Phytochemical Composition and Biological Activities of Extracts from Early, Mature, and Germinated Somatic Embryos of Cotyledon orbiculata L.

Cotyledon orbiculata L. (Crassulaceae)—round-leafed navelwort—is used worldwide as a potted ornamental plant, and it is also used in South African traditional medicine. The current work aims to assess the influence of plant growth regulators (PGR) on somatic embryogenesis (SE) in C. orbiculata; compare the metabolite profile in early, mature, and germinated somatic embryos (SoEs) by utilizing ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS); and determine the antioxidant and enzyme inhibitory potentials of SoEs. A maximum SoE induction rate of 97.2% and a mean number of SoEs per C. orbiculata leaf explant of 35.8 were achieved on Murashige and Skoog (MS) medium with 25 µM 2,4-Dichlorophenoxyacetic acid and 2.2 µM 1-phenyl-3-(1,2,3,-thiadiazol-5-yl)urea. The globular SoEs were found to mature and germinate best on MS medium with gibberellic acid (4 µM). The germinated SoE extract had the highest amounts of both total phenolics (32.90 mg gallic acid equivalent/g extract) and flavonoids (1.45 mg rutin equivalent/g extract). Phytochemical evaluation of SoE extracts by UHPLC-MS/MS reveals the presence of three new compounds in mature and germinated SoEs. Among the SoE extracts tested, germinated SoE extract exhibited the most potent antioxidant activity, followed by early and mature somatic embryos. The mature SoE extract showed the best acetylcholinesterase inhibitory activity. The SE protocol established for C. orbiculata can be used for the production of biologically active compounds, mass multiplication, and conservation of this important species.


Introduction
Cotyledon orbiculata L.-a member of the Crassulaceae-is commonly called roundleafed navelwort or pig's ear, is native to South Africa, and is typically found in Southern Africa [1]. C. orbiculata is widely used as a potted plant worldwide due to its attractive bellflowers along with its leaves and low-care requirements. In South African traditional medicine, leaves collected from the wild populations of C. orbiculata are used to treat deworming, earache, inflammation, neurological problem, skin infection, and wounds [2,3]. The crude extracts obtained from the aerial parts of C. orbiculata have been shown to possess anticancer [4], anticonvulsant [1], anti-inflammatory [5,6], antimicrobial [2,7], antinociceptive [5], antioxidant [6], and anthelmintic [2,8] activities. Several bufadienolides, including Healthy, young shoots isolated from greenhouse-raised Cotyledon orbiculata (L.) plants were soaked in a mild detergent solution and kept under running tap water for 30 min. Shoots were disinfected in ethanol (70%, 90 s), then mercuric chloride (0.1%, 15 min), followed by three washes (60 s per wash) in sterilized distilled water and air-dried. Leaves were dissected, cut into 0.6-1.0 cm long segments, and placed in a sterilized culture bottle (500 mL) containing Murashige and Skoog [32] (MS) medium with 8 g/L agar, 30 g/L sucrose, and 0-30 µM of 2,4-Dichlorophenoxyacetic acid (2,4-D), along with indole-3-aceticacid (IAA), indole-3-butyric acid (IBA), and α-naphthalene acetic acid (NAA) or 1. induction percentage was calculated as the number of leaf segments with SoEs divided by the total number of leaf segments cultured × 100 [33]. Globular SoEs were subcultured into the MS medium with 0, 1, 2, 4, or 8 µM 6-BA or gibberellic acid (GA 3 ), then proceeded to further development and germination. The cultures were kept under a 16-h photoperiod (20-25 µMol s −1 m −2 ) at temperatures from 23 to 26 • C. Fifty globular SoEs were used per treatment, with three repetitions. After eight weeks, the SoE conversion percentage was calculated as the number of germinated SoEs divided by the total number of SoEs cultured × 100 [34].

