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Article

Phytochemical Composition, Antioxidant Capacity, and Enzyme Inhibitory Activity in Callus, Somaclonal Variant, and Normal Green Shoot Tissues of Catharanthus roseus (L) G. Don

by
O. New Lee
1,
Gunes Ak
2,
Gokhan Zengin
2,
Zoltán Cziáky
3,
József Jekő
3,
Kannan R.R. Rengasamy
4,
Han Yong Park
1,
Doo Hwan Kim
5 and
Iyyakkannu Sivanesan
5,*
1
Department of Bioindustry and Bioresource Engineering, Sejong University, 209 Neungdong-ro, Gwangjin-gu, Seoul 05006, Korea
2
Department of Biology, Faculty of Science, Selcuk University, Konya 42130, Turkey
3
Agricultural and Molecular Research and Service Institute, University of Nyíregyháza, 4400 Nyíregyháza, Hungary
4
Indigenous Knowledge Systems Centre, Faculty of Natural and Agricultural Sciences, North-West University, Private Bag X2046, Mmabatho 2745, North West, South Africa
5
Department of Bioresources and Food Science, Institute of Natural Science and Agriculture, Konkuk University, Seoul 05029, Korea
*
Author to whom correspondence should be addressed.
Molecules 2020, 25(21), 4945; https://doi.org/10.3390/molecules25214945
Submission received: 28 September 2020 / Revised: 20 October 2020 / Accepted: 21 October 2020 / Published: 26 October 2020
(This article belongs to the Special Issue Plant In Vitro Systems as an Effective Tool for In Vivo Biocatalysis of Bioactive Natural Products)
Browse Figure
Versions Notes

Abstract

:
This study aimed to investigate the impact of plant growth regulators, sucrose concentration, and the number of subcultures on axillary shoot multiplication, in vitro flowering, and somaclonal variation and to assess the phytochemical composition, antioxidant capacity, and enzyme inhibitory potential of in vitro-established callus, somaclonal variant, and normal green shoots of Catharanthus roseus. The highest shoot induction rate (95.8%) and highest number of shoots (23.6), with a mean length of 4.5 cm, were attained when the C. roseus nodal explants (0.6–1 cm in length) were cultivated in Murashige and Skoog (MS) medium with 2 µM thidiazuron, 1 µM 2-(1-naphthyl) acetic acid (NAA), and 4% sucrose. The in vitro flowering of C. roseus was affected by sucrose, and the number of subcultures had a significant effect on shoot multiplication and somaclonal variation. The highest levels of phenolics and flavonoids were found in normal green shoots, followed by those in somaclonal variant shoots and callus. The phytochemicals in C. roseus extracts were qualified using liquid chromatography–tandem mass spectrometry. A total of 39, 55, and 59 compounds were identified in the callus, somaclonal variant shoot, and normal green shoot tissues, respectively. The normal green shoot extracts exhibited the best free radical scavenging ability and reducing power activity. The strongest acetylcholinesterase inhibitory effects were found in the callus, with an IC50 of 0.65 mg/mL.

Academic editors: Christophe Hano; Bilal Haider Abbasi; Marcello Iriti

1. Introduction

Catharanthus roseus (L) G. Don (Family: Apocynaceae), also known as periwinkle, is an attractive, evergreen herb. It grows to approximately 100 cm in height and is native to Madagascar. Periwinkle is a source of commercial bioactive alkaloids, including vinblastine and vincristine, which have anti-cancer activities [1,2]. It also contains several important bioactive compounds, such as anthocyanins, flavonol glycosides, phenolic acids, saponins, steroids, and terpenoids, that exhibit antidiarrheal, antidiabetic, antihypoglycemic, antimicrobial, wound healing, and antioxidant activities [3,4,5,6,7,8,9]. C. roseus blooms throughout the year with pink, purple, or white fragrant flowers, which have high ornamental value. It is commonly cultivated as an ornamental and medicinal plant in Africa, Australia, China, Europe, and the United States [10]. It is naturally propagated by seeds or cuttings, but a shortage of healthy seeds and cuttings has affected its extensive propagation. Additionally, the large-scale commercial production of new cultivars with medicinal or ornamental value, raised by traditional methods, is time-consuming. Furthermore, the marketable production of C. roseus metabolites is often restricted by low levels of medicinal compounds. However, the limitations of conventional propagational methods may be overcome by in vitro culturing. Micropropagation is an effective in vitro technique for the rapid commercial production of plantlets and bioactive metabolites. Several studies have attempted to micropropagate C. roseus using plant tissue culture [9,11].
The production of C. roseus phytochemicals has been accomplished using callus, cell suspension, somatic embryo, and transformed or non-transformed root and shoot cultures by optimizing the chemical and physical parameters [7,9,12,13]. Several alkaloids, such as ajmalicine, vindoline, catharanthine, vinblastine, and vincristine, were successfully obtained from C. roseus shoot cultures [14,15,16,17,18,19,20]. Phenolics are essential secondary metabolites obtained from various plant parts and have a wide range of biological activities [21]. Several phenolic compounds have been obtained in vitro, mostly from callus and cell suspension cultures of C. roseus [3,7,9]. However, information on the production of phenolics from C. roseus shoot cultures has never been reported, except for the identification of 2,3-dihydroxybenzoic acid from C. roseus shoot cultures [22]. To date, the phenolic profile of C. roseus shoot cultures has not been documented. Therefore, it is necessary to develop effective analysis procedures for bioactive compounds, including phenolics, in C. roseus shoot cultures, for the large-scale commercial production of phytochemicals. The mass production of shoots in vitro often depends on explant type, plant growth regulators (PGRs), sucrose, and the number of subcultures [23].
Explants with vegetative meristems are often suitable for axillary shoot multiplication and clonal propagation. Direct multiple shoot regeneration has been achieved using nodal segments, shoot tips, and axillary buds from C. roseus seedlings and mature plants [11,20,24,25,26,27,28]. Cytokinins play an essential role in shoot development. N6-benzyladenine (BA) [19,25,26,27], N6-furfuryladenine (Kinetin) [19,24,26,27], and thidiazuron (1-phenyl-3-(1,2,3,-thiadiazol-5-yl)urea, TDZ) [19] are used to induce multiple shoots in C. roseus. TDZ (substituted phenyl urea) is more efficient at multiple shoot formation in several shrubs, including C. roseus [19,29,30]. Moreover, TDZ supplementation increases the phytochemical content of in vitro cultures by altering various physiological activities [30,31]. However, high-dose or continuous TDZ exposure results in growth inhibition, leaf chlorosis, and hyperhydricity in explants containing media [29,32]. Thus, identifying the optimal dose of TDZ is necessary for healthy mass shoot production.
Sucrose is a frequent carbon source in tissue culture media that plays an important role in culture initiation and development and metabolite production [33]. High-level sucrose supplementation (6%) enhances the biomass and phytochemical content of C. roseus cell suspension cultures [34,35], while low-dose sucrose supplementation (2%) has been used in woody plant medium for adventitious shoot regeneration in C. roseus [36]. Other carbon sources also affect the somatic embryo maturation of C. roseus [37]. To the best of our knowledge, the effects of sucrose on axillary shoot proliferation in C. roseus have not been reported.
Variations in plant in vitro cultures are called somaclonal variation (SV). SV is a severe problem for the extensive micropropagation of elite genotypes but can also be used in plant improvement programs. The incidence of SV is higher in callus and indirectly regenerated shoots than in axillary shoot cultures. However, the rate of SV in in vitro cultures depends on the plant species, cultivar, culture conditions, growth media components, and the number of subcultures [38,39,40,41]. The effects of subculturing on C. roseus shoot multiplication have received little attention and the SV of multiple C. roseus shoot cultures is unreported.
Prior studies of the in vitro micropropagation of C. roseus have shown that axillary shoot multiplication depends on the explant source, genotype, plant growth regulators, and the components of the culture media. To date, the simultaneous detection of important phytochemicals, such as alkaloids and phenolics, in C. roseus callus and shoot cultures has not been documented. The objectives of this study were (1) to evaluate the effects of the plant growth regulators, the sucrose concentration, and the number of subcultures on in vitro micropropagation, (2) to document SV in axillary shoot cultures, (3) to assess the phytochemical composition of in vitro-established callus, somaclonal variant, and normal green shoots, and (4) to evaluate the antioxidant capacity and enzyme inhibitory potential of C. roseus.

2. Results

2.1. In Vitro Micropropagation

The surface disinfection technique produced 91% germ-free explants. Nodal explants of C. roseus were cultivated on Murashige and Skoog (MS) medium containing 0–16 µM of cytokinin for axillary shoot multiplication. Shoot initiation was observed within 14 days of cultivation. The cytokinins, their concentration, and the interactions significantly (p ≤ 0.001) affected the induction and development of axillary shoots (Table 1). The presence of 1–16 µM BA in the medium improved the axillary shoot multiplication compared to control (devoid of BA). The rate of shoot initiation (66.4%) and the number of shoots (6.3) in the MS medium with 4 µM BA were higher than the other BA treatments. The longest shoot length (3.1 cm) was attained on basal medium with 2 µM BA (Table 1).
The addition of 1–16 µM kinetin also promotes multiple shoot production in C. roseus, the rate of shoot induction ranged from 25.7% to 59.5%, and the number of shoots produced ranged from 2.3 to 5.7, with an average length of 1.5–3.6 cm (Table 1). The inclusion of 1–16 µM TDZ in the medium increased the percentage of multiple shoot regeneration compared to the control (without TDZ). The shoot induction rate (75.2%), the number of shoots (10.1), and shoot elongation (5.0 cm) in the MS medium with 2 µM TDZ was higher than in other TDZ treatments (Table 1).
Amongst the three cytokinins used, TDZ induced a higher percentage of multiple shoot induction (59%) compared to that in BA (49.9%) and kinetin (44.2%) (Table 2). Of the five different concentrations used, 4 µM cytokinin induced the highest rate of shoot formation (64.4%) and the maximum number of shoots (6.6). The longest shoot length (3.4 cm) was attained on basal medium with 2 µM cytokinin (Table 2). These results suggest that increasing the cytokinin concentration beyond the optimum level decreases the rates of shoot initiation, multiplication, and elongation.
C. roseus nodal segments were inoculated on MS medium with 2, 4, or 8 µM TDZ and 0.5, 1, or 2 µM indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), or NAA initiated shoot multiplication within a week of incubation. Both the TDZ and auxin levels were important for enhancing axillary shoot multiplication in C. roseus. The medium with 2 µM TDZ and 1 µM auxin (IAA, IBA, or NAA) had the best shoot induction percentages (Table 3). A higher shoot induction rate, higher number of shoots, and shoot growth were attained when the C. roseus nodal explants were cultivated on MS medium with 2 µM TDZ and 1 µM NAA (Figure 1a, Table 3). Lower shoot formation was observed on medium with higher TDZ levels with NAA, and callus induction was observed at the base of the C. roseus nodal explants. The highest rate of callus induction (100%) was obtained on medium with 8 µM TDZ and 2 µM NAA (data not shown).
Nodal explants developed shoots in the presence of sucrose (2–5%) and failed to produce shoots on the sucrose-free MS medium (Table 4). The highest shoot induction rate (95.8%) and highest number of shoots (23.6), with a mean length of 4.5 cm, were attained when the C. roseus nodal explants were cultivated on MS medium with 2 µM TDZ, 1 µM NAA, and 4% sucrose (Table 4). High sucrose concentrations (5%) inhibited the rate of shoot initiation and the number and length of induced axillary shoots. The shoots formed on MS medium containing 2 µM TDZ, 1 µM NAA, and sucrose (2–3%) failed to develop flowers after 45 days of cultivation. Higher sucrose concentrations (4 and 5%) promoted flowering within 30 days of incubation. The maximum rate of flowering (35.3%), with a mean of 2.9 flowers, was attained on MS medium containing 2 µM TDZ, 1 µM NAA, and 5% sucrose (Figure 1b, Table 4).
The number of subcultures had a significant effect on shoot multiplication and somaclonal variation in C. roseus (Table 5). The frequency of shoot induction increased with the number of subcultures, from zero to two, and then remained unchanged after six subcultures. The mean number of shoots increased up to three subcultures and significantly decreased thereafter (Table 3). The greatest number of shoots (39/nodal explant) was attained at the third subculture. Morphological changes in the shoots were observed after the third subculture; albino shoots were detected during the fourth subculture (Figure 1c), and the highest number of variant shoots (13.4) was attained at the sixth subculture (Table 5). The somaclonal variant shoots proliferated on MS medium with 2 µM TDZ and 1 µM NAA (Figure 1d) and were used for phytochemical analysis and biological assays. Seeds obtained from ex vitro acclimatized somaclonal variant and normal green plantlets were germinated on MS nutrient medium and displayed normal and variant shoots (Figure 1e,f).
The shoots developed roots after 14 days of culturing on half-strength MS medium containing 2–8 µM IBA (Table 6). The highest rooting response (90.9%) and highest number of roots (9.3), with a mean length of 6.2 cm, were attained on half-strength MS medium with 4 µM IBA after 35 days of culture (Figure 1g, Table 6). The lowest percentage of root induction (39.8%) was observed on half-strength MS medium with 4 µM IBA and no sucrose. Sucrose in the culture medium enhances the rooting response of shoots. However, the percentage of root induction and the number of roots varied with the concentration of sucrose (Table 7). The highest rate of root induction (96.7%) and number of roots (15.2), with a mean length of 8.3 cm, were observed on half-strength MS medium with 2% sucrose and 4 µM IBA. Higher sucrose concentrations (3–5%) reduced the percentage of root induction and the number of induced roots (Table 7). The in vitro-induced shoots (≥2 cm in length) developed on MS medium containing 2 µM TDZ and 1 µM NAA grew flowers within 20 days of cultivation on half-strength MS medium with 3–5% sucrose and 4 µM IBA (Figure 1h,i). The greatest rate of flowering (67.6%), with a mean number of 3.9 flowers, was obtained in a medium with 5% sucrose and 4 µM IBA after 35 days of cultivation (Table 7). The in vitro-developed C. roseus plantlets were acclimatized in a greenhouse with 98% survival; the acclimatized plants grew well without any morphological variations (data not shown).

