Tapping the potential of Calotropis procera hairy roots for cardiac glycosides production and their identification using UHPLC/QTOF-MS

Abstract

The present work deals with the establishment of hairy root cultures from different explants of C. procera using Agrobacterium rhizogenes strain A4. A high transformation frequency (95%) was obtained from leaves followed by cotyledons (81.6%) and hypocotyls (38.3%). Genetic transformation of hairy roots was confirmed through PCR by amplifying a 400 bp fragment of the rolB gene. Hairy roots were highly branched, possessed plagiotropic and rapid growth on hormone-free ½ B5 medium. Ten cardiac glycosides, including calotropagenin, calotoxin, frugoside, coroglaucigenin, calotropin, calactin, uzarigenin, asclepin, uscharidin, and uscharin, based on their specific masses and fragmentation properties were identified in ethanolic extracts of hairy roots by ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry UHPLC/QTOF-MS. This protocol could be used as a powerful tool for large-scale in vitro production of highly valued cardiac glycosides and for further transcriptomics or metabolomics studies.

Introduction

Medicinal plants have been used for centuries in the cure and treatments of many diseases and illnesses with their specific metabolites and bioactive compounds (Gaobotse et al. 2023; Moiketsi et al. 2023; Nkwe et al. 2021). Cardiac glycosides are bioactive natural metabolites produced by different species of plants with potential health benefits. The chemical structure of these compounds is very comparable. They contain a steroid ring, a lactone ring with five or six carbons, and a sugar moiety (Botelho et al. 2019). These specialized metabolites have been used to treat heart failure and arrhythmias (Bartnik and Facey, 2017; Jensen et al. 2011; Kumavath et al. 2021; Schneider et al. 2017). In addition, some cardiac glycosides, such as digitoxin, digoxin, ouabain, oleandrin, calactin, calotropin, and bufalin in addition to their cardiotonic effect, have shown a cytotoxic activities against a number of cancer cell lines through inhibition of Na⁺/K⁺-ATPase and induction of immunogenic cell death (Hou et al. 2021; Kumavath et al. 2021; Petschenka et al. 2018). These are biosynthesized by plants belonging to Apocynaceae, Asparagaceae, Plantaginaceae, and Moraceae family such as Digitalis purpurea, Antiaris toxicaria, Asclepias sp., Bowiea volubilis, Calotropis procera, Calotropis gigantea, Nerium oleander, and Thevetia peruviana (Patel 2016).

Calotropis procera (Aiton) W.T. Aiton (Family: Apocynaceae), commonly known as kranka (Algerian arabic) and tourha (Tamahaq) in Algeria (Hammiche and Maiza 2006), is a xerophytic perennial medicinal shrub or small tree (Hassan et al. 2015). It is also commonly known as Giant milkweed, Sodom apple, Madar, Calotrope, or Ushar (Kumari and Chaudhary 2021). It is distributed in the arid and semi-arid areas in Africa and Asia (Boutraa 2010) and grown widely in these regions without irrigation, chemical fertilizers, pesticides, or other agronomic practices (Erdman 1983). C. procera is recognized for its therapeutic properties and it has been highly used in traditional medicines in Algeria and throughout the world.

This plant species is known to possess wide range of biological properties as reviewed recently by Dogara (2023) and Wadhwani et al. (2021). Different parts like roots, root bark, leaves, flowers, and latex of this plant species have been used to prevent dermatosis, infected sores, syphilis, respiratory diseases, cough, tonsillitis, jaundice, helminthiases, bilharziose, dysentery, constipation, fever, rheumatism, asthma, epilepsy, pains, tonic, abortive, piles, infected wounds, psoriasis, leishmaniasis, headache, digestive disorders, eye diseases, and as antiseptic (Gamal et al. 2010; Hammiche and Maiza 2006; Kumari and Chaudhary 2021; Lal and Yadav 1983).

