Induction of Antioxidant Metabolites in Moringa oleifera Callus by

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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

Induction of Antioxidant Metabolites in Moringa oleifera Callus by Abiotic Stresses Letizia Zanella,† Angelo Gismondi,† Gabriele Di Marco,† Roberto Braglia,† Francesco Scuderi,† Enrico L. Redi,† Andrea Galgani,‡ and Antonella Canini*,† †

Department of Biology, University of Rome “Tor Vergata”, Via della Ricerca Scientifica 1, Rome 00133, Italy Interdepartmental Centre for Animal Technology, University of Rome “Tor Vergata”, Rome 00133, Italy

‡

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S Supporting Information *

ABSTRACT: Moringa oleifeira has recently been subjected to numerous scientific studies pursuing its biological properties. However, biotechnological approaches promoting the synthesis of pharmacological compounds in this species are still scarce, despite the fact that moringa metabolites have shown significant nutraceutical effects. For this reason, in vitro cultures of moringa callus, obtained from leaf explantation, were subjected to various abiotic stresses such as temperature, salicylic acid, and NaCl, to identify the best growth conditions for the production of high levels of antioxidant molecules. Temperature stresses (exposure to 4 and 45 °C) led to no significant variation in moringa callus, in terms of antiradical metabolites, whereas salicylic acid (200 μM) and NaCl (50−100 μM) affected an increase of total phenolic compounds, after 15 and 30 days of treatment. Overall, the treatment with 100 μM NaCl for 30 days showed the highest free radical scavenging activity, comparable to that measured in moringa leaf. In addition, high doses of NaCl (200 μM) inhibited callus growth and reduced the amount and bioactivity of the secondary metabolites of callus. This study provides useful information to standardize growth conditions for the production of secondary metabolites in moringa in vitro cultures, a biotechnological system that could be employed for a rapid, controlled, and guaranteed production of antioxidant molecules for pharmaceutical purposes.

phenomena match completely the theory that plant metabolites carry out a protective role against environmental biotic and abiotic factors.18 In this context, the literature showed that EtOH extracts of M. oleifera callus are effective against various human diseases.19,20 Specifically, the biological properties of these preparations may be related to the high content in flavonoids of moringa cell cultures, which Talreja21 reported to be even higher than in flowers. Although the data strongly support the development of in vitro systems to propagate moringa cells and produce bioactive natural compounds, little research in this area has been done. In light of the above-mentioned evidence, the effects of four abiotic stimuli (high temperature, low temperature, the presence of salicylic acid, and the presence of sodium hypochlorite) on in vitro cultures of moringa callus were investigated in terms of production of secondary metabolites for the first time. The free radical scavenging activity of the extracts derived from these stimulated plant cells was evaluated in order to identify the treatment that could better trigger the synthesis of antioxidant molecules exploitable for nutraceutical purposes.

Moringa oleifera Lam. (commonly known as moringa) is one of the most interesting African plants due to its nutritional and pharmaceutical features.1 The phytocomplex of moringa leaves was widely associated with various beneficial effects for human health, including hypocholesterolemic,2,3 hypotensive,4 antiatherosclerotic,3 antioxidant,5 antineoplastic,6 and hepatoprotective activities.7 Moringa derivatives have recently been recognized as food ingredients, dietary supplements, and cosmetic components.8 Although moringa originated from India’s sub-Himalayan region, this species is nowadays cultivated throughout the subtropical areas, including West Africa and Fiji.9 Moringa seed production varies considerably depending on factors such as location, soil type, vegetation, and climate conditions.10 The germination rate has been documented as being quite low. For this reason, several research groups have applied in vitro culture techniques on M. oleifera mainly for conservation purposes.11−13 Only in a few cases has an in vitro culture technique been used to promote the production of antioxidant compounds in moringa cells.14,15 Indeed, in the last decades, in vitro growth has been widely proposed as a means for inducing plant secondary metabolism, especially under stimulation by elicitors and stress conditions.16 For instance, Shaaban and Maher17 verified that drought, simulated by different concentrations of mannitol, increased total phenolic content in moringa callus. These © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 20, 2018

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DOI: 10.1021/acs.jnatprod.8b00801 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Chemical structures of the various plant compounds analyzed by the HPLC system. A, gallic acid; B, chlorogenic acid; C, caffeic acid; D, p-coumaric acid; E, 3,3-dimethylallyl caffeate; F, phenyl caffeate; G, 3-O-α-L-arabinopyranosylquercetin; H, rutin; I, quercetin; J, myricetin; K, 3-Oβ-D-glucopyranosylkaempferol; L, genistein; M, kaempferol; N, apigenin; O, chrysin; P, galangin.