2.2. Phytochemical Composition

The total content of phenolics and flavonoids in the extracts was measured using colorimetric methods (results shown in Table 8). The normal green shoots showed the highest level of phenolics (30.58 mg GAE/g), followed by the somaclonal variant shoots (26.45 mg GAE/g) and callus (14.66 mg GAE/g). The same order was observed for total flavonoids (normal green shoots (2.47 mg RE/g) > somaclonal variant shoots (1.21 mg RE/g) > callus (0.28 mg RE/g)).
Ultra-high-performance liquid chromatography–electrospray ionization–tandem mass spectrometry (UHPLC/ESI-MS/MS) was used for the rapid qualitative determination and identification of unknown compounds from different extracts and detailed results (retention time, protonated or deprotonated molecular ions, main fragment ions) are presented in Table 9, Table 10 and Table 11. MS/MS spectra contain rich structural information; however, because of the structural diversity of the molecules in the extracts, mass spectra were collected in positive and negative ionization modes separately. Some compounds were identified based on the retention times of the reference standards, protonated or deprotonated molecule ions, and characteristic fragment ions. In other cases, the unknown components were tentatively identified by their molecular ions and analyses of the UHPLC-MS/MS fragmentation data compared to published literature and/or our previous results (Figures S1–S9).
Fifty-five compounds were identified in the somaclonal variant shoot tissues, thirty-nine in the callus, and fifty-nine compounds in the normal green shoots. Similar components were found in the somaclonal variant and normal green shoot tissues.
Several groups of natural phenols, such as phenolic acids, O-caffeoylquinic acids, O-feruloylquinic acids, coumarin, quercetin, kaempferol, isorhamnetin derivatives, and other alkaloids, were identified in the samples. A wide range of low-molecular-weight polar compounds, e.g., methylcoumarin (MW: 178) and ajmalicine, a monomeric indole alkaloid (MW: 352), and higher molecular mass compounds, e.g., quercetin-O-dirhamnosylhexoside (MW: 756), were identified. Moreover, several known Catharanthus alkaloids, including vindolinine (Rt: 17.11 min), 19-S-vindolinine (Rt: 18.16 min), catharanthine (21.76 min), vindoline (Rt: 24.80 min), and vindolidine (Rt: 25.23 min), were chromatographically separated and characterized (Figures S10–S13).

2.3. Antioxidant Effects

The results are presented in Table 12. DPPH and ABTS were used to determine the scavenging ability of natural products or synthetics. As shown in Table 12, the normal green shoots exhibited better ability in both assays (IC50: 1.57 and 1.44 mg/mL for DPPH and ABTS, respectively). The weakest scavenging ability was observed in callus (IC50: >3 and 1.85 mg/mL for DPPH and ABTS, respectively). Similarly, the reducing power assays (CUPRAC and FRAP) indicated that the order of the samples was normal green shoots > somaclonal variant shoots > callus, reflecting the electron-donation abilities of the antioxidant compounds.

2.4. Enzyme Inhibitory Properties

We tested the enzyme inhibitory effects of C. roseus extracts against cholinesterases (AChE and BChE), tyrosinase, and amylase, and the results are reported in Table 13. The best AChE inhibitory effect was found in callus with an IC50 value of 0.65 mg/mL, followed by the normal green (IC50: 0.72 mg/mL) and somaclonal variant (IC50: 0.74 mg/mL) shoots. The samples had similar BChE inhibition values and the differences were non-significant. Similar results were also observed for amylase inhibition and the extracts exhibited close inhibition ability. As seen in Table 13, the best tyrosinase inhibitory effects were observed in the normal green (IC50: 0.83 mg/mL) and somaclonal variant shoots (IC50: 0.86 mg/mL).

3. Discussion

Multiple shoots initiated after nodal explants incubated on MS medium supplemented with cytokinins. The optimal BA concentration for axillary shoot multiplication from nodal explants of C. roseus was 4 µM (Table 1). The ability of BA to promote the formation of multiple C. roseus shoots was also observed in previous reports [19,25,26,28]. Pati et al. [19] reported that the nodal segments of C. roseus produced the maximum number of shoots (7.87) on MS liquid medium with 5 µM BA. In contrast, a growth medium containing 4.4 µM BA induced meager shoot formation (1.07) from nodal explants of C. roseus [25]. Amiri et al. [28] reported that the inclusion of 4.4 µM BA led to maximum shoot establishment (43%). However, 98% of the C. roseus nodal explants developed a mean of 7.12 shoots on MS medium with 4.4 µM BA [26]. These differences in shoot formation may be due to the different genotypes and explant sources. Kinetin also promotes multiple shoot production in C. roseus [19,24,26,27]. The percentage of shoot formation (59.4%), the number of shoots (5.7), and shoot elongation (3.6 cm) on MS medium with 4 µM kinetin were higher than in the other kinetin treatments and the control (Table 1). Similarly, Mehta et al. [26] reported that C. roseus nodal segments inoculated on medium with 4.4 µM kinetin developed 6.67 shoots, with a mean length of 2.7 cm. Pati et al. [19] reported that C. roseus single nodes inoculated in liquid medium with 5 µM kinetin formed 4.55 shoots, with an average length of 4.1 cm. Amongst the three cytokinins used, TDZ was the most effective in producing multiple shoots. TDZ, a plant growth regulator, has been shown to increase multiple shoot regeneration in a wide range of plants [29,30]. TDZ may enhance axillary shoot multiplication by varying the endogenous levels of growth regulators [30], and cytokinin concentration requirements differ for shoot induction and shoot elongation.
A combination of plant growth regulators (PGRs), such as cytokinin and auxin, was used to obtain a higher frequency of multiple shoot formation. Several studies have shown that media with cytokinin and auxin enhance shoot proliferation in C. roseus [9,16,25,26,28]. Satdive et al. [16] reported that the morphogenetic response (73.33%) and the number of shoots (9–13 per cotyledonary leaf) were highest in medium with 11.4 µM kinetin and 0.27 µM NAA. Kumar et al. [25] reported that the multiplication rate (4.01 shoots/nodal segment) and shoot length (2.07 cm) were highest in medium with 4.4 µM BA and 1.08 µM NAA, and Mehta et al. [26] reported that the shooting response (99%), number of shoots (7.3/node), and shoot length (5.97 cm) were highest with 2.2 µM BA and 10.8 µM NAA. Amiri et al. [28] reported that the multiplication rate (5.2 shoots/nodal segment) and shoot length (6.3 cm) were highest with 6.6 µM BA and 2.5 µM IBA. Although TDZ has both auxin and cytokinin activities, the addition of TDZ medium with auxin often improves the in vitro shoot production of a wide range of plants [29,30,46]. In this study, a higher shoot induction rate (91.1%) and higher number of shoots (19.2), with a mean length of 4.9 cm, were attained when the C. roseus nodal explants were cultivated on MS medium with 2 µM TDZ and 1 µM NAA (Table 3).
The impact of sucrose on multiple shoot production in C. roseus is unreported. In this study, sucrose had a significant effect on multiple shoot formation. The supplementation of sucrose or sugar is essential to stimulate axillary bud growth in vitro [47]. Sucrose in the cultivation media may increase the endogenous levels of carbohydrates, such as sucrose, glucose, fructose, and starch [48,49], and plant hormones, such as IAA, isopentenyl adenine riboside 5′-monophosphate, isopentenyl adenine riboside, isopentenyl adenine, zeatin riboside 5′-monophosphate, and zeatin riboside [50], that are important for various phases of plant growth. Starch accumulation is a prerequisite for shoot initiation in numerous plants [51]. Endogenous glucose levels improve the PGR-induced growth response. Glucose may affect the auxin biosynthetic YUCCA gene family members, auxin transporter PIN proteins, receptor TIR1, and the members of several gene families, including AUX/IAA, GH3, and SAUR, that are involved in auxin signaling [52]. Genes involved in cytokinin biosynthesis, such as AHK2, AtCKX4, AtCKX5, AtHXK4, ARR10, ARR1, ARR2, ARR6, ARR8, ARR11, CRF1, CRF2, CRF3, and IP3, are also regulated by glucose [53]. The highest multiple shoot production was attained when the C. roseus nodal explants were cultivated on MS medium with 2 µM TDZ, 1 µM NAA, and 4% sucrose (Table 4). However, the presence of 5% sucrose inhibited the rate of shoot initiation (67.8%). Sucrose, either alone or via interaction with other plant hormones, can induce or suppress many of the growth-related genes [50,54], which subsequently enhances or reduces the shooting response.
Flowering is regulated by internal plant factors and environmental signals [55]. The in vitro flower induction depends on culture environment, PGRs, media composition, and sucrose level [56]. The in vitro flowering of C. roseus is also affected by sucrose (Table 4), which promotes in vitro flowering in many plants [40,57,58]. Recently, C. roseus in vitro flowering has been achieved by using silver nitrate [27]. However, to our knowledge, the influence of sucrose on the in vitro flowering of C. roseus is unreported. In this study, including 4% and 5% sucrose in the MS nutrient medium promoted flowering in C. roseus (Table 4). Similar results have been reported for Ceropegia rollae [58], Scrophularia takesimensis [40], and Withania somnifera [57]. The in vitro flowering procedure established in this study can be utilized in bioactive compounds, mainly alkaloid [27] production, and in vitro breeding of C. roseus.
The continuous exposure of explants to shoot induction medium during several subcultures decreased the morphogenetic potential (Table 5). Thus, a secondary medium (TDZ-free MS) was required to maintain the morphogenetic potential of the nodal explants, where multiple shoots induced after the third subculture were elongated (Table 5). Several studies have shown that TDZ is slowly metabolized by plants and affects shoot formation [30,32]. The adverse effects of continued TDZ presence on shoot multiplication have also been reported in several plants [29,30]. In this study, somaclonal variants (albino shoots) were detected during the fourth subculture. Continuous exposure to TDZ also resulted in leaf chlorosis in Astragalus schizopterus [59], Philodendron cannifolium [60], and Sphagneticola trilobata [61]. Dewir et al. [32] reported that the TDZ-induced SV may be a valuable source of new genetic material. In this study, seeds obtained from the somaclonal plantlets were successfully germinated on MS nutrient medium and several seedlings exhibited a similar morphology. The somaclonal variants obtained in this study will be useful for new cultivar development.
There was no root formation in the absence of IBA; similar results have been reported in C. roseus [19,28]. Rooting of the in vitro-developed shoots of C. roseus was observed on auxin-free medium [25,26,36]. Differences in the rooting ability of micro shoots may be due to the endogenous levels of PGRs. When cytokinins were applied to induce shoot multiplication, they often inhibited the subsequent rooting of in vitro-regenerated shoots [19,28]. The rooting ability of micro shoots also depends on the type and concentration of cytokinins used in the shoot induction medium. TDZ has high cytokinin activity and strongly inhibits the activity of cytokinin oxidase, which increases the endogenous levels of natural cytokinins [29]. Thus, TDZ inhibits adventitious root formation. IBA has also been used for in vitro rooting in C. roseus [19,25,26]. In this study, higher IBA concentrations (12 µM) significantly diminished the rate of rooting (26.7%), number of roots (2.9), and elongation of the roots (2.1 cm) (Table 6). This is consistent with an earlier study of C. roseus [25]. In contrast, the highest rooting response (80%) and number of roots (7.0), with a mean length of 1.66 cm, were achieved in MS liquid medium containing 10 µM IBA [19]. The highest rate of root induction (90%) and number of roots (3.6), with a mean length of 1.68 cm, were achieved in quarter-strength MS medium with 24.6 µM IBA [26]. Root formation is an energy-consuming process that requires a source of carbon [62]. Sucrose is an important sugar that is frequently used in plant tissue culture medium as a source of energy and osmoticum. A culture medium with low osmotic potential is often preferred for the induction of roots and the osmotic potential is mostly maintained by sucrose. Low sucrose concentrations (2%) in the medium may decrease the osmotic potential and improve the rooting response of C. roseus (Table 7).
Different results have been reported in previous studies evaluating the total bioactive compounds of C. roseus. These differences may be explained based on differences in culture conditions, harvest times, or mineral intake [8,63]. Nonetheless, spectrophotometric methods have some drawbacks. For example, phenolics and other compounds (e.g., proteins) could interact with the Folin–Ciocalteu reagent and interfere with the results [64]. Moreover, some phytochemicals may form a complex with AlCl3 [65]. Thus, the identification, qualification, and quantification of phytochemicals should be confirmed using chromatographic methods, such as high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and gas chromatography (GC), for more accurate results. In this study, the phytochemicals in C. roseus extracts were qualified using UHPLC-MS/MS.
The term “antioxidant” has gained interest because it may play a role in preventing chronic and degenerative diseases. Several investigations have suggested that an imbalance between oxidants and antioxidants is the main reason for disease progression. Thus, many attempts have been made to find novel and safe antioxidants, and most have involved plants or plant products [66]. In light of the facts mentioned above, the antioxidant properties of the C. roseus samples were tested via different chemical methods, including free radical scavenging, reducing power, metal chelating, and phosphomolybdenum. In this study, the best antioxidant properties were obtained in the normal green shoot, followed by somaclonal variant shoot and callus extracts. These results can be attributed to the levels of phenolics in the extracts, as suggested by several researchers [67,68], who reported a positive correlation between the concentration of phenolics and their antioxidant properties. The metal chelating ability was ranked as somaclonal variant shoots > normal green shoots > callus. These contradictory results may be due to the non-phenolic chelators in the somaclonal variant shoots [5]. Some authors have also suggested that metal chelation plays a minor role in the antioxidant abilities of phenolic compounds. Studies on the antioxidant properties of C. roseus have yielded variable results. For example, Moon et al. [63] reported that the reducing power activity (FRAP and CUPRAC assays) of C. roseus samples was affected by culture conditions. Pham et al. [8] investigated the bioactivity and observed activity of C. roseus stem extracts and found that they were dependent on the solvents used. Finally, Pereira et al. [69] grew C. roseus roots in a 25 °C growth chamber for 16 h, and the root extracts exhibited significant free radical scavenging abilities in the in vitro assays. Taken together, these results suggest that C. roseus may be a natural raw material for novel antioxidants in the pharmaceutical and nutraceutical industries.
In the 21st century, some diseases are considered epidemiological pandemics and have created global crises. Alzheimer’s disease, diabetes mellitus, and obesity are such diseases [70,71], which require effective therapeutic strategies. One of the approaches to tackling this issue is the inhibition of enzymes that play roles in disease progression. Keeping this in mind, several key enzymes have been targeted. Carbohydrate-hydrolyzing enzymes (amylase and glucosidase) are the main targets for managing and preventing diabetes mellitus; their inhibition could retard the increase in blood glucose levels after a carbohydrate-rich diet [72]. Cholinesterases (especially acetylcholinesterase) are important factors in neurotransmission across synaptic gaps, and the inhibition of these may enhance the cognitive functioning in patients with Alzheimer’s disease [73]. Based on these facts, some compounds are produced as effective inhibitors in the pharmaceutical industry. However, most of these compounds have undesirable side effects [72,74]. Thus, novel and safe inhibitors from natural sources are needed to ameliorate the above-mentioned diseases. In the present study, the enzyme inhibitory effects of C. roseus extracts were investigated using different enzymes. We observed different results for each enzyme inhibition ability. To date, there have been few reports on the enzyme inhibitory effects of C. roseus. Pereira et al. [75] reported significant inhibitory effects of C. roseus root alkaloids against acetylcholinesterase, and vindoline and serpentine exhibited good anti-cholinesterase inhibition effects. These alkaloids were also found in our study and the combined results suggested that the cholinesterase inhibitory effects may be due to the presence of these alkaloids. Several other researchers have also reported alkaloids as effective inhibitors of cholinesterases. Moreover, some of the alkaloids from C. roseus exhibit significant antidiabetic effects in vivo. Tyrosinase is the main enzyme of melanin synthesis and is important for controlling hyperpigmentation problems [76]. In this study, the best tyrosinase inhibitory effect was detected in the normal green shoot extracts of C. roseus. From a pharmacological perspective, C. roseus may be an effective weapon against global health problems.

