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Secondary Metabolites of Various Eleuthero (Eleutherococcus senticosus/Rupr. et Maxim./Maxim) Organs Derived from Plants Obtained by Somatic Embryogenesis

  • Katarzyna Bączek
  • Anna Pawełczak
  • Jarosław L. Przybył
  • Olga Kosakowska
  • Zenon WęglarzEmail author
Living reference work entry
  • 115 Downloads
Part of the Reference Series in Phytochemistry book series (RSP)

Abstract

Eleuthero is a thorny shrub native to Far East Asia, where for centuries it has been used as a medicinal plant. Due to strong adaptogenic activity, it has recently received considerable attention from both consumers and scientists. Although eleuthero is a rare, and in certain countries protected, species, its raw materials, i.e., underground organs, are still exclusively collected from natural sites. Thus, introduction into cultivation gives the chance for its survival in natural habitat. Note that eleuthero is characterized by a relatively low reproductive capacity; both generative and simple vegetative propagations are ineffective. The most efficient method to increase the reproductive potential of this plant on the purpose of its cultivation is application of in vitro techniques. In the case of eleuthero, the phenomenon of direct somatic embryogenesis is observed. The most effective explants for establishing the formation of somatic embryos are apical buds and hypocotyl fragments of plantlets obtained from extremely immature zygotic embryos that were isolated from seeds. In such cases, somatic embryos are directly formed on the explant tissues without the callus phase. Plants that are obtained from somatic embryos adapt relatively well to ex vitro conditions. In subsequent years of cultivation (4-year cycle), they can be an effective source of biologically active compounds, such as eleutherosides B and E, which have been used to standardize the raw material. The content of eleutherosides and other active compounds, such as phenolic acids, changes with the age of plants and depends on developmental phase of the plant, as well as on the plant organs (rhizomes, roots, shoots, or bark of these organs). The anatomical studies of plant tissues indicate that these compounds accumulate in the form of heterogeneous secretion in schizogenous reservoirs as well as in the vacuoles of epithelial and parenchymal cells of the secondary phloem.

Keywords

Eleuthero Diversity Reproduction ability In vitro propagation Solid medium Somatic embryos Eleutherosides Phenolic acids Schizogenous reservoirs 

Abbreviations

BA

6-Benzyladenine (cytokinin)

DW

Dry weight

EMA

European Medicines Agency

ESCOP

European Scientific Cooperative on Phytotherapy

hLf

Human lactoferrin

HMPs

Herbal medicinal products

HPLC

High-performance liquid chromatography

LTB

Escherichia coli heat-labile toxin

MS

Murashige and Skoog medium

MS/B5

Murashige and Skoog/Gamborg 5B medium

NAA

1-Naphthaleneacetic acid (auxin)

PgSS1

Panax ginseng squalene synthase gene

TCM

Traditional Chinese Medicine

Tr.

Trace amount

WHO

World Health Organization

WR

Without growth regulators

1 Introduction

Eleuthero (Eleutherococcus senticosus/Rupr. et Maxim./Maxim.; syn. Acanthopanax senticosus Rupr. et Maxim.) is a wild shrub that grows in Eastern Russia, Northern China, Korea, and Japan. It has been used for centuries in Far East medicine, particularly in Traditional Chinese Medicine (TCM) [1]. Since the 1960s, eleuthero has received considerable attention from Western countries, when new alternative herbal raw materials that could replace well-known but very deficient ginseng were being explored [2]. At that time, plants growing in Far Eastern Asia such as Schisandra chinensis (Turcz.) Baill., Rhodiola rosea (L.), and E. senticosus were specifically examined. They became objects of detailed studies, first carried out in the Soviet Union, followed by the Western countries. Studies, including clinical trials, revealed the specific activity of these species. They limited the negative effects of harmful factors of different origins (biological, chemical, and physical) affecting organisms, including viral and bacterial infections, radiation, toxic substances, noise, as well as extremely low and high temperatures or pressure, which have been the cause of aging and many civilization diseases. Such substances, which normalize dysfunctions, enhance stamina, and improve the body’s resistance against harmful, non-specific stressors of both the healthy and diseased organisms, are nowadays classified as adaptogens [3, 4, 5]. The studies focused on eleuthero clearly showed that the adaptogenic activity of the plant is similar or even stronger than that of ginseng’s (Panax ginseng C.A. Meyer) [1]. Thus, additional studies were performed on the chemical structure of the substances that are responsible for the pharmacological activity of raw materials obtained from this plant. Nowadays, eleuthero monographs can be found not only in Chinese, Russian, and Japanese but also in European Pharmacopoeia as well as in EMA (European Medicines Agency) and WHO (World Health Organization) reports [6, 7, 8, 9, 10, 11].