The above-mentioned biological properties of C. procera are attributed to the presence of various specialized metabolites including sterols, cardiac glycosides, alkaloids, tannins, flavonoids, triterpenes, coumarins, and saponins (Mossa et al. 1991). These activities were already confirmed with scientific experiments such as larvicidal (Markouk et al. 2000), antibacterial (Mako et al. 2012), anthelmintic (Iqbal et al. 2005), acaricidal (Al‐Rajhy et al. 2003), schizontocidal (Sharma and Sharma 2000), antinociceptive (Soares et al. 2005), antioxidant (Kumar et al. 2013), spasmolytic (Iwalewa et al. 2005), immunomodulatory (Seddek Abdel Latif Shaker et al. 2000), antipyretic, analgesic, purgative (Mossa et al. 1991), wound healing (Rasik et al. 1999), hepatoprotective (Setty et al. 2007), antiulcer (Basu et al. 1997), antifertility (Kamath and Rana 2002), anti-inflammatory (Teixeira et al. 2011), anti-diarrheal (Kumar et al. 2001), and anticancer activities (Sweidan et al. 2021).

A number of bioactive compounds such as calotropagenin, calotoxin, frugoside, coroglaucigenin, calotropin, calactin, uzarigenin, asclepin, uscharidin, and uscharin have been reported from C. procera. Specifically calotropin and related cardiac glycosides have been found to possess cardiac properties (Farr et al. 2002; Petschenka et al. 2018) similar to the medications digitoxin and digoxin (Koch et al. 2020), as well as cytotoxic activities against a number of cancer cell lines (Huang et al. 2018; Li et al. 2009; Zheng et al. 2021) and autoimmune disorders (Liu et al. 2018).

Actually, plant tissue (restricted resource) is used for industrial production of pharmaceutically important cardiac glycosides on a large scale. Complicate structure of these glycosides makes their chemical synthesis unfeasible, thus relying only on the plants for the production of these valuable compounds (Bhusare et al. 2021). Determining the entire biosynthetic pathway is essential to engineering the synthesis of cardiac glycosides in heterologous organisms for medicinal application (Hoopes et al. 2018).

To produce useful secondary metabolites, in vitro cultures are being investigated as an alternative to agricultural processes for medicinal plants and their active constituents (Malik et al. 2009, 2011, 2014a, b, 2016, Gallego et al. 2017). Hairy root (HR) cultures have demonstrated promising biosynthetic potential for specialized metabolite production independent of environmental variations (Makhzoum et al. 2015, 2013; Makhzoum and Hefferon 2022; Moussous et al. 2018; Kim et al. 2002). HRs induced by A. rhizogenes (recently known as Rhizobium rhizogenes) infection are characterized by their rapid growth on hormone-free medium and high genetic stability (Benyammi et al. 2016; Habibi et al. 2017; Makhzoum et al. 2011; Singhabahu et al. 2017). Furthermore, when compared to undifferentiated cell cultures, HRs always have comparable biosynthetic potential to native plant roots and typically accumulate bioactive secondary compounds at higher levels (Chandra and Chandra 2011). Hairy roots cultures have also been used to produce recombinant proteins and other pharmaceutical compounds and molecules (Gaobotse et al. 2022; Makhzoum et al. 2014a, b; Moustafa et al. 2016; Tremouillaux-Guiller et al. 2020).

HR cultures in C. procera have been studied recently in response to nano-elicitors for the enhanced production of essential oil and antioxidant properties (Adabavazeh et al. 2022, 2023). There is no study conducted so far related to the analysis and characterization of calotropin and related cardiac glycosides from HR cultures. It is well known that transformation and HR induction depend on the type of explant used. The present study aims to (a) see the potential of different explants, viz., leaves, hypocotyls, and cotyledons in C. procera for inducing HRs by following the procedures developed previously in the laboratory (Amdoun et al. 2009; Benyammi et al. 2016), and (b) analyze and phytochemically characterize the cardiac glycosides from HRs using UHPLC/QTOF-MS.

Materials and methods

Plant material and growth conditions

Seeds of C. procera were collected from the National Institute for Agricultural Research station (Adrar, Algeria). Healthy plants of C. procera were used as seed source. Mature fruits were opened in the lab in order to release their seeds. Undamaged seeds were selected and cleaned from silk fibers before their use. Seeds of C. procera were disinfected by immersion in ethanol 70% (v/v) for 1 min, followed by soaking in a hypochlorite sodium solution (NaClO 2.6% chlorine) for 15 min, then rinsed three times with sterile distilled water (Benmahioul et al. 2009). The seeds are plated into test tubes containing 20 ml of Murashige and Skoog (MS) medium (Murashige and Skoog 1962) supplemented with 7 g/L (w/v) of agar and 20 g/L (w/v) sucrose. The test tubes were then placed in the growth chamber at a temperature of 26 ± 1 °C and a photoperiod of 16 h. Hypocotyls, cotyledons, and leaves were excised aseptically from seedlings after 30 days of culture and used as explants for the induction of hairy roots.

