Introduction

Panax quinquefolium (American ginseng) is a dicotyledonous plant belonging to the Araliaceae family and is one of several known species of ginseng, the others being P. ginseng, P. notoginseng, and P. vietnamensis. These species are commonly cultivated and used for therapeutic purposes such as improving physical and mental efficiency, stimulating cell metabolism, and acting on the nervous, cardiovascular, and immune systems (Yuan et al. 2010). Ginseng has also demonstrated anticancer, antiobesity, and radioprotective properties (Xie et al. 2007; Lee et al. 2010).

The main active compounds of ginseng, ginsenosides, are responsible for most of its medicinal activity. Ginsenosides are triterpene saponins that can be divided into two major groups: 20(S)-protopanaxadiol derivatives (Rb group) and 20(S)-protopanaxatriol derivatives (Rg group). Metabolites Rb1, Rb2, Rc, and Rd belong to the Rb group; Rg1 and Re belong to the Rg group. Ginsenosides are present not only in the roots, which are recorded as a source in many pharmacopoeias, but also in the aerial parts of the plant. For example, ginseng leaf appeared as a new crude drug in the 2005 version of China Pharmacopoeia (Pharmacopoeia of the People’s Republic of China 2005) and is used as a tea or a component of dietary supplements in the Western market. Moreover, ginseng leaves are added into skin-care products and cosmetics due to their antioxidant properties (Zhang et al. 2014). However, obtaining ginseng from field cultivation is problematic because it takes 3–4 yr and is expensive. An alternative source of ginseng secondary metabolites may be in vitro shoot culture. Previous research on in vitro shoot cultures of P. quinquefolium focused on the multiplication and germination of somatic embryos and their further development (Tirajoh et al. 1998; Zhao and Brown 2006; Uchedu et al. 2011). Meanwhile, reports on the content of secondary metabolites in somatic embryo-derived plantlets of ginseng are limited, with most studies of antioxidant capacity and the phenolic content of ginseng confined to reports on field-grown plants. (Jung et al. 2005; Zhang et al. 2014). Therefore, the present study was intended not only to obtain somatic embryo cultures of P. quinquefolium that were capable of creating shoots but also to determine the levels of ginsenosides and polyphenolic compounds and their antioxidant activity in these cultures.

Materials and Methods

Plant materials.

Callus and regenerated shoot cultures. Callus cultures of P. quinquefolium were initiated from the epicotyl of seedlings obtained from plants grown on a plantation of the University of Agriculture in Lublin (Poland). The seedlings were washed under running water with detergent, disinfected using 1% (v/v) sodium hypochlorite for 10 min, and cut into 1-cm pieces. Aseptic pieces of epicotyl were rinsed three times in sterile distilled water. The medium for callus initiation was composed of MS (Murashige and Skoog 1962) basal medium containing 3% (w/v) sucrose (Avantor™ Performance Materials, Gliwice, Poland), 0.7% (w/v) agar (Difco™ Bacto™ Agar; Sigma-Aldrich®, St. Louis, MO), and a combination of auxins (1 mg L−1 2,4-dichlorophenoxyacetic acid [2,4-D] and 1 mg L−1 1-naphthaleneacetic acid [NAA]) and cytokinin (0.2 mg L−1 6-benzylaminopurine [BAP]) (all from Sigma-Aldrich®). Medium was sterilized for 25 min at 121°C after the medium pH was adjusted to 5.8.

The callus cultures were placed in plant culture tubes (19 × 2.5 cm, containing 25 mL of medium), at 26 ± 2°C, in the dark. The calluses were transferred into fresh medium every 5 wk. The developed embryoid structures were transferred to semi-solid half-strength MS medium supplemented with gibberellic acid (GA3; 0.5, 1, or 10 mg L−1) (Sigma-Aldrich®) and either BAP (0.5 or 1 mg L−1) or kinetin (kin; 1 mg L−1) (Sigma-Aldrich®). Shoot regeneration was observed after 4 wk of culture, and these shoots were rooted in semi-solid half-strength B5 medium (Gamborg et al. 1968) with 0.2 mg L−1 3-(benzo [b] selenyl) acetic acid (BSAA, Thermo SCI Acros Organics™, Geel, Belgium), 2 mg L−1 indole-3-acetic acid (IAA) (Sigma-Aldrich®), and 50 g L−1 sucrose. The cultivation of cultures during regeneration and the rooting of shoots took place in plant culture tubes under continuous light (40 μm m−2 s−1) (white light, fluorescent lamp, Polamp, Pabianice, Poland), at 26 ± 2°C.

Shoots from field cultivation.

The aerial parts of 4-yr-old plants from field cultivation were obtained from the plantation of the University of Agriculture in Lublin. Fresh plant material was dried on absorbent paper at room temperature and then processed (described below) to estimate antioxidant ability and ginsenoside content.