The primary raw material collected from eleuthero are underground organs, i.e., rhizomes and roots, treated as a homogeneous product, commonly named root (Eleutherococci radix). According to the abovementioned Pharmacopoeias, it is standardized based on the sum of eleutherosides B and E (not<0.8%); however, the raw material is characterized by a large variety of secondary metabolites belonging to different chemical groups, such as phenylpropanoid derivatives, lignans, sterols, coumarins, and saponins [2]. According to the EMA monograph [10], the eleuthero root was classified as a traditional herbal medicinal product, indicated for use in symptoms of asthenia such as fatigue and weakness. It is used mainly in a simple herbal form for preparing teas or as a liquid or as dry extracts.

The raw material is exclusively collected from wild-growing plants, mainly on the territory of Russia and China and exactly in Primorsky and Khabarovsk Krai and in Chinese Manchuria [12, 13]. Although there are sites where the plant grows naturally, low reproduction rate and excessive harvest of raw materials have significantly threatened the existence of this species, which is also affected by adverse changes in the natural environment related to both the climate change and expansion of human civilization. In fact, this threats have already led to the legal protection of this plant in some countries [14].

In this chapter, we present the reproductive problems of the species, using in vitro propagation as one of the most effective method to obtain a good-quality plant material to establish plantations. We also discussed the accumulation of biologically active compounds (in plants originated from in vitro cultures) in various organs collected in different developmental stages. The anatomical structures in which these compounds are accumulated are also shown.

2 Plant Development

Eleuthero is a shrub that grows up to a height of 2–6 m (Figs. 1 and 2). The system of underground organs located in the top layer of soil is composed of a branched, knotty rhizomes with numerous roots growing horizontally to >10 m in length [15]. The aboveground parts are shoots covered with light gray or light brown bark with ~5-mm-long thorny bristles that are directed downward. The leaves are palmate, with five leaflets (Fig. 3). The flowers are gathered in spherical umbels. Usually, 4–6 umbels are arranged at the top of the shoots to form a cyme (Figs. 4 and 5). The inflorescence axis is 5–7 cm long, whereas the peduncle is 1–2 cm long [12, 15, 16, 17]. Inflorescences contain fivefold flowers of three types: male, female, and bisexual. Male flowers are white or pale violet and produce 58,000–81,000 grains of pollen. Female flowers are usually yellowish and have sterile pollen, whereas bisexual ones are grayish or pale violet with semi-sterile pollen [4, 18]. Moreover, the flowers are protandric which prevents self-pollination. The most effective pollinators of eleuthero are Bumpus, Halictus, Megachile, Vespa, and Apis [19]. The flowering period falls during summer, and the first flowers bloom in the outer part of the umbel. The fruits are dark blue berries, ripening in autumn. They have a spherical shape and a diameter of 0.8–1 cm. They form a compact umbels, the mass of which reaches ~50 g (Fig. 6). Fresh berries are eaten and spread by birds [16, 20, 21, 22]. Each fruit usually contains five seeds, and only ~ 40% of them are completely developed (Table 1).
Fig. 1

4-year-old plants at experimental plantation (full vegetation)

Fig. 2

A 4-year-old plant (winter dormancy)

Fig. 3

Leaves

Fig. 4

Inflorescence

Fig. 5

Flowers

Fig. 6

Unripen fruits

Table 1

Characteristics of eleuthero fruits and seeds

Investigated traits

Top umbels

Lateral umbels

Fresh mass of one umbel (g)

48.53

16.02

Fresh mass of one fruit (g)

0.72

0.49

Number of fruits per umbel

72.25

38.77

Number of seeds per umbel

355.43

175.68

1000 seed weight (g)

10.132

9.209

Completely developed seeds per umbel (%)