Bacterial strains and growth media

Agrobacterium rhizogenes strain A4 was kindly provided by Professor Zoulikha Krimi (Laboratoire de Valorisation des Ressources Agrobiologiques, Université de Blida 1, Algérie). The A4 strain was used to induce hairy roots in C. procera. The bacteria was cultured for 48h onto a solid yeast extract mannitol medium (YEM) (Wise et al. 2006) under dark conditions at 28 °C. A single colony of bacteria was picked and allowed to grow in YEM liquid medium for 72 h at 100 rpm at 28 °C (till the optic density “OD600” of 0.6–0.8 was achieved). This bacterial suspension was used for the infection of plant material.

Establishment of hairy root cultures

HRs were induced by infecting explants (hypocotyls, cotyledons, and leaves) taken from in vitro seedlings of C. procera with A. rhizogenes strain. Experiment was performed using forty explants and each experiment was repeated thrice. Hypocotyl segments were wounded and then infected with bacterial suspension and cultivated onto solidified half strength B5 (½ B5) medium (Gamborg et al. 1968) at 26 ± 1 °C in darkness. However, cotyledons and leaves were immersed in the bacterial suspension for 30 min, dry blotted on sterile filter paper, and co-cultivated at 26 ± 1 °C for 48 h on solidified MS medium. After 2 days of co-cultivation, bacteria were removed from the explants by washing with sterile water and then transferred onto solidified ½ strength B5medium supplemented with 250 mg/L cefotaxime and were incubated at 26 ± 1°C under dark conditions till the emergence of HRs. YEM liquid medium without bacteria was applied to the explants and served as a control.

Confirmation of the transgenic nature of the established HR

The integration of A. rhizogenes T-DNA in explants of C. procera was confirmed by polymerase chain reaction (PCR) using a BIO-RAD^® CFX96 thermal cycler. Genomic DNA was extracted from the HR1 and from those non-treated (control) using NucleoSpin DNA Plant Mini kit (Macherey–Nagel, Düren, Germany) according to the manufacturer's instructions. The plasmid DNA isolated from cultures of A. rhizogenes strain A4 was used as a positive control; it was isolated using an alkaline lysis Miniprep. The PCR was performed to amplify internal rol B gene fragment using specific primers (F-5’-GCGACAACGATTCAACCATATCG-3’ and R-5’-TTTACTGCAGCAGGCTTCATGAC-3’). Each PCR reaction was carried out in a 20 µL volume, containing 2 µL genomic DNA, 0.8 µL of each rolB gene specific primer (forward and reverse), 2 µL of 10 × Taq polymerase buffer, 0.4 µL dNTP mix, and 0.4 µL of Taq DNA polymerase. The PCR was performed under the following conditions: initial denaturation temperature of 94 °C for 4 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 57 °C for 1 min and extension at 72 °C for 1 min, and ended with a final extension of 72 °C for 7 min. The PCR results were checked with a 1000 bp DNA marker using agarose gel electrophoresis (1.2%), which was stained with ethidium bromide solution and examined under UV light.

Cardiac glycosides analysis

Sample preparation

Cardiac glycosides extraction was performed according to an earlier method described by (Pandey et al. 2016) with minor changes. Briefly, 500 mg of sample was dissolved in 10 ml absolute ethanol and left shaking mechanically for 48 h. The mixture was centrifuged (Sigma) at 3500 rpm for 1 min. The resulted supernatant was collected and dried under nitrogen evaporator (Liebisch). After that, 5 mL of MeOH (LC–MS grade) was added to the residue. The obtained solution was mixed on vortex (VWR), sonicated (Fisher Scientific) for 5 min at 40 °C, and filtered using a 45 μm sterile syringe microfilter for UPLC-QTOF/MS analysis.

UHPLC/QTOF-MS analysis: instrumentation and chromatographic conditions

The Waters ultra-performance liquid chromatography system consisted of two binary solvent manager Acquity™ LC pumps, a Sample manager Acquity™ autosampler and a Column manager Acquity™ oven. Mass spectrometry data were acquired on a XEVO G2XS QTOF (Waters) instrument controlled with UNIFY software. Mass calibration was maintained via infusion of calibration solution after every fifth injection.

