ozone treatment on specific Appl. Sci. 2022, 12, x FOR PEER REVIEW 6 of 12 cific days are marked with small letters, and differences between dose of ozone (ppm) measure- days are marked with small letters, and differences between dose of ozone (ppm) measurements are ments are marked with capital letters; significance level is defined as p < 0.05. marked with capital letters; significance level is defined as p < 0.05. 3.2. Ascorbic Acid Content 5 ppm 10 ppm The content of vitamin C in Kalanchoe daigremontiana significantly affects the quality 134.16bA of the raw herbal material. The concentration of ascorbic acid in plant material depends on the plan 125.14aA t spec 125.14aA ies, the conditions of cultivation, as well as the activity of abiotic factors 126.19bA 121.57bA occurring during plant growth. One of the abiotic factors affecting vitamin C content is gaseous ozone; applied in an appropriate way, it modifies the levels of this compound in 112.25aB the raw material [23]. 110.39aA Ozone treatment applied to alligator plants affected the content of vitamin C in the leaves (Figure 3). The value, however, was varied relative to the concentration of gaseous 97.78aA ozone applied and the duration of alligator plant exposure to its activity. Ozone treatment applied at a low rate of 5 ppm, irrespective of the process duration, contributed to a de- crease in vitamin C content in the leaves. This decrease may have resulted from the acti- vation of ascorbate oxidase, which is responsible for the degradation of ascorbic acid [33]. On the other hand, the samples treated with gaseous ozone at a higher rate of 10 ppm for 5 or 10 min were found with higher contents of vitamin C. However, the highest in- 0 1 5 10 crease of 7%, compared to the content in the leaves of untreated plants (0 ppm 0 min), was Time of ozonation [min] observed in the samples exposed to ozone for 10 min. Figure 3. Ascorbic acid content in Kalanchoe daigremontiana leaves depending on the duration of Figure 3. Ascorbic acid content in Kalanchoe daigremontiana leaves depending on the duration of The effect of ozonation on ascorbic acid content was also reported by other research- ozone treatment (n = 20): differences between the results for duration of ozone treatment on specific ozone treatment (n = 20): differences between the results for duration of ozone treatment on specific ers. Onopiuk et al. [34] exposed raspberries to gaseous ozone, with a concentration of 0.3 days are marked with small letters, and differences between dose of ozone (ppm) measurements days are marked with small letters, and differences between dose of ozone (ppm) measurements are −1 and 0.9 mg L for 60 and 120 min. The researchers found that the contents of vitamin C in are marked with capital letters; significance level is defined as p < 0.05. marked with capital letters; significance level is defined as p < 0.05. the fruit decreased in storage as a result of L-ascorbic acid oxidation into dehydroascorbic acid. However, the decrease was significantly lower in the fruit subjected to treatment 3.3. Rea On ctiv the e Oxygen other hand, Species Ac the samples cumulatio treated n with gaseous ozone at a higher rate of 10 ppm with gaseous ozone. for 5 or 10 min were found with higher contents of vitamin C. However, the highest Reactive oxygen species (ROS) are naturally produced in plant cells involved in pho- increase of 7%, compared to the content in the leaves of untreated plants (0 ppm 0 min), tosynthesis, and they participate in signal transduction and in response to the adverse was observed in the samples exposed to ozone for 10 min. effects of abiotic factors [35], such as pathogen attack [36], or exposure to ozone [37]. Oxi- The effect of ozonation on ascorbic acid content was also reported by other researchers. dative stress, occurring as a consequence of such stressors, is induced by the imbalance Onopiuk et al. [34] exposed raspberries to gaseous ozone, with a concentration of 0.3 and between the increased production of, and the ability to rapidly detoxify, ROS in plant cells 0.9 mg L for 60 and 120 min. The researchers found that the contents of vitamin C in [38]. the fruit decreased in storage as a result of L-ascorbic acid oxidation into dehydroascorbic In the present study, the ability of Kalanchoe daigremontiana plants to produce ROS acid. However, the decrease was significantly lower in the fruit subjected to treatment with varied depending on the conditions of the process (Figure 4). After 1 min of the ozone gaseous ozone. treatment, carried out at a rate of both 5 and 10 ppm, there was a decrease in the concen- tration of ROS in plant tissue, compared to the control sample (not exposed to ozone). The longer duration of ozone treatment led to an increased production of ROS in cells, and this effect was most visible when ozone was applied at a rate of 10 ppm. The increased production of ROS in plant cells treated with ozone at higher rates may be linked to the fact that a greater amount of the gas entered the mesophyll through the stomata, and then penetrated the cell wall and plasmalemma, where it was converted into ROS [39]. Fur- thermore, ozone entering through the stomata may have impaired the respiratory chain (gas exchange), and consequently may have affected the respiratory process by increasing the speed of ROS production in the mitochondria of plants exposed to higher doses of the gas [35]. The activation of this loop, enhancing the production of cellular ROS in plants as a result of exposure to higher doses of gaseous ozone, has also been shown by other re- searchers [40]. Piechowiak et al. [41] demonstrated that ozone treatment applied to rasp- •− berries that had been kept in storage led to higher levels of O2 and H2O2 (ROS) in the mitochondria of fruit treated with ozone, compared to a control sample (untreated fruit). On the other hand, Pellinen et al. [42] showed that cells of Betula pendula leaves exposed to ozone contained a significant accumulation of H2O2, which was a direct symptom of oxidative stress induced by ozone. Ascorbic acid content -1 Total phenolic content [mgGA · g d.m.] -1 [mg kg d.m.] Appl. Sci. 2022, 12, 8934 6 of 11 3.3. Reactive Oxygen Species Accumulation Reactive oxygen species (ROS) are naturally produced in plant cells involved in photosynthesis, and they participate in signal transduction and in response to the adverse effects of abiotic factors [35], such as pathogen attack [36], or exposure to ozone [37]. Oxidative stress, occurring as a consequence of such stressors, is induced by the imbalance between the increased production of, and the ability to rapidly detoxify, ROS in plant cells [38]. In the present study, the ability of Kalanchoe daigremontiana plants to produce ROS varied depending on the conditions of the process (Figure 4). After 1 min of the ozone treat- ment, carried out at a rate of both 5 and 10 ppm, there was a decrease in the concentration of ROS in plant tissue, compared to the control sample (not exposed to ozone). The longer duration of ozone treatment led to an increased production of ROS in cells, and this effect was most visible when ozone was applied at a rate of 10 ppm. The increased production of ROS in plant cells treated with ozone at higher rates may be linked to the fact that a greater amount of the gas entered the mesophyll through the stomata, and then penetrated the cell wall and plasmalemma, where it was converted into ROS [39]. Furthermore, ozone entering through the stomata may have impaired the respiratory chain (gas exchange), and consequently may have affected the respiratory process by increasing the speed of ROS production in the mitochondria of plants exposed to higher doses of the gas [35]. The activation of this loop, enhancing the production of cellular ROS in plants as a result of exposure to higher doses of gaseous ozone, has also been shown by other researchers [40]. Piechowiak et al. [41] demonstrated that ozone treatment applied to raspberries that had been kept in storage led to higher levels of O and H O (ROS) in the mitochondria of 2 2 2 fruit treated with ozone, compared to a control sample (untreated fruit). On the other hand, Pellinen et al. [42] showed that cells of Betula pendula leaves exposed to ozone contained a Appl. Sci. 2022, 12, x FOR PEER REVIEW 7 of 12 significant accumulation of H O , which was a direct symptom of oxidative stress induced 2 2 by ozone. 