Chemical analysis of callus extracts from toxic and non-toxic varieties of Jatropha curcas L.

Introduction

Jatropha curcas L. (Euphorbiaceae) is a multipurpose plant native to Mesoamerica, and it is important because of its usefulness as raw material in biofuels production (Salvador-Figueroa et al., 2015) as well as in veterinary and human traditional medicine (Zhang et al., 2017). Several compounds with different biological activities have been isolated from different species of Jatropha (Ferreira-Rodrigues et al., 2016; Katagi et al., 2016). The identification of biologically active compounds extracted from different organs of this plant has been reported (Prasad, Izam & Khan, 2012; Sharma, Dhamija & Parashar, 2012). Isolated compounds or whole plant extracts have been studied because of their potential pharmacological activity (Cocan et al., 2018). Biological effects of J. curcas include antibacterial (Rampadarath, Puchooa & Jeewon, 2016), cytotoxic (Katagi et al., 2016), anti-inflammatory (Salim et al., 2018), and antifungal effects (Abdelgader, Suleiman & Ali, 2019; Srinivasan, Palanisamy & Mulpuri, 2019). Most research on J. curcas have been done with toxic varieties; toxicity is referred to phorbol esters content in seeds.

In Mexico, Brazil and India, non-toxic varieties of this species have been identified with very low or non-detectable levels of phorbol esters (PEs) in seeds (Laviola et al., 2010; Martínez-Herrera, Chel-Guerrero & Martínez-Ayala, 2004; Kumar, Anand & Reddy, 2011). PEs are known as Jatropha factors because each one of them has the same nucleus diterpene moiety, namely, 12-deoxy-16-hydroxy-phorbol (DHP) which is coupled to unstables intramolecular diterpenes (named C₁–C₆ factors) (Hirota et al., 2017).

Plants are the most successful source of chemical compounds, and their potential mode of action makes them an alternative phytomedicinal drug, since several natural products have shown benefits against human diseases (Aye et al., 2019). Several compounds are tissue-specific accumulated, and are usually structurally complex (Armaly et al., 2015). Therefore, it is necessary the use of chemical analysis techniques to isolate and identify the extracted plant metabolites (Hernandez & Sarlah, 2019). There are a few cases where the use of the plant cell culture of Jatropha curcas has allowed the production of bioactive compounds (Alvero-Bascos & Ungson, 2012; Mahalakshmi, Eganathan & Parida, 2013; Nassar et al., 2013; Zaragoza-Martínez et al., 2016); the study of the culture at different stages of toxic and non-toxic varieties generates the opportunity to design biotechnological models for production of bioactive compounds i.e., terpenoids, alkaloids, flavonoids (Abdelgadir & Van Staden, 2013; Sabandar et al., 2013), providing opportunities for new drugs discovery.

Secondary metabolites are generally in complex matrices at very low concentrations in plant organs, and lower in dedifferentiated cells. These compounds have a wide range of polarities; therefore, it is necessary the use of solvents with different polarity to obtain the extracts (Chemat et al., 2019). The aim of this work was to characterize the chemical profile of the extracts from callus of both varieties (toxic and non-toxic) of Jatropha curcas, through the cell culture, to verify the presence of important compounds in dedifferentiated cells and consider the possibility of using these cultures for the massive production of bioactive compounds.

Materials & Methods

Results

Establishment of callus culture

Dedifferentiation cell was not observed in leaf blade explants. Petiole explants showed tissue dedifferentiation since seventh day of culture and complete process was evident at the day 21 (Fig. 1). Friable and light green callus was obtained on MS media added with both combinations: NAA (1.5 mg L⁻¹), BAP (1.5 mg L⁻¹), and NAA (3.0 mg L⁻¹) and BAP (3.0 mg L⁻¹).

Thin layer chromatography (TLC) analysis

TLC showed differences in compounds accumulation during time culture (2, 6, 10, 14, 18, 22, 26, 30, 34 and 38 d). Regard diterpenes, spots with Rf of 0.71 and 0.27 showed higher intensity along this period (Fig. 2A), unlike flavonoids in which spots with Rf of 0.77 and 0.58, decreased throughout the same culture period (Fig. 2B). These results suggest that the accumulation of diterpenes and flavonoids was inversely related during callus development. To obtain diterpenes the Soxhlet-methanol extraction was more efficient than sonication-ethanol 80%. TLC analysis of extracts obtained by both methods evidenced differences in the size and intensity of spots in regard to: extraction method, variety (toxic and non-toxic), and plant material (seeds, leaves and callus) (Fig. S1).