Determination of ginsenosides and total phenolic content.

The procedures for extraction and quantitative analysis of ginsenosides were described in a previous report (Kochan et al. 2014). The total phenolic content (TPC) was determined using the Folin–Ciocalteu reagent according to Singleton and Rossi (1965). Briefly, a 400-μL extract of each sample was oxidized with 2 mL of Folin–Ciocalteu reagent (Avantor™ Performance Materials). This reagent had been previously diluted tenfold with distilled water. After 5 min at room temperature, the reaction was neutralized with 1.6 mL of 7.5% sodium carbonate. The mixture was then incubated for 30 min at room temperature. The absorbance of the resulting blue color was measured at 765 nm on a Rayleigh UV-1601 spectrophotometer (Beijing Reyleigh Corp., Beijing China) versus a blank sample. TPC was expressed as milligrams of gallic acid equivalents (GAE) per gram of dry extract. The calibration curve was obtained by preparing gallic acid solution at concentrations ranging from 1 to 400 mg L−1. The results are presented as means of triplicate analyses.

Determination of antioxidant activities.

ABTS (radical scavenging assay). The antioxidant activity was determined using the ABTS radical cation decolorization test according to Re et al. (1999). An ABTS stock solution contained 7 mM 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2.45 mM potassium persulfate (both from Sigma-Aldrich®). This mixture was kept in the dark at room temperature for 12–16 h. Then, 2 mL of ABTS solution was mixed with 2 mL of methanol and the absorbance was measured as a negative control (determined to be 0.7 ± 0.05 at 734 nm). The assay was performed by adding 2 mL of extract at concentrations of 2, 20, 100, 200, and 500 μg mL−1 to 2 mL of ABTS solution. The absorbance at 734 nm was measured after 10 min. The results were expressed as EC50 (μg mL−1): the concentration of the sample at which 50% maximum scavenging activity was recorded. Additionally, the results were calculated as (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) (Sigma-Aldrich®) micromolar equivalents per gram of dry extract using a calibration curve obtained by measuring a Trolox standard at concentrations from 1 to 20 μM.

Ferric reducing antioxidant power (FRAP) assay.

The FRAP assay is a method of measuring the ability of reductants (antioxidants) to reduce Fe3+ to Fe2+. The formation of blue Fe2+-TPTZ complex (Fe2+-2,4,6-tripyridyl-s-triazine) increases absorbance at 593 nm. The method of Pulido et al. (2000) was used with some modifications. The FRAP reagent was freshly prepared by mixing 2.5 mL TPTZ solution (10 mM TPTZ in 40 mM HCl), 2.5 mL of FeCl3·6H2O solution (20 mM), and 45 mL of acetate buffer (300 mM at pH 3.6). The FRAP reagent (3 mL) was added to 300 μL redistilled water and 100 μL of the sample methanol extract. The reaction mixture was incubated at 37°C for 15 min. After this time, the absorbance was measured at 593 nm relative to a blank: a sample containing methanol instead of extract. The antioxidant potential of the sample was determined against a standard FeSO4·7H2O value calculated from a calibration curve plotted with concentrations from 0 to 2000 μM.

Statistical analysis.

All treatments were performed in triplicate. Data were analyzed using the Kruskal–Wallis test. Differences between values were considered significant at p ≤ 0.05. Statistica Version 10 software was used for all statistical analyses (StatSoft www.statsoft.com/Products/STATISTICA-Features/Version-10).

Results and Discussion

Development of embryoids and regenerated shoots.

The pieces of sterile epicotyl derived from seedlings placed in MS medium with the addition of 1 mg L−1 2,4-D, 1 mg L−1 NAA, and 0.2 mg L−1 BAP were covered with callus tissue after 2 wk of culture. Initially, the callus tissue was opaque, light-yellowish, and friable, without any structures visible under macroscopic observation. After three subcultures, spherical structures developed spontaneously on the surface of the callus. Spontaneous development of embryoid structures was also observed by Tang (2000) and Choi et al. (1997) for other species of ginseng. These authors suggest that the presence of 2,4-D in the medium induces the formation of high-quality somatic embryos of P. ginseng (Tang 2000) and Acanthopanax koreanum (Choi et al. 1997). In the present study, the embryoid structures of P. quinquefolium initially appeared as small yellowish-white globular masses (Fig. 1). The frequency of spherical structure formation was 65%, and the competence for structure formation increased with the age of the culture, whereas Tirajoh et al. (1998) showed lower frequencies (40, 30, and 15.6%) of somatic embryo formation on calluses derived respectively from roots, mature leaves, and seedling-leaf explants of American ginseng. Similarly, Punja et al. (2004) observed that the type of explant influenced callus development and formation of embryonic structures. These authors stated that 11 lines of American ginseng calluses, which derived from 11 seed sources from different geographic regions and were incubated in MS medium containing 10 μM NAA and 9 μM 2,4-D, significantly differed in their formation of embryonic structures.