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RESULTS AND DISCUSSION In vitro plant cultures represent an efficient way to produce several valuable natural products.22 Indeed plant cells can be used as sources of bioactive phytochemicals, overcoming their conventional ornamental use. In vitro culture biotechnology, independent of geographical, seasonal, and environmental variations, makes it possible to produce a large amount of plant material in a relatively short time, with uniform quality and yield and without the use of pesticides and/or herbicides.23−25 Therefore, considering the remarkable nutritional and nutraceutical properties of M. oleifera, this work improves the knowledge about the production of antioxidant substances in this species by means of in vitro cultures. Since moringa is a plant widely adaptable to extreme climates, such as the African equatorial one, the effect of temperature stresses on growth rate and on secondary metabolite production on one-month-old calli was monitored. The first stimulus applied to calli was cold stress (4 °C; CS), an environmental condition to which M oleifera is not subjected in nature. It was assumed that this temperature variation could induce modifications in secondary metabolite

synthesis in moringa cells, which are evolutionary-adapted to face warm and humid climates. On the other hand, the effect of high temperature (45 °C; HS) on moringa calli was also tested, to monitor possible variations of its secondary metabolism. No significant variation of total phenolic content was observed in plant material exposed to both cold and heat treatment (Figure S1, Supporting Information). In all cases, chromatographic analysis of the secondary metabolite content for temperaturestressed callus did not show any variation in the level of detected plant compounds (Figure 1 and Table S1, Supporting Information). However, it is important to note that chlorogenic acid (Figure S2-B) and caffeic acid (Figure S2C) were the most abundant molecules in HS calli, compared to control and CS cells, where 3,3-dimethylallyl caffeate (Figure S2-E) and phenyl caffeate (Figure S2-F) were predominant. Secondary metabolites play a key role in plant adaptation to abiotic stresses such as temperature variations, hence justifying and explaining the increase of specific secondary metabolites under hot conditions.26−28 According to this evidence, it was hypothesized that the caffeic acid esters might be less active against heat alterations but useful to compensate for cold B



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stress. The wide geographical distribution of M. oleifera29 suggests the adaptability of this species to different climate conditions, evidence supported by our data. The antioxidant power of plant extracts depends on both amount and chemical nature of the antiradical compounds they contain. Thus, in order to characterize the bioactivity of the MeOH extracts obtained from our samples and exploit their potential application as nutraceutical products, two different in vitro antiradical assays, the ferric reducing antioxidant power (FRAP) and 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid (ABTS) assays, were carried out. In all cases, in line with previous spectrophotometric and chromatographic results, no significant changes in free radical scavenging activity between temperature treatment and respective control were observed, as shown in Figure S2 (Supporting Information). The second type of stimulus applied to M. oleifera callus was the exposure to salicylic acid. This natural compound, synthesized throughout the plant kingdom via the phenylpropanoid pathway,30 is strongly involved in the response of plants to abiotic stresses.29 However, the effects of salicylic acid can be contradictory due to the fact that plant resistance pathways are both dependent on and independent of salicylic acid.29,31 Treatments, for 15 and 30 days, with low doses of salicylic acid (100 μM) did not modify the total concentration of phenolic compounds in plant tissues, compared to controls. On the contrary, on both treatment times, callus grown in the presence of salicylic acid at high concentration (200 μM) showed a 30% increase in the phenolic content (Figure 2).