10 ppm 5 ppm 1531.22bC 1300.65aB 1225.61aB 1234.70aB 1129.23aA 1129.23aA 728.45aA 760.80aA 0 1 5 10 Time of ozonation [min] Figure 4. ROS generation in Kalanchoe daigremontiana leaves depending on the duration of ozone Figure 4. ROS generation in Kalanchoe daigremontiana leaves depending on the duration of ozone treatment (n = 20): differences between the results for duration of ozone treatment on specific days treatment (n = 20): differences between the results for duration of ozone treatment on specific days are are marked with small letters, and differences between dose of ozone (ppm) measurements are marked with small letters, and differences between dose of ozone (ppm) measurements are marked marked with capital letters; significance level is defined as p < 0.05. with capital letters; significance level is defined as p < 0.05. 3.4. Antioxidant Enzyme Activity 3.4. Antioxidant Enzyme Activity Fumigation with gaseous ozone applied to Kalanchoe daigremontiana plants led to an Fumigation with gaseous ozone applied to Kalanchoe daigremontiana plants led to an increase in reactive oxygen species (ROS), such as superoxide anions (O •− ) and hydrogen increase in reactive oxygen species (ROS), such as superoxide anions (O 22 ) and hydrogen peroxide (H O ) in plant cells (Figure 4). The increased production of ROS coincided 2 2 peroxide (H2O2) in plant cells (Figure 4). The increased production of ROS coincided with with the activation of the antioxidant system in the plant cells, consisting of a number of the activation of the antioxidant system in the plant cells, consisting of a number of en- zymes responsible for the decomposition of ROS, as well as small-molecule compounds easily reacting with free radicals. The enzymatic antioxidant system includes enzymes such as superoxide dismutase (SOD) and catalase (CAT) [32,43]. The findings of the study show a visible increase in the activity of SOD (Figure 5A) and CAT (Figure 5B) after 1 min of ozone treatment, irrespective of the ozone concentra- tion applied; it is likely that this effect, in combination with the increased level of low- molecular weight antioxidants (Figure 2), contributed to the decreased production of ROS in cells of Kalanchoe daigremontiana plants (Figure 4). The decrease in the production of ROS in cells of plants treated with ozone for a short time (1 min) possibly resulted from the fact that superoxide anion radicals were transformed by SOD into hydrogen peroxide and oxygen [44], which is a natural mechanism of initial defence against oxidative stress. Subsequently, hydrogen peroxide is decomposed by CAT to H2O and O2, which conse- quently leads to a greater tolerance in Kalanchoe daigremontiana plants to stress induced by gaseous ozone [45]. It should also be emphasised that the activation of these enzymes in the chloroplasts of plants treated with ozone contributed to the lack of defects in these plants due to the activity of this reactive gas. The increased activity of SOD and CAT, resulting from the rapid production of ROS in cells affected by a stressor (ozone, drought, pathogen attack), was also observed in other studies [46]. The researchers found that the exposure of three varieties of P. vulgaris to ozone led to a significantly increased activity of CAT, compared to the value identified in the control plants. The authors, however, showed that the activity of CAT was different, relative to the variety of P. vulgaris, which means that in the case of the two other varieties, seedlings exposed to O3 did not exhibit significant changes in the activity of CAT, compared to their respective control samples [46]. A longer duration of ozone treatment applied to Kalanchoe daigremontiana, in partic- ular at a rate of 10 ppm, produced phytotoxic effects, because the activity of SOD and CAT was gradually decreased (Figure 5A,B), whereas the level of ROS was increased (Figure 4). Phytotoxic effects of high doses of ozone, resulting from the decreased expression of ROS generation -1 -1 [ΔF min g ] Appl. Sci. 2022, 12, 8934 7 of 11 enzymes responsible for the decomposition of ROS, as well as small-molecule compounds easily reacting with free radicals. The enzymatic antioxidant system includes enzymes such as superoxide dismutase (SOD) and catalase (CAT) [32,43]. The findings of the study show a visible increase in the activity of SOD (Figure 5A) and CAT (Figure 5B) after 1 min of ozone treatment, irrespective of the ozone concentration applied; it is likely that this effect, in combination with the increased level of low-molecular weight antioxidants (Figure 2), contributed to the decreased production of ROS in cells of Kalanchoe daigremontiana plants (Figure 4). The decrease in the production of ROS in cells of plants treated with ozone for a short time (1 min) possibly resulted from the fact that super- oxide anion radicals were transformed by SOD into hydrogen peroxide and oxygen [44], which is a natural mechanism of initial defence against oxidative stress. Subsequently, hydrogen peroxide is decomposed by CAT to H O and O , which consequently leads to a 2 2 greater tolerance in Kalanchoe daigremontiana plants to stress induced by gaseous ozone [45]. It should also be emphasised that the activation of these enzymes in the chloroplasts of plants treated with ozone contributed to the lack of defects in these plants due to the activity of this reactive gas. The increased activity of SOD and CAT, resulting from the rapid production of ROS in cells affected by a stressor (ozone, drought, pathogen attack), Appl. Sci. 2022, 12, x FOR PEER REVIEW 8 of 12 was also observed in other studies [46]. The researchers found that the exposure of three varieties of P. vulgaris to ozone led to a significantly increased activity of CAT, compared to the value identified in the control plants. The authors, however, showed that the activity of CAT was different, relative to the variety of P. vulgaris, which means that in the case of CAT and SOD, were also demonstrated by other researchers. Ueda et al. [37] showed that the two other varieties, seedlings exposed to O did not exhibit significant changes in the as a result of ozone-induced oxidative stress there was a decrease in the expression of activity of CAT, compared to their respective control samples [46]. enzymes, mainly SOD, in the leaves of Oryza sativa L. Figure 5. SOD (A) and CAT (B) activity in Kalanchoe daigremontiana leaves depending on the dura- Figure 5. SOD (A) and CAT (B) activity in Kalanchoe daigremontiana leaves depending on the duration tion of ozone treatment (n = 20): differences between the results for duration of ozone treatment on of ozone treatment (n = 20): differences between the results for duration of ozone treatment on specific specific days are marked with small letters, and differences between dose of ozone (ppm) measure- days are marked with small letters, and differences between dose of ozone (ppm) measurements are ments are marked with capital letters; significance level is defined as p < 0.05. marked with capital letters; significance level is defined as p < 0.05. 3.5. Mechanical Properties A longer duration of ozone treatment applied to Kalanchoe daigremontiana, in particular at a rate The fof umi 10gat ppm, ion of prall oduced igator phytotoxic plants with ef ga fects, seous because ozone impac the activity ted selec ofted SOD mec and hanical CAT properties of the leaves measured using an indentation test 10 days after the treatment was gradually decreased (Figure 5A,B), whereas the level of ROS was increased (Figure 4). (Ta Phytotoxic ble 1). Th efe fects values of high of d doses estrucof tive ozone, force resulting Fmax in th fre om cas the e of decr leav eased es from expr ozon ession e-tof reated CAT pla and ntSOD, s were wer lower e also thdemonstrated an Fmax values i by den other tified re in sear un chers. treated Ueda leaves et al. (0 min [37] showed 0 ppm). that The d as ifa - result of ozone-induced oxidative stress there was a decrease in the expression of enzymes, ferences, however, were not statistically significant, and the values ranged from 3.06 N in mainly SOD, in the leaves of Oryza sativa L. leaves from the control sample (0 min 0 ppm) to 2.83 N in the case of leaves treated for 1 min with ozone at a rate of 10 ppm. The lower Fmax values identified in ozone-treated leaves of alligator plant most possibly can be linked to the fact that ozone penetrated through the cuticle and the upper epidermis into the deeper layers of leaves which re- sulted in a slight decrease in their resistance to the operation of external forces. Neverthe- less, ozone penetrating into the deeper layers did not destroy the leaves since no necroses nor surface damage were observed in the ozone-treated leaves of Kalanchoe daigremontiana. The process, however, caused oxidative stress which induced an elevated production of secondary metabolites and small-molecule antioxidants (Figures 1 and 2). An additional effect produced may involve a change in the activity