4. Materials and Methods

4.1. In Vitro Micropropagation

4.1.1. Plant Materials and Surface Decontamination

Actively growing shoots were collected from 6-month-old C. roseus plants cultivated in a greenhouse. The shoots were thoroughly rinsed under running tap water for 20 min, soaked in Tween 20 (0.1%, v/v) for 12 min, and then rinsed with distilled water. The shoots were surface decontaminated in 70% (v/v) ethanol (Daejung, Siheung-si, Gyeonggi-do, Korea) for 30 s, 5% (v/v) sodium hypochlorite (Daejung, Siheung-si, Gyeonggi-do, Korea) solution containing 3–6 drops of Tween 20 for 15 min, and 70% ethanol for 60 s. Each treatment was followed by 3–5 rinses using sterilized distilled water containing 0.1% (w/v) polyvinylpyrrolidone (Duchefa, Haarlem, The Netherlands).

4.1.2. Axillary Shoot Multiplication

The decontaminated shoots were cut into single nodal segments (0.6–1 cm) cultured in MS [77] medium fortified with 0, 1, 2, 4, 8, or 16 µM BA, kinetin, or TDZ and 2, 4, or 8 µM TDZ plus 0.5, 1, or 2 µM 2-(1-naphthyl) acetic acid (NAA), indole-3-butyric acid (IBA), or indole-3-acetic acid (IAA) for axillary shoot multiplication. To study the effects of sucrose on multiple shoot induction and flowering, nodal explants were inoculated on MS medium with optimal plant growth regulators (2 µM TDZ and 1 µM NAA) plus 0, 2, 3, 4, or 5% (w/v) sucrose. To study the effects of subculturing on shoot multiplication and SV, nodal explants derived from the in vitro multiple shoots (each subculture) were inoculated on MS medium with the optimal plant growth regulators and 4% sucrose. The shoot induction medium consisted of MS basal nutrients and vitamins with 3% sucrose (unless otherwise specified) and solidified with 0.8% (w/v) plant agar. The pH of the cultivation medium was adjusted to 5.6–5.8 before autoclaving at 121 °C for 20 min. The cultures were kept for 45 days at 23 ± 1 °C in a 16/8 light/dark photoperiod (50 µmol m−2 s−1), provided by cool white fluorescent tubes. The experiments were conducted as a completely randomized design; ten explants were used in each treatment, with three replications, and all experiments were performed twice. The shoot induction rate, total number of shoots, shoot length, percentage of flowering, total number of flowers, and total number of variant shoots were assessed after 45 days.

4.1.3. Rooting and Acclimatization

For root induction, in vitro-induced shoots (≥2 cm in length) were separated from the shoot clusters and inoculated on 1/2 MS medium with 0, 1, 2, or 4 µM IBA. To study the effects of sucrose on root induction and flowering, shoots were cultured on 1/2 MS medium fortified with 0, 2, 3, 4, or 5% sucrose and 4 µM IBA. For acclimatization, the rooted shoots were removed from the 1/2 MS medium, rinsed in tap water, and transplanted into plastic cups (200 mL) containing autoclaved peat moss, perlite, and vermiculite (1:1:1, v/v/v). The shoots were irrigated at four-day intervals with a 1/4 MS basal nutrient solution. The experiments were conducted as a completely randomized design; ten explants were used in each treatment, with three replications, and all experiments were performed twice. The rate of root induction, total number of roots, root length, percentage of flowering, total number of flowers, and plantlet survival were recorded after 35 days. The data were subjected to analysis of variance tests (ANOVA) in SAS (Release 9.1, SAS Institute, NC, USA).

4.2. Phytochemical Analysis

4.2.1. Extract Preparation

Callus (obtained from MS with 8 µM TDZ and 2 µM NAA), somaclonal variant, and normal green shoots (collected from MS with 2 µM TDZ and 1 µM NAA) were obtained from 45-day-old in vitro cultures, cut into small pieces, stored at −70 °C for 16 h, and then lyophilized. The freeze-dried samples (0.5 g) were extracted with methanol (80%) using an ultraturrax at 6000× g for 20 min. The extracts were filtered, and the solvents were removed using a rotary evaporator. All extracts were stored at 4 °C until further analysis.

4.2.2. Identification and Quantification of the Phytochemicals

Gradient reversed-phase ultra-high-performance liquid chromatography (UHPLC) separations with electrospray tandem mass spectrometry (MS/MS) detection (both positive and negative ion modes) were used for the structural characterization of the compounds in the extracts. The UHPLC system consisted of a Dionex Ultimate 3000RS UHPLC instrument coupled to a Thermo Q Exactive Orbitrap mass spectrometer. Chromatographic separation was achieved on a reversed-phase column Thermo Accucore C18 (100 mm × 2.1 mm i.d., 2.6 µm) [78]. Analytical details are presented in the Supplementary Materials.

4.2.3. Determination of Total Phenolics and Flavonoids

The total phenolic content was determined via the Folin–Ciocalteu method, as described by Slinkard and Singleton [79], and calculated as the gallic acid equivalent (GAE). The total flavonoid content was determined using the aluminum chloride (AlCl3) method, according to Zengin et al. [80], and was expressed as the rutin equivalent (RE).

4.3. Biological Activities

4.3.1. Antioxidant Activity

The antioxidant potential of the extracts was measured using several assay models, as previously described by Uysal et al. [81]. These include the radical scavenging assays for ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals, the redox assays for FRAP (ferric reducing antioxidant power) and CUPRAC (cupric reducing antioxidant capacity), and phosphomolybdenum total antioxidant capacity (TAC). Metals may catalyze the oxidation reactions; therefore, a metal chelating assay was also performed. Trolox and EDTA (for the chelating assay) were used as reference antioxidant compounds.

4.3.2. Enzyme Inhibition Assay

The extracts were tested for possible enzyme inhibition activity against several drug targets of different human diseases. Their activity was expressed in comparison to known drug inhibitors; acarbose for amylase, galantamine for acetylcholinesterase (AChE) and butylcholinestrase (BChE), and kojic acid for tyrosinase. All assay procedures were conducted according to methods described by Uysal et al. [81].

5. Conclusions

A competent in vitro propagation system through axillary shoot multiplication was established for C. roseus. This study showed that the PGRs and sucrose are significant factors affecting shoot bud initiation and multiplication from nodal segments. High levels of sucrose in the shoot induction or rooting medium have positive effects on in vitro flowering. SV was observed after the third subculture. In vitro flowering and SV may be exploited for C. roseus improvement. Phytochemical analysis indicated the presence of several phenolics and alkaloids in the callus, normal green, and somaclonal variant shoot extracts of C. roseus. Additionally, the extracts possessed potent antioxidant and enzyme inhibitory activities. These findings suggest that in vitro-derived callus, somaclonal variant, and normal green shoots may serve as alternative sources of bioactive metabolites with antioxidant and enzyme inhibitory activities. However, further experimental studies, such as in vivo animal models and toxicological assays, are recommended.