Flavonoids analysis

On the other hand, the main group of compounds in Jatropha leaf extracts are flavonoids, among them the apigenin, nevertheless, it is important to refer that the natural condition of flavonoids in the plants is in glycosylated form. On this regard, another of the highest signals observed at the chromatograms was the m/z of 381 [M+H]⁺ ion, the MS-MS experiment of this signal and the proposed structures obtained by CFM-ID platform allowed to relate that molecular ion (m/z 381 [M+H]⁺) to the fragmentation profiles of apigenin 6-C-α-L-arabinopyranosyl-8-C- β-D-xylopyranoside, and of apigenin 4′-O-rhamnoside (Fig. 5). The fragmentation signals and their corresponding predictive structure were also related for vitexin (m/z 433 [M+H]⁺), vitexin 4′-O-glucoside-2″-O-rhamnoside (m/z 741 [M+H]⁺), vicenin-2 (m/z 595 [M+H]⁺), and vicenin-2,6″-O-glucoside m/z 757 [M+H]⁺ (Fig. S3). Table 1 shows the relative abundance of six tentatively identified compounds by relating their molecular ions on 14 d and 38 d callus extracts from both varieties. Inversely to observed on DHP related signal (m/z 365 [M+H]⁺), the intensity of the molecular ion related with glycosylated apigenin (m/z 381 [M+H]⁺) diminished (Fig. 6).

Table 1:

Tentative compounds identified by ESI-MS in hydroalcoholic extracts from seeds, leaves, and callus of 14 and 38 d of culture from both toxic and non-toxic Jatropha curcas L. varieties.

Compound type/ name	Elemental composition	Mass	Fragment ions in positive ion mode (m/z)	Plant material	Time of culture (d)	Variety	Relative abundance (%)
Phorbol
12-deoxy-16-hydroxy-phorbol	C20H28O6	364.4	127, 185, 203, 234, 276, 295
				Seeds		T	64.70
						NT	21.05
					Callus	14	T	14.28
							NT	8.69
				38		T	30.00
NT	25.00
Glycosylated Flavonoids
Apigenin 6-C- α-L-arabinopyranosyl-8-C- β-D-xylopyranoside (m/z 535 [M+H]+), and Apigenin 4′-O-rhamnoside (m/z 417 [M+H]+)	C25H26O13 and C21H20O9	534.47 and 416.4	381
				Leaves		T	45.83
						NT	100
					Callus	14	T	100
							NT	100
				38		T	100
NT	62.50
Vitexin (m/z 433 [M+H]+)	C21H20O10	432.37	415
				Leaves		T	27.7
						NT	70.00
					Callus	14	T	10.34
							NT	4.76
				38		T	11.11
NT	<12.50
Vitexin 4′-O-glucoside-2″-O-rhamnoside (m/z 741 [M+H]+)	C33H40O19	740.7	577
				Leaves		T	33.3
						NT	42.84
					Callus	14	T	1.42
							NT	6.36
				38		T	12.50
NT	8.75
Vicenin-2 (m/z 595 [M+H]+)	C27H30O15	594.5	503
				Leaves		T	25.00
						NT	29.16
					Callus	14	T	7.14
							NT	4.00
				38		T	6.25
NT	3.75
Vicenin-2,6″-O-glucoside (m/z 757 [M+H]+)	C33H40O20	756.7	757
				Leaves		T	<9.09
						NT	8.00
					Callus	14	T	1.42
							NT	1.73
				38		T	2.22
NT	1.25

DOI: 10.7717/peerj.10172/table-1

Notes:

T

Toxic

NT

Non-toxic

Discussion

The highest callus induction (95.5%) was observed in petiole explants on MS medium added with NAA (3.0 mg L⁻¹) and BAP (3.0 mg L⁻¹), the second best result (87.7%) was obtained with NAA (1.5 mg L⁻¹) and BAP (1.5 mg L⁻¹), in contrast to reported by Nassar et al. (2013), who observed dedifferentiation with NAA and BAP at 0.5 mg L⁻¹ of each one plant growth regulator. Explants dedifferentiation reported in this work was similar to reported by Kumar et al. (2015). The follow up of the explants dedifferentiation process, every 7 d showed callus formation on explants starting on the seventh day. Dedifferentiation began at the cutting sites as expected (Sujatha, Makkar & Becker, 2005; Nogueira et al., 2011; Ovando-Medina et al., 2016). The callus obtained was light green and friable, similar to reported by Hernández et al. (2015). It has been reported that high auxins concentrations could affect production and accumulation of secondary metabolites (Kim et al., 2007), hence, according to our results, it is suggested the use of the lowest effective concentration, 1.5 mg L⁻¹ for both growth regulators. Muñoz-Valverde et al. (2003) concluded that BAP is determinant to induce callus formation in foliar explants of J. curcas. Likewise, Suárez & Salgado (2008) reported that the presence of NAA induce callus formation in Stevia rebaudiana, and this effect could be increased when adding BAP. On the other hand, Solange et al. (2002) determined that the use of NAA and BAP in equal proportion induces callus formation from leaf explants of Tridax procumbens. Coutiño-Cortés et al. (2013) reported the callus induction in J. curcas leaf explants at 10 d of culture, and total explant-cell dedifferentiation at 20 d using 2, 4-D, BAP and KIN, while in this work, petioles dedifferentiation started at 7 d and total explant-cell dedifferentiation was achieved at 21 d. These results support that synergy between NAA and BAP is essential to achieve a high dedifferentiation degree. Stable callus culture conditions for the two varieties of Jatropha curcas were established.