of proteolytic enzymes, which di- rectly affects the condition of cell walls. As shown by Stephenson and Harwood [47] and Martínez-Gallegos [48], these enzymes directly affect the lysis of polysaccharides, which are the main building blocks of the cell wall; this may lead to changes in the mechanical properties of ozone-treated plants. A similar effect, observed in the case of flour treated with ozone [49], impacted the rheological properties of baked products made from this type of flour. The current findings also show a negative correlation between values of destructive force and total polyphenol contents, as well as antioxidant potential identified in leaves treated with the specific doses of ozone investigated in this study. A relationship between ozone treatment applied to plant material and Fmax value measured using an indentation test was also reported by other researchers. This effect, however, was varied depending on the plant organ which was subjected to ozone treat- ment. Zardzewiały et al. [50] treated rhubarb petioles with ozone and found an increase in their resistance to external force measured during the indentation test and a process of Appl. Sci. 2022, 12, 8934 8 of 11 3.5. Mechanical Properties The fumigation of alligator plants with gaseous ozone impacted selected mechanical properties of the leaves measured using an indentation test 10 days after the treatment (Table 1). The values of destructive force F in the case of leaves from ozone-treated plants max were lower than F values identified in untreated leaves (0 min 0 ppm). The differences, max however, were not statistically significant, and the values ranged from 3.06 N in leaves from the control sample (0 min 0 ppm) to 2.83 N in the case of leaves treated for 1 min with ozone at a rate of 10 ppm. The lower F values identified in ozone-treated leaves of alligator max plant most possibly can be linked to the fact that ozone penetrated through the cuticle and the upper epidermis into the deeper layers of leaves which resulted in a slight decrease in their resistance to the operation of external forces. Nevertheless, ozone penetrating into the deeper layers did not destroy the leaves since no necroses nor surface damage were observed in the ozone-treated leaves of Kalanchoe daigremontiana. The process, however, caused oxidative stress which induced an elevated production of secondary metabolites and small-molecule antioxidants (Figures 1 and 2). An additional effect produced may involve a change in the activity of proteolytic enzymes, which directly affects the condition of cell walls. As shown by Stephenson and Harwood [47] and Martínez-Gallegos [48], these enzymes directly affect the lysis of polysaccharides, which are the main building blocks of the cell wall; this may lead to changes in the mechanical properties of ozone-treated plants. A similar effect, observed in the case of flour treated with ozone [49], impacted the rheological properties of baked products made from this type of flour. The current findings also show a negative correlation between values of destructive force and total polyphenol contents, as well as antioxidant potential identified in leaves treated with the specific doses of ozone investigated in this study. Table 1. Value of destructive force F (N), deformation dL at F (mm), elastic modulus E max max mod (MPa) and work performed to achieve maximum destructive force W to F (Nmm) in leaves of max Kalanchoe daigremontiana in process of puncture test performed with indenter, diameter of ' = 4 mm (n = 30; SD). F dL at F E W to F max max mod max Ozone Dose (N) (mm) (MPa) (Nmm) a a a a 0 ppm 0 min 3.06  0.28 0.76  0.16 0.0009  0.0002 1.13  0.27 a a a a 5 ppm 1 min 2.99  0.36 0.74  0.32 0.0010  0.0003 1.02  0.48 a a a a 5 ppm 5 min 2.91  0.27 0.68  0.20 0.0010  0.0003 0.95  0.24 a a a a 5 ppm 10 min 2.94  0.33 0.69  0.16 0.0010  0.0002 1.01  0.23 a a a a 10 ppm 1 min 2.83  0.44 0.62  0.12 0.0011  0.0002 0.87  0.21 a a a a 10 ppm 5 min 3.04  0.46 0.78  0.19 0.0009  0.0002 1.09  0.25 a a a a 10 ppm 10 min 3.04  0.32 0.75  0.27 0.0011  0.0003 1.03  0.25 Note: Significant differences between values of the selected mechanical parameters of leaves from alligator plants in indentation test are marked with different letters, at significance level defined as = 0.05. A relationship between ozone treatment applied to plant material and F value max measured using an indentation test was also reported by other researchers. This effect, however, was varied depending on the plant organ which was subjected to ozone treatment. Zardzewiały et al. [50] treated rhubarb petioles with ozone and found an increase in their re- sistance to external force measured during the indentation test and a process of compression of independent specimens. Similar findings were reported by Zapałowska et al. [23], who demonstrated that a higher dose of ozone applied corresponds to an increase in destructive force, as identified in the case of sea buckthorn Hippophae rhamnoides L. On the other hand, when ozone treatment was applied to apples of the Ligol variety, the researchers found that the mean F value measured using an indentation test was related to the colour of max the fruit. The measurement showed that the F value was higher on the blush side in max Appl. Sci. 2022, 12, 8934 9 of 11 the case of ozone-treated fruit, whereas on the opposite side there was a decrease in F max values corresponding to exposure to gaseous ozone [51]. Similar relationships were observed between the dose of ozone and mean deformation value, elastic modulus and work performed to achieve maximum destructive force in the leaves of Kalanchoe daigremontiana in a process of indentation (Table 1). Alligator plants are used in the processing industry where isolates, such as juice, are extracted [52]. The amount of juice (isolate) obtained depends on the parameters of mechanical resistance. Plants with lower parameters related to destructive force F are max more effectively used in the process of acquiring juice by pressing. It should be noted that proposed solutions have a large commercialization potential. This is related to the great interest in some of the biological properties of the Kalanchoe daigremontiana, as well as the availability of industrial solutions that allow the ozonation process to be applied on a large scale [53]. The proposed technological solutions are protected by a patent application [no P.441824]. 4. Conclusions The findings show that the use of controlled conditions of ozone treatment (10 ppm; 1 min) resulted in increased contents of selected bioactive compounds (enhancement of total polyphenols 79%, enhancement of antioxidant potential ABTS 55.6% and potential DPPH 65.8%) in the ozone-treated raw material, with no phytotoxic effects of the process observed. This was caused by placing plants in a state of controlled oxidative stress. The ozonation process caused the activation of the plant’s defence mechanisms, which led to an increased content of low-molecular antioxidants and an increase in the activity of selected enzymes. It should be noted that short a duration of ozone in controlled conditions is related to the increased activity of SOD (max 44%) and CAT (max 18.8%), as well as higher contents of antioxidants, which contributes to the lower production of ROS in cells of Kalanchoe daigremontiana. Ozonation also significantly changed the mechanical properties of the leaves. The destructive force of Kalanchoe daigremontiana leaves was maximally reduced by 5%. This observation is extremely important to increase the value of this medicinal plant. 5. Patent Matłok, N.; Balawejder, M.; Zardzewiały, M. and Piechowiak, T. Method of modifica- tion of biological properties of alligator plants (patent application no P.441824). Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/app12188934/s1, S1: Total ascorbic acid assay; S2: ROS generation assay; S3: SOD and CAT activities assay. Author Contributions: Conceptualization, methodology, investigation, visualization and writing— original draft preparation, N.M.; investigation, T.P. and M.Z.; writing—original draft preparation, M.B. All authors have read and agreed to the published version of the manuscript. Funding: This research project was funded by the program of the Minister of Science and Higher Educa- tion named “Regional Initiative of Excellence” in the years 2019–2023, project number 026/RID/2018/19, the amount of financing PLN 9 542 500.00. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in this article. Conflicts of Interest: The authors declare no conflict of interest. Appl. Sci. 2022, 12, 8934 10 of 11 References 1. El Abdellaoui, S.; Destandau, E.; Toribio, A.; Elfakir, C.; Lafosse, M.; Renimel, I.; André, P.; Cancellieri, P.; Landemarre, L. 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Effect of Ozone Treatment on the Contents of Selected Bioactive Phytochemicals in Leaves of Alligator Plant Kalanchoe daigremontiana