Supplementary Materials

Supplementary Materials are available online. Figure S1: Total ion chromatogram of the albino shoot sample in positive mode; Figure S2: Total ion chromatogram of the albino shoot sample in positive mode in 13–28 min; Figure S3: Total ion chromatogram of albino shoot sample in negative mode; Figure S4: Total ion chromatogram of callus sample in positive mode; Figure S5: Total ion chromatogram of callus sample in positive mode in 11–28 min; Figure S6: Total ion chromatogram of callus sample in negative mode; Figure S7: Total ion chromatogram of the normal-green shoot sample in positive mode; Figure S8: Total ion chromatogram of the normal-green shoot sample in a positive mode in 14–28 min; Figure S9: Total ion chromatogram of the normal-green shoot sample in negative mode; Figure S10: The typical extracted ion chromatogram (m/z 337.1916) in positive ion mode; Figure S11: MS2 spectrum of Catharantine at retention time 21.76 min; Figure S12: The typical extracted ion chromatogram (m/z 427.2233) in positive ion mode; Figure S13: MS2 spectrum of Vindolidine at retention time 25.23 min

Author Contributions

Conceptualization, O.N.L., D.H.K., and I.S.; methodology, G.A., G.Z., Z.C., J.J., and I.S.; investigation, G.A., G.Z., Z.C., J.J., and I.S.; data curation, K.R.R.R. and H.Y.P; writing—original draft preparation, G.A., G.Z., Z.C., J.J., O.N.L., and I.S.; writing—review and editing, G.Z., Z.C., K.R.R.R., H.Y.P., D.H.K., and I.S.; funding acquisition, O.N.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1F1A1075790).