Although with limited significance, the exposure to salicylic acid showed peculiar variations of plant molecules in the callus matrix, according to HPLC-DAD data (Figure 1 and Table S1, Supporting Information). In particular, among the levels of phenolic acids, gallic acid (Figure 1-A), p-coumaric acid (Figure 1-D), and phenyl caffeate showed the highest variations compared to the controls. By contrast, the chlorogenic acid and caffeic acid contents significantly increased in treated callus, when compared to controls. Among the flavonoids, the rutin (Figure 1-H) content was greater in the control than in treated callus at both 15 and 30 days. 3-O-α-L-Arabinopyranosylquercetin (Figure 1-G) was synthesized only in callus treated for 15 days with 200 μM salicylic acid and, in equal measure, in both samples subjected to salicylic acid (SA) for 30 days. This phenomenon, or rather the absence of some specific molecules in the controls and the increase of their synthesis in stressed tissues, can be explained by the fact that plant calli are made up of undifferentiated cells, where secondary metabolism is limited and the absence of stimuli (control conditions) does not favor its induction. On the other hand, controls showed higher levels of kaempferol (Figure 1-M) than treatments at 15 and 30 days. Finally, the genistein (Figure 1-L) content increased after 15 days of treatment with 100 μM SA, compared to the control. The antioxidant effect of the extracts produced by calli treated for 15 and 30 days with SA did not considerably vary with respect to the control ones, against both FRAP and ABTS radicals (Figure 3). The different concentrations of phenolic compounds detected in control and salicylic acid-treated cells were not significant in terms of biological properties, since the antioxidant activities of these samples were similar. This observation was also confirmed in 200 μM salicylic acid treatment, although its total phenolic content was higher than the other samples. This effect could be explained by the fact that several classes of plant metabolites possess antiradical properties and that salicylic acid maybe was not able to induce the synthesis of compounds with strong free radical scavenging activity. Saline stress is one of the most widespread forms of plant abiotic stress; thus, plants evolved many different strategies for tolerating high salt concentrations.32,33 Salt induces three types of stress patterns: osmotic stress, ionic stress, and secondary stress. These critical conditions generally determine accumulation of high concentrations of toxic compounds, such as reactive oxygen species (ROS), which induce cell damage.34 Consequently, to mitigate oxidative stress, plants activate both enzymatic and nonenzymatic antioxidant systems.35 In

Figure 2. Total phenol analysis performed on moringa callus treated with different concentrations of salicylic acid (0−100−200 μM) for 15 and 30 days. Data are reported as μg GAE/g FW (**p ≤ 0.01, vs all other samples).

Figure 3. Antiradical activity of the extracts obtained from moringa callus exposed to salicylic acid (0−100−200 μM) for 15 and 30 days. Results of FRAP (A) and ABTS (B) antioxidant assays are shown. Data are reported as μg AAE/g FW. C



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μM) the biochemical profiles of the samples proved to be similar to those of the control, a peculiar tendency in line with previous spectrophotometric data. In particular, chlorogenic acid, as already observed in salicylic acid treatment, was totally absent in the controls, while it showed a significant peak after 15 and 30 days of treatment with 100 μM NaCl. Rutin proved to be the most abundant molecule in stressed callus, especially in the presence of 100 μM NaCl at both 15 and 30 days. 3-Oα-L-Arabinopyranosylquercetin was significantly detectable in callus only after exposure to 100 μM NaCl at both times of treatment. Salt treatment seemed to be the type of stress most effective in inducing synthesis of antioxidant compounds, as demonstrated by FRAP and ABTS tests (Figure 5). Indeed, both assays revealed a significant increase in the antioxidant power of moringa extract obtained from 50 and 100 μM NaCltreated callus, at 15 and 30 days, compared to the control. As expected, the highest concentration of salt, at both 15 and 30 days of treatment, caused a reduction in the free radical scavenging properties of moringa extracts comparable to the respective controls. All these data clearly demonstrate that NaCl was the best inducer of secondary metabolites in M. oleifera callus grown under in vitro conditions, compared to salicylic acid and temperature variation. However, it is important to note that the highest concentration of salt does not have the same effects as low amounts of the same compound. This anomaly might be explained twofold: the first assumes that high doses of NaCl could inhibit secondary metabolite production, while the second hypothesizes that elevated doses of salt exert a toxic effect on plant cells, a known phenomenon already described in literature,41,42 thus leading to a decrease of the total antioxidant content. To clarify the presence of any inhibitory effects of the salt on callus growth, both the fresh material weight and the browning rate of moringa cells cultured with different NaCl concentrations, for 15 and 30 days, were monitored. The measurements revealed that, with respect to control cells, which presented a linear growth rate, all treatments, independent of their concentration, inhibited plant tissue proliferation, showing at 30 days the same fresh weight of calli recorded at time 0 (Figure 6). At the same time, consistent with growth analysis, the browning rate of the callus significantly increased in all treatments, in a dose- and timeindependent manner, compared to the controls (Figure 7). These observations indicated that salt stress, even at small intensity, determined cell growth arrest. Moreover, together