Phytochemical Analysis 2.2.1. Extract Preparation
Early (globular), mature (torpedo), and germinated (cotyledonary) SoEs were lyophilized. The extracts were obtained using a homogenizer-assisted extraction. In the procedure, C. orbiculata samples (50 mg) were extracted with 80% methanol using an Ultraturrax at 6000 g for 30 min. After filtration, the extracts were dried using a rotary vacuum evaporator before being stored at 4 • C until further analysis.

Estimation of Total Phenolics Content (TPC) and Flavonoids Content (TFC)
The TPCs of C. orbiculata SoEs extracts were determined using the Folin-Ciocalteu reagent described by Slinkard and Singleton [35], and the results were expressed in terms of mg of gallic acid equivalent (GAE). The TFCs of C. orbiculata SoEs extracts were determined using the aluminum chloride (AlCl 3 ) method described by Zengin et al. [36] and calculated in terms of mg of rutin equivalent (RE).

Chemical Characterization
A previously optimized and described UHPLC/MS/MS technique was used to screen the chemical compositions of three extracts containing phenolic and flavonoid compounds. Mass spectrometry was conducted using an electrospray ionization source (ESI) operating in both negative and positive ion modes. Mass spectra were recorded as full MS between m/z 100 and 1500 atomic mass units and MS/MS mode using a Q-Exactive (Thermo Fisher Scientific) Orbitrap mass spectrometer. These data can be examined to detect and confirm analytes in complex matrices. The detected compounds were identified through comparison with authentic standards, their MS/MS spectra and fragmentation patterns, and their HRMS spectral information. All data were processed using the TraceFinder software and tentatively identified by comparing their retention time (R t ) and mass spectrum with the reported data and our spectral library. The difference between the mass of measured and calculated* exact protonated or deprotonated molecular ions was less than 5 ppm [37].

Enzyme Inhibition Assay
The amylase, acetylcholinesterase (AChE), tyrosinase, and butyrylcholinesterase (BChE) inhibitory effects of C. orbiculata SoEs extracts were each conducted in triplicate according to the procedures described by Uysal et al. [38]. The results are given as IC 50 values (mg/mL).

Statistical Analysis
Data were subjected to analysis of variance (ANOVA), and significant differences (p < 0.05) among means were determined by Duncan's multiple range test (DMRT) using SAS version 9.4 (SAS Institute, Cary, NC, USA).

Somatic Embryogenesis (SE)
3.1.1. Influence of Auxins on SE in C. orbiculata The surface sterilization of C. orbiculata shoots with ethanol and mercuric chloride resulted in 100% sterile leaf culture. The explants cultivated on MS PGR-free medium (control) did not produce SoEs or callus. On the other hand, the explants of C. orbiculata developed callus, root, or SoE within 45 days of culture on an auxin-containing medium. However, the addition of auxin at 5 or 10 µM in the cultivation medium did not support SE. The SoE formation occurred at the cut edges of C. orbiculata leaf segments in the presence of 15-30 µM auxin (Table 1). After eight weeks of cultivation, pale green globular SoEs were detected ( Figure 1a). The ANOVA showed that auxin type, auxin concentration, and the interaction of type and concentration of auxin all had significant (p < 0.001) effects on SoE induction and the number of SoE developed per explant (Table 1). Of the studied auxin types, a high rate of SoE induction was obtained on 2,4-D (25.6%), followed in descending order by NAA (16.9%), IBA (13.9%), and IAA (11.7%). Similarly, 2,4-D produced the highest mean number of SoEs (6.3), followed in descending order by NAA (4.1), IBA (2.6), and IAA (2.2). Of the studied auxin concentrations, a high incidence of SoE induction was obtained on 25 µM (34.7%), followed in descending order by 20 µM (31.3%), 30 µM (23.9%), and 15 µM (13.4%). Lastly, 25 µM produced the highest mean number of SoEs (8.0), followed in descending order by 20 µM (6.7), 30 µM (4.7), and 15 µM (3.4). The maximum SoE induction rate (60.6%) and mean number of SoEs per C. orbiculata leaf explant (14.9) were achieved on an MS medium with 25 µM of 2,4-D (Table 1). Thus, 25 µM of 2,4-D was selected for the additional SE experiments. Table 1. Impact of auxins on SE in C. orbiculata.
Means ± SDs within columns (3 and 4) followed by different alphabets ( a-k ) are significantly different according to DMRT at p < 0.05. *-Interaction 3.1.3. Effect of 6-BA and GA3 on Conversion of C. orbiculata SoEs Within eight weeks, globular SoEs matured and germinated. Only a few SoEs germinated on the control (MS) medium. The conversion of globular SoEs was boosted by supplementing MS medium with 6-BA and GA3 (1-8 μM). The frequency of SoE conversion ranged from 23.7% to 100%. Among the two tested PGRs, GA3 proved to be the most effective for converting C. orbiculata SoEs. The highest rate of SoE conversion (100%) was attained on a medium with 4 μM GA3 ( Figure 2).