Acknowledgments

This article was supported by the KU Research Professor Program of Konkuk University.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Alam, M.M.; Naeem, M.; Khan, M.M.A.; Uddin, M. Vincristine and Vinblastine Anticancer Catharanthus Alkaloids: Pharmacological Applications and Strategies for Yield Improvement. In Catharanthus Roseus; Springer: Cham, Switzerland, 2017; Volume 15, pp. 277–307. [Google Scholar]
  2. Qu, Y.; Safonova, O.; De Luca, V. Completion of the canonical pathway for assembly of anti-cancer drugs vincristine/vinblastine in Catharanthus roseus. Plant J. 2019, 97, 257–266. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  3. Mustafa, N.R.; Verpoorte, R. Phenolic compounds in Catharanthus roseus. Phytochem. Rev. 2007, 6, 243–258. [Google Scholar] [CrossRef] [Green Version]
  4. Piovan, A.; Filippini, R. Anthocyanins in Catharanthus roseus in vivo and in vitro: A review. Phytochem. Rev. 2007, 6, 235–242. [Google Scholar] [CrossRef]
  5. Ferreres, F.; Pereira, D.M.; Valentão, P.; Andrade, P.B.; Seabra, R.M.; Sottomayor, M. New Phenolic Compounds and Antioxidant Potential Catharanthus roseus. J. Agric. Food Chem. 2008, 56, 9967–9974. [Google Scholar] [CrossRef] [PubMed]
  6. Verma, P.; Mathur, A.K.; Masood, N.; Luqman, S.; Shanker, K. Tryptophan over-producing cell suspensions of Catharanthus roseus (L) G. Don and their up-scaling in stirred tank bioreactor: Detection of phenolic compound with antioxidant potential. Protoplasma 2013, 250, 371–380. [Google Scholar] [CrossRef] [PubMed]
  7. Senbagalakshmi, P.; Rao, M.V.; Kumar, T.S. In Vitro Studies, Biosynthesis of Secondary Metabolites and Pharmacological Utility of Catharanthus roseus (L.) G. Don.: A Review. In Catharanthus Roseus; Springer Science: Cham, Switzerland, 2017; Volume 1, pp. 153–199. [Google Scholar]
  8. Pham, H.N.T.; Sakoff, J.A.; Vuong, Q.V.; Bowyer, M.C.; Scarlett, C.J. Screening phytochemical content, antioxidant, antimicrobial and cytotoxic activities of Catharanthus roseus (L.) G. Don stem extract and its fractions. Biocatal. Agric. Biotechnol. 2018, 16, 405–411. [Google Scholar] [CrossRef]
  9. Das, A.; Sarkar, S.; Bhattacharyya, S.; Gantait, S. Biotechnological advancements in Catharanthus roseus (L.) G. Don. Appl. Microbiol. Biotechnol. 2020, 104, 4811–4835. [Google Scholar] [CrossRef] [PubMed]
  10. Nejat, N.; Valdiani, A.; Cahill, D.; Tan, Y.-H.; Mahmood, M.; Abiri, R. Ornamental Exterior versus Therapeutic Interior of Madagascar Periwinkle (Catharanthus roseus): The Two Faces of a Versatile Herb. Sci. World J. 2015, 2015, 1–19. [Google Scholar] [CrossRef] [Green Version]
  11. Pietrosiuk, A.; Furmanowa, M.; Łata, B. Catharanthus roseus: Micropropagation and in vitro techniques. Phytochem. Rev. 2007, 6, 459–473. [Google Scholar] [CrossRef]
  12. Aslam, J.; Ajaz, S.; Nadim, M.M. Pharmacognosy, phytochemistry pharmacological and biotechnological approaches of Catharanthus roseus (L.) G. Don. In Recent Trends In Biotechnology and Therapeutic Application of Medicinal Plants; Shahid, M., Shahzad, A., Malik, A., Sahai, A., Eds.; Springer: New York, NY, USA, 2013; pp. 189–210. [Google Scholar]
  13. Barrales-Cureño, H.J.; Andrade-Hoyos, P.; Luna-Cruz, A.; Reyes, C.; Chávez-Salinas, S.; López-Valdez, L.G. In Vitro Biotechnological Production and Pharmacological Studies of Antileukemic Alkaloids of Catharanthus roseus. In Catharanthus roseus; Naeem, M., Aftab, T., Khan, M., Eds.; Springer: Cham, Switzerland, 2017; pp. 17–34. [Google Scholar]
  14. Miura, Y.; Hirata, K.; Kurano, N.; Miyamoto, K.; Uchida, K. Formation of Vinblastine in Multiple Shoot Culture of Catharanthus roseus. Planta Medica 1988, 54, 18–20. [Google Scholar] [CrossRef]
  15. Hirata, K.; Horiuchi, M.; Asada, M.; Ando, T.; Miyamoto, K.; Miura, Y. Stimulation of dimeric alkaloid production by near-ultraviolet light in multiple shoot cultures of Catharanthus roseus. J. Ferment. Bioeng. 1992, 74, 222–225. [Google Scholar] [CrossRef]
  16. Satdive, R.K.; Fulzele, D.P.; Eapen, S. Studies on Production of Ajmalicine in Shake Flasks by Multiple Shoot Cultures of Catharanthus roseus. Biotechnol. Prog. 2003, 19, 1071–1075. [Google Scholar] [CrossRef] [PubMed]
  17. Hernandez-Dominguez, E.; Campos-Tamayo, F.; Vázquez-Flota, F. Vindoline synthesis in in vitro shoot cultures of Catharanthus roseus. Biotechnol. Lett. 2004, 26, 671–674. [Google Scholar] [CrossRef]
  18. Campos-Tamayo, F.; Hernández-Domínguez, E.; Vázquez-Flota, F. Vindoline Formation in Shoot Cultures of Catharanthus roseus is Synchronously Activated with Morphogenesis Through the Last Biosynthetic Step. Ann. Bot. 2008, 102, 409–415. [Google Scholar] [CrossRef] [Green Version]
  19. Pati, P.K.; Kaur, J.; Singh, P. A liquid culture system for shoot proliferation and analysis of pharmaceutically active constituents of Catharanthus roseus (L.) G. Don. Plant Cell, Tissue Organ Cult. 2011, 105, 299–307. [Google Scholar] [CrossRef]
  20. Sharma, A.; Mathur, A.K.; Ganpathy, J.; Joshi, B.; Patel, P. Effect of abiotic elicitation and pathway precursors feeding over terpenoid indole alkaloids production in multiple shoot and callus cultures of Catharanthus roseus. Biology 2019, 74, 543–553. [Google Scholar] [CrossRef]
  21. Khan, M.; Liu, H.; Wang, J.; Sun, B. Inhibitory effect of phenolic compounds and plant extracts on the formation of advance glycation end products: A comprehensive review. Food Res. Int. 2020, 130, 108933. [Google Scholar] [CrossRef]
  22. Moreno, P.R.H.; Van Der Heijden, R.; Verpoorte, R. Elicitor-mediated induction of isochorismate synthase and accumulation of 2,3-dihydroxy benzoic acid in Catharanthus roseus cell suspension and shoot cultures. Plant Cell Rep. 1994, 14, 188–191. [Google Scholar] [CrossRef]
  23. Park, H.Y.; Kim, D.H.; Sivanesan, I. Micropropagation of Ajuga species: A mini review. Biotechnol. Lett. 2017, 39, 1291–1298. [Google Scholar] [CrossRef]
  24. Bakrudden, A.A.A.; Shanthi, G.S.; Gouthaman, T.; Kavitha, M.S.; Rao, M.V. In vitro micropropagation of Catharanthus roseus—An anti-cancer medicinal plant. Acta Bot. Hung. 2011, 53, 197–209. [Google Scholar] [CrossRef]
  25. Kumar, A.; Prakash, K.; Sinha, R.K.; Kumar, N. In Vitro Plant Propagation of Catharanthus roseus and Assessment of Genetic Fidelity of Micropropagated Plants by RAPD Marker Assay. Appl. Biochem. Biotechnol. 2013, 169, 894–900. [Google Scholar] [CrossRef]
  26. Mehta, J.; Upadhyay, D.; Paras, P.; Ansari, R.; Rathore, S.; Tiwari, S. Multiple shoots regeneration of (anti-cancer plant) Catharanthus roseus—An important medicinal plant. American J. PharmTech Res. 2013, 3, 785–793. [Google Scholar]
  27. Panigrahi, J.; Dholu, P.; Shah, T.J.; Gantait, S. Silver nitrate-induced in vitro shoot multiplication and precocious flowering in Catharanthus roseus (L.) G. Don, a rich source of terpenoid indole alkaloids. Plant Cell Tissue Organ Cult. 2018, 132, 579–584. [Google Scholar] [CrossRef]
  28. Amiri, S.; Fotovat, R.; Tarinejhad, A.; Panahi, B.; Mohammadi, S.A. In vitro regeneration of periwinkle (Catharanthus roseus L.) and fidelity analysis of regenerated plants with ISSR markers. J. Plant Physiol. Breed. 2019, 9, 129–138. [Google Scholar]
  29. Huetteman, C.A.; Preece, J.E. Thidiazuron: A potent cytokinin for woody plant tissue culture. Plant Cell Tissue Organ Cult. 1993, 33, 105–119. [Google Scholar] [CrossRef]
  30. Guo, B.; Bilal, H.A.; Zeb, A.; Xu, L.L.; Wei, Y.H. Thidiazuron: A multi-dimensional plant growth regulator. Afr. J. Biotechnol. 2011, 10, 8984–9000. [Google Scholar] [CrossRef] [Green Version]
  31. Unal, B.T. Thidiazuron as an Elicitor in the Production of Secondary Metabolite. In Thidiazuron: From Urea Derivative to Plant Growth Regulator; Ahmad, N., Faisal, M., Eds.; Springer: Singapore, 2018. [Google Scholar]
  32. Dewir, Y.H.; Nurmansyah; Naidoo, Y.; Da Silva, J.A.T. Thidiazuron-induced abnormalities in plant tissue cultures. Plant Cell Rep. 2018, 37, 1451–1470. [Google Scholar] [CrossRef] [PubMed]
  33. Jeong, B.R.; Sivanesan, I. Impact of light quality and sucrose on adventitious shoot regeneration and bioactive compound accumulation in Ajuga multiflora Bunge. Sci. Hortic. 2018, 236, 222–228. [Google Scholar] [CrossRef]
  34. Knobloch, K.-H.; Berlin, J. Influence of Medium Composition on the Formation of Secondary Compounds in Cell Suspension Cultures of Catharanthus roseus (L.) G. Don. Zeitschrift für Naturforschung C 1980, 35, 551–556. [Google Scholar] [CrossRef]
  35. Scragg, A.H.; Ashton, S.; York, A.; Stepan-Sarkissian, G.; Grey, D. Growth of Catharanthus roseus suspension from maximum biomass and alkaloids accumulation. Enzym. Microb. Technol. 1990, 12, 292–298. [Google Scholar] [CrossRef]
  36. Swanberg, A.; Dai, W. Plant Regeneration of Periwinkle (Catharanthus roseus) via Organogenesis. HortScience 2008, 43, 832–836. [Google Scholar] [CrossRef] [Green Version]
  37. Junaid, A.; Mujib, A.; Bhat, M.; Sharma, M. Somatic embryo proliferation, maturation and germination in Catharanthus roseus. Plant Cell Tissue Organ Cult. 2006, 84, 325–332. [Google Scholar] [CrossRef]
  38. Bairu, M.W.; Aremu, A.O.; Van Staden, J. Somaclonal variation in plants: Causes and detection methods. Plant Growth Regul. 2011, 63, 147–173. [Google Scholar] [CrossRef]
  39. Sivanesan, I.; Jeong, B.R. Identification of somaclonal variants in proliferating shoot cultures of Senecio cruentus cv. Tokyo Daruma. Plant Cell Tissue Organ Cult. 2012, 111, 247–253. [Google Scholar] [CrossRef]
  40. Jeong, B.R.; Sivanesan, I. Direct adventitious shoot regeneration, in vitro flowering, fruiting, secondary metabolite content and antioxidant activity of Scrophularia takesimensis Nakai. Plant Cell Tissue Organ Cult. 2015, 123, 607–618. [Google Scholar] [CrossRef]
  41. Kim, D.H.; Kang, K.W.; Sivanesan, I. Influence of auxins on somatic embryogenesis in Haworthia retusa Duval. Biol. 2018, 74, 25–33. [Google Scholar] [CrossRef]
  42. Ferreres, F.; Pereira, D.M.; Valentão, P.; Oliveira, J.M.; Faria, J.; Gaspar, L.; Sottomayor, M.; Andrade, P.B. Simple and reproducible HPLC–DAD–ESI-MS/MS analysis of alkaloids in Catharanthus roseus roots. J. Pharm. Biomed. Anal. 2010, 51, 65–69. [Google Scholar] [CrossRef]
  43. Zhou, H.; Tai, Y.; Sun, C.; Pan, Y. Rapid identification of vinca alkaloids by direct-injection electrospray ionisation tandem mass spectrometry and confirmation by high-performance liquid chromatography-mass spectrometry. Phytochem. Anal. 2005, 16, 328–333. [Google Scholar] [CrossRef]
  44. Heijden, R.; Jacobs, D.; Snoeijer, W.; Hallard, D.; Verpoorte, R. The Catharanthus Alkaloids:Pharmacognosy and Biotechnology. Curr. Med. Chem. 2004, 11, 607–628. [Google Scholar] [CrossRef]
  45. Chen, Q.; Lu, X.; Guo, X.-R.; Guo, Q.; Li, D. Metabolomics Characterization of Two Apocynaceae Plants, Catharanthus roseus and Vinca minor, Using GC-MS and LC-MS Methods in Combination. Molecules 2017, 22, 997. [Google Scholar] [CrossRef] [Green Version]
  46. Park, H.; Kim, D.H.; Saini, R.K.; Gopal, J.; Keum, Y.S.; Sivanesan, I. Micropropagation and Quantification of Bioactive Compounds in Mertensia maritima (L.) Gray. Int. J. Mol. Sci. 2019, 20, 2141. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  47. Mason, M.G.; Ross, J.J.; Babst, B.A.; Wienclaw, B.N.; Beveridge, C.A. Sugar demand, not auxin, is the initial regulator of apical dominance. Proc. Natl. Acad. Sci. USA 2014, 111, 6092–6097. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  48. Aragão, V.P.M.; Ribeiro, Y.R.D.S.; Reis, R.S.; Macedo, A.F.; Floh, E.I.S.; Silveira, V.; Santa-Catarina, C. In vitro organogenesis of Cedrela fissilis Vell. (Meliaceae): The involvement of endogenous polyamines and carbohydrates on shoot development. Plant Cell Tissue Organ Cult. 2015, 124, 611–620. [Google Scholar] [CrossRef]