Analytical separation was conducted with ACQUITY HSS C18 column (150 × 2 mm, 1.8 µm) (Waters), an oven temperature of 50 °C, and mobile phases including ammonium formate buffer 5mM, pH 3 (A2), and acetonitrile in 1% formic acid (B2); flow rate of 0.4 ml/min was used. Initial concentration of mobile phase B2 (3%) held until 0.5 min, increased to 40% at 25 min, then to 100% at 26 min, and held until 30 min.

Mass spectrometric conditions were as follows: interface, positive electrospray ionization (ESI +); source temperature 140 °C; and ion spray voltage 20V. Conditions for the time-of-flight mass spectrometer (TOF MS^e) scan mode were as follows: scan range 100–1000 m/z for the function 1 and 50–1000 m/z, with a collision energy ramp from 10 to 40 eV for the function 2.

Statistical analysis

All data were analyzed statistically using IBM, SPSS statistics (Version 20.0). Analysis of variance was performed to calculate statistical significance of the treatment effects and means ± standard deviation (SD) that differed significantly were compared using Newman–Keuls test at the α = 0.05 level.

Results and discussion

Establishment of hairy root cultures

Hypocotyls, cotyledons, and leaves were cultivated with Agrobacterium rhizogenes. The effect of explant type on frequency of transformation was investigated during five weeks. After two weeks of cultivation, roots emerged from wounded parts of explants (Fig. 1a–c). The minimum period required for initiation of hairy roots was 13 days. In all, the most appreciable number of roots emerged at infection site was derived from explants of leaves and cotyledons (about 14). No hairy roots or calli formation was observed from control C. procera explants (untreated). Results showed a direct emergence of roots from wounded sites of explants. The roots presented the typical traits of hairy roots such as rapid growth, lateral branching, hormone autotrophy, and plagiotropism.

Fig. 1.

Establishment and growth of hairy roots from C. procera in vitro explants by A. rhizogenes strain A4. a–c Roots at the wounding site after A. rhizogenes infection from hypocotyls (G =  × 10), cotyledons (G =  × 10), and leaves explants, respectively, d HR line phenotype, e Proliferation of HR on semi-solid ½ B5 medium after 30 days, f Fast growing HR1 on ½ B5 liquid medium after 20 days under dark conditions. Scale bars correspond to 1 cm (c–f)

Hairy roots induction was observed after 35 days of co-cultivation. The frequency (%) of HR induction was calculated as the percentage of inoculated explants forming roots to the total number of explants used for the HR induction. Hairy roots induced from all the explants tested; however, the highest frequency of hairy roots induction in C. procera was observed in leaves (95%) after five weeks of incubation followed by 81.67% and 38.33% from cotyledon and hypocotyl explants, respectively (Fig. 2). A maximum of transformation frequency was recorded in leaf explants (Fig. 2).

Fig. 2.

Percentage of hairy root induction from explants of C. procera by direct infection with Agrobacterium rhizogenes strain A4. Observations were recorded after 5 weeks. Each treatment involved 40 explants. The experiments were repeated three times. Values are given as the mean ± SD (standard deviation). Values of bars followed by the same letter are not significantly different at the 0.05 level using Newman–Keuls test

The nature of the explant plays a very important role in the success of genetic transformation. In the present study, leaves were found to be the best material for hairy root induction followed by cotyledons and hypocotyls. The rooting was observed from the cut surfaces. This result is in accordance with previous studies obtained in Papaver bracteatum, where leaves showed high rate of hairy root induction than hypocotyl (Sharafi et al. 2013), in Cucumis anguria, leaves induced higher number of hairy roots compared to cotyledon (Yoon et al. 2015). Also, leaf explants give high induction of hairy root (91.5%) in Indian Momordica charantia related to cotyledons (75%) and hypocotyls explants (25%) (Thiruvengadam et al. 2014).