Phytochemical Analysis
In the present study, we determined the total amounts of phenolic and flavonoids of C. orbiculata extracts. The results are presented in Table 3. Among the tested samples, the highest levels of total phenolics and flavonoids were determined in the extract of germinated somatic embryos (32.90 mg GAE/g extract and 1.45 mg RE/g extract, respectively).

Phytochemical Analysis
In the present study, we determined the total amounts of phenolic and flavonoids of C. orbiculata extracts. The results are presented in Table 3. Among the tested samples, the highest levels of total phenolics and flavonoids were determined in the extract of germinated somatic embryos (32.90 mg GAE/g extract and 1.45 mg RE/g extract, respectively). The extracts from early and mature somatic embryos contained almost the same contents of total phenolics and flavonoids. The characterized compounds are listed in Table 4. The chromatographic and mass spectrometric data (retention times, protonated or deprotonated molecular ions, fragment ions) and assigned identities for compounds were given Tables S1-S3. In total, 38 compounds were identified in the extracts. A total of 32 compounds were found in early somatic embryo extract, 33 were found in mature somatic embryo extract, and 32 were found in germinated somatic embryo extract (Figures S1-S3). The structures of some compounds are given in Figure 3.

Antioxidant Abilities
We determined the antioxidant properties of C. orbiculata extracts, and the results are presented in Table 5. Among the antioxidant assays, DPPH and ABTS are the most popular for evaluating plant extracts' radical scavenging ability. As can be seen in Table 5, the most active extract was germinated somatic embryos with an IC50 of 0.62 mg/mL, followed by early and mature somatic embryos. However, Trolox showed a stronger ability to scavenge free radicals compared to the tested extracts. The transformations of cupric to cuprous and ferric to ferrous reflect the electron-donating ability of antioxidant compounds, and the mechanism is known to be the reduction in power. For this purpose, we performed CUPRAC and FRAP assays. In both assays, the best reduction ability was provided by germinated somatic embryos (CUPRAC: 0.92 mg/mL; FRAP: 0.55 mg/mL). However, all extracts were less active than the standard antioxidant, Trolox. Phosphomolybdenum (PBD) assay is one of the total antioxidant assays, and all antioxidant compounds could play an effective role in the assay. As presented in Table 5, the tested samples were in descending order of germinated > early >mature. The chelation of transition metals can negative ion mode for the determination of the chemical structure of the compounds. In many cases, the sensitivity was higher and more fragment ions could be detected in positive mode; examples include Nicotinic acid and its derivatives, oxybutanedioic acid derivatives, hydroxy-, dihydroxy-and trihydroxy-methoxy/dimethyoxy/trimethoxy(iso)flavones. The major advantage of negative ion mode (ESI − ) is the reduced background noise.