particular, into this latter group fall secondary metabolites, such as phenols, terpenoids, and alkaloids.36−38 Therefore, NaCl is often used as an elicitor to enhance metabolite production using plant cell tissue and organ cultures.39,40 For this reason, the last part of our work focused on the effect of NaCl (0, 50, 100, and 200 μM) on moringa callus cultures, for 15 and 30 days. In general, the presence of NaCl in the growth medium led to a large increase of total phenol content in moringa callus. As shown in Figure 4, at both 15 and 30 days of

Figure 4. Total phenol analysis performed on moringa callus treated with different concentrations of NaCl (0−50−100−200 μM) for 15 and 30 days. Data are reported as μg GAE/g FW (*p ≤ 0.05 and **p ≤ 0.01 vs all other samples).

treatment, plant cells exposed to 50 and 100 μM NaCl showed a higher content of phenols than controls. In particular, 100 μM NaCl induced the greater increase of these plant molecules, with respect to the control, at about 4- and 5-fold after 15 and 30 days of treatment, respectively. By contrast, the highest salt concentration did not modify the callus phenol content, compared to the control (Figure 4). Comparable levels of total phenol content were obtained by Shaaban and Maher17 using different concentrations of mannitol on M. oleifera callus. Under saline stress, the concentration of secondary metabolites in callus varied according to the amount of NaCl used in the treatment, at both 15 and 30 days, as revealed by chromatographic evidence (Figure 1 and Table S2, Supporting Information). In particular, our results showed a dose-dependent trend, or rather the quantity of plant compounds increased in moringa cells in parallel with salt levels. However, at the highest concentration of NaCl (200

Figure 5. Antiradical activity of the extracts obtained from moringa callus exposed to NaCl (0−50−100−200 μM) for 15 and 30 days. Results of FRAP (A) and ABTS (B) antioxidant assay are shown. Data are reported as μg AAE/g FW (*p ≤ 0.05 and **p ≤ 0.01 vs control and other treatments). D



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Table 1. Concentrations of Secondary Metabolite in Moringa Callus Treated with Different Concentrations of Salicylic Acid (0−100−200 μM) for 15 and 30 Daysa 15 d Phenolic acids gallic acid chlorogenic acid caffeic acid p-coumaric acid 3,3-dimethylallyl caffeate phenyl caffeate Flavonoids rutin 3-O-α-L-arabinopyranosylquercetin myricetin 3-O-β-D-glucopyranosylkaempferol quercetin genistein kaempferol apigenin chrysin galangin

30 d

control

100 μM SA

200 μM SA

control

100 μM SA

200 μM SA

1.15 ± 0.02** n.d. n.d. 0.12 ± 0.002 n.d. 0.05 ± 0.0007

0.08 ± 0.001 0.7 ± 0.01 0.42 ± 0.01 0.07 ± 0.001 n.d. n.d.

0.07 ± 0.001 1.31 ± 0.02 0.45 ± 0.006 0.5 ± 0.007 n.d. 0.04 ± 0.0005

1.34 ± 0.01** n.d. 0.14 ± 0.001 0.38 ± 0.003 n.d. n.d.

0.19 ± 0.004 n.d. 0.61 ± 0.01 n.d. n.d. 0.06 ± 0.001

0.16 ± 0.01 n.d. 0.51 ± 0.02 n.d. n.d. n.d.

0.52 ± 0.008** n.d. 0.06 ± 0.001 0.61 ± 0.01* 0.009 ± 0.0001 0.09 ± 0.001 0.007 ± 0.0001 0.02 ± 0.0003 0.15 ± 0.002 0.02 ± 0.002

0.1 ± 0.002 n.d. 0.07 ± 0.001 0.53 ± 0.01 n.d. 0.32 ± 0.006** n.d. n.d. n.d. 0.08 ± 0.001

0.09 ± 0.001 0.25 ± 0.003 0.11 ± 0.001 0.37 ± 0.005 0.01 ± 0.0002 0.02 ± 0.0002 n.d. 0.02 ± 0.0002 0.10 ± 0.001 0.01 ± 0.0001