60.71

45.18

Germination capacity (%)

42.23

12.35

*P<0.05 (in rows), Tukey’s test

2.1 Generative and Vegetative Reproduction Problems

Plants of the genus Araliaceae, including E. senticosus, are characterized by relatively low reproductive capacity [15]. Both generative and simple vegetative propagation of eleuthero is ineffective [23]. In natural conditions, the seeds usually germinate poorly, after a few years of natural stratification [23, 24, 25, 26, 27]. However, the germination capacity of eleuthero seeds depends on multiply factors, including seed’s setting on a plant. The weight of the umbels and the number of fruits and seeds in one umbel are higher in the top umbels (Fig. 7) compared to the lateral ones. The percentage of completely developed seeds (Fig. 8) and their weight is higher when originating from the top umbels, as well (Table 1). These seeds were characterized by a higher germination capacity; however, stratification was necessary. According to our results, the best model of stratification for eleuthero is the one previously developed for P. ginseng, i.e., with the seeds maintained in wet sand, at 20 °C for 10 weeks, followed by 14 weeks at 2 °C. After 6 months of such stratification, the germination capacity of eleuthero seeds from top umbels reached ~40%, whereas those obtained from lateral umbels was only 12% (Table 1). The importance of stratification of eleuthero seeds has been reported earlier by Isoda and Shoji [27], who observed a positive effect of this treatment after maintaining the seeds at 10 °C for 18 months. The residual effect of seeds setting on a plant was also observed concerning seedlings derived from these seeds. The plants (Figs. 9 and 10) obtained from top umbel’s seeds were shapely and adapted better to the field growth compared to the plants from lateral ones.
Fig. 7

Top umbels with ripen fruits

Fig. 8

Seeds

Fig. 9

Germinating seeds

Fig. 10

Seedlings

The attempts of simple vegetative reproduction of eleuthero, using various types of cuttings prepared from aboveground and underground parts of the plants, indicate that the ability to rooting shows only cuttings from underground organs and, particularly, root cuttings with primary developed shoots, which grew better after they were planted in the field (Table 2). However, when considering large-scale cultivation, vegetative propagation using such techniques seems to be ineffective.
Table 2

Rooting of cuttings (n = 50, three replications)

Type of cuttings

% of rooted cuttings

% of plants adopted to field conditions

Roots with primary shoots

68.0a

88.8a

Roots

38.7b

79.1a

Semi-wooden shoots

6.6c

88.9a

Primary developed shoots

6.8c

50.3b

Values marked in column with different letters differ at P < 0.05, Tukey’s test

2.2 Production of Plantlets with Application of In Vitro Techniques

The most effective methods to propagate rare, endangered plants on a larger scale involve application of plant tissue culture techniques. This is particularly important for plants that do not form seeds or have seeds with very low viability and weak germination. However, a very important reason for propagating medicinal plants using in vitro techniques is the possibility to produce a large number of genetically homogeneous plants with a desired chemical profile in a short time. This helps meet the newest requirements of ESCOP (European Scientific Cooperative on Phytotherapy) and pharmacopoeia commissions that herbal medicinal products (HMPs) should be obtained only from a standardized raw material [28]. Moreover, it helps meet the important requirements of the phytopharmaceutical industry about the quality of processed raw materials, such as the minimum content of biologically active compounds.

In this chapter, we focus on the production of eleuthero plant material using direct somatic embryogenesis with a solid medium.

2.2.1 Zygotic Embryos

As mentioned above, the germination capacity of eleuthero seeds is very low. It depends on the maturity of embryo in the seeds (extremely immature) and endosperm-related factors, which are responsible for the dormancy of seeds. Such a phenomenon was observed in the embryos isolated from the seeds of different age, i.e., fresh (directly after harvest), stored at 22 °C for 1 and 2 years, and germinated on a solid medium (modified MS/B5 medium, prepared according to Murashige and Skoog [29] and according to Gamborg [30] with agar (6.5 g L−1) and sucrose (30 g L−1)). The germination of zygotic embryos isolated from fresh seeds exceeds 56%; however, only 40% of them developed well, i.e., created normal roots and two cotyledons (Figs. 11 and 12). Note that most embryos obtained from seeds stored for 1 or 2 years died (84.5% and 99.0%, respectively) (Table 3). The application of both stratification (Table 4) and growth regulators, i.e., BA (6-benzyladenine) and NAA (1-naphthaleneacetic acid) in a solid medium (Table 4), resulted in more effective germination of zygotic embryos. BA and NAA stimulated also their development. However, the reaction of embryos strictly depends on the concentration of growth regulators. The best results were observed when the concentration was relatively low (Tables 5 and 6).
Fig. 11