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applied sciences Article Effect of Ozone Treatment on the Contents of Selected Bioactive Phytochemicals in Leaves of Alligator Plant Kalanchoe daigremontiana 1 , 2 1 2 Natalia Matłok * , Tomasz Piechowiak , Miłosz Zardzewiały and Maciej Balawejder Department of Food and Agriculture Production Engineering, University of Rzeszow, St. Zelwerowicza 4, 35-601 Rzeszow, Poland Department of Chemistry and Food Toxicology, University of Rzeszow, St. Cwiklinskiej ´ 1a, 35-601 Rzeszow, Poland * Correspondence: nmatlok@ur.edu.pl Abstract: The study investigated the effect of ozone treatment applied to Kalanchoe daigremontiana plants on the contents of selected phytochemicals and on markers of oxidative stress in the leaves. For this purpose, alligator plants were exposed to the gaseous ozone applied at a rate of 5 and 10 ppm for 1, 5, and 10 min. Subsequently, tests were performed to assess the ozone-treated plants for the ability to produce reactive oxygen species (ROS), and for the activation of superoxide dismutase (SOD) and catalase (CAT), i.e., enzymes responsible for the decomposition of ROS. Measurements were also carried out to determine antioxidant potential, total contents of polyphenols, and vitamin C in plants as well as their mechanical properties. The findings show that the use of controlled conditions of ozone treatment (10 ppm; 1 min) resulted in increased contents of selected bioactive compounds (enhancement of total polyphenols 79%, enhancement of antioxidant potential ABTS 55.6% and DPPH 65.8%) in the ozone-treated raw material, with no phytotoxic effects of the process Citation: Matłok, N.; Piechowiak, T.; observed. It was shown that a short duration of ozone treatment is related to the increased activity of Zardzewiały, M.; Balawejder, M. SOD (max 44%) and CAT (max 18.8%), which contributes to the lower production of ROS in cells of Effect of Ozone Treatment on the Kalanchoe daigremontiana. Contents of Selected Bioactive Phytochemicals in Leaves of Alligator Plant Kalanchoe Keywords: Kalanchoe daigremontiana; ozone; antioxidant activity; polyphenols; mechanical properties; daigremontiana. Appl. Sci. 2022, 12, ROS generation; SOD activity; COT activity 8934. https://doi.org/10.3390/ app12188934 Academic Editor: Monica Gallo 1. Introduction Received: 8 August 2022 The genus Kalanchoe (Crassulaceae) comprises succulent plants [1,2] native to subtropi- Accepted: 2 September 2022 cal and tropical regions of Asia, Africa, and America, as well as Australia and Madagascar. Published: 6 September 2022 Most commonly grown in Europe, K. daigremontiana is an ornamental house plant [3,4], Publisher’s Note: MDPI stays neutral well known for its aesthetic attributes. Regarding its characteristics, the plant has a single with regard to jurisdictional claims in straight stalk, up to 100 cm in height, with fleshy, spiky lanceolate-shaped leaves and published maps and institutional affil- purple to brown spots on the underside [5]. iations. The multi-coloured leaves of Kalanchoe daigremontiana are a good source of phenolic compounds, bufadienolides and vitamins, including ascorbic acid, vitamins B1, B2, and B3, vitamin E, and macronutrients such as calcium, potassium, and sodium [6]. Moreover, alligator plants also contain organic acids, such as chlorogenic, ferulic, gallic, caffeic, p- Copyright: © 2022 by the authors. coumaric, protocatechuic, syringic, b- and g-resorcylic, and malic acids [7]. Owing to the Licensee MDPI, Basel, Switzerland. ample contents of bioactive compounds, Kalanchoe plants are used in herbal medicine. This article is an open access article Traditional medicine mainly makes use of juice made from the leaves, applied to promote distributed under the terms and the healing of wounds and skin damage [8]. Nowadays, extracts obtained from plants in conditions of the Creative Commons the genus Kalanchoe are used in the treatment of a number of conditions, including asthma, Attribution (CC BY) license (https:// gastric ulcers, diarrhoea, and diabetes. creativecommons.org/licenses/by/ 4.0/). Appl. Sci. 2022, 12, 8934. https://doi.org/10.3390/app12188934 https://www.mdpi.com/journal/applsci Appl. Sci. 2022, 12, 8934 2 of 11 Studies investigating the biological activity of isolates acquired from Kalanchoe daigre- montiana showed their antiviral [9], antioxidant, cytotoxic and insecticidal properties [10]. Moreover, the juice obtained from Kalanchoe plants was found to have anti-inflammatory and antiseptic activity [1,11,12]. The medicinal properties of isolates acquired from these plants are linked to the high contents of bioactive compounds with antioxidant activity; these include flavonols, phenolic acid glycosides [13], and quercetin [14]. Research focusing on the modification of plants rich in bioactive compounds has shown that the controlled exposure of such plants to stressors induces a defence re- sponse, [15] which, under appropriate conditions, leads to the increased production of compounds classified as small-molecule antioxidants, and to the activation of selected groups of enzymes. These stressors include abiotic elicitors such as ozone, which by its activity induces changes in the chemical composition of plant material [16]. Exposure to ozone induces oxidative stress in plants [17]. It has been demonstrated that, by inducing a partial reduction in molecular oxygen (O ), this factor is involved in the production of reac- tive oxygen species (ROS), such as hydrogen peroxide H O , hydroxyl radical OH , singlet 2 2 oxygen O , and superoxide anion radical O [18]. Notably, however, severe oxidative 2 2 stress results in the excessive production of reactive oxygen species, and in a disturbed antioxidant balance, which adversely affects redox homeostasis in the organism [19]. How- ever, by adequately selecting the parameters of ozone treatment, it is possible to control the severity of the stress [15]. Correctly conducted ozone treatment leads to increased contents of antioxidants representing various groups of chemical compounds [15]. This happens owing to the activation of an antioxidant defence mechanism, which makes it possible to effectively and quickly remove reactive oxygen species [20]. The system successfully removes oxygen free radicals generated by the ozone from cell organelles [21]. For this purpose, cells use enzymatic systems directly scavenging ROS, i.e., glutathione peroxidases (SOD), catalases (CAT), peroxidases (POX), and Halliwell–Asada cycle enzymes [22], as well as non-enzymatic antioxidants such as glutathione (GSH), vitamins C and E, as well as cysteine, homoglutathione, carotenoids, polyphenols, and mannitol. Under controlled stress, it is possible to increase the contents of these factors in plant material subjected to ozone treatment. It has also been shown that ozonation may affect mechanical properties of plant material [23]. The study aims to define advantageous conditions for ozone treatment to be applied to alligator plant Kalanchoe daigremontiana, making it possible to expose the plant to controlled oxidative stress in order to directly modify the contents of bioactive compounds and the activity of selected enzymes, as well as to affect the mechanical properties of the leaves. It is hypothesised that gaseous ozone applied in correctly defined conditions stimulates Kalanchoe daigremontiana plants to increase the production of small-molecule antioxidants and to change the mechanical properties of leaves so that they are more manageable for technological processes, making it possible to isolate bioactive substances from these plants. 2. Materials and Methods 2.1. Plant Materials The material used in the experiment comprised Kalanchoe daigremontiana plants com- mercially grown in pots. Dry matter content in the leaves of alligator plant amounted to 92  3%. 2.2. Determination of Antioxidant Activity The antioxidant activity of Kalanchoe daigremontiana leaves was assayed using ABTS (2,2 -Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) described by R. Re et al. [24] and DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical described by Brand-Williams et al. [25] and Matłok et al. [26]. The results are expressed with Trolox equivalent (mg) per 1 kg of plant. Appl. Sci. 2022, 12, 8934 3 of 11 2.3. Total Phenolic Content Assay The level of total phenolic content was measured following the method presented by Matłok et al. [27]. The results are expressed as gallic acid equivalent (mg) per 1 g of leaf dry matter. 