Fig. 1
figure 1

The development of somatic embryos of P. quinquefolium.

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Most (91%) of the spherical structures of P. quinquefolium cultivated in MS medium with the addition of 1 mg L−1 GA3 and 0.5 mg L−1 BAP regenerated into multiple or single shoots, frequently without roots. Some of the shoots displayed an abnormal morphology: they were slender and elongated, sometimes hyperhydric and with an abnormal shape to the lamina. The shoots with normal morphology were rooted; of these, only 11% (17 shoots) developed roots. Numerous previous reports indicated that the development of embryoids was sometimes abnormal and that plantlets regenerated from such embryoids lacked roots or had a poorly developed root system (Wang 1990; Choi et al. 1998; Tirajoh et al. 1998). Choi et al. (1998) suggested that the inability of regenerated plants to form roots might be related to embryonic structural abnormalities within morphologically abnormal embryoids (e.g., multicotyledonary or multiple embryoids). In contrast, Zhao and Brown (2006) reported that about 85% of germinated embryos of P. quinquefolium converted into plants with well-developed taproot systems and appeared normal when transplanted to growth chambers and field plots. According to that study, such results were possible through the use of higher concentrations of GA3 (10–20 mg L−1) in the germination medium and further development of shoot cultures in half-strength SH basal medium supplemented with 0.5% charcoal.

Ginsenoside content.

Saponin production was studied in somatic embryo-derived shoots after 4 wk of cultivation as well as in the aerial parts of 4-yr-old P. quinquefolium plants cultivated in the field. The somatic somatic embryo-derived shoots contained a total of nearly 9.8 mg g−1 DW of the six measured saponins (Table 1 and Fig. 2). Wang et al. (1999a) reported a lower level (6.6 mg g−1 DW) of total ginseng saponins from embryo-derived plantlets of P. quinquefolium, whereas Asaka et al. (1994) reported higher levels (18.78–21.35 mg g−1 DW) from such plantlets of P. ginseng. The present study indicated that the aerial parts of field-grown P. quinquefolium contained about 13 mg g−1 DW of the six measured saponins. Kim et al. (2014b) presented comparable levels (10.92 mg g−1 DW) of ginsenosides in the leaves of 3-yr-old plants of P. ginseng from hydroponic cultures. By comparison, ginsenoside accumulation in callus was significantly lower (3.26 mg g−1 DW; Fig. 2).

Table 1 Ginsenoside in P. quinquefolium derived from in vitro culture and field cultivation
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Fig. 2
figure 2

Ginsenoside and total phenolic content (TPC) levels in in vitro-derived and field-grown Panax quinquefolium. Rb group contains protopanaxadiol derivatives (Rb1, Rb2, Rc and Rd). Rg group contains protopanaxatriol derivatives (Rg1 and Re). Total contains all six ginsenosides. Rb/Rg is the ratio of Rb group contents to Rg group contents. Data are presented as milligrams per gram of DW, except for TPC (mg GAE g−1) and Rb/Rg (dimensionless). Bars indicate mean ± SD.

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The ratio of protopanaxadiol derivatives to protopanaxatriol derivatives (Rb group/Rg group ratio) is used as an indicator for qualitative evaluation of the ginseng material. In methanolic extracts obtained from in vitro cultures, the ratios of Rb group to Rg group ginsenosides were 1.67 and 3.44, respectively, for somatic embryo-derived shoots and callus cultures. Rb group saponins constituted about 66.3% of the total amount of examined ginsenosides from those cultures. In contrast to these results, the ratio of Rb group to Rg group saponins in the aerial parts of field-cultivated plants was 0.78. Protopanaxatriol derivatives (Rg group) quantitatively predominated in these extracts (Fig. 2). Several reports (Asaka et al. 1994; Wang et al. 1999a; Kim et al. 2014b) showed that the Rb group/Rg group ratio in extracts from plantlets derived from somatic embryos as well as in leaves of field-grown plants was <1.

The contents of six individual saponins (Rb1, Rb2, Rc, Rd, Re, and Rg1) were also compared (Table 1). Their relative levels differed in in vitro shoot cultures and the aerial parts of field-grown P. quinquefolium. Ginsenosides Rb1 and Re predominated quantitatively in shoots regenerated from embryoids, and their levels were similar, each about 3 mg g−1 DW. The amount of metabolite Re in regenerated shoots was about ninefold that of Rg1 saponin, another ginsenoside belonging to the Rg group. Wang et al. (1999a) noted that metabolite Rd dominated quantitatively among Rb group derivatives in somatic embryo-derived plantlets, and Asaka et al. (1994) reported that the three ginsenosides they examined, Rb2, Rc, and Rd, were present in larger quantities in plantlets regenerated from somatic embryoids of P. ginseng than were callus and emryoid cultures. These reports (Asaka et al. 1994; Wang et al. 1999a) confirm that Re ginsenoside is the main component of the derivatives in the Rg group.