Plantlets from zygotic embryos, grown in vitro (30 days from embryos isolation)

Fig. 12

Plantlets from zygotic embryos, grown in vitro (150 days from embryos isolation)

Table 3

Effect of seeds storage on the germination of isolated zygotic embryos and plantlets vigor (%) (n = 100; four replications)

 

Origin of zygotic embryos

Seeds directly after harvest

Seeds stored in room temperature per 1 year

Seeds stored in room temperature per 2 years

Well-developed plantlets

40.0a

13.5b

0.0c

Abnormal or died plantlets

16.0a

2.0b

1.0b

Died embryos

44.0b

84.5a

99.0a

Values marked in rows with different letters differ at P < 0.05, Tukey’s test

Table 4

Effect of seeds stratification (after 2-year storage) on the germination of isolated zygotic embryos (%) (n = 100, four replications)

 

Origin of zygotic embryos

Non-stratified seeds

Stratified seeds

Well-developed plantlets

0

28

Abnormal or died plantlets

8

18

Died embryos

92

54

P < 0.05 (in rows), Tukey’s test

Table 5

Influence of growth regulators on germination of zygotic embryos isolated from seeds directly after their harvest and plantlets vigor (%) (n = 25, three replications)

 

Concentration of growth regulators in solid medium

 

WR

0.01 mg L−1 BA 0.001 mg L−1 NAA

0.1 mg L−1 BA 0.001 mg L−1 NAA

0.001 mg L−1 NAA

0.01 mg L−1 BA

0.1 mg L−1 BA

Well-developed plantlets

40.5b

59.0a

30.0c

26.5c

33.5c

31.0c

Abnormal or died plantlets

18.5b

19.0b

45.0a

25.0b

26.0b

27.5b

Died embryos

41.0a

22.0b

25.0b

48.5a

40.5a

41.5a

Without growth regulators; values marked in rows with different letters differ at P < 0.05, Tukey’s test

Table 6

Influence of growth regulators on mass increment of plantlets (g plant−1) (n = 25, three replications)

Days from embryos isolation

Concentration of growth regulators in solid medium

WR

0.01 mg L−1 BA 0.001 mg L−1 NAA

0.1 mg L−1 BA 0.001 mg L−1 NAA

0.001 mg L−1 NAA

0.01 mg L−1 BA

0.1 mg L−1 BA

30

0.010b

0.011b

0.010b

0.070a

0.064a

0.014b

90

0.055b

0.069ab

0.112a

0.118a

0.073ab

0.105a

150

0.190ab

0.238a

0.175ab

0.171ab

0.120b

0.189ab

Without growth regulators; values marked in rows with different letters differ at P < 0.05, Tukey’s test

The isolation of zygotic embryos created the possibility to obtain plantlets (Fig. 12) without long-term seeds stratification and allowed the selection of genetic plant materials, which was also an effective method to obtain explants for further studies on micropropagation. However, a disadvantage of this method was the necessity of using laboratory equipment and incurring related costs.