2.4. Total Ascorbic Acid Assay The content of ascorbic acid in the tissue was determined using 2,6-Dichloropheno- lindophenol titration, in line with the protocol proposed by Piechowiak et al. [15]. See Supplementary Materials: S1. 2.5. ROS The level of reactive oxygen species in plant tissues was measured using a fluorimetric 0 0 method and 2 ,7 -dichlorodihydrofluorescein diacetate. The results are expressed as an in- crease in fluorescence during 1 min in 1 g of tissue [15,28]. See Supplementary Materials: S2. 2.6. SOD and CAT The activity of superoxide dismutase was assessed using a calorimetric method, mak- ing it possible to determine the degree of inhibition of adrenalin auto-oxidation by SOD present in the plant extract. A unit of SOD activity was defined as the amount of enzyme inhibiting adrenaline oxidation by 50%. The activity of catalase was assessed in accordance with the methodology presented by Piechowiak et al. [29,30], which involved a colorimetric estimation of H O residue in the enzyme mixture comprising catalase from plant extract. A 2 2 unit of CAT activity is defined as the amount of the enzyme which results in neutralisation of 1 mmol H O during 1 min. See Supplementary Materials: S3. 2 2 2.7. Mechanical Properties The mechanical parameters of Kalanchoe daigremontiana leaves were assessed by per- forming an epidermis and flesh puncture test using a Zwick/Roell 2000 testing machine, as described by Gorzelany et al. [31]. 2.8. Statistical Analysis Multi-dimensional ANOVA analysis of variance of results were conducted at the significance level = 0.05, and utilized STATISTICA 13.1 software (TIBCO Software Inc., Hillview Avenue, Palo Alto, CA, USA). The mean values calculated from the three indepen- dent replications were analyzed statistically by comparing the results between the variants of the experiment. 3. Results and Discussion 3.1. Antioxidant Activity and Total Phenolic Content Ozone treatment applied to Kalanchoe daigremontiana plants affected the contents of bioactive compounds. An increase in the contents of bioactive compounds, measured one day after the treatment, was observed in the case of each dose of ozone applied. The findings show that samples treated with ozone at a rate to 10 ppm for 1 min had the highest antioxidant potentials, assayed using ABTS (2,2 -Azino-bis-(3-ethylbenzothiazoline- 6-sulfonic acid) and DPPH (2,2-Diphenyl-1-picrylhydrazyl) (Figure 1). An assessment based on the ABTS method showed an increase of 54% compared to the antioxidant potential of the control samples (untreated alligator plants). A similar increase of 65% was identified in a DPPH assay. This change most possibly was an effect of oxidative burst in the apoplastic space of cells in the ozone-treated plants. This led to a greater activation of the mechanisms involved in the biosynthesis of low-molecular-weight antioxidants easily reacting with free radicals [32]. Increased antioxidant potential in plant materials exposed to gaseous ozone was also observed by other researchers [15]. Importantly, once a certain dose of ozone applied to plant materials is exceeded, the gas starts to induce catabolism which eventually leads to a significant decrease in antioxidant potential. In the case of alligator plants, this Appl. Sci. 2022, 12, x FOR PEER REVIEW 4 of 12 to gaseous ozone was also observed by other researchers [15]. Importantly, once a certain dose of ozone applied to plant materials is exceeded, the gas starts to induce catabolism which eventually leads to a significant decrease in antioxidant potential. In the case of alligator plants, this effect was also observed when a dose of 10 ppm was applied; when the duration of the ozone treatment exceeded 1 min, antioxidant potential was considera- bly lower, and in samples treated for 10 min its value was similar to that in the control samples (untreated). On the other hand, when ozone was applied at a rate of 5 ppm, no Appl. Sci. 2022, 12, 8934 4 of 11 significant effect of the treatment on the antioxidant potential of alligator plants was ob- served, irrespective of the duration of the treatment. The antioxidant potential of plant material is a combined effect produced by the ac- effect was also observed when a dose of 10 ppm was applied; when the duration of the tivity of a few groups of chemical compounds, most importantly including polyphenols. ozone treatment exceeded 1 min, antioxidant potential was considerably lower, and in In the case of alligator plants, there was a strong relationship between total polyphenol samples treated for 10 min its value was similar to that in the control samples (untreated). contents and total antioxidant potential (Figure 2). The highest polyphenol contents were On the other hand, when ozone was applied at a rate of 5 ppm, no significant effect of the observed in plants exposed to gaseous ozone at a rate of 10 ppm for 1 min. The value was treatment on the antioxidant potential of alligator plants was observed, irrespective of the 79% higher than the total polyphenol contents identified in the control plants (0 ppm, 0 duration of the treatment. min). Figure 1. Antioxidant activity ABTS (2,2′ -Azin 0 o-bis-(3-ethylbenzothiazoline-6-sulfonic acid) test (A) Figure 1. Antioxidant activity ABTS (2,2 -Azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) test and DPPH (2,2-Diphenyl-1-picrylhydrazyl) test (B) in Kalanchoe daigremontiana leaves depending on (A) and DPPH (2,2-Diphenyl-1-picrylhydrazyl) test (B) in Kalanchoe daigremontiana leaves depending the duration of ozone treatment (n = 20): differences between the results for duration of ozone treat- on the duration of ozone treatment (n = 20): differences between the results for duration of ozone ment on specific days are marked with small letters, and differences between dose of ozone (ppm) treatment on specific days are marked with small letters, and differences between dose of ozone measurement are marked with capital letters; significance level is defined as p < 0.05. (ppm) measurement are marked with capital letters; significance level is defined as p < 0.05. The antioxidant potential of plant material is a combined effect produced by the activity of a few groups of chemical compounds, most importantly including polyphenols. In the case of alligator plants, there was a strong relationship between total polyphenol contents and total antioxidant potential (Figure 2). The highest polyphenol contents were observed in plants exposed to gaseous ozone at a rate of 10 ppm for 1 min. The value was 79% higher than the total polyphenol contents identified in the control plants (0 ppm, 0 min). 3.2. Ascorbic Acid Content The content of vitamin C in Kalanchoe daigremontiana significantly affects the quality of the raw herbal material. The concentration of ascorbic acid in plant material depends on the plant species, the conditions of cultivation, as well as the activity of abiotic factors occurring during plant growth. One of the abiotic factors affecting vitamin C content is gaseous ozone; applied in an appropriate way, it modifies the levels of this compound in the raw material [23]. Ozone treatment applied to alligator plants affected the content of vitamin C in the leaves (Figure 3). The value, however, was varied relative to the concentration of gaseous ozone applied and the duration of alligator plant exposure to its activity. Ozone treatment applied at a low rate of 5 ppm, irrespective of the process duration, contributed to a decrease in vitamin C content in the leaves. This decrease may have resulted from the activation of ascorbate oxidase, which is responsible for the degradation of ascorbic acid [33]. Appl. Sci. 2022, 12, 8934 5 of 11 Appl. Sci. 2022, 12, x FOR PEER REVIEW 5 of 12 5 ppm 10 ppm 966.27bB 699.55bA 650.74aA 648.55aA 623.04aA 579.10aA 538.48aA 538.48aA 0 1 5 10 Time of ozonation [min] Figure 2. Total polyphenolic content in Kalanchoe daigremontiana leaves depending on the duration Figure 2. Total polyphenolic content in Kalanchoe daigremontiana leaves depending on the duration of of ozone treatment (n = 20): differences between the results for duration of ozone treatment on spe- ozone treatment (n = 20): differences between the results for duration of ozone treatment on specific Appl. Sci. 2022, 12, x FOR PEER REVIEW 6 of 12 cific days are marked with small letters, and differences between dose of ozone (ppm) measure- days are marked with small letters, and differences between dose of ozone (ppm) measurements are ments are marked with capital letters; significance level is defined as p < 0.05. marked with capital letters; significance level is defined as p < 0.05. 