The PEs are responsible for the toxicity in the plant (Devappa, Makkar & Becker, 2011; Sabandar et al., 2013; Zhang et al., 2017). There are varieties of Jatropha curcas denominated as toxic and non-toxic (Makkar et al., 1997). The non-toxic varieties have PEs concentration lower than 0.86 mg/g of seed on dry basis (He et al., 2011). Martínez-Herrera et al. (2006) detected high levels of PEs in seed oil from the municipality of Coatzacoalcos, Veracruz, México, but did not detect PEs in seeds from the municipality of Yautepec, Morelos, México. This corroborates the differences between the seeds of the two varieties used in this study.

Regard, to TLC profile analysis, it has been reported that methanolic extraction from seed-oil facilitates separation and availability of methyl ester type compounds, mainly phorbol esters (PEs) (Demissie & Lele, 2010; Devappa, Bingham & Khanal, 2013). The detection by TLC of PEs in seed methanolic extracts from toxic and non-toxic J. curcas varieties was reported (Devappa, Makkar & Becker (2012), they reported higher spots intensity from toxic variety than from non-toxic, when plates were observed at 366 nm UV light, this result is similar to that observed in this work (Fig. S1). Makkar & Becker (2009) detected higher PEs accumulation in seeds than in leaves extracts. Similar results were obtained in this work, even with different method of extraction. Nevertheless, these results are different of that obtained by Martínez-Herrera, Chel-Guerrero & Martínez-Ayala (2004), because they reported 96% of PEs extraction through hydroalcoholic extraction, quantified by HPLC; while, in this work the intensity of the spots was higher on Soxhlet-methanol extracts than hydroalcoholic extraction (Fig. S1). Using TLC, differences between dedifferentiated cell extracts of both varieties of Jatropha curcas were evidenced.

On the other hand, Hirota et al. (2017) reported the identification of DHP as the fundamental structural core which is derived from 12-deoxy-16-hydroxy-phorbol-4′-[12′,14′-butadienyl]-6′-[16′,18′,20′-nonatrienyl]- bicycle [3.1.0] hexane-(13-O)-2′-[carboxylate]- (16-O)-3′- [8′-butenoic-10′]ate (DHPB or Jatropha factor C₁), identified as DHPB-Na adduct m/z 733 [M+Na]⁺. Furthermore, DHPB m/z 711 [M+H]⁺ and DHP m/z of 365 [M+H]⁺ were also reported in J. curcas seeds (Wink et al., 2000). Even more so, Haas, Sterk & Mittelbach (2002) reported the identification of diterpenes named Jatropha factors C₂ to C ₆through ESI-MS m/z 711 [M+H]⁺ and of DHP (m/z of 365 [M+H] ⁺). Furthermore, Nishshanka et al. (2016) identified six phorbol esters in J. curcas seeds by LC-MS, which have the same core (DHP), at the so named Jatropha factors (C₁ to C₆).

Regard to PEs identification by ESI-MS analysis, Verardo et al. (2019) identified six phorbol esters in J. curcas seeds with m/z of 711 [M+H]⁺, which have the same fundamental structural core (DHP) m/z of 365 [M+H]⁺ which is coupled to diterpenes of 24 carbon structures named Jatropha factors from C₁ (DHPB) to C₆. The relative intensity of the molecular ion m/z 365 [M+H] ⁺ was higher in seeds extracts from toxic variety, than in seed extracts from non-toxic variety (Data not shown). While in callus, the relative intensity is higher in toxic and non-toxic varieties callus of 38 d of culture (Figs. 4B, and 4D, than in toxic and non-toxic varieties callus of 14 d of culture (Figs. 4A, and 4C). These results could suggest the presence of PEs coupled to DHP in the samples analyzed and that their accumulation is differential in regard to the variety-derived cell culture and throughout the time of culture. These results suggest that their accumulation of DHP is time dependent. This ESI-MS analysis allowed to corroborate the results obtained by TLC (Fig. 2A). Nevertheless, the relative intensity of the signals observed in extracts from callus were lower than that obtained from seeds extracts as reported by Demissie & Lele (2010). By ESI-MS, differences in the relative intensity of the signal corresponding to DHP were observed between the callus extracts of both varieties, being higher in the toxic variety in addition to that in calluses at 38 d of culture, it was higher than in the 14 d.