Metabolite Rb2 was found to be the most plentiful saponin of the Rb group in the aerial parts of field plants (Table 1). The levels of Rb1, Rc, and Rd ginsenosides were significantly lower than in the shoots obtained from in vitro cultures. Metabolite Re predominated quantitatively and represented almost 50% of the total examined saponins, and the Rg1 content in the aerial parts was higher than that observed in in vitro cultures. Searels et al. (2013) and Li et al. (2012) reported Rd, Re, and Rg1 to be the most prevalent saponins in Korean ginseng leaves regardless of the age of the plants.

A comparative analysis of data described in the literature and presented in this paper reveals significant differences in the contents of total and individual saponins in shoots or plantlets regenerated from somatic embryoids as well as in the leaves or the aerial parts of ginseng plants cultivated in the field. This may be due to several factors, such as the species of ginseng, growing conditions (field cultivation or in vitro culture), and the type of explant from which the callus or somatic embryoids are derived.

Total content of phenolics.

TPC was measured in the callus, the shoots regenerated from somatic embryo and the aerial parts of field-cultivated plants by using the Folin–Ciocalteu method. As shown in Fig. 2, TPC for shoot cultures was 12.75 mg GAE g−1 of extract, whereas the value for the aerial parts of field-cultivated P. quinquefolium (6.36 mg GAE g−1 of extract) was half that amount. These TPC levels were, respectively, four and two times the levels in ginseng root reported by Hwang et al. (2010).

A comparative analysis of TPC and total saponin contents showed that their levels were inversely related. Higher TPC levels were associated with lower total ginsenoside levels; conversely, lower TPC levels corresponded to higher total ginsenoside levels (Fig. 2). Similarly, protopanaxatriol derivative (Rg group) content was inversely related to both Rb group/Rg group ratio and TPC content of the studied extracts (Fig. 2).

Antioxidant activities.

The antioxidant potentials of methanolic extracts of P. quinquefolium shoots regenerated from somatic embryos were compared with those of the aerial parts of field-cultivated plants. Two methods based on different reaction mechanisms, the FRAP and ABTS assays, were used to measure antioxidant activity (Table 2). In the FRAP assay, the extracts of callus tissue demonstrated the strongest antioxidant capacity (181 μmol Fe (II) g−1 DW of extract). Furthermore, the shoot cultures had 43% more Fe-reducing ability than the aerial parts had. The ABTS assay (Table 2) also showed the methanolic extract of shoots regenerated from somatic embryoids to be a more potent scavenger than that from the aerial parts of field-grown plants. However, it should be noted that the effectiveness of the extracts as antioxidants was relatively low in comparison to those of well-known products such as blueberries or blackberries (respectively, 257 and 344 μmol Fe (II) g−1 DW; Henríquez et al. 2011) or cranberry powder (242–319 μmol Fe (II) g−1 DW; Kim et al. 2014a). On the other hand, the antioxidant properties of the extracts described in this paper were stronger than those of other medicinal plants such as Aloe vera (for fresh leaf extract, 37 μmol Fe (II) g−1 DW) or Coriandrum sativum (for fruit extract, 23 μmol Fe (II) g−1 DW) (Guleria et al. 2013).

Table 2 Antioxidant abilities of methanolic extracts of P. quinquefolium derived from in vitro culture and field cultivation
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The results reported here demonstrated a relationship between antioxidant activity and total phenolic level: the extracts found to have higher antioxidant activity, as confirmed by both FRAP and ABTS assays, were those with a higher TPC (Table 2 and Fig. 2). This observation was not surprising and has been described for other species (Henríquez et al. 2011; Guleria et al. 2013; Kim et al. 2014a). On the other hand, an inverse correlation was found between antioxidant capacity and the amount of total saponins. These results differed from those of Zhang et al. (2014), who showed a strong correlation between antioxidant capacities and the contents of ginsenosides in leaf extracts from 1- to 13-yr-old ginseng samples.

Conclusions

Embryogenic callus and somatic embryo-derived shoot cultures were obtained from P. quinquefolium. In the studied extracts, higher total saponin levels, higher protopanaxatriol (Rg group) derivative content, and Rb group/Rg group ratio corresponded to lower TPC levels. Stronger antioxidant activities (according to both FRAP and ABTS) were observed in extracts with a higher total phenol content. On the other hand, saponin content was inversely related to the free-radical scavenging capacity of the studied extracts.