3.2. Ascorbic Acid Content 5 ppm 10 ppm The content of vitamin C in Kalanchoe daigremontiana significantly affects the quality 134.16bA of the raw herbal material. The concentration of ascorbic acid in plant material depends on the plan 125.14aA t spec 125.14aA ies, the conditions of cultivation, as well as the activity of abiotic factors 126.19bA 121.57bA occurring during plant growth. One of the abiotic factors affecting vitamin C content is gaseous ozone; applied in an appropriate way, it modifies the levels of this compound in 112.25aB the raw material [23]. 110.39aA Ozone treatment applied to alligator plants affected the content of vitamin C in the leaves (Figure 3). The value, however, was varied relative to the concentration of gaseous 97.78aA ozone applied and the duration of alligator plant exposure to its activity. Ozone treatment applied at a low rate of 5 ppm, irrespective of the process duration, contributed to a de- crease in vitamin C content in the leaves. This decrease may have resulted from the acti- vation of ascorbate oxidase, which is responsible for the degradation of ascorbic acid [33]. On the other hand, the samples treated with gaseous ozone at a higher rate of 10 ppm for 5 or 10 min were found with higher contents of vitamin C. However, the highest in- 0 1 5 10 crease of 7%, compared to the content in the leaves of untreated plants (0 ppm 0 min), was Time of ozonation [min] observed in the samples exposed to ozone for 10 min. Figure 3. Ascorbic acid content in Kalanchoe daigremontiana leaves depending on the duration of Figure 3. Ascorbic acid content in Kalanchoe daigremontiana leaves depending on the duration of The effect of ozonation on ascorbic acid content was also reported by other research- ozone treatment (n = 20): differences between the results for duration of ozone treatment on specific ozone treatment (n = 20): differences between the results for duration of ozone treatment on specific ers. Onopiuk et al. [34] exposed raspberries to gaseous ozone, with a concentration of 0.3 days are marked with small letters, and differences between dose of ozone (ppm) measurements days are marked with small letters, and differences between dose of ozone (ppm) measurements are −1 and 0.9 mg L for 60 and 120 min. The researchers found that the contents of vitamin C in are marked with capital letters; significance level is defined as p < 0.05. marked with capital letters; significance level is defined as p < 0.05. the fruit decreased in storage as a result of L-ascorbic acid oxidation into dehydroascorbic acid. However, the decrease was significantly lower in the fruit subjected to treatment 3.3. Rea On ctiv the e Oxygen other hand, Species Ac the samples cumulatio treated n with gaseous ozone at a higher rate of 10 ppm with gaseous ozone. for 5 or 10 min were found with higher contents of vitamin C. However, the highest Reactive oxygen species (ROS) are naturally produced in plant cells involved in pho- increase of 7%, compared to the content in the leaves of untreated plants (0 ppm 0 min), tosynthesis, and they participate in signal transduction and in response to the adverse was observed in the samples exposed to ozone for 10 min. effects of abiotic factors [35], such as pathogen attack [36], or exposure to ozone [37]. Oxi- The effect of ozonation on ascorbic acid content was also reported by other researchers. dative stress, occurring as a consequence of such stressors, is induced by the imbalance Onopiuk et al. [34] exposed raspberries to gaseous ozone, with a concentration of 0.3 and between the increased production of, and the ability to rapidly detoxify, ROS in plant cells 0.9 mg L for 60 and 120 min. The researchers found that the contents of vitamin C in [38]. the fruit decreased in storage as a result of L-ascorbic acid oxidation into dehydroascorbic In the present study, the ability of Kalanchoe daigremontiana plants to produce ROS acid. However, the decrease was significantly lower in the fruit subjected to treatment with varied depending on the conditions of the process (Figure 4). After 1 min of the ozone gaseous ozone. treatment, carried out at a rate of both 5 and 10 ppm, there was a decrease in the concen- tration of ROS in plant tissue, compared to the control sample (not exposed to ozone). The longer duration of ozone treatment led to an increased production of ROS in cells, and this effect was most visible when ozone was applied at a rate of 10 ppm. The increased production of ROS in plant cells treated with ozone at higher rates may be linked to the fact that a greater amount of the gas entered the mesophyll through the stomata, and then penetrated the cell wall and plasmalemma, where it was converted into ROS [39]. Fur- thermore, ozone entering through the stomata may have impaired the respiratory chain (gas exchange), and consequently may have affected the respiratory process by increasing the speed of ROS production in the mitochondria of plants exposed to higher doses of the gas [35]. The activation of this loop, enhancing the production of cellular ROS in plants as a result of exposure to higher doses of gaseous ozone, has also been shown by other re- searchers [40]. Piechowiak et al. [41] demonstrated that ozone treatment applied to rasp- •− berries that had been kept in storage led to higher levels of O2 and H2O2 (ROS) in the mitochondria of fruit treated with ozone, compared to a control sample (untreated fruit). On the other hand, Pellinen et al. [42] showed that cells of Betula pendula leaves exposed to ozone contained a significant accumulation of H2O2, which was a direct symptom of oxidative stress induced by ozone. Ascorbic acid content -1 Total phenolic content [mgGA · g d.m.] -1 [mg kg d.m.] Appl. Sci. 2022, 12, 8934 6 of 11 3.3. Reactive Oxygen Species Accumulation Reactive oxygen species (ROS) are naturally produced in plant cells involved in photosynthesis, and they participate in signal transduction and in response to the adverse effects of abiotic factors [35], such as pathogen attack [36], or exposure to ozone [37]. Oxidative stress, occurring as a consequence of such stressors, is induced by the imbalance between the increased production of, and the ability to rapidly detoxify, ROS in plant cells [38]. In the present study, the ability of Kalanchoe daigremontiana plants to produce ROS varied depending on the conditions of the process (Figure 4). After 1 min of the ozone treat- ment, carried out at a rate of both 5 and 10 ppm, there was a decrease in the concentration of ROS in plant tissue, compared to the control sample (not exposed to ozone). The longer duration of ozone treatment led to an increased production of ROS in cells, and this effect was most visible when ozone was applied at a rate of 10 ppm. The increased production of ROS in plant cells treated with ozone at higher rates may be linked to the fact that a greater amount of the gas entered the mesophyll through the stomata, and then penetrated the cell wall and plasmalemma, where it was converted into ROS [39]. Furthermore, ozone entering through the stomata may have impaired the respiratory chain (gas exchange), and consequently may have affected the respiratory process by increasing the speed of ROS production in the mitochondria of plants exposed to higher doses of the gas [35]. The activation of this loop, enhancing the production of cellular ROS in plants as a result of exposure to higher doses of gaseous ozone, has also been shown by other researchers [40]. Piechowiak et al. [41] demonstrated that ozone treatment applied to raspberries that had been kept in storage led to higher levels of O and H O (ROS) in the mitochondria of 2 2 2 fruit treated with ozone, compared to a control sample (untreated fruit). On the other hand, Pellinen et al. [42] showed that cells of Betula pendula leaves exposed to ozone contained a Appl. Sci. 2022, 12, x FOR PEER REVIEW 7 of 12 significant accumulation of H O , which was a direct symptom of oxidative stress induced 2 2 by ozone. 10 ppm 5 ppm 1531.22bC 1300.65aB 1225.61aB 1234.70aB 1129.23aA 1129.23aA 728.45aA 760.80aA 0 1 5 10 Time of ozonation [min] Figure 4. ROS generation in Kalanchoe daigremontiana leaves depending on the duration of ozone Figure 4. ROS generation in Kalanchoe daigremontiana leaves depending on the duration of ozone treatment (n = 20): differences between the results for duration of ozone treatment on specific days treatment (n = 20): differences between the results for duration of ozone treatment on specific days are are marked with small letters, and differences between dose of ozone (ppm) measurements are marked with small letters, and differences between dose of ozone (ppm) measurements are marked marked with capital letters; significance level is defined as p < 0.05. with capital letters; significance level is defined as p < 0.05. 