Antioxidant Abilities
We determined the antioxidant properties of C. orbiculata extracts, and the results are presented in Table 5. Among the antioxidant assays, DPPH and ABTS are the most popular for evaluating plant extracts' radical scavenging ability. As can be seen in Table 5, the most active extract was germinated somatic embryos with an IC 50 of 0.62 mg/mL, followed by early and mature somatic embryos. However, Trolox showed a stronger ability to scavenge free radicals compared to the tested extracts. The transformations of cupric to cuprous and ferric to ferrous reflect the electron-donating ability of antioxidant compounds, and the mechanism is known to be the reduction in power. For this purpose, we performed CUPRAC and FRAP assays. In both assays, the best reduction ability was provided by germinated somatic embryos (CUPRAC: 0.92 mg/mL; FRAP: 0.55 mg/mL). However, all extracts were less active than the standard antioxidant, Trolox. Phosphomolybdenum (PBD) assay is one of the total antioxidant assays, and all antioxidant compounds could play an effective role in the assay. As presented in Table 5, the tested samples were in descending order of germinated > early >mature. The chelation of transition metals can hinder the production of hydroxyl radicals via the Fenton reaction and, therefore, be considered an important antioxidant mechanism. In contrast to other assays, the extracts of early and germinated somatic embryos exhibited similar chelating abilities. However, the extract of mature somatic embryos showed the weakest chelating ability. Moreover, EDTA was shown to be an excellent chelator with the lowest IC 50 value (0.02 mg/mL).

Enzyme Inhibition Effects
The present study reported the enzyme inhibitory properties of C. orbiculata extracts against AChE, BChE, tyrosinase, and amylase. The results are listed in Table 6. In the AChE inhibition assay, the mature somatic embryo extract provided the best inhibition with the lowest IC50 value (0.75 mg/mL). The early and germinated somatic embryo extracts had almost the same inhibitory potency. Regarding the BChE inhibition assay, the best effect was found in the germinated somatic embryo extract, but the ability was close to that of the mature somatic embryo extract. The extract of early somatic embryos was found to have the weakest ability to inhibit BChE. Tyrosinase is a key enzyme in melanogenesis, and its inhibition is important for controlling hyperpigmentation problems. As listed in Table 6, the tested extracts showed similar tyrosinase inhibitory activities, and the most active one was from the germinated somatic embryos. However, kojic acid was the superior inhibitor with the lowest IC50 (0.08 mg/mL). Amylase is the main enzyme involved in the hydrolysis of carbohydrates, and its inhibition can control blood sugar levels in diabetics. The highest amylase inhibition was achieved by early somatic embryos, followed by germinated and mature somatic embryos. All extracts also showed weaker abilities compared to acarbose (IC 50 : 0.68 mg/mL).