Varied response of different plant tissues to A. rhizogenes transformation has been previously listed (Sujatha et al. 2013). It is evident that A. rhizogenes is able to modulate defense pathways in transformed plant cells by expressing T-DNA oncogenes (Kiselev et al. 2006). The effect of rol genes in alteration of the cytokinin/auxin balance has been reported to be the origin of the variations in explant responses (Hamill 1993). Each explant type express rol genes differently (Park 2021; Sudha et al. 2013; Winans 1992) This advises that the predisposition of explants to Agrobacterium is dependent on the physiological state of different tissues in the same plant.

The induced hairy root was cut off (5–6 cm in length) and placed on fresh ½ strength B5 agar-gelled medium containing 250 mg/L cefotaxime. Among 30 hairy root lines were chosen from all hairy root lines generated based on their normal growth, degree of lateral ramification, lack of callus formation, and increase of biomass. The selected HR were subcultured regularly after every 4 weeks on a fresh medium and the cefotaxime concentration was gradually reduced and finally removed after three subcultures. During the present study, hairy root line HR1 showed an excellent and fast growth rate on ½ B5 semi-solid medium. Therefore, HR1 line was selected for further experimentation (Fig. 1d–f).

Confirmation of the transgenic nature of established HRs

In order to confirm the integration of the A. rhizogenes Ri plasmid T-DNA into the HR genomic DNA (selected HR line), PCR amplification was achieved using forward and reverse primers of rolB gene. The fragment of rolB gene was observed in the amplified DNA from the HR line induced by an infection with A. rhizogenes (Fig. 3). However, no amplification of the rolB gene was detected in the roots of the untreated plant material (negative control; Fig. 3). The fragments corresponding to rolB gene were amplified in the plasmid DNA of A4 strain of A. rhizogenes used as positive control (Fig. 3). These results confirmed the transgenic nature of the selected HR1 line. rolB gene has been routinely used to confirm the transgenic nature of several plant species (Nakasha et al. 2017; El-Esawi et al. 2017; Ya-ut et al. 2011; Aoki and Syōno 1999). Besides a major role of rol genes in the development of hairy roots, they can increase production of secondary metabolites in hairy roots (Shkryl et al. 2008).

Fig. 3.

Agarose gel image of PCR amplification of rolB gene in selected C. procera HR line resulted from transformation with A. rhizogenes strain A4. HR1 HR line of C. procera; M Molecular weight marker; C- Negative control (C. procera roots); C + A4 Positive control of bacterial strain A4 of A. rhizogenes

Identification and characterization of cardiac glycosides in transgenic HRs

Ethanolic extract of HR1 line was characterized for the determination of cardiac glycosides within of C. procera HR. UHPLC/QTOF-MS (in positive ionization mode) analysis revealed the presence of ten compounds based on the interpretation of their mass spectra, retention time, precursor ion [M + H]⁺ (m/z), diagnostic fragment ions (m/z), and by taking into consideration the data from the literature and databases. The exhaustive information of identified compounds (with numbers from 1 to 10 indicating the elution order) is listed in Table 1.

Table 1.

Cardiac glycosides detected by positive-ion UHPLC/QTOF-MS in Calotropis procera HRs

Peak	Identification of compound	Rt (min)	Molecular formula	Exp. m/z	Calc. m/z	Major fragment ions (m/z)	Chemical structure
1	Calotropagenin	3.85	C23H32O6	405.2270	405.2275	323.2002–339.1956–351.1953–369.2056–387.2163
2	Calotoxin	4.54	C29H40O10	549.2685	549.2693	325.2160–373.2373–391.2478–513.2484
3	Frugoside	5.63	C29H44O9	537.3068	537.3066	325.2166–337.2165– 339.2318–373.2378
4	Coroglaucigenin	5.76	C23H34O5	391.2481	391.2485	325.2166–337.2165–373.2378
5	Calotropin	6.66	C29H40O9	533.2746	533.2750	177.0548–323.2007–355.2263–497.2530– 515.2641
6	Calactin	7.30	C29H40O9	533.2739	533.2747	323.2005–339.1968–497.2528–515.2637
7	Uzarigenin	8.35	C23H34O4	375.2422	375.2529	205.1223–293.2258–321.2209–339.2316– 357.2422
8	Asclepin	8.88	C31H42O10	575.2844	575.2853	311.2011–497.2525– 539.2620–557.2735
9	Uscharidin	9.56	C29H38O9	531.2353	531.2353	193.0861–267.1591– 362.2467
10	Uscharin	9.72	C31H41NO8S	588.2636	588.2637	161.1326–323.2009– 341.2115–552.2424– 570.2525