2.2.2 Somatic Embryos

Somatic embryogenesis is a phenomenon that can be used for both the basic studies of plant morphogenesis and clonal reproduction of plants. Somatic embryos may develop by direct embryogenesis from primary explant cells having specific competence or through indirect embryogenesis from explant cells, which must acquire the competence for embryogenesis (most often by the dedifferentiation of tissues), followed by callus induction [31, 32, 33]. In turn, callus can be used to obtain suspension cultures grown in bioreactors for producing somatic embryos. This phenomenon allows multiplying particularly valuable plants in a short time, because the induction of root formation is not necessary. Moreover, somatic embryos may be a material for the production of artificial seeds [34, 35]. To date, reproduction protocols using indirect somatic embryogenesis have been developed for many species that are difficult to propagate both via seeds and vegetatively [36, 37, 38]. In turn, direct somatic embryogenesis is not so frequent. In this process, the age of the explant tissues used to induce the culture is crucial. In the case of Dalbergia latifolia Roxb. (rosewood), somatic embryos directly formed on the cotyledons of plantlets obtained from the immature zygotic embryos [39]. Both the number of somatic embryos obtained and the time required for their induction depended on the maturity of zygotic embryos. A similar tendency was observed for E. senticosus, which was previously described by Choi et al. [25]. The efficiency of somatic embryo formation depends on the age of the plantlets and the organ from which the explants originated. According to our results, the most effective eleuthero explants to induce somatic embryogenesis are hypocotyl fragments and apical buds (cultured on MS solid medium of 1 mg L−1 2,4D), obtained from plantlets originating from extremely immature zygotic embryos. In such cases, somatic embryos directly form on the explant tissues, without the callus phase. However, these results were obtained when the plantlets, being a source of explants, were cultured on a solid medium without growth regulators. The explants originated from plantlets grown on a solid medium with BA and NAA developed callus more frequently (Table 7).
Table 7

Influence of the type of explants on the formation of somatic embryos (n = 30; three replications)

Type of explant

% of explants with regenerated somatic embryos

% of explants with callus

% of necrotic explants

Number of regenerated somatic embryos per explant

Explants obtained from plantlets grown on a solid mediuma without growth regulators

Apical buds

35

22

45

38.1

Cotyledon fragments

0

0

100

0

Hypocotyl fragments

69

20

11

63.9

Root fragments

9

19

72

21.3

Explants obtained from plantlets grown on a solid mediuma with growth regulators (0.01 mg mL−1 BA and 0.001 mg L−1 NAA)

Apical buds

0

59

41

0

Cotyledon fragments

0

0

100

0

Hypocotyl fragments

19

53

72

38.5

Root fragments

0

40

60

0

aSolid medium, prepared according to Murashige and Skoog [29] and according to Gamborg [30] with agar (6.5 g L−1) and sucrose (30 g L−1)

Plant tissue cultures are also used as a source of biologically active compounds. The biotechnological studies on the isolation of eleutherosides B and E from such cultures, including somatic embryos, of some Eleutherococcus species has been undertaken for many years [40]. However, Shohael et al. [41] reported that the content of eleutherosides is significantly higher in field-grown plants compared to different stages of somatic embryos. According to our results, eleutherosides B, E, and E1 were not present in the callus, somatic embryos, or in young (6-week-old) plantlets obtained from these embryos (Figs. 13, 14, 15, and 16). However, in all the abovementioned eleuthero tissue cultures, phenolic acids were detected, namely, chlorogenic, rosmarinic, caffeic, and ferulic acids. Their content was the highest in the plantlets and the lowest in the callus maintained in the dark (Table 8). These compounds were also present in the field-grown plants, both in the aboveground and underground organs, but in distinctly higher amounts (see Sect. 3.1). In turn, according to Park et al. [42], the content of eleutherosides B and E in E. koreanum plantlets obtained in bioreactors is considerably high; moreover the plantlets may be an efficient source of extracts. It is worth noting that the accumulation of biomass, as well as content and composition of biologically active compounds in eleuthero suspension cultures, depends on multiple factors, including bioreactor type, medium components, aeration volume, temperature, light quality, or gaseous factors present in the bioreactor (oxygen, carbon dioxide, and ethylene) [43].
Fig. 13

Callus maintained in the dark

Fig. 14

Callus maintained in the light

Fig. 15

Somatic embryos

Fig. 16

Plantlets obtained from somatic embryos

Table 8

Phenolic acids in plant tissue cultures (mg 100 g−1 DW)

Phenolic acids

Callus maintained in the dark

Callus maintained in the light

Somatic embryos

Plantlets obtained from somatic embryos

Chlorogenic

5.24c

58.98b

157.33a

165.56a

Rosmarinic

1.44c

11.11b

8.83b

33.11a

Caffeic

0.25b

2.41a

0.92b

3.03a

Ferulic

0.47c

7.54b

11.37b

20.46a

Analysis performed using HPLC, method described in Bączek et al. [50]; values marked in rows with different letters differ at P < 0.05, Tukey’s test