  49. De Conti, D.; Corredor-Prado, J.P.; Junior, D.R.; Suzuki, R.M.; Guerra, M.P.; Pescador, R. Determination of endogenous IAA and carbohydrates during the induction and development of protocorm-like bodies of Cattleya tigrina A. Richard. Acta Sci. Biol. Sci. 2018, 40, 37874. [Google Scholar] [CrossRef] [Green Version]
  50. Barbier, F.; Péron, T.; Lecerf, M.; Perez-Garcia, M.-D.; Barrière, Q.; Rolčík, J.; Boutet-Mercey, S.; Citerne, S.; Lemoine, R.; Porcheron, B.; et al. Sucrose is an early modulator of the key hormonal mechanisms controlling bud outgrowth in Rosa hybrida. J. Exp. Bot. 2015, 66, 2569–2582. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  51. Calamar, A.; De Klerk, G.-J. Effect of sucrose on adventitious root regeneration in apple. Plant Cell Tissue Organ Cult. 2002, 70, 207–212. [Google Scholar] [CrossRef]
  52. Mishra, B.S.; Singh, M.; Aggrawal, P.; Laxmi, A. Glucose and Auxin Signaling Interaction in Controlling Arabidopsis thaliana Seedlings Root Growth and Development. PLoS ONE 2009, 4, e4502. [Google Scholar] [CrossRef]
  53. Sami, F.; Siddiqui, H.; Hayat, S. Interaction of glucose and phytohormone signaling in plants. Plant Physiol. Biochem. 2019, 135, 119–126. [Google Scholar] [CrossRef]
  54. Barbier, F.F.; Dun, E.A.; Kerr, S.C.; Chabikwa, T.G.; Beveridge, C.A. An Update on the Signals Controlling Shoot Branching. Trends Plant Sci. 2019, 24, 220–236. [Google Scholar] [CrossRef]
  55. Vaz, A.P.; Figueiredo-Ribeiro, R.D.C.L.; Kerbauy, G.B. Photoperiod and temperature effects on in vitro growth and flowering of P. pusilla, an epiphytic orchid. Plant Physiol. Biochem. 2004, 42, 411–415. [Google Scholar] [CrossRef]
  56. Da Silva, J.A.T.; Nhut, D.T. Thin Cell Layers and Floral Morphogenesis, Floral Genetics and in Vitro Flowering. In Thin Cell Layer Culture System: Regeneration and Transformation Applications; Springer: Dordrecht, The Netherlands, 2003; pp. 285–342. [Google Scholar]
  57. Sivanesan, I.; Park, S.W. Optimizing factors affecting adventitious shoot regeneration, in vitro flowering and fruiting of Withania somnifera (L.) Dunal. Ind. Crop. Prod. 2015, 76, 323–328. [Google Scholar] [CrossRef]
  58. Upadhye, A.S.; Datar, M.N.; Waghamode, P.B.; Rajopadhye, A.A. In vitro flowering and fruiting of critically endangered plant Ceropegia rollae Hemadri. Ind. J. Biotechnol. 2016, 15, 112–115. [Google Scholar]
  59. Yorgancilar, M.; Erisen, S. The effect of thidiazuron (TDZ) on shoot regeneration of Astragalus schizopterus. J. Animal Plant Sci. 2011, 21, 519–524. [Google Scholar]
  60. Han, B.-H.; Park, B.-M. In vitro micropropagation of Philodendron cannifolium. J. Plant Biotechnol. 2008, 35, 203–208. [Google Scholar] [CrossRef] [Green Version]
  61. Sivanesan, I.; Park, S.W. Effect of thidiazuron on axillary shoot multiplication and subsequent rooting of Sphagneticola trilobata (L.) Pruski. Propag. Ornam. Plants 2014, 14, 147–151. [Google Scholar]
  62. Khan, P.S.V.; Hausman, J.; Rao, K. Effect of Agar, MS Medium Strength, Sucrose and Polyamines on in vitro Rooting of Syzygium alternifolium. Biol. Plant. 1999, 42, 333–340. [Google Scholar] [CrossRef]
  63. Moon, S.H.; Pandurangan, M.; Kim, D.H.; Venkatesh, J.; Patel, R.V.; Mistry, B.M. A rich source of potential bioactive compounds with anticancer activities by Catharanthus roseus cambium meristematic stem cell cultures. J. Ethnopharmacol. 2018, 217, 107–117. [Google Scholar] [CrossRef]
  64. Sánchez-Rangel, J.C.; Benavides, J.; Heredia, J.B.; Cisneros-Zevallos, L.; Jacobo-Velázquez, D.A. The Folin–Ciocalteu assay revisited: Improvement of its specificity for total phenolic content determination. Anal. Methods 2013, 5, 5990–5999. [Google Scholar] [CrossRef]
  65. Fernandes, A.J.D.; Ferreira, M.R.A.; Randau, K.P.; De Souza, T.P.; Soares, L.A.L. Total Flavonoids Content in the Raw Material and Aqueous Extractives fromBauhinia monandraKurz (Caesalpiniaceae). Sci. World J. 2012, 2012, 1–7. [Google Scholar] [CrossRef] [Green Version]
  66. Neha, K.; Haider, R.; Pathak, A.; Yar, M.S. Medicinal prospects of antioxidants: A review. Eur. J. Med. Chem. 2019, 178, 687–704. [Google Scholar] [CrossRef]
  67. Sarikurkcu, C.; Andrade, J.C.; Ozer, M.S.; Silva, J.M.F.D.L.; Ceylan, O.; De Sousa, E.O.; Coutinho, H.D.M. LC-MS/MS profiles and interrelationships between the enzyme inhibition activity, total phenolic content and antioxidant potential of Micromeria nervosa extracts. Food Chem. 2020, 328, 126930. [Google Scholar] [CrossRef] [PubMed]
  68. Vidal-Gutiérrez, M.; Robles-Zepeda, R.E.; Vilegas, W.; Gonzalez-Aguilar, G.A.; Torres-Moreno, H.; López-Romero, J.C. Phenolic composition and antioxidant activity of Bursera microphylla A. Gray. Ind. Crop. Prod. 2020, 152, 112412. [Google Scholar] [CrossRef]
  69. Pereira, D.M.; Faria, J.; Gaspar, L.; Ferreres, F.; Valentão, P.; Sottomayor, M.; Andrade, P.B. Exploiting Catharanthus roseus roots: Source of antioxidants. Food Chem. 2010, 121, 56–61. [Google Scholar] [CrossRef]
  70. Rauf, A.; Jehan, N. Natural Products as a Potential Enzyme Inhibitors from Medicinal Plants. In Enzyme Inhibitors and Activators; IntechOpen: Rijeka, Croatia, 2017; pp. 165–177. [Google Scholar]
  71. Ramsay, R.R.; Tipton, K.F. Assessment of Enzyme Inhibition: A Review with Examples from the Development of Monoamine Oxidase and Cholinesterase Inhibitory Drugs. Molecules 2017, 22, 1192. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  72. Chinsembu, K.C. Diabetes mellitus and nature’s pharmacy of putative antidiabetic plants. J. Herb. Med. 2019, 15, 100230. [Google Scholar] [CrossRef]
  73. Mishra, P.; Kumar, A.; Panda, G. Anti-cholinesterase hybrids as multi-target-directed ligands against Alzheimer’s disease (1998–2018). Bioorg. Med. Chem. 2019, 27, 895–930. [Google Scholar] [CrossRef] [PubMed]
  74. Prasansuklab, A.; Brimson, J.M.; Tencomnao, T. Potential Thai medicinal plants for neurodegenerative diseases: A review focusing on the anti-glutamate toxicity effect. J. Tradit. Complement. Med. 2020, 10, 301–308. [Google Scholar] [CrossRef]
  75. Pereira, D.M.; Ferreres, F.; Oliveira, J.M.; Gaspar, L.; Faria, J.; Valentão, P.; Sottomayor, M.; Andrade, P.B. Pharmacological effects of Catharanthus roseus root alkaloids in acetylcholinesterase inhibition and cholinergic neurotransmission. Phytomedicine 2010, 17, 646–652. [Google Scholar] [CrossRef]
  76. Mukherjee, P.K.; Biswas, R.; Sharma, A.; Banerjee, S.; Biswas, S.; Katiyar, C. Validation of medicinal herbs for anti-tyrosinase potential. J. Herb. Med. 2018, 14, 1–16. [Google Scholar] [CrossRef]
  77. Murashige, T.; Skoog, F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol. Plant 1962, 15, 473–497. [Google Scholar] [CrossRef]
  78. Dall’Acqua, S.; Uysal, A.; Diuzheva, A.; Gunes, E.; Jekő, J.; Cziáky, Z.; Picot-Allain, C.M.N.; Mahomoodally, M.F. Characterization of phytochemical components of Ferula halophila extracts using HPLC-MS/MS and their pharmacological potentials: A multi-functional insight. J. Pharm. Biomed. Anal. 2018, 160, 374–382. [Google Scholar] [CrossRef]
  79. Slinkard, K.; Singleton, V.L. Total phenol analysis: Automation and comparison with manual methods. Am. J. Enol. Viticult. 1977, 28, 49–55. [Google Scholar]
  80. Zengin, G.; Sarikurkcu, C.; Aktumsek, A.; Ceylan, R. Sideritis galatica Bornm.: A source of multifunctional agents for the management of oxidative damage, Alzheimer’s’s and diabetes mellitus. J. Funct. Foods 2014, 11, 538–547. [Google Scholar] [CrossRef]
  81. Uysal, S.; Zengin, G.; Locatelli, M.; Bahadori, M.B.; Mocan, A.; Bellagamba, G.; De Luca, E.; Mollica, A.; Aktumsek, A. Cytotoxic and Enzyme Inhibitory Potential of Two Potentilla species (P. speciosa L. and P. reptans Willd.) and Their Chemical Composition. Front. Pharmacol. 2017, 8, 290. [Google Scholar] [CrossRef] [PubMed]
Sample Availability: Samples are not available from the authors.
Molecules 25 04945 g001 550
Figure 1. In vitro propagation of Catharanthus roseus. (a) Multiple shoots produced from nodal segments of C. roseus cultivated on MS medium with 2 µM TDZ and 1 µM NAA after 45 days; (b) in vitro flowers produced from the multiple shoots regenerated on MS medium with 2 µM TDZ, 1 µM NAA and 5% sucrose; (c) somaclonal variation in C. roseus; (d) multiple albino (variant) shoots produced from nodal segments isolated from the variant shoot cultivated on MS medium with 2 µM TDZ and 1 µM NAA; (e) seeds obtained from normal green plantlets were germinated on MS nutrient medium; (f) seeds obtained from somaclonal variant plantlets were germinated on MS nutrient medium; (g) root induction from a shoot cultivated on half-strength MS medium with 4 µM IBA; in vitro flowers produced from the rooted shoot cultivated on half-strength MS medium with 4 µM IBA plus (h) 3% sucrose and (i) 5% sucrose.
Figure 1. In vitro propagation of Catharanthus roseus. (a) Multiple shoots produced from nodal segments of C. roseus cultivated on MS medium with 2 µM TDZ and 1 µM NAA after 45 days; (b) in vitro flowers produced from the multiple shoots regenerated on MS medium with 2 µM TDZ, 1 µM NAA and 5% sucrose; (c) somaclonal variation in C. roseus; (d) multiple albino (variant) shoots produced from nodal segments isolated from the variant shoot cultivated on MS medium with 2 µM TDZ and 1 µM NAA; (e) seeds obtained from normal green plantlets were germinated on MS nutrient medium; (f) seeds obtained from somaclonal variant plantlets were germinated on MS nutrient medium; (g) root induction from a shoot cultivated on half-strength MS medium with 4 µM IBA; in vitro flowers produced from the rooted shoot cultivated on half-strength MS medium with 4 µM IBA plus (h) 3% sucrose and (i) 5% sucrose.
Molecules 25 04945 g001
Table
Table 1. Effect of cytokinins on multiple shoot regeneration from nodal explants of Catharanthus roseus.
Table 1. Effect of cytokinins on multiple shoot regeneration from nodal explants of Catharanthus roseus.
CytokininConc. (µM)Shoot Induction (%)Shoot NumberShoot Length (cm)
Control (MS)018.3 ± 2.7 j1.3 ± 0.3 i1.0 ± 0.5 h,i
BA123.1 ± 4.8 i2.6 ± 0.7 h2.5 ± 0.4 d-f
255.8 ± 3.9 d4.0 ± 1.0 e,f3.1 ± 0.4 d
466.4 ± 3.7 b6.3 ± 1.3 c2.9 ± 0.6 d,e
860.3 ± 2.9 c3.7 ± 1.0 e-g2.4 ± 0.4 e-g
1644.2 ± 4.0 f2.7 ± 1.2 g,h1.5 ± 0.3 h
Kinetin125.7 ± 2.8 i2.3 ± 0.9 h1.8 ± 0.5 g,h
232.7 ± 2.8 h3.1 ± 0.8 f-h2.1 ± 0.4 f,g
459.5 ± 2.4 c5.7 ± 1.0 c3.6 ± 0.8 c
854.2 ± 3.0 d4.4 ± 1.1 d,e2.2 ± 0.4 f,g
1648.7 ± 3.3 e2.9 ± 1.1 g,h1.5 ± 0.3 h
TDZ137.8 ± 2.8 g4.3 ± 0.9 d,e4.4 ± 1.1 b
275.2 ± 3.7 a10.1 ± 1.5 a5.0 ± 0.7 a
467.1 ± 3.4 b7.7 ± 1.2 b3.0 ± 0.4 d
861.7 ± 5.5 c5.3 ± 0.7 c,d1.4 ± 0.3 h
1653.3 ± 3.1 d3.7 ± 1.0 e-g0.8 ± 0.2 i
f-value
F-testCytokinin196.683.018.7
Conc. 393.460.573.8
Cytokinin * Conc. 50.815.830.8
p-value
Cytokinin<0.001<0.001<0.001
Conc. <0.001<0.001<0.001
Cytokinin * Conc. <0.001<0.001<0.001
Mean ± S.D., followed by the same letters within a column, were not significantly different p < 0.05. BA, N6-benzyladenine; TDZ, thidiazuron; Conc., concentration.
Table
Table 2. Effect of cytokinins and their concentration on multiple shoot regeneration from nodal explants of Catharanthus roseus.