Rt Retention time, Exp experimented, Calc calculated

In QTOF/MS analysis, compound (1) eluted at retention time (RT) 3.85 min showed an [M + H]⁺ ion peak at m/z 405.2275 in positive ion mode. This compound, which corresponds to the formula C₂₃H₃₂O₆, was identified as Calotropagenin. The mass spectrum of this compound yielded a major fragment at m/z 323.2002. At the same time other peaks were shown at m/z 387.2163, 369.2056, 351.1953, and 339.1956 (Table 1; Fig. 4a). These results are in agreement with the identification suggested by (Pandey et al. 2016). This compound has been previously identified from C. procera (Hassall and Reyle 1959; Kumar et al. 2019). Calotropagenin is a cardiac glycoside, knowing for its potent anticancer effect due to its HIF-1 (a transcription factor highly involved in cancer development) (Lopez-Lazaro 2009) transcriptional inhibitory activity against T47D cell line (Zheng et al. 2021). According to previous studies, calotropagenin demonstrated significant antihepatocytotoxic potential. The IC50 for calotropagenin was found to be 10.40 ± 0.98 µg/mL (Al-Taweel et al. 2017).

Fig. 4.

Mass spectra of cardiac glycosides (a–j) in positive ion mode identified from C. procera HRs. a Mass spectrum of calotropagenin at m/z 405.2275, b Mass spectrum of calotoxin at m/z 549.2693, c Mass spectrum of frugoside at m/z 537.3066, d Mass spectrum of coroglaucigenin at m/z 391.2485, e Mass spectrum of calotropin at m/z 533.2750, f Mass spectrum of calactin at m/z 533.2747, g Mass spectrum of uzarigenin at m/z 375.2529, h Mass spectrum of asclepin at m/z 575.2853, i Mass spectrum of uscharidin at m/z 531.2353, j Mass spectrum of uscharin at m/z 588.2637

Compound (2) was eluted at RT = 4.54 min and gave a precursor ion [M + H]⁺ at m/z 549.2693. This compound has a major fragment at m/z 373.2373. Other characteristic picks appeared at m/z 513.2484, 391.2478, and 325.2160 (Table 1; Fig. 4b). This compound with molecular formula (C₂₉H₄₀O₁₀) was characterized as calotoxin and it has already been reported in C. procera (Kumar et al. 2019; Seiber et al. 1982; Zheng et al. 2021). This compound has been also suggested to have a potent anticancer effect (Zheng et al. 2021). Calotoxin can be used for the development of novel Interleukin-2-inducible T cell kinase (ITK) inhibitors, which may have vast therapeutic applications as immune-suppressants and as anticancer drugs (Parthasarathy et al. 2021).

Compound (3) (RT = 5.63 min) produced a precursor ion [M + H]⁺ at m/z 537.3066 with major fragments ions at m/z 339.2318 and 337.2165. MS spectrum gave other fragments at m/z 373.2378 and 325.2166 (Table 1; Fig. 4c). The compound (3) was, therefore, recognized as frugoside, having molecular formula (C₂₉H₄₄O₉). These findings corroborate with those published by (Pandey et al. 2016). Frugoside is previously isolated from roots of C. procera and displayed important in vitro growth inhibitory activity against human cancer cells (Ibrahim et al. 2014; Zheng et al. 2021). It was demonstrated that frugoside inhibited the activity of peroxiredoxins by downregulating sulfiredoxin expression. Resulting accumulation of reactive oxygen species (ROS) and stimulated p-p38 activation led to mitochondria-mediated death of two human melanoma cell lines (M14 and A375). They reverted this phenotype by sulfiredoxin overexpression or antioxidants stimulation. The effect on tumor growth was confirmed in xenograft assays (Song et al. 2019).