According to previously reported studies, Eleutherococcus species are strongly endangered. Thus, tissue culture techniques were used for producing artificial seeds both for long-term conservation and cultivation. The first study on eleuthero encapsulated somatic embryos was published in 2002 by Choi and Jeong [40]. They described the process of artificial seeds production and the development of the seeds into well-grown plants. Additional studies on artificial seeds were related to their long-term conservation at low temperatures [44], as well as application of carbon and starch in the encapsulating matrix for the enhancement of post-germinative growth of embryos [45]. Note that eleuthero was used in Agrobacterium-mediated transformations, e.g., human lactoferrin (hLf) gene was transformed into eleuthero cells, which were able to produce this important glycoprotein [46, 47], while Seo et al. [48] used squalene synthase-encoding gene (PgSS1) from P. ginseng to obtain transgenic eleuthero plants producing distinctly higher amount of phytosterols and triterpenes than non-transformed ones. Furthermore, eleuthero transgenic somatic embryos were used for producing Escherichia coli heat-labile toxin (LTB) subunit B, which was applied for developing edible vaccines [49].

2.2.3 Adaptation of Plantlets to Ex Vitro Conditions

As mentioned above, in vitro techniques allow rapid multiplication of individual plants of a desired chemical profile, which is particularly interesting for the phytopharmaceutical industry. According to our results, eleuthero somatic embryos may be a useful material for obtaining homogeneous propagation material for field cultivation. We were able to obtain 3.4–6.5 of well-formed plantlets, i.e., with roots, stems, and leaves, from 1 g of embryos produced on a solid medium (Table 9, Figs. 1517). Gui et al. [51] reported that 75% of eleuthero somatic embryos developed into normal plantlets. However, one of the most difficult stages in the production of material for establishing plantations is the adaptation of plants to ex vitro conditions. It depends on multiple factors, such as the type of soil, its pH, and photoperiod used. According to our observations, the highest number of well-developed eleuthero plants was obtained on a substrate with a pH of 5.5 (80.2%). Plantlets planted on a substrate with higher pH, i.e., 6.0 and 6.5, did not adapt so well. Moreover, the adaptation of plants to ex vitro conditions was also influenced by the length of the day. The highest number of well-formed plants, ready for field cultivation, was obtained using a 16/8 photoperiod (Table 10, Fig. 18).
Table 9

Characteristics of 2-month-old plantlets obtained from somatic embryos (n = 50; three replications)

Investigated traits

Concentration of growth regulators in solid medium

WR

0.01 mg L−1 BA 0.001 mg L−1 NAA

0.01 mg L−1 BA

Number of well-developed plantlets obtained from 1 g of somatic embryos

5.2ab

3.4b

6.5a

Fresh mass of one plantlet (g)

0.39

0.34

0.58

Without growth regulators; values marked in rows with different letters differ at P < 0.05, Tukey’s test

Table 10

Plantlets adaptation to ex vitro depending on photoperiod and substrate pH (%) (n = 30; three replications)

Photoperiod

Substrate pH

Mean

6.5

6.0

5.5

16/8

75.0b

70.5b

90.0a

78.5

14/10

55.5ab

45.0b

75.5a

58.7

12/12

65.0ab

55.0b

75.0a

65.0

Mean

65.2

56.8

80.2

 

Values marked in rows with different letters differ at P < 0.05, Tukey’s test

Fig. 17

Plantlets obtained from somatic embryos (before ex vitro adaptation)

Fig. 18

Plants adopted to ex vitro, which were grown at different photoperiods

3 Eleuthero Raw Materials and Their Chemical Profile

The medicinal raw materials described in the European Pharmacopoeia [9] are rhizomes with roots, commonly named roots (Eleutherococci radix). Note that rhizomes are either gray-brown or dark brown and have irregular, strongly knotty shape with a diameter of 1.5–10 cm (Fig. 19). The bark, ~2–3 mm thick, strongly adheres to the pale yellowish wood. Regardless of the age of plants (in cultivation), rhizomes constitute ~60% of the total mass of underground organs. The roots are also twisted and cylindrical, with a diameter of 0.3–2 cm (Fig. 20). The roots bark is ~0.5–1 mm thick. Underground organs are usually collected in November, during the autumn season. The raw material is dried at 70–82 °C. Its odor is characteristic, slightly acrid, whereas its taste is bitter-sweet and astringent. Another raw material collected from eleuthero plants is stem bark [52]; however, probably because of difficulties associated with its harvesting (stems are covered with thorns) and low yield (~100 g DW per plant for 4-year-old plans) [53], it is used less frequently. Generally, stem bark is obtained from 2–3-year-old shoots. In addition to underground organs and shoots, the leaves are used, and the raw materials are obtained primarily in Russia and China [52, 54, 55].
Fig. 19