Table 2. Effect of cytokinins and their concentration on multiple shoot regeneration from nodal explants of Catharanthus roseus.
FactorsShoot Induction (%)Shoot NumberShoot Length (cm)
BA49.9 ± 17.1 b3.8 ± 1.5 b2.5 ± 0.6 b
Kinetin44.2 ± 14.4 c3.7 ± 1.4 b2.2 ± 0.8 b
TDZ59.0 ± 14.3 a6.2 ± 2.7 a2.9 ± 1.8 a
1 µM28.9 ± 7.8 e3.1 ± 1.1 d2.9 ± 1.3 b
2 µM54.6 ± 21.3 c5.7 ± 3.8 b3.4 ± 1.5 a
4 µM64.4 ± 4.2 a6.6 ± 1.0 a3.1 ± 0.4 ab
8 µM58.7 ± 4.0 b4.5 ± 0.8 c1.9 ± 0.5 c
16 µM48.7 ± 4.6 d3.1 ± 0.5 d1.3 ± 0.4 d
Mean ± S.D., followed by the same letters within a column, were not significantly different p < 0.05. The means in Table 2 refer to all concentrations and effects observed in Table 1. BA, N6-benzyladenine; TDZ, thidiazuron.
Table
Table 3. Effect of TDZ plus auxins on multiple shoot induction from nodal explants of Catharanthus roseus.
Table 3. Effect of TDZ plus auxins on multiple shoot induction from nodal explants of Catharanthus roseus.
Concentration (µM)Shoot Induction (%)Shoot NumberShoot Length (cm)
TDZIAAIBANAA
000018.3 ± 2.7 r1.3 ± 0.3 r1.0 ± 0.5 k
20.50079.1 ± 2.4 c,d11.0 ± 1.2 e,f3.8 ± 0.5 c,d
40.50069.2 ± 3.4 h-k8.0 ± 1.7 j-l3.3 ± 0.6 e,f
80.50066.3 ± 3.3 k,l5.2 ± 1.3 n-p1.7 ± 0.5 j
210082.3 ± 2.7 b,c12.9 ± 1.5 c,d4.1 ± 0.6 b,c
410080.4 ± 4.7 c10.0 ± 1.7 f,g2.8 ± 0.4 g
810070.1 ± 3.5 g-j7.3 ± 1.2 k-m1.9 ± 0.4 h-j
220063.6 ± 3.2 l,m8.9 ± 1.6 g-j3.0 ± 0.2 f,g
420043.7 ± 3.2 p6.3 ± 0.9 m,n2.8 ± 0.3 g
820038.2 ± 3.5 q4.1 ± 1.2 p,q1.1 ± 0.3 k
200.5080.8 ± 4.1 c13.3 ± 1.7 c,d4.5 ± 0.4 b
400.5070.9 ± 2.9 g-i9.4 ± 1.3 f-j3.6 ± 0.3 d,e
800.5067.3 ± 4.4 j,k7.1 ± 1.5 k-m1.8 ± 0.2 i,j
201084.1 ± 2.8 b14.9 ± 1.8 b4.1 ± 0.3 b,c
401076.4 ± 3.4 d,e10.4 ± 1.2 f,g4.4 ± 0.5 b
801073.1 ± 3.6 f,g8.1 ± 1.1 i-l2.3 ± 0.3 h
202068.2 ± 3.5 i-k6.8 ± 1.2 l,m3.3 ± 0.4 e,f
402060.4 ± 2.2 m,n5.9 ± 0.8 n-o2.2 ± 0.4 h,i
802045.8 ± 3.8 p4.7 ± 1.2 o-q1.0 ± 0.3 k
2000.579.0 ± 3.3 c,d14.3 ± 1.7 b,c5.1 ± 0.3 a
4000.575.2 ± 3.7 e,f10.2 ± 1.6 f,g4.4 ± 0.5 b
8000.567.1 ± 4.0 j,k8.4 ± 1.7 h-k2.8 ± 0.4 g
200191.1 ± 2.7 a19.2 ± 2.0 a4.9 ± 0.4 a
400179.3 ± 2.9 c,d12.3 ± 2.1 d,e3.5 ± 0.5 d,e
800169.9 ± 3.6 g-j9.7 ± 1.9 f-i2.7 ± 0.3 g
200272.6 ± 2.6 f-h9.6 ± 1.9 f-j3.3 ± 0.4 e,f
400260.1 ± 3.3 g-j9.5 ± 1.8 f-j2.1 ± 0.2 h,i
800252.7 ± 2.7 o3.2 ± 1.6 q1.1 ± 0.3 k
Mean ± S.D., followed by the same letters within a column, were not significantly different p < 0.05. TDZ, thidiazuron; IAA, indole-3-acetic acid; IBA, indole-3-butyric acid; NAA, 2-(1-Naphthyl)acetic acid.
Table
Table 4. Effect of sucrose on in vitro multiple shoot induction and flowering of Catharanthus roseus.
Table 4. Effect of sucrose on in vitro multiple shoot induction and flowering of Catharanthus roseus.
Sucrose (%)Shoot Induction (%)Shoot NumberShoot Length (cm)Flowering (%)Flower Number
00.0 ± 0.0 e0.0 ± 0.0 e0.0 ± 0.0 d0.0 ± 0.0 c0.0 ± 0.0 c
285.6 ± 2.9 c13.7 ± 1.3 d3.6 ± 0.5 c0.0 ± 0.0 c0.0 ± 0.0 c
391.1 ± 2.7 b19.2 ± 2.0 b4.9 ± 0.4 a0.0 ± 0.0 c0.0 ± 0.0 c
495.8 ± 2.0 a23.6 ± 2.7 a4.5 ± 0.4 b23.8 ± 3.7 b2.0 ± 0.7 b
567.8 ± 3.4 d15.8 ± 2.3 c3.9 ± 0.7 c35.3 ± 4.0 a2.9 ± 1.2 a
Mean ± S.D., followed by the same letters within a column, were not significantly different p < 0.05. Medium: Murashige and Skoog with 2 µM thidiazuron and 1 µM 2-(1-Naphthyl)acetic acid.
Table
Table 5. Effect of subculture on shoot multiplication and somaclonal variation in Catharanthus roseus.
Table 5. Effect of subculture on shoot multiplication and somaclonal variation in Catharanthus roseus.
No. of SubcultureShoot Induction (%)Normal Shoot NumberVariant Shoot Number
095.8 ± 2.0 c23.6 ± 2.7 d0.0 ± 0.0 d
198.3 ± 1.5 b28.3 ± 2.7 c0.0 ± 0.0 d
2100 ± 0.0 a31.7 ± 3.3 b0.0 ± 0.0 d
3100 ± 0.0 a39.0 ± 2.9 a0.0 ± 0.0 d
4100 ± 0.0 a20.4 ± 2.6 e7.4 ± 1.7 c
5100 ± 0.0 a14.8 ± 1.9 f11.8 ± 1.6 b
6100 ± 0.0 a7.9 ± 1.1 g13.4 ± 2.1 a
Mean ± S.D., followed by the same letters within a column, were not significantly different p < 0.05. Medium: Murashige and Skoog with 2 µM thidiazuron and 1 µM 2-(1-Naphthyl)acetic acid and 4% sucrose.
Table
Table 6. Effect of IBA on in vitro rooting of Catharanthus roseus.
Table 6. Effect of IBA on in vitro rooting of Catharanthus roseus.
IBA (µM)Rooted Shoot (%)Number of RootsRoot Length (cm)
00.0 ± 0.0 e0.0 ± 0.0 e0.0 ± 0.0 d
257.7 ± 6.3 c5.1 ± 1.4 c3.5 ± 0.8 b
490.9 ± 5.2 a9.3 ± 1.3 a6.2 ± 1.8 a
878.4 ± 6.2 b6.4 ± 1.4 b5.2 ± 1.1 a
1226.7 ± 4.9 d2.9 ± 1.1 d2.1 ± 0.8 c
Mean ± S.D., followed by the same letters within a column, were not significantly different p < 0.05. Medium: Half-strength Murashige and Skoog with 3% sucrose. IBA, indole-3-butyric acid.
Table
Table 7. Effect of sucrose on in vitro rooting and flowering of Catharanthus roseus.
Table 7. Effect of sucrose on in vitro rooting and flowering of Catharanthus roseus.
Sucrose (%)Rooted Shoot (%)Number of RootsRoot Length (cm)Flowering (%)Flower Number
039.8 ± 7.2 e2.7 ± 1.2 e2.9 ± 0.9 d0.0 ± 0.0 d0.0 ± 0.0 d
296.7 ± 3.8 a15.2 ± 2.5 a8.3 ± 1.4 a0.0 ± 0.0 d0.0 ± 0.0 d
390.9 ± 5.2 b9.3 ± 1.3 b6.2 ± 1.9 b32.6 ± 9.6 c1.3 ± 0.5 c
479.6 ± 5.7 c6.1 ± 1.9 c4.9 ± 1.5 bc56.4 ± 7.9 b2.3 ± 0.7 b
567.3 ± 6.2 d4.4 ± 1.3 d4.4 ± 1.7 c67.6 ± 6.2 a3.9 ± 1.2 a
Mean ± S.D., followed by the same letters within a column, were not significantly different p < 0.05. Medium: Half-strength Murashige and Skoog with 4 µM indole-3-butyric acid.
Table
Table 8. Total phenolic and flavonoid content in the extracts.
Table 8. Total phenolic and flavonoid content in the extracts.
Total Phenolic Content (mg GAE/g)Total Flavonoid Content (mg RE/g)
Somaclonal variant shoot26.45 ± 0.171.21 ± 0.07
Callus14.66 ± 0.060.28 ± 0.09
Normal green shoot30.58 ± 0.662.47 ± 0.07
Values are expressed as mean ± S.D. of three parallel measurements. GAE, gallic acid equivalent; RE, rutin equivalent.
Table
Table 9. Chemical composition of somaclonal variant shoot tissues of Catharanthus roseus.
Table 9. Chemical composition of somaclonal variant shoot tissues of Catharanthus roseus.
No.NameFormulaRt[M + H]+[M – H]−Fragment 1Fragment 2Fragment 3Fragment 4Fragment 5References
1Neochlorogenic acid (5-O-Caffeoylquinic acid)C16H18O910.12355.10291 163.0387145.0283135.0440117.033789.0389
2 1Chlorogenic acid (3-O-Caffeoylquinic acid)C16H18O914.85355.10291 163.0388145.0283135.0440117.033589.0389
33-O-Feruloylquinic acid cis isomerC17H20O914.87 367.10291193.0498191.0550173.0443134.0362
43-O-Feruloylquinic acidC17H20O915.11 367.10291193.0498191.0556173.0443134.036093.0330
5Methylcoumarin isomer 1C10H8O215.71161.06026 133.0647105.0701103.054591.054579.0547
6Loganic acidC16H24O1015.72 375.12913213.0761169.0858151.0753113.022969.0329
7Chryptochlorogenic acid (4-O-Caffeoylquinic acid)C16H18O916.09355.10291 163.0387145.0283135.0440117.033689.0388
8VindolinineC21H24N2O217.11337.19161 320.1641276.1383177.0909144.0807117.0700[42,43]
9SecologanosideC16H22O1117.27 389.10839345.1190209.0448165.0545121.064469.0329
10Unidentified alkaloidC20H24N2O218.08325.19161 307.1801277.1320186.0914174.0912138.0913
1119-S-VindolinineC21H24N2O218.16337.19161 320.1640276.1380177.0908144.0807117.0700[42,43]
12Unidentified alkaloidC20H22N2O218.17323.7596 248.1437219.1039173.1072144.080779.0548
13DihydrositsirikineC21H28N2O318.37357.21782 339.2061311.1382251.1178234.0910136.1120[44]
145-O-Feruloylquinic acidC17H20O918.47 367.10291193.0499191.0552173.0442134.036093.0329
15Unidentified alkaloidC21H28N2O318.94357.21782 253.1694226.1434214.1435144.0807110.0966
164-O-Feruloylquinic acidC17H20O918.99 367.10291193.0498191.0551173.0443134.036093.0329
17LoganinC17H26O1019.05391.16043 229.1067197.0820179.0703151.0752109.0649[45]
18Methylcoumarin isomer 2C10H8O219.06161.06026 133.0648105.0702103.054591.054779.0546
19Unidentified alkaloidC20H22N2O219.67323.17596 216.1017184.0758156.0807129.0699
20Antirhine isomerC19H24N2O19.83297.19669 280.1698236.1428166.1221154.1225144.0807
2111-Hydroxycyclolochnerine or LochneridineC20H24N2O319.91341.18652 323.1751281.1640264.1386218.0808200.0703[44]
22VinervineC20H22N2O320.07339.17087 307.1435279.1484250.1215185.0704
23PanarineC20H22N2O220.32323.17596 305.1643166.0860156.0804148.1119144.0806
24SecologanolC17H26O1020.36391.16043 229.1068211.0963193.0859179.0701167.0702
255-O-Feruloylquinic acid cis isomerC17H20O920.51 367.10291193.0490191.0552173.0445134.036093.0330
26AmmocallineC19H22N220.64279.18612 248.1431219.1039149.0232144.0807107.0858[44]
27AntirhineC19H24N2O20.82297.19669 280.1689196.1122166.1224154.1225144.0807[44]
28Unidentified alkaloidC21H24N2O220.95337.19161 305.1639222.1276180.1018156.0806144.0807
29Quercetin-O-dirhamnosylhexosideC33H40O2021.05 755.20347301.0354300.0275299.0198271.0247255.0296
30Cathenamine or VallesiachotamineC21H22N2O321.26351.17087 321.1592289.1330247.1226233.1069182.0836[44]
3111-Hydroxycyclolochnerine or LochneridineC20H24N2O321.48341.18652 323.1749279.1491264.1381198.0913138.1277[44]
32Cathenamine or VallesiachotamineC21H22N2O321.59351.17087 321.1590289.1333247.1225196.0752168.0805[44]
33AkuammicineC20H22N2O221.60323.17596 294.1484291.1487280.1330263.1538234.1279[44]
34CatharanthineC21H24N2O221.76337.19161 173.1071165.0907144.0806133.064893.0702[43,45]
35AjmalicineC21H24N2O322.06353.18652 321.1593222.1113210.1121178.0862144.0807[44]
363-epi-Ajmalicine or 19-epi-3-iso-AjmalicineC21H24N2O322.34353.18652 321.1593222.1112210.1121178.0859144.0806[44]
377-Deoxyloganic acidC16H24O922.35 359.13421197.0810153.0907135.0803109.064389.0228
38Kaempferol-O-dirhamnosylhexosideC33H40O1922.40 739.20856285.0402284.0325283.0244255.0294227.0343
39CoronaridineC21H26N2O222.42339.20725 307.1795262.1585209.1072144.0807130.0653[44]
40Akuammicine isomerC20H22N2O222.75323.17596 294.1487291.1490280.1330263.1538234.1289
41StrictosidineC27H34N2O922.80531.23426 514.2064352.1535334.1432165.0545144.0806[44]
42TubotaiwineC20H24N2O222.95325.19161 293.1643265.1333236.1421222.1271194.0958[44]
43Unidentified alkaloidC21H24N2O323.06353.18652 321.1593228.1015214.0859196.0754168.0805
44Unidentified alkaloidC20H24N2O223.50325.19161 296.1642293.1644236.1427216.1016156.0806
453-epi-Ajmalicine or 19-epi-3-iso-AjmalicineC21H24N2O323.71353.18652 321.1605222.1113210.1121178.0862144.0806[44]
46Tabersonine or isomerC21H24N2O223.76337.19161 305.1646277.1695228.1016196.0756168.0806[42]
47Serpentine or AlstonineC21H20N2O323.80349.15522 317.1280263.0811261.0653235.0862206.0829[45]
48Serpentine or AlstonineC21H20N2O324.44349.15522 317.1280263.0810261.0654235.0861206.0832[45]
49Tabersonine or isomerC21H24N2O224.61337.19161 305.1642277.1693228.1016196.0758168.0807[42]
50VindolineC25H32N2O624.80457.23387 439.2197397.2116337.1886222.1125188.1068[43,45]
51VindolidineC24H30N2O525.23427.22330 409.2113367.2011158.0963143.0730 [43,44]
52Isorhamnetin-O-hexosideC22H22O1225.29 477.10330315.0512314.0434285.0407271.0250243.0293
53Isorhamnetin-3-O-rutinoside (Narcissin)C28H32O1625.56 623.16122315.0508314.0432300.0276299.0196271.0246
54RosicineC19H20N2O329.32325.15522 293.1281265.1328249.1381230.1171170.0962[44]
55 1Isorhamnetin (3′-Methoxy-3,4′,5,7-tetrahydroxyflavone)C16H12O730.41 315.05048300.0270151.0026107.0123
1 Confirmed by standard.
Table
Table 10. Chemical composition of callus of Catharanthus roseus.
Table 10. Chemical composition of callus of Catharanthus roseus.
No.NameFormulaRt[M + H]+[M – H]−Fragment 1Fragment 2Fragment 3Fragment 4Fragment 5References
1Pantothenic acidC9H17NO56.11220.11850 202.1073184.0968174.1122116.034490.0553
2 1TryptamineC10H12N29.65161.10788 144.0807143.0730117.0701103.054691.0547