Compound (4), appeared at RT = 5.76 min, gives a molecular ion [M + H]⁺ at m/z 391.2485, which corresponded to a molecular formula C₂₃H₃₄O₅. The MS spectrum presented major fragments ions at m/z 339.2318 and 337.2166, daughter ions at m/z 373.2378 and 325.2166 (Table 1; Fig. 4d). This compound has been tentatively assigned as coroglaucigenin. These results were also corroborating with those obtained by Pandey et al. (2016). This molecule was isolated and identified in different parts of C. procera (Kumar et al. 2019; Rajagopalan et al. 1955). In the literature coroglaucigenin is reported to prevent colorectal cancer cell proliferation associated with cell cycle arrest and senescence, both in vitro than in vivo (Huang et al. 2018). At the same time, it is a very promising potential sensitizer for cancer radiotherapy, which not only inhibits cancer cell proliferation but also improves cancer cell radiosensitivity by suppressing the expression of antioxidant molecules (Sun et al. 2017).

Compound (5), eluted at RT = 6.66 min, gives a precursor ion [M + H]⁺ at m/z 533.2750. In MS spectra, major fragments were yielded at m/z 515.2641, 323.2007, and 177.0548. Other daughter ions appeared at m/z 497.2530 and 355.2263 (Table 1; Fig. 4e). Accordingly, compound (5) with molecular formula C₂₉H₄₀O₉ was characterized as Calotropin. Data of present study were compatible with those obtained by Crout et al. (1964) and Pandey et al. (2016). This compound was revealed from different parts of C. procera in previous studies (Alzahrani et al. 2017; Brüschweiler et al. 1969; Kumar et al. 2019; Seiber et al. 1982). Calotropin is reported to possess a significant anti-tumor activity (Ibrahim et al. 2014; Li et al. 2009; Zheng et al. 2021). Cytotoxicity of calotropin against various cell lines of human and murine origin was verified. It presented similar cell line selectivity for cardiac glycosides such as digoxin and ouabain (Kiuchi et al. 1998). Zhou et al. (2019) reveal that calotropin prevents tumor growth and activates a protective role of Yes-associated protein (YAP) in colorectal cancer cells, suggesting that a potential approach to the treatment of colorectal cancer may involve the combination of calotropin and YAP targeting medicines. It can be also used for the treatment of autoimmune disorders as a medicinal compound (Liu et al. 2018).

Compound (6), appeared at RT = 7.30 min, yielded a precursor ion [M + H]⁺ at m/z 533.2747, corresponding to molecular formula C₂₉H₄₀O_9. The major fragments in the MS spectrum for this compound appeared at m/z 515.2637 and 323.2005. Daughter ions appeared as well at m/z 497.2528 and 339.1968 (Table 1; Fig. 4f). This compound was characterized as Calactin which was previously reported in C. procera (Baabad et al. 2018). These findings are similar to previous published letters (Kanojiya and Madhusudanan 2012). Calactin was reported to present high growth inhibitory activity against human A549 and Hela cell lines with IC50s values of (0.036 µM, A-549) and (0.083 µM, Hela) (Mohamed et al. 2015). It is able to inhibit Na + /K + -ATPase stronger than ouabain (Petschenka et al. 2018). This substance can be employed as lead molecule for the creation of novel ITK inhibitors, which may have a wide range of therapeutic uses as immune-suppressants and anticancer medications (Parthasarathy et al. 2021).

Compound (7) eluted at RT = 8.35 min having molecular formula C₂₃H₃₄O₄ was considered as Uzarigenin, a cardiac glycoside, yielding a precursor ion [M + H]⁺ at m/z 375.2529 and major fragments at m/z 339.2316 and 205.1223. Other characteristic fragments appeared at m/z 357.2422, 321.2209, and 293.2258 (Table 1; Fig. 4g). These results are consistent with the reported literature (Bader et al. 2021; Pandey et al. 2016). Uzarigenin was already isolated from C. procera (Shaker et al. 2010) and was reported for its ability to inhibit Na + /K + -ATPase in the same way as its 5β isomer digitoxigenin (Farr et al. 2002; Petschenka et al. 2018). This makes uzarigenin one of the best drugs for the treatment of heart disease (Tomilova et al. 2022). At a concentration of 50 µM, uzarigenin decreases the metabolic activity of HT29 and HepG2 cells by 59% and 35%, respectively (Shaker et al. 2010). According to observations made by Navarro et al. (1985), uzarigenin has a positive inotropic efficacy that is comparable to digitoxigenin’s (Navarro et al. 1985).