Discussion
The surface sterilization of explants (plant materials) is an essential aspect of establishing in vitro aseptic culture [39]. In this study, disinfection of C. orbiculata shoots resulted in a 100% sterile in vitro culture. A similar disinfection method was also used to obtain sterile explants of C. orbiculata [7]. The control (MS) medium and MS medium supplemented with lower levels (5 and 10 µM) of auxin failed to promote SE in C. orbiculata. However, incorporating high levels (above 10 µM) of auxin resulted in SoE induction from leaf explants of C. orbiculata (Table 1). In many species, the presence of auxin-often at high concentrations-is required to induce SoEs [26,40,41]. In this study, 2,4-D (25 µM) proved to be significantly (p < 0.001) superior in inducing SE from leaf explants of C. orbiculata than NAA, IBA, and IAA, which is likely attributable to the fact that the degradation rate of 2,4-D is lower than those of other studied auxins. The effectiveness of 2,4-D for stimulating SE has already been disclosed in various species [24,26,33,[40][41][42]. The 2,4-D and cytokinin combination was frequently used to enhance SoE induction in most species. The addition of cytokinin (6-BA, KN, or TDZ at 1.2-4.4 µM) to the SE-promoting level (25 µM) of 2,4-D significantly enhanced the formation of SoEs ( Table 2). The combination of 2,4-D and 6-BA has been shown to be effective for the induction of SoEs in Ananas comosus [15], Betula platyphalla [43], Campanula punctata [24], Crassula ovata [27], Orostachys japonicus [29], and Picea pungens [44]. Similarly, a combination of 2,4-D and KN was effective for the induction of SoEs in chrysanthemum 'Hornbill Dark' [45], Trachyspermum ammi [46], and Viola canescens [47]. Likewise, a combination of 2,4-D and TDZ was found to be the best for the induction of SoEs in Camellia oleifera [48], Hippeastrum [49], Prunus dulcis [50], and Tulipa gesneriana [51]. Among the texted cytokinins, the highest rate of SoE induction with the maximum number of SoEs per C. orbiculata leaf explant was achieved using the optimal SE medium with TDZ (Table 2).
TDZ is a PGR that is often used for the induction of SoEs and callus, adventitious shoot regeneration, and multiple shoot induction in various plants [52]. It is often combined with other PGRs to achieve the best in vitro culture results. However, the ratio of auxin and cytokinin significantly affects SE. In this study, the best rate of SoE formation (97.2%) with a maximum number of SoEs per C. orbiculata leaf explant (35.8) was obtained in the MS medium containing 2,4-D (25 µM) and TDZ (2.2 µM). Similarly, the presence of a high level of auxin (22.5 µM of 2,4-D) and a low level of cytokinin (2.2 µM of 6-BA) was found to be effective for SE in Orostachys japonicus [29]. By contrast, a low level of auxin (2.3 µM of 2,4-D) and a high level of cytokinin (4.4 µM of 6-BA) were found to be the best conditions for SE in Crassula ovata [27]. Therefore, the requirement of the PGRs ratio for SE in Crassulaceae varies according to genus. The globular-, heart-, and torpedo-stage SoEs were formed when the C. orbiculata leaf explants were cultured on optimal SE induction medium with TDZ (Figure 1a-c). However, only a few globular SoEs matured, and germination was not accomplished. Similar results have also been reported in another Crassulaceae member, Orostachys japonicus [29]. SoE maturation and subsequent plantlet conversion are often affected by the presence of PGRs in the SE medium. Globular SoE conversion (maturation and germination) has commonly been achieved on PGR-free medium [22,41]; however, in some species, the addition of cytokinins [21,29,49], abscisic acid [44,53], or GA 3 [33,34] is needed for the development and germination of SoE. In this study, the highest conversion of C. orbiculata SoE was accomplished on a medium with 4 µM GA 3 . GA 3 has been reported to have positive effects on SoE conversion in Haworthia retusa [33], Hosta minor [34], and Juglans regia [54].
Over the last decade, phenols have attracted more interest in nutraceutical and pharmaceutical applications due to their promising biological activities [55]. In this sense, when the content of phenols in an extract is detected, it is a significant indicator of its biological effects. In the current work, the extract of germinated somatic embryos was found to have the highest total phenolic and flavonoid content. In a previous study conducted by Ondua et al. [6], the total phenolic level of C. orbiculata extracts varied from 1.34 (in n-hexane extract) to 23.93 mg GAE/g (in methanol extract), which is lower than that of the extract from germinated somatic embryos tested in the study. Although the spectrophotometric methods could provide initial insight into the pharmacological value of plant extracts, certain concerns have recently arisen from the assays. Due to the complex nature of plant extracts, not only will certain compounds of interest react with the reagent used in the assays, but so will other phytochemicals. Therefore, the results of these assays could be suspect. Keeping this in mind, chromatographic techniques are needed to obtain more accurate chemical profiles of plant extracts. In the present study, the chemical composition of the tested extracts was characterized using the UHPLC/MS/MS technique, and the results are listed in Table 2. The extracts had a similar chemical composition, and, interestingly, new compounds were identified in mature SoE (33)(34)(35) and germinated SoE (36-38) (Figure 4).  