0.33 ± 0.002* n.d. 0.13 ± 0.001 0.91 ± 0.007** 0.03 ± 0.0002 0.07 ± 0.0005 0.02 ± 0.0001 0.005 ± 0.0001 0.32 ± 0.002 0.06 ± 0.0004

0.25 ± 0.005 0.38 ± 0.008 0.23 ± 0.005 0.66 ± 0.01 0.03 ± 0.0007 0.07 ± 0.001 0.009 ± 0.0002 0.05 ± 0.001 n.d. 0.16 ± 0.003

0.14 ± 0.006 0.38 ± 0.02 0.16 ± 0.01 0.37 ± 0.02 0.027 ± 0.001 0.1 ± 0.004 0.05 ± 0.002 n.d. 0.4 ± 0.02 n.d.

a Data are expressed as μg of metabolite for g of sample. Results represent the mean of three independent measurements ± standard deviation (*p ≤ 0.05 and **p ≤ 0.01 vs the other samples on the same day of treatment).

Figure 7. Representative images of moringa calli grown in the absence of salt (A and E) or presence of 50 μM NaCl (B and F), 100 μM NaCl (C and G), and 200 μM NaCl (D and H) for 15 (A−D) and 30 (E−H) days are shown. The mean color intensity, expressed in arbitrary units, is reported per each sample. Measurements are indicated with relative standard deviation (obtained analyzing at least 6 replicates) (*p ≤ 0.05 vs treated callus). Black bars indicate 1 cm.

Figure 6. Callus growth in the presence of different concentrations of NaCl (0−50−100−200 μM) after 15 and 30 days of treatment. Results are reported as grams of fresh plant material and expressed as the mean of three independent measurements ± standard deviation. (*p ≤ 0.05 vs the sample at time 0 and all treated calli for the same time; **p ≤ 0.01 vs the sample at time 0 and all treated calli for the same time period).

germination of seeds in in vitro cultures. Moringa leaf extract showed a total phenolic content of 1812.93 μg gallic acid equivalent/g sample fresh weight (GAE/g FW) and an HPLCDAD profile (Figure 1 and Table S2, Supporting Information) significantly richer than those measured in all callus samples. In particular, rutin was the most abundant metabolite in leaves, followed by chlorogenic acid and 3-O-α-L-arabinopyranosylquercetin. However, some compounds identified in the callus were completely missing in the leaves (e.g., 3,3-dimethylallyl caffeate; gallic acid). Although leaves showed a higher concentration of secondary metabolites than callus (treated or not), surprisingly, FRAP and ABTS assays revealed a leaf antioxidant power of 2.22 μg of ascorbic acid equivalent/g sample fresh weigh (AAE/g FW) and 14.5 μg AAE/g FW, respectively, which was strongly comparable to that measured in salt-stressed callus. These findings suggest that moringa callus exposed to low-median saline stress is able to produce secondary metabolites, other than those identified in the

with previous data, they also suggested that low doses of NaCl (50−100 μM) triggered secondary metabolite synthesis in moringa callus, indicating that these plant cells were still alive although not proliferating. On the contrary, high concentrations of salt (200 μM) did not modify the production of antioxidant compounds in plant tissues, indicating that metabolism had probably been blocked. Indeed, the trypan blue exclusion test verified that after saline treatment moringa cells remained alive (data not shown); this stress did not induce cell death but only inhibited the cell growth. Similarly, Alharby and colleagues43 observed inhibition of tomato callus proliferation in the presence of NaCl (3 g/L); a similar growth inhibition was reported by Gao and colleagues42 in potato seedlings exposed to 200 μM NaCl. The same parameters previously taken into account were used to analyze young M. oleifera leaves obtained by E



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Table 2. Concentrations of Secondary Metabolite in Moringa Callus Treated with Different Concentrations of NaCl (0−50− 100−200 μM) for 15 and 30 Daysa 15 d

Phenolic acids gallic acid chlorogenic acid caffeic acid p-coumaric acid 3,3-dimethylallyl caffeate phenyl caffeate Flavonoids rutin 3-O-α-Larabinopyranosylquercetin myricetin 3-O-β-Dglucopyranosylkaempferol quercetin genistein kaempferol apigenin chrysin galangin

30 d

control

50 μM NaCl

100 μM NaCl

200 μM NaCl

control

50 μM NaCl

100 μM NaCl

200 μM NaCl

1.29 ± 0.08 n.d. 0.11 ± 0.008 0.26 ± 0.03 1.15 ± 0.04 n.d.