3Unidentified alkaloidC20H24N2O213.25325.19161 307.1801277.1329160.1120152.1068135.1041
4Norharman (β-Carboline)C11H8N214.52169.07658 115.0542 [44]
5Loganic acidC16H24O1015.70 375.12913213.0760169.0857151.0750113.022869.0329
6VindolinineC21H24N2O217.02337.19161 320.1639276.1384177.0908144.0807117.0700[42,43]
7SecologanosideC16H22O1117.25 389.10839345.1187209.0444165.0544121.064369.0329
8Sweroside or isomerC16H22O918.00359.13421 197.0807179.0702151.0751127.0390111.0806
919-S-VindolinineC21H24N2O218.05337.19161 320.1640276.1383177.0909144.0807117.0700[42,43]
10Unidentified alkaloidC20H24N2O218.08325.19161 307.1802277.1330186.0913174.0912138.0914
11Unidentified alkaloidC25H32N2O618.74457.23387 439.1856295.1801277.1703185.1084144.0814
12LoganinC17H26O1019.02391.16043 229.1068197.0818179.0703151.0752109.0651[45]
13Unidentified alkaloidC25H32N2O619.36457.23387 325.1898307.1802270.1330174.0914122.0963
14Unidentified alkaloidC20H22N2O219.60323.17596 216.1016184.0755156.0806129.0700
15Harmine isomerC13H12N2O19.63213.10279 198.0786170.083388.0760
16VinervineC20H22N2O320.02339.17087 307.1436279.1490250.1216185.0705
17PanarineC20H22N2O220.25323.17596 305.1641166.0861156.0805148.1120144.0807
18SecologanolC17H26O1020.34391.16043 229.1065211.0961193.0858179.0700167.0700
19Unidentified alkaloidC21H24N2O220.35337.19161 305.1643277.1690234.1276196.0995144.0805
20AntirhineC19H24N2O20.83297.19669 280.1689196.1122166.1227154.1225144.0807[44]
21Unidentified alkaloidC21H24N2O220.93337.19161 305.1647277.1700222.1274180.1019156.0807
22Cathenamine or VallesiachotamineC21H22N2O321.22351.17087 321.1590289.1335247.1226233.1069182.0838[44]
2311-Hydroxycyclolochnerine or LochneridineC20H24N2O321.46341.18652 323.1748279.1488264.1354198.0911 [44]
24Cathenamine or VallesiachotamineC21H22N2O321.56351.17087 321.1593289.1331247.1226168.0804 [44]
25AkuammicineC20H22N2O221.65323.17596 294.1487291.1487280.1311263.1538234.1280[44]
26CatharanthineC21H24N2O221.82337.19161 173.1071165.0906144.0806133.064893.0702[43,45]
27AjmalicineC21H24N2O322.01353.18652 321.1586222.1112210.1121178.0860144.0807[44]
287-Deoxyloganic acidC16H24O922.33 359.13421197.0811153.0907135.0801109.064489.0227
293-epi-Ajmalicine or 19-epi-3-iso-AjmalicineC21H24N2O322.36353.18652 321.1593222.1112210.1122178.0860144.0806[44]
30StrictosidineC27H34N2O922.63531.23426 514.2069352.1545334.1433165.0544144.0807[44]
31TubotaiwineC20H24N2O222.83325.19161 293.1643265.1325236.1427222.1264194.0966[44]
32Tabersonine or isomerC21H24N2O223.68337.19161 305.1645277.1696228.1014196.0759168.0805[42]
33Serpentine or AlstonineC21H20N2O323.70349.15522 317.1278263.0810261.0652235.0862206.0832[45]
34Serpentine or AlstonineC21H20N2O324.40349.15522 317.1279263.0810261.0653235.0860206.0827[45]
35VindolineC25H32N2O624.84457.23387 439.2195397.2118337.1883222.1122188.1069[43,45]
36Unidentified alkaloidC21H24N2O324.93353.18652 321.1592293.1629250.1233212.0932199.0865
37VindolidineC24H30N2O525.37427.22330 409.2098367.2010158.0962143.0727 [43,44]
38Unidentified alkaloidC21H24N2O326.34353.18652 321.1595278.1180210.1122170.0959144.0807
39RosicineC19H20N2O329.33325.15522 293.1280265.1329249.1381230.1171170.0962[44]
1 Confirmed by standard.
Table
Table 11. Chemical composition of normal green shoot tissues of Catharanthus roseus.
Table 11. Chemical composition of normal green shoot tissues of Catharanthus roseus.
No.NameFormulaRt[M + H]+[M – H]−Fragment 1Fragment 2Fragment 3Fragment 4Fragment 5References
1Neochlorogenic acid (5-O-Caffeoylquinic acid)C16H18O910.12355.10291 163.0387145.0283135.0440117.033689.0388
2Unidentified alkaloidC20H24N2O213.27325.19161 307.1799277.1329160.1117152.1068135.1042
33-O-Feruloylquinic acid cis isomerC17H20O914.84 367.10291193.0498191.0550173.0444134.0360
4 1Chlorogenic acid (3-O-Caffeoylquinic acid)C16H18O914.87355.10291 163.0387145.0283135.0440117.033789.0389
53-O-Feruloylquinic acidC17H20O915.09 367.10291193.0497191.0552173,0443134.036093.0329
6Loganic acidC16H24O1015.70 375.12913213.0760169.0858151.0751113.022969.0329
7Chryptochlorogenic acid (4-O-Caffeoylquinic acid)C16H18O916.11355.10291 163.0388145.0284135.0441117.033689.0389
8VindolinineC21H24N2O217.02337.19161 320.1640276.1380177.0910144.0807117.0700[42,43]
9SecologanosideC16H22O1117.25 389.10839345.1189209.0446165.0543121.064369.0329
105-O-(4-Coumaroyl)quinic acidC16H18O817.40 337.09235191.0552173.0443163.0388119.048793.0329
114-O-Feruloylquinic acid cis isomerC17H20O917.59 367.10291193.0496191.0556173.0443134.036093.0329
12Sweroside or isomerC16H22O917.98359.13421 197.0807179.0701151.0752127.0390111.0806
13Unidentified alkaloidC20H24N2O217.99325.19161 307.1800277.1325186.0914174.0912138.0913
144-O-(4-Coumaroyl)quinic acidC16H18O818.04 337.09235191.0550173.0443163.0387119.048693.0329
1519-S-VindolinineC21H24N2O218.07337.19161 320.1640276.1385177.0908144.0807117.0700[42,43]
16Unidentified alkaloidC20H22N2O218.10323.17596 248.1431219.1040173.1070144.080679.0547
175-O-Feruloylquinic acidC17H20O918.45 367.10291193.0499191.0552173.0443134.035993.0329
18Unidentified alkaloidC21H28N2O318.88357.21782 253.1695226.1434214.1434144.0806110.0966
194-O-Feruloylquinic acidC17H20O918.95 367.10291193.0497191.0548173.0443134.036093.0329
20Unidentified alkaloidC20H22N2O219.59323.17596 216.1016184.0757156.0806129.0702
215-O-(4-Coumaroyl)quinic acid cis isomerC16H18O819,63 337.09235191.0552173.0440163.0391119.048793.0328
22Antirhine isomerC19H24N2O19.77297.19669 280.1697236.1425166.1225154.1224144.0807
2311-Hydroxycyclolochnerine or LochneridineC20H24N2O319.84341.18652 323.1751281.1640264.1386218.0808200.0703[44]
24VinervineC20H22N2O319.98339.17087 307.1436279.1487250.1258185.0707
25Methyl caffeoylquinateC17H20O919.99 367.10291193.0499179.0340173.0443161.0232135.0438
26PanarineC20H22N2O220.26323.17596 305.1644166.0860156.0805148.1119144.0807
27SecologanolC17H26O1020.33391.16043 229.1069211.0963193.0859179.0702167.0703
285-O-Feruloylquinic acid cis isomerC17H20O920.48 367.10291193.0499191.0552173.0448134.036193.0329
29AmmocallineC19H22N220.54279.18612 248.1429219.1041149.0231144.0806107.0858[44]
30AntirhineC19H24N2O20.75297.19669 280.1687196.1117166.1225154.1225144.0807[44]
31Unidentified alkaloidC21H24N2O220.81337.19161 305.1640222.1275180.1017156.0807144.0806
32Quercetin-O-dirhamnosylhexosideC33H40O2021.02 755.20347301.0352300.0275299.0216271.0247255.0294
33Cathenamine or VallesiachotamineC21H22N2O321.17351.17087 321.1596289.1333247.1226233.1069182.0837[44]
3411-Hydroxycyclolochnerine or LochneridineC20H24N2O321.38341.18652 323.1749279.1491264.1279198.0913138.1277[44]
35Cathenamine or VallesiachotamineC21H22N2O321.42351.17087 321.1592289.1331247.1223196.0756168.0806[44]
36AkuammicineC20H22N2O221.49323.17596 294.1485291.1487280.1332263.1538234.1279[44]
37CatharanthineC21H24N2O221.57337.19161 173.1071165.0908144.0806133.064893.0702[43,45]
38DesacetylvindolineC23H30N2O521.91415.22330 397.2130365.1854355.2009188.1069173.0830
39AjmalicineC21H24N2O321.99353.18652 321.1596222.1119210.1122178.0863144.0807[44]
40CoronaridineC21H26N2O222.29339.20725 307.1802262.1590209.1062144.0808130.0646[44]
417-Deoxyloganic acidC16H24O922.33 359.13421197.0811153.0907135.0802109.064389.0228
42Kaempferol-O-dirhamnosylhexosideC33H40O1922.38 739.20856285.0403284.0326283.0246255.0295227.0341
43Akuammicine isomerC20H22N2O222.70323.17596 294.1486291.1487280.1325263.1538234.1281
44StrictosidineC27H34N2O922.77531.23426 514.2066352.1537334.1431165.0543144.0807[44]
45TubotaiwineC20H24N2O222.89325.19161 293.1642265.1330236.1440222.1274194.0963[44]
46Unidentified alkaloidC21H24N2O323.02353.18652 321.1590228.1014214.0865196.0755168.0805
47Unidentified alkaloidC20H24N2O223.45325.19161 296.1636293.1644236.1434216.1016156.0806
48Serpentine or AlstonineC21H20N2O323.71349.15522 317.1280263.0810261.0653235.0863206.0832[45]
49Tabersonine or isomerC21H24N2O223.72337.19161 305.1646277.1698228.1016196.0755168.0806[42]
50Unidentified alkaloidC21H24N2O324.13353.18652 336.1828308.1645229.1096165.0908144.0807
51Serpentine or AlstonineC21H20N2O324.32349.15522 317.1278263.0810261.0654235.0862206.0825[45]
52Tabersonine or isomerC21H24N2O224.54337.19161 305.1643277.1692228.1017196.0753168.0807[42]
53VindolineC25H32N2O624.65457.23387 439.2211397.2116337.1901222.1122188.1068[43,45]
54VindolidineC24H30N2O524.96427.22330 409.2123367.2012158.0963143.0727 [43,44]
55Isorhamnetin-O-hexosideC22H22O1225.28 477.10330315.0509314.0433285.0404271.0247243.0294
56Isorhamnetin-3-O-rutinoside (Narcissin)C28H32O1625.56 623.16122315.0511314.0433300.0275299.0197271.0249
57Methoxy-trihydroxyflavanoneC16H14O627.83303.08686 179.0337177.0546163.0394153.0181145.0284
58RosicineC19H20N2O329.33325.15522 293.1284265.1332249.1384230.1173170.0962[44]
59 1Isorhamnetin (3′-Methoxy-3,4′,5,7-tetrahydroxyflavone)C16H12O730.40 315.05048300.0270283.0252271.0257151.0022107.0122
1 Confirmed by standard.
Table
Table 12. Antioxidant parameters of the tested extracts (IC50 (mg/mL)).
Table 12. Antioxidant parameters of the tested extracts (IC50 (mg/mL)).
DPPHABTSCUPRACFRAPPBDChelating
Somaclonal variant shoot1.65 ± 0.051.45 ± 0.011.33 ± 0.011.00 ± 0.011.44 ± 0.040.69 ± 0.02
Callus>31.85 ± 0.052.41 ± 0.011.35 ± 0.02>30.96 ± 0.02
Normal green shoot1.57 ± 0.081.44 ± 0.031.16 ± 0.010.97 ± 0.011.13 ± 0.060.82 ± 0.02
Trolox0.06 ± 0.010.09 ± 0.010.11 ± 0.010.04 ± 0.010.52 ± 0.02nt
EDTAntntntntnt0.02 ± 0.001
Values are expressed as mean ± S.D. of three parallel measurements. nt, not tested; ethylenediaminetetraacetic acid: EDTA; DPPH: 2,2-diphenyl-1-picrylhydrazyl; ABTS: 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); CUPRAC: Cupric reducing antioxidant capacity; FRAP: Ferric reducing antioxidant power; PBD, phosphomolybdenum.
Table
Table 13. Enzyme inhibitory effects of the tested extracts (IC50 (mg/mL)).
Table 13. Enzyme inhibitory effects of the tested extracts (IC50 (mg/mL)).
AChEBChETyrosinaseAmylase
Somaclonal variant shoot0.74 ± 0.011.02 ± 0.030.86 ± 0.021.39 ± 0.02
Callus0.65 ± 0.011.04 ± 0.031.05 ± 0.031.29 ± 0.07
Normal green shoot0.72 ± 0.010.96 ± 0.060.83 ± 0.011.30 ± 0.01
Galantamine0.003 ± 0.0010.007 ± 0.002ntnt
Kojic acidntNt0.08 ± 0.001nt
AcarbosentNtnt0.68 ± 0.01
Values are expressed as mean ± S.D. of three parallel measurements. nt, not tested.
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Lee, O.N.; Ak, G.; Zengin, G.; Cziáky, Z.; Jekő, J.; Rengasamy, K.R.R.; Park, H.Y.; Kim, D.H.; Sivanesan, I. Phytochemical Composition, Antioxidant Capacity, and Enzyme Inhibitory Activity in Callus, Somaclonal Variant, and Normal Green Shoot Tissues of Catharanthus roseus (L) G. Don. Molecules 2020, 25, 4945. https://doi.org/10.3390/molecules25214945

AMA Style

Lee ON, Ak G, Zengin G, Cziáky Z, Jekő J, Rengasamy KRR, Park HY, Kim DH, Sivanesan I. Phytochemical Composition, Antioxidant Capacity, and Enzyme Inhibitory Activity in Callus, Somaclonal Variant, and Normal Green Shoot Tissues of Catharanthus roseus (L) G. Don. Molecules. 2020; 25(21):4945. https://doi.org/10.3390/molecules25214945

Chicago/Turabian Style

Lee, O. New, Gunes Ak, Gokhan Zengin, Zoltán Cziáky, József Jekő, Kannan R.R. Rengasamy, Han Yong Park, Doo Hwan Kim, and Iyyakkannu Sivanesan. 2020. "Phytochemical Composition, Antioxidant Capacity, and Enzyme Inhibitory Activity in Callus, Somaclonal Variant, and Normal Green Shoot Tissues of Catharanthus roseus (L) G. Don" Molecules 25, no. 21: 4945. https://doi.org/10.3390/molecules25214945

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