Figure 4). Over the last century, most people have come to be familiar with the term antiox dant. The term denotes protection against free radical attacks that affect the progressio of serious health problems such as cancer, diabetes, or obesity. Several studies have r ported that antioxidant intake is inversely associated with the development of these di eases [56,57]. With this in mind, we determined the antioxidant properties of C. orbicula extracts, and the results are presented in Table 5. The germinated somatic embryo extra generally showed stronger antioxidant ability than other tested extracts. Ondua et al. [6 reported that the IC50 values of the methanol extract of C. orbiculata were 3.76 g/mL an 3.35 g/mL for DPPH and ABTS, respectively. Based on their results, our extracts showe weaker free radical scavenging ability than their tested extracts. From Table 5, when th combined scavenger and reduction performance results were obtained, we found an a most similar order. The obtained results almost agreed with the results of the total ph nolic and flavonoid content of the tested extracts; therefore, we concluded that phenolic made the main contribution to the radical scavenging and reducing ability. Moreove some compounds have only been detected in germinated SoE extract, and these are als Over the last century, most people have come to be familiar with the term antioxidant. The term denotes protection against free radical attacks that affect the progression of serious health problems such as cancer, diabetes, or obesity. Several studies have reported that antioxidant intake is inversely associated with the development of these diseases [56,57]. With this in mind, we determined the antioxidant properties of C. orbiculata extracts, and the results are presented in Table 5. The germinated somatic embryo extract generally showed stronger antioxidant ability than other tested extracts. Ondua et al. [6] reported that the IC 50 values of the methanol extract of C. orbiculata were 3.76 g/mL and 3.35 g/mL for DPPH and ABTS, respectively. Based on their results, our extracts showed weaker free radical scavenging ability than their tested extracts. From Table 5, when the combined scavenger and reduction performance results were obtained, we found an almost similar order. The obtained results almost agreed with the results of the total phenolic and flavonoid content of the tested extracts; therefore, we concluded that phenolics made the main contribution to the radical scavenging and reducing ability. Moreover, some compounds have only been detected in germinated SoE extract, and these are also known to be powerful antioxidants [58,59].
Enzymes are pharmaceutical targets for treating various health problems, including Alzheimer's disease, obesity and diabetes. In particular, the inhibition of key enzyme abilities might alleviate the symptoms of the diseases mentioned above [60]. For this purpose, several compounds have been manufactured as enzyme inhibitors, and most of them are presented on pharmacy shelves. However, synthetic compounds exhibit unpleasant side effects, including gastrointestinal problems and toxicity [61][62][63]. Therefore, several studies have focused on replacing synthetic inhibitors with natural ones. The tested extracts showed remarkable inhibitory effects on AChE, BChE, tyrosinase, and amylase.
The observed capabilities of the tested samples can be explained by the presence of some chemical compounds. As listed in Table 6, some compounds have been reported to serve as enzyme inhibitors [64][65][66]. The current work is the first report examining the enzymeinhibitory effect of C. orbiculata. Thus, these results could establish future directions for studies using C. orbiculata to develop functional applications.
Supplementary Materials: The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/plants12051065/s1, Table S1: Chemical composition of early somatic embryo extract; Table S2: Chemical composition of mature; somatic embryo extract Table S3: Chemical composition of germinated somatic embryo extract; Figure S1: Total ion chromatogram of early somatic embryos extract of C. orbiculata in positive (a) and negative (b) mode; Figure S2: Total ion chromatogram of mature somatic embryos extract of C. orbiculata in positive (a) and negative (b) mode; Figure S3: Total ion chromatogram of germinated somatic embryos extract of C. orbiculata in positive (a) and negative (b) mode.