3.4. Antioxidant Enzyme Activity 3.4. Antioxidant Enzyme Activity Fumigation with gaseous ozone applied to Kalanchoe daigremontiana plants led to an Fumigation with gaseous ozone applied to Kalanchoe daigremontiana plants led to an increase in reactive oxygen species (ROS), such as superoxide anions (O •− ) and hydrogen increase in reactive oxygen species (ROS), such as superoxide anions (O 22 ) and hydrogen peroxide (H O ) in plant cells (Figure 4). The increased production of ROS coincided 2 2 peroxide (H2O2) in plant cells (Figure 4). The increased production of ROS coincided with with the activation of the antioxidant system in the plant cells, consisting of a number of the activation of the antioxidant system in the plant cells, consisting of a number of en- zymes responsible for the decomposition of ROS, as well as small-molecule compounds easily reacting with free radicals. The enzymatic antioxidant system includes enzymes such as superoxide dismutase (SOD) and catalase (CAT) [32,43]. The findings of the study show a visible increase in the activity of SOD (Figure 5A) and CAT (Figure 5B) after 1 min of ozone treatment, irrespective of the ozone concentra- tion applied; it is likely that this effect, in combination with the increased level of low- molecular weight antioxidants (Figure 2), contributed to the decreased production of ROS in cells of Kalanchoe daigremontiana plants (Figure 4). The decrease in the production of ROS in cells of plants treated with ozone for a short time (1 min) possibly resulted from the fact that superoxide anion radicals were transformed by SOD into hydrogen peroxide and oxygen [44], which is a natural mechanism of initial defence against oxidative stress. Subsequently, hydrogen peroxide is decomposed by CAT to H2O and O2, which conse- quently leads to a greater tolerance in Kalanchoe daigremontiana plants to stress induced by gaseous ozone [45]. It should also be emphasised that the activation of these enzymes in the chloroplasts of plants treated with ozone contributed to the lack of defects in these plants due to the activity of this reactive gas. The increased activity of SOD and CAT, resulting from the rapid production of ROS in cells affected by a stressor (ozone, drought, pathogen attack), was also observed in other studies [46]. The researchers found that the exposure of three varieties of P. vulgaris to ozone led to a significantly increased activity of CAT, compared to the value identified in the control plants. The authors, however, showed that the activity of CAT was different, relative to the variety of P. vulgaris, which means that in the case of the two other varieties, seedlings exposed to O3 did not exhibit significant changes in the activity of CAT, compared to their respective control samples [46]. A longer duration of ozone treatment applied to Kalanchoe daigremontiana, in partic- ular at a rate of 10 ppm, produced phytotoxic effects, because the activity of SOD and CAT was gradually decreased (Figure 5A,B), whereas the level of ROS was increased (Figure 4). Phytotoxic effects of high doses of ozone, resulting from the decreased expression of ROS generation -1 -1 [ΔF min g ] Appl. Sci. 2022, 12, 8934 7 of 11 enzymes responsible for the decomposition of ROS, as well as small-molecule compounds easily reacting with free radicals. The enzymatic antioxidant system includes enzymes such as superoxide dismutase (SOD) and catalase (CAT) [32,43]. The findings of the study show a visible increase in the activity of SOD (Figure 5A) and CAT (Figure 5B) after 1 min of ozone treatment, irrespective of the ozone concentration applied; it is likely that this effect, in combination with the increased level of low-molecular weight antioxidants (Figure 2), contributed to the decreased production of ROS in cells of Kalanchoe daigremontiana plants (Figure 4). The decrease in the production of ROS in cells of plants treated with ozone for a short time (1 min) possibly resulted from the fact that super- oxide anion radicals were transformed by SOD into hydrogen peroxide and oxygen [44], which is a natural mechanism of initial defence against oxidative stress. Subsequently, hydrogen peroxide is decomposed by CAT to H O and O , which consequently leads to a 2 2 greater tolerance in Kalanchoe daigremontiana plants to stress induced by gaseous ozone [45]. It should also be emphasised that the activation of these enzymes in the chloroplasts of plants treated with ozone contributed to the lack of defects in these plants due to the activity of this reactive gas. The increased activity of SOD and CAT, resulting from the rapid production of ROS in cells affected by a stressor (ozone, drought, pathogen attack), Appl. Sci. 2022, 12, x FOR PEER REVIEW 8 of 12 was also observed in other studies [46]. The researchers found that the exposure of three varieties of P. vulgaris to ozone led to a significantly increased activity of CAT, compared to the value identified in the control plants. The authors, however, showed that the activity of CAT was different, relative to the variety of P. vulgaris, which means that in the case of CAT and SOD, were also demonstrated by other researchers. Ueda et al. [37] showed that the two other varieties, seedlings exposed to O did not exhibit significant changes in the as a result of ozone-induced oxidative stress there was a decrease in the expression of activity of CAT, compared to their respective control samples [46]. enzymes, mainly SOD, in the leaves of Oryza sativa L. Figure 5. SOD (A) and CAT (B) activity in Kalanchoe daigremontiana leaves depending on the dura- Figure 5. SOD (A) and CAT (B) activity in Kalanchoe daigremontiana leaves depending on the duration tion of ozone treatment (n = 20): differences between the results for duration of ozone treatment on of ozone treatment (n = 20): differences between the results for duration of ozone treatment on specific specific days are marked with small letters, and differences between dose of ozone (ppm) measure- days are marked with small letters, and differences between dose of ozone (ppm) measurements are ments are marked with capital letters; significance level is defined as p < 0.05. marked with capital letters; significance level is defined as p < 0.05. 3.5. Mechanical Properties A longer duration of ozone treatment applied to Kalanchoe daigremontiana, in particular at a rate The fof umi 10gat ppm, ion of prall oduced igator phytotoxic plants with ef ga fects, seous because ozone impac the activity ted selec ofted SOD mec and hanical CAT properties of the leaves measured using an indentation test 10 days after the treatment was gradually decreased (Figure 5A,B), whereas the level of ROS was increased (Figure 4). (Ta Phytotoxic ble 1). Th efe fects values of high of d doses estrucof tive ozone, force resulting Fmax in th fre om cas the e of decr leav eased es from expr ozon ession e-tof reated CAT pla and ntSOD, s were wer lower e also thdemonstrated an Fmax values i by den other tified re in sear un chers. treated Ueda leaves et al. (0 min [37] showed 0 ppm). that The d as ifa - result of ozone-induced oxidative stress there was a decrease in the expression of enzymes, ferences, however, were not statistically significant, and the values ranged from 3.06 N in mainly SOD, in the leaves of Oryza sativa L. leaves from the control sample (0 min 0 ppm) to 2.83 N in the case of leaves treated for 1 min with ozone at a rate of 10 ppm. The lower Fmax values identified in ozone-treated leaves of alligator plant most possibly can be linked to the fact that ozone penetrated through the cuticle and the upper epidermis into the deeper layers of leaves which re- sulted in a slight decrease in their resistance to the operation of external forces. Neverthe- less, ozone penetrating into the deeper layers did not destroy the leaves since no necroses nor surface damage were observed in the ozone-treated leaves of Kalanchoe daigremontiana. The process, however, caused oxidative stress which induced an elevated production of secondary metabolites and small-molecule antioxidants (Figures 1 and 2). An additional effect produced may involve a change in the activity of proteolytic enzymes, which di- rectly affects the condition of cell walls. As shown by Stephenson and Harwood [47] and Martínez-Gallegos [48], these enzymes directly affect the lysis of polysaccharides, which are the main building blocks of the cell wall; this may lead to changes in the mechanical properties of ozone-treated plants. A similar effect, observed in the case of flour treated with ozone [49], impacted