Rhizomes of 3-year-old plants

Fig. 20

Roots of 3-year-old plants

The underground organs are characterized by various secondary metabolites belonging to different chemical groups, such as phenylpropanoids, lignans, coumarins, phenolic acids, sterols, and a small amount of essential oils [2, 10, 21]. Among phenylpropanoids, the most important is eleutheroside B (syn. syringin; 4-O-β-d-glucoside of the sinapyl alcohol). Moreover, among lignans, the most valuable is eleutheroside E (syn. liriodendrin; (+)-syringaresinol-4,4-O-β-d-diglucoside) (Fig. 21a and b). According to European Pharmacopoeia [9], the sum of both compounds in dry underground organs should not be <0.08%, determined by HPLC (high-performance liquid chromatography). Other constituents among monomeric phenylpropanoids identified in this raw material are coniferyl alcohol-4-O-β-d-glucoside, coniferyl aldehyde, sinapyl alcohol, as well as sinapyl aldehyde and their glucosides [2, 21]. In plant tissues, coniferyl alcohol is present only in a bound form as a glycoside, whereas sinapyl alcohol is an aglicon of eleutheroside B [56]. Because of their biogenetic origin, certain phenolic acids present in the raw materials of eleuthero are classified as phenylpropanoids derivatives, as well. The most common ones are chlorogenic, rosmarinic, and caffeic acids (Fig. 21d, f, g). Furthermore, ferulic (Fig. 21h), protocatechuic (Fig. 21e), p-hydroxybenzoic, syringic, and p-coumaric acids have been identified in the underground organs in smaller amounts [21, 57, 58]. According to Anetai et al. [59], the dominant one is chlorogenic acid. Moreover, the synthesis of coumarin compounds is related to the biogenetic pathway of phenylpropane derivatives [56]. To date, the following coumarins have been isolated from eleuthero’s underground organs: isofraxidin, its 7-O-glucoside (syn. eleutheroside B1), and 4-O-ethylumbelliferone [2].
Fig. 21

Chemical structures of biologically active compounds identified in eleuthero: (a) eleutheroside B, (b) eleutheroside E, (c) eleutheroside E1, (d) chlorogenic acid, (e) protocatechuic acid, (f) rosmarinic acid, (g) caffeic acid, (h) ferulic acid

In addition to phenylpropanoids, another important group of compounds in the underground organs of eleuthero are lignans, which, biogenetically, are linked to the biosynthesis of lignin. In Eleuthero, they occur in both underground and aboveground organs. As mentioned above, the most valuable lignan compound is eleutheroside E. Its derivatives, i.e., syringaresinol-4,4-O-β-d-monoglucoside (syn. eleutheroside E1) and syringaresinol-4,4′-O-diglucoside (syn. eleutheroside D), are present in the raw material in significantly lower amounts. Another important compound belonging to this group is (+)-sesamin (syn. eleutheroside B4) [2, 21, 60].

In the underground organs of eleuthero, certain sterol compounds have been identified, namely, β-sitosterol and eleutheroside A (syn. daucosterol; sitosterol-3-O-β-d-glucoside) [21]. The raw material contains up to 0.8% of essential oil, as well [4]. The oil comprises monoterpenes (56%–16%) and sesquiterpenes (36%–25%), with the dominant compounds as anethole (1.0%–27.9%), limonene (0.9%–9.8%), α-longipinene (0.8%–8.9%), spathulenol (5.4%–7.2%), and linalool (0.3%–4.0%) [61]. Furthermore, certain active saccharides are present in the underground organs, too. In fact, the raw material contains monosaccharides such as α- and β-glucose, galactose, methyl-α-d-galactoside (syn. eleutheroside C); disaccharides such as α- and β-maltose; and polysaccharides [62, 63].