By other hand, phenolic compounds are ubiquitously produced by plants (Kumar & Goel, 2019), the main role of phenols in plants is to protect them from biotic or abiotic stress (Pereira, 2016). These properties include antimicrobial, insecticidal, antiparasitic, antiviral, anti-ulcerogeBynic, cytotoxic, antioxidant, anti-hepatotoxic, anti-hypertensive and anti-inflammatory activities (Oskoueian et al., 2011; Papalia, Barreca & Panuccio, 2017). Flavonoids are recognized as polyphenols. Several of them have been identified in Jatropha genus, such as apigenin glycosides, vitexin, and isovitexin which have been considered as chemiotaxonomic compounds from the genus (Abdelgadir &Van Staden, 2013; Huang et al., 2014).

The tentative identification of glycosylates-flavonoids through microQTOF-QII has been already reported (Pezzini et al., 2019). In this regard Xie et al. (2003) reported the apigenin 6-C- α-L-arabinopyranosyl-8-C- β-D-xylopyranoside m/z 535 [M+H]⁺. Likewise, this result may be related to that obtained by Abd-Alla et al. (2009) who identified apigenin and its aglycone as majoritarian flavonoids in J. curcas leaves, as well as, that obtained by Reena, Nand & Sharma (2008) who reported to apigenin as major flavonoid in the same species. Those reports differ from that published by Papalia, Barreca & Panuccio (2017) who identified to vitexin and vicenin-2 as the majoritarian flavonoids.

The results obtained by microQTOF-QII of the molecular ion m/z 381 [M+H]⁺ through the MS/MS experiment, and the predictive structures obtained through the CFM-ID platform allowed to suggest the relation of the structures from the molecular ion m/z 381 [M+H]⁺ with the fragmentation profile from apigenin 6-C- α-L-arabinopyranosyl-8-C- β-D-xylopyranoside m/z 535 [M+H]⁺, which was identified through ESI-MS in Viola yedoensis (Xie et al., 2003) and apigenin 4′-O-rhamnoside m/z 417 [M+H] ⁺, which was identified in Olea europaea (Pieroni et al., 1996).

Based on the molecular ion, MS-MS fragmentation profile and the predictive structures obtained by CFM-ID platform, it is suggested the tentative identification of vitexin m/z of 433 [M+H]⁺, vicenin-2 m/z of 595 [M+H]⁺, and vitexin 4′-O-glucoside-2″-O-rhamnoside m/z of 741 [M+H]⁺ in leaves and callus from both varieties. These results are similar to obtained by Huang et al. (2014) who identified vitexin m/z of 433 [M+H]⁺ in J. curcas leaves. This flavonoid was also identified by ESI-MS in Parkinsonea aculeata m/z of 431 [M-H]⁻ (Hassan, Abdelaziz & Al Yousef, 2019). In this work it is also suggested the tentative identification of vicenin-2,6″-O-glucoside m/z 757 [M+H]⁺ which has not been reported to Jatropha curcas, but to Stell aria holostea (Bouillant et al. 1984) (Fig. S2). By the MS-MS fragmentation profile, the identification of six glycosylated flavonoids is suggested, it was observed that relative intensities signals related to flavonoid related molecular ion m/z 381 [M+H]⁺ in callus of 14 d was higher than callus of 38 d, differences that were not observed between calluses of the different varieties.

Conclusions

Stable dedifferentiated cells culture from petiole explants of Jathopha curcas, were stablished from toxic and non-toxic varieties on MS medium added with NAA and BAP. Thin layer chromatography and mass spectrometry, suggest an inverse relationship between phorbol esters and flavonoids accumulation in callus throughout the time of culture. The tentative identification of diterpene type compounds such as 12-deoxy-16-hydroxy-phorbol and Jatropha factors by ESI-MS in seed and callus (14 and 38 d), as well as, the presence of six flavonoids glycosides in leaf and callus, in extracts from both toxic and non-toxic varieties of J. curcas, is suggested. Both of them, groups of compounds reported with bioactive activity with pharmaceutic/agroindistrial potential.

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