the rheological properties of baked products made from this type of flour. The current findings also show a negative correlation between values of destructive force and total polyphenol contents, as well as antioxidant potential identified in leaves treated with the specific doses of ozone investigated in this study. A relationship between ozone treatment applied to plant material and Fmax value measured using an indentation test was also reported by other researchers. This effect, however, was varied depending on the plant organ which was subjected to ozone treat- ment. Zardzewiały et al. [50] treated rhubarb petioles with ozone and found an increase in their resistance to external force measured during the indentation test and a process of Appl. Sci. 2022, 12, 8934 8 of 11 3.5. Mechanical Properties The fumigation of alligator plants with gaseous ozone impacted selected mechanical properties of the leaves measured using an indentation test 10 days after the treatment (Table 1). The values of destructive force F in the case of leaves from ozone-treated plants max were lower than F values identified in untreated leaves (0 min 0 ppm). The differences, max however, were not statistically significant, and the values ranged from 3.06 N in leaves from the control sample (0 min 0 ppm) to 2.83 N in the case of leaves treated for 1 min with ozone at a rate of 10 ppm. The lower F values identified in ozone-treated leaves of alligator max plant most possibly can be linked to the fact that ozone penetrated through the cuticle and the upper epidermis into the deeper layers of leaves which resulted in a slight decrease in their resistance to the operation of external forces. Nevertheless, ozone penetrating into the deeper layers did not destroy the leaves since no necroses nor surface damage were observed in the ozone-treated leaves of Kalanchoe daigremontiana. The process, however, caused oxidative stress which induced an elevated production of secondary metabolites and small-molecule antioxidants (Figures 1 and 2). An additional effect produced may involve a change in the activity of proteolytic enzymes, which directly affects the condition of cell walls. As shown by Stephenson and Harwood [47] and Martínez-Gallegos [48], these enzymes directly affect the lysis of polysaccharides, which are the main building blocks of the cell wall; this may lead to changes in the mechanical properties of ozone-treated plants. A similar effect, observed in the case of flour treated with ozone [49], impacted the rheological properties of baked products made from this type of flour. The current findings also show a negative correlation between values of destructive force and total polyphenol contents, as well as antioxidant potential identified in leaves treated with the specific doses of ozone investigated in this study. Table 1. Value of destructive force F (N), deformation dL at F (mm), elastic modulus E max max mod (MPa) and work performed to achieve maximum destructive force W to F (Nmm) in leaves of max Kalanchoe daigremontiana in process of puncture test performed with indenter, diameter of ' = 4 mm (n = 30; SD). F dL at F E W to F max max mod max Ozone Dose (N) (mm) (MPa) (Nmm) a a a a 0 ppm 0 min 3.06  0.28 0.76  0.16 0.0009  0.0002 1.13  0.27 a a a a 5 ppm 1 min 2.99  0.36 0.74  0.32 0.0010  0.0003 1.02  0.48 a a a a 5 ppm 5 min 2.91  0.27 0.68  0.20 0.0010  0.0003 0.95  0.24 a a a a 5 ppm 10 min 2.94  0.33 0.69  0.16 0.0010  0.0002 1.01  0.23 a a a a 10 ppm 1 min 2.83  0.44 0.62  0.12 0.0011  0.0002 0.87  0.21 a a a a 10 ppm 5 min 3.04  0.46 0.78  0.19 0.0009  0.0002 1.09  0.25 a a a a 10 ppm 10 min 3.04  0.32 0.75  0.27 0.0011  0.0003 1.03  0.25 Note: Significant differences between values of the selected mechanical parameters of leaves from alligator plants in indentation test are marked with different letters, at significance level defined as = 0.05. A relationship between ozone treatment applied to plant material and F value max measured using an indentation test was also reported by other researchers. This effect, however, was varied depending on the plant organ which was subjected to ozone treatment. Zardzewiały et al. [50] treated rhubarb petioles with ozone and found an increase in their re- sistance to external force measured during the indentation test and a process of compression of independent specimens. Similar findings were reported by Zapałowska et al. [23], who demonstrated that a higher dose of ozone applied corresponds to an increase in destructive force, as identified in the case of sea buckthorn Hippophae rhamnoides L. On the other hand, when ozone treatment was applied to apples of the Ligol variety, the researchers found that the mean F value measured using an indentation test was related to the colour of max the fruit. The measurement showed that the F value was higher on the blush side in max Appl. Sci. 2022, 12, 8934 9 of 11 the case of ozone-treated fruit, whereas on the opposite side there was a decrease in F max values corresponding to exposure to gaseous ozone [51]. Similar relationships were observed between the dose of ozone and mean deformation value, elastic modulus and work performed to achieve maximum destructive force in the leaves of Kalanchoe daigremontiana in a process of indentation (Table 1). Alligator plants are used in the processing industry where isolates, such as juice, are extracted [52]. The amount of juice (isolate) obtained depends on the parameters of mechanical resistance. Plants with lower parameters related to destructive force F are max more effectively used in the process of acquiring juice by pressing. It should be noted that proposed solutions have a large commercialization potential. This is related to the great interest in some of the biological properties of the Kalanchoe daigremontiana, as well as the availability of industrial solutions that allow the ozonation process to be applied on a large scale [53]. The proposed technological solutions are protected by a patent application [no P.441824]. 4. Conclusions The findings show that the use of controlled conditions of ozone treatment (10 ppm; 1 min) resulted in increased contents of selected bioactive compounds (enhancement of total polyphenols 79%, enhancement of antioxidant potential ABTS 55.6% and potential DPPH 65.8%) in the ozone-treated raw material, with no phytotoxic effects of the process observed. This was caused by placing plants in a state of controlled oxidative stress. The ozonation process caused the activation of the plant’s defence mechanisms, which led to an increased content of low-molecular antioxidants and an increase in the activity of selected enzymes. It should be noted that short a duration of ozone in controlled conditions is related to the increased activity of SOD (max 44%) and CAT (max 18.8%), as well as higher contents of antioxidants, which contributes to the lower production of ROS in cells of Kalanchoe daigremontiana. Ozonation also significantly changed the mechanical properties of the leaves. The destructive force of Kalanchoe daigremontiana leaves was maximally reduced by 5%. This observation is extremely important to increase the value of this medicinal plant. 5. Patent Matłok, N.; Balawejder, M.; Zardzewiały, M. and Piechowiak, T. Method of modifica- tion of biological properties of alligator plants (patent application no P.441824). Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/app12188934/s1, S1: Total ascorbic acid assay; S2: ROS generation assay; S3: SOD and CAT activities assay. Author Contributions: Conceptualization, methodology, investigation, visualization and writing— original draft preparation, N.M.; investigation, T.P. and M.Z.; writing—original draft preparation, M.B. All authors have read and agreed to the published version of the manuscript. Funding: This research project was funded by the program of the Minister of Science and Higher Educa- tion named “Regional Initiative of Excellence” in the years 2019–2023, project number 026/RID/2018/19, the amount of financing PLN 9 542 500.00. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in this article. Conflicts of Interest: The authors declare no conflict of interest. Appl. Sci. 2022, 12, 8934 10 of 11 References 1. El Abdellaoui, S.; Destandau, E.; Toribio, A.; Elfakir, C.; Lafosse, M.; Renimel, I.; André, P.; Cancellieri, P.; Landemarre, L. 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Journal

Applied Sciences – Multidisciplinary Digital Publishing Institute

Published: Sep 6, 2022

Keywords: Kalanchoe daigremontiana; ozone; antioxidant activity; polyphenols; mechanical properties; ROS generation; SOD activity; COT activity

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