1.84 ± 0.07 2.93 ± 0.12 0.56 ± 0.03 0.4 ± 0.03 1.76 ± 0.06 n.d.

2.03 ± 0.02 5.17 ± 0.36* 1.22 ± 0.18 0.57 ± 0.08 1.92 ± 0.05 n.d.

1.39 ± 0.06 1.07 ± 0.09 0.4 ± 0.01 0.31 ± 0.004 1.33 ± 0.07 n.d.

1.25 ± 0.09 n.d. 0.10 ± 0.006 0.27 ± 0.02 1.18 ± 0.04 n.d.

2.03 ± 0.08 3.09 ± 0.1 0.78 ± 0.03 0.49 ± 0.05 1.89 ± 0.05 n.d.

3.69 ± 0.14 6.97 ± 0.12** 1.88 ± 0.12 0.87 ± 0.04 2.35 ± 0.08 n.d.

1.39 ± 0.07 1.12 ± 0.07 0.54 ± 0.06 0.42 ± 0.01 1.32 ± 0.02 n.d.

0.25 ± 0.02 n.d.

4.11 ± 0.3 n.d.

7.84 ± 0.4* 2.3 ± 0.13

2.17 ± 0.1 n.d.

0.26 ± 0.02 n.d.

6.43 ± 0.2 n.d.

10.31 ± 1.02** 4.29 ± 0.2***

3.42 ± 0.16 n.d.

0.10 ± 0.01 0.84 ± 0.02

1.22 ± 0.05 2.24 ± 0.09

2.59 ± 0.26 3.83 ± 0.05

0.70 ± 0.03 1.37 ± 0.06

0.12 ± 0.008 0.93 ± 0.07

2.20 ± 0.13 3.34 ± 0.1

3.23 ± 0.14 5.49 ± 0.16

0.99 ± 0.09 2.31 ± 0.11

0.02 ± 0.003 0.15 ± 0.02 0.01 ± 0.002 0.01 ± 0.004 0.28 ± 0.03 0.10 ± 0.006

0.70 ± 0.05 0.63 ± 0.02 0.41 ± 0.01 1.89 ± 0.11 0.55 ± 0.03 2.12 ± 0.006

1.15 ± 0.13 0.9 ± 0.05 0.55 ± 0.07 2.83 ± 0.2 0.81 ± 0.1 3.48 ± 0.12

0.36 ± 0.04 0.32 ± 0.03 0.2 ± 0.01 1.48 ± 0.1 0.32 ± 0.02 1.09 ± 0.07

0.02 ± 0.005 0.18 ± 0.02 0.01 ± 0.002 0.01 ± 0.002 0.31 ± 0.02 0.15 ± 0.01

1.42 ± 0.1 0.78 ± 0.03 0.77 ± 0.03 3.46 ± 0.18 0.77 ± 0.07 3.37 ± 0.14

1.96 ± 0.3 1.45 ± 0.09 1.32 ± 0.06 4.1 ± 0.06 1.39 ± 0.08 4.55 ± 0.08

0.63 ± 0.04 0.4 ± 0.006 0.31 ± 0.003 1.34 ± 0.05 0.4 ± 0.02 0.88 ± 0.04

Data are expressed as μg of metabolite for g of sample. These values were obtained measuring the area of the relative HPLC-DAD peak and comparing it with that corresponding to its pure standard. All concentrations represent the mean of three independent measurements ± standard error (*p ≤ 0.05 and **p ≤ 0.01 vs the other samples on the same day of treatment; ***p ≤ 0.001 vs the same treatment at the other time of sampling). a