Compound (8), recorded at RT = 8.88, produced a precursor ion [M + H]⁺ at m/z 575.2853 along with major fragments at m/z 497.2525 and 311.2011, and other characteristic fragments at m/z 557.2735 and 539.2620. The compound (8) having molecular formula C₃₁H₄₂O₁₀ was identified as Asclepin due to its mass spectrum (Table 1; Fig. 4h). These data were similar to those obtained by Bader (Bader et al. 2021). Asclepin was already identified in methanol extract of C. procera latex (Kumar et al. 2019). It has been observed in earlier studies that asclepin possesses cardiac effects and it was found to be more active than g-strophanthin, digoxin, digitoxin, and digitoxigenin (Patnaik and Köhler 1978). This compound has been presented as a strong cytotoxic activity with an IC50 value of 0.02 µM against two cancer cell lines (HepG2 and Raji) (Li et al. 2009).

Compound (9) with RT = 9.56 min yielded a precursor ion [M + H]⁺ at m/z 531.2353, while characteristic fragments were appeared at m/z 362.2467, 267.1591, and 193.0861 (Table 1; Fig. 4i). MS spectrum data suggest that compound (9) with molecular formula C₂₉H₃₈O₉ is determined as uscharidin, a cardiac glycoside already reported in C. procera by Crout et al. (1964); Kumar et al. (2019). In earlier studies, this compound has shown also a significant anti-tumor activity, exhibiting cytotoxic activity with an IC50 value of 0.38 µM and 0.02 µM against HepG2 and Raji cell lines respectively (Li et al. 2009).

Compound (10) with RT = 9.72 min produced a precursor ion [M + H]⁺ at m/z 588.2637, major fragments at m/z 570.2525, 341.2115, and 161.1326, and other characteristic peaks at m/z 552.2424 and 323.2009 (Table 1; Fig. 4j). Based on fragment interpretation, the compound (10) was identified as uscharin having formula C₃₁H₄₁NO₈S. Similar findings have been approved by Bader et al. (2021). Previous research has detected this compound in various parts of C. procera (Brüschweiler et al. 1969; Kumar et al. 2019). Uscharin has been shown to have a significant inhibitory effect on HIF-1 activity more potent than digoxin (Parhira et al. 2014). This molecule exhibited substantially more strong cytotoxic effects (IC50 of 16.61–73.48 nM) on different cancer cell lines including HCT116, HeLa, HepG2, A549, MCF-7, A2780, and MDA-MB-231, which suggests that it exhibits an anticancer potential (Zheng et al. 2021). It was reported also that uscharin displayed potential treatment of neurodegenerative diseases through the acceleration of neuronal differentiation of neural stem cells (NSCs) (Yoneyama et al. 2017). Likewise, this compound presented potent molluscicidal effect against land snails. It was demonstrate that uscharin can be 128 times more poisonous than methomyl against the land snail Theba pisana (Hussein et al. 1994).

The ability of HR of C. procera to synthesize calotropin and related cardiac glycosides have been reported for the first time in this study. These biomolecules are known to have cardiac properties and anti-tumor potential (Koch et al. 2020). These findings showed that C. procera HR can be used as a model in basal research and for various biotechnological applications.

Conclusion

This paper reports the production of the transgenic HR in C. procera. There is a variability in transformation percentages due to differences in nature of explants. Leaves appear to be the leading explant for hairy root induction compared to hypocotyls and cotyledons. The genetic transformation and expression of rolB in transgenic roots of C. procera were validated by PCR.

This study demonstrates the utility of UHPLC/QTOF-MS for cardiac glycoside analysis and identification. This achievement can serve in isolation and determination of the biological activities of C. procera roots metabolites. On the other hand, this study shows the diversity of cardiac glycosides which can be produced biotechnologically. This study opens up, thus, new perspectives and new hopes in the fight against serious human diseases such as cancers.

We aim in the future to produce biotechnologically and increase the most important secondary metabolites content (cardiac glycosides) by using transgenic C. procera HRs in different ways.

Author contributions

A.D. and L.K. conceived and designed research. A.D. conducted experiments. A.D. and Y.B. contributed to UHPLC/QTOF-MS. A.D. wrote the manuscript. L.K. supervised the experiments and research documentation, and corrected the manuscript. M. A. and S. M and A. M. provided comments and revised the manuscript. All authors read and approved the manuscript.

Data availability

No applicable.

Declarations

Conflict of interest

The authors declare there is no conflict of interest.