washes with sterile distilled water, seeds were sown in magenta boxes filled with a solid culture substrate containing Murashige and Skoog (MS) basal medium (pH 5.8),44 3% sucrose, and 0.6% agar and previously sterilized by autoclaving at 121 °C for 20 min. Seed cultures were maintained at 22 °C in a growth chamber characterized by a 16/8 h light/dark cycle. After 15 days of culture, leaves of moringa sprouts were sliced into uniform pieces (about 0.5 cm2 in size) and placed with their abaxial page in contact with solid medium whose composition was the same as that reported above with the addition of 0.5 mg/L of 2,4-dichlorophenoxyacetic acid, as suggested by Khalafalla et al.19 and Oriabi.20 Callus induction was performed by placing samples in the dark in a growth chamber at 22 °C for one month. After this period, the percentage of callus generation was measured as follows: induction rate (%) = (number of explants producing callus/number of total explants) × 100. Callus Treatments. Cold stress was applied to a one-month-old callus, setting the culture at 4 °C for 6 h, 16 h, 24 h, 48 h, and 7 days. Heat stress was done on a one-month-old callus, incubating the culture at 45 °C for 2 h. For saline and elicitor induction, the onemonth-old callus was transferred under sterile conditions, for 15 and 30 days, on fresh solid culture medium supplemented with 0, 100, or 200 μM salicylic acid and 0, 50, 100, or 200 μM NaCl, respectively. Plant Extraction. At the end of the treatments, plant materials were weighed and ground with liquid nitrogen, with a pestle and mortar. For spectrophotometric and chromatographic analyses, M. oleifera calli were processed according to the method of Atawodi et al.45 In detail, the powdered sample (0.5 g) was dissolved in 5 mL of MeOH, for 48 h under agitation, and centrifuged at 7.000g for 10 min. The supernatant was recovered and stored at 4 °C, until use. Total Phenolic Content. Total phenols were determined by the Folin Ciocalteu method, as reported in McDonald et al.46 and Di Marco et al.47 In brief, 0.5 mL of plant extract was completely dried using a speed-vacuum system (Eppendorf AG 22331 Hamburg, Concentration Plus) and, then, resuspended in 0.5 mL of doubly distilled water. The whole volume of plant extract was mixed with 5 mL of Folin Ciocalteu reagent (previously diluted 1:10, v/v, in distilled water) and 4 mL of 1 M Na2CO3. The mixture was incubated for 60 min in the dark and subjected to spectrophotometric analysis at

present study (e.g., alkaloids) and showing extraordinary antiradical property. In conclusion, in the present work the response of M. oleifera callus to different abiotic stresses was analyzed, with the aim to identify the best in vitro conditions for inducing the synthesis of antiradical secondary metabolites. Temperature variations and the presence of salicylic acid did not lead to an increase of the antioxidant behavior. By contrast, the treatments with NaCl, in particular at 100 μM concentration, caused an increase of total phenol content and antioxidant capacity in plant cells, compared to the untreated callus. Surprisingly, these values also appeared comparable to those measured in leaves of moringa plants grown under in vitro cultures. This last result is particularly interesting because it opens the way to potential industrial applications where moringa callus cultures, treated with 100 μM NaCl, might be used for a rapid, controlled, and guaranteed production of antioxidant molecules for pharmaceutical purposes. Moreover, liquid cultures of moringa cells exposed to stress conditions could promote the significant production of bioactive compounds in a short time, as the growth rate of suspension cells is usually higher than solid culture cells. Finally, the treatment of moringa cultures with multiple stresses could prove a further promising strategy for increasing secondary metabolite production in plant cells.

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EXPERIMENTAL SECTION

Plant Material and Callus Induction. Moringa oleifera seeds were collected from mature pods harvested before they spilt from trees grown in the District of Dschang in West Cameroon by the Cooperative of Medical Plant Producers SOCOPOMO. Seeds were preserved at the Botanical Garden of Rome “Tor Vergata” at 2 °C and in the presence of 20% humidity. Before use, M. oleifera seeds were deprived of the external envelope, sterilized by immersion in 70% EtOH for 1 min, washed with sterile distilled water, and soaked for 10 min in a solution of 2.5% NaOCl and a drop of Tween 20. After three F



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765 nm (Varian Cary, 50 Bio UV−vis, Italy). A standard curve was prepared using 0−250 mg L−1 solutions of gallic acid (Sigma-Aldrich, Milan, Italy) in water (equation: y = 0.1085x + 0.0525; R2 = 0.9944). Results are expressed as μg of gallic acid equivalent per g of sample fresh weight (μg GAE/g FW). Antiradical Assays. The FRAP assay is a colorimetric method based on the reduction of the Fe3+ TPTZ (2,4,6-tripyridyl-S-triazine) colorless complex to Fe2+ TPTZ colored molecule, in the presence of antioxidants. It was performed according to the Benzie and Strain48 method after adequate modifications, as reported by Gismondi et al.17 A fresh FRAP reagent was prepared by mixing 10 volumes of 300 mM acetate buffer (pH 3.6), 1 volume of 10 mM TPTZ resuspended in 40 mM HCl, and 1 volume of 20 mM FeCl3. A 200 μL amount of plant extract was mixed with 200 μL of EtOH and 1 mL of the FRAP reagent. The reaction mixture was incubated at 37 °C for 10 min, and its absorbance spectrophotometrically determined at 593 nm. The free radical scavenging capacity of the plant extracts was also studied using the 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) assay, or ABTS test, as described in Gismondi et al.49 The radical cation solution (ABTS+•) was prepared by dissolving 7 mM ABTS and 2.45 mM K2S2O8 in doubly distilled water. An appropriate solvent blank reading was taken (AB). After addition of 100 μL of plant extract, or MeOH (blank sample), to 3 mL of ABTS+• solution and incubation for 10 min at room temperature, the absorbance was read at 734 nm. Results are expressed as nM of ascorbic acid equivalents per g of sample fresh weight (μM AAE/g FW), according to a calibration curve adequately produced using pure ascorbic acid (range: 0−50 μM L−1; equation: y = 0.002x + 0.113 and R2 = 0.992 for FRAP; equation: y = −0.0007x + 1.5731 and R2 = 0.995 for ABTS). Concentrations of Secondary Metabolites in Moringa Callus. The biochemical profile of M. oleifera calli was carried out based on the modified method of Gismondi et al.50 The analyses were performed using an HPLC system associated with an LC-20AD pump, a CBM-20A controller, a SIL-20a HT autosampler, and an SPD-M20A diode array detector (DAD) (Shimadzu, Kyoto, Japan). Chromatographic separation was achieved by a Phenomenex Luna 3u C18(2) column (150 mm × 4.60 mm × 3 μm) using mobile phases consisting of 1% formic acid (v/v) (phase A) and MeOH (phase B), at a flow rate of 0.95 mL per minute. The elution started at 15% B solvent; this condition was maintained for 20 min and was linearly increased up to 35% B solvent in 20 min and up to 90% in 60 min. The injection volume was 50 μL and column temperature was set at 40 °C. Data acquisition was performed using LAB-SOLUTION software (Shimadzu). Qualitative and quantitative determination of specific secondary metabolites in the samples was carried out comparing their retention times (minutes), UV-light absorption spectra, and peak areas of pure standard molecules (gallic acid; chlorogenic acid; caffeic acid; p-coumaric acid; 3,3-dimethylallyl caffeate; phenyl caffeate; 3-O-α-L-arabinopyranosylquercetin; rutin; quercetin; myricetin; 3-O-β-D-glucopyranosylkaempferol; genistein; kaempferol; apigenin; chrysin; galangin; Figure 1; Sigma-Aldrich, Milan, Italy) relative to each identified plant compound. Results are expressed as μg of standard equivalent per g of sample fresh weight (μg SE/g FW). Browning Rate Measurement. Images of stressed callus were acquired, and the degree of browning of plant material was measured as mean intensity value of the whole area. Quantitation was performed using ImageJ software (National Institute of Health, Bellevue, WA, USA); results were expressed in arbitrary units (from 0 to 255), where lower values corresponded to darker colors. Measurements were collected on at least six images of replicates per sample. Statistical Analysis. All experiments were performed, at least, in triplicate, and results were reported as a mean of these independent replicates ± standard deviation. Statistical analysis was performed using the ANOVA test followed by the Tukey test using GraphPad Prism 6 software.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.8b00801.

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Concentrations of secondary metabolite in moringa callus exposed to different temperature stress and in young leaves obtained by germination of seeds; total phenol content in moringa callus exposed to different temperature stress; antiradical activity of the extracts from moringa callus exposed to different temperature stress (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel (A. Canini): +39 06 7259 4333. Fax: +39 06 20 25 300. E-mail: [email protected]. ORCID

Antonella Canini: 0000-0003-1132-8899 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Mr. Martin Bennet for revising the English syntax and grammar of the manuscript. REFERENCES

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Induction of Antioxidant Metabolites in Moringa oleifera Callus by

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