3.1 Chemical Diversity of Raw Materials

The accumulation of biologically active compound in eleuthero is clearly associated with the age of plants and depends on both the development phase of the plant and the plant organ. Generally, the biologically active compounds occur in the whole plant; however, some group of substances are limited only to certain organs. For example, the presence of eleutherosides B and E, i.e., the most important compounds when considering the quality of raw materials, is observed in underground organs, i.e., in rhizomes and roots, as well as in shoots or fruits; however they are not detected in the leaves. In turn, the leaves act as a rich source of rutoside and hyperoside [50, 53], which are characteristics for the aboveground organs of a number of different plant species [56].

Eleutheroside B clearly dominates in the external tissues of rhizomes, roots, and shoots; however, shoots bark is evidently the richest source of this compound (Table 11, Fig. 21a–f). When considering seasonal changes, this compound, both in underground organs and shoots bark, accumulates in the highest amounts at the beginning of winter dormancy (November) (83.73 and 380.00 mg 100 g−1 DW, respectively) compared to the full flowering period (41.91 and 131.50 mg 100 g−1 DW, respectively) [50]. Its content is similar in 2- and 3-year-old plants and the highest during the 4th year of vegetation (Table 11).
Table 11

Accumulation of eleutherosides B and E in different plant organs collected during winter dormancy (mg 100g−1 DW)1

 

Age of plants (years)

 

Raw materials

1st

2nd

3rd

4th

Mean

Eleutheroside B

Rhizomes

16.47c

56.27b

52.82b

89.16a

53.68

Rhizomes bark

201.47b

231.84b

371.98a

268.43

Roots

29.58c

50.25b

47.76b

93.34a

63.78

Roots bark

209.14

191.29

228.67

209.70

Shoots without bark

54.23b

49.22b

82.13a

61.86

Shoots bark

319.09b

323.36b

360.20a

334.22

Eleutheroside E

Rhizomes

54.55b

65.02ab

63.64ab

88.35a

67.89

Rhizomes bark

66.38b

65.62b

107.88a

79.96

Roots

37.50b

44.85ab

45.67ab

58.73a

46.69

Roots bark

68.12b

77.99b

93.56a

79.89

Shoots without bark

64.19b

71.14b

124.14a

86.49

Shoots bark

29.09

21.24

20.73

23.69

–In the 1st year of vegetation, the mass of these organs was too low to collect raw materials

Values marked in rows with different letters differ at P < 0.05, P < 0.05 (in column), Tukey’s test

1Analysis performed using HPLC, method described in Bączek et al. [50]

Compared to eleutheroside B, the content of eleutheroside E in the bark of rhizomes and roots (79.96 and 79.98 mg 100 g−1 DW, respectively) is only slightly higher than that in all these organs (67.89 and 46.69 mg 100 g−1 DW, respectively). In turn, in the shoots bark, its amount is four times lower than in the shoots without bark (Table 10). As in the case of eleutheroside B, its content increases with the age of plants and is significantly higher in plants during winter dormancy than during the vegetation period [50].

For both groups of raw materials, i.e., in the aboveground and underground organs, chlorogenic, rosmarinic, caffeic, ferulic, and protocatechuic acids are present, although chlorogenic acid has the higher concentration. The highest content of chlorogenic, ferulic, and protocatechuic acids is observed in the roots compared to rhizomes and shoots. Moreover, their amount is higher in roots bark than in the whole roots (Table 12, Fig. 22a–f). When considering seasonal differences, higher content of these compounds is observed in 2-year-old compared to 4-year-old plants [50]. The opposite trend is observed in the case of rosmarinic acid, whose content was higher in the whole roots and rhizomes than in their bark, and it was increasing from the second to the fourth year of plant vegetation (Table 12). All of the abovementioned phenolic acids were present in distinctly higher quantities during the winter dormancy compared to the full vegetation stage [50].
Table 12

Accumulation of phenolic acids in different plant organs collected during winter dormancy (mg 100g−1 DW)1

Raw materials

Age of plants (years)

Mean

1st

2nd

3rd

4th

Chlorogenic acid

Rhizomes

81.49

418.64b

576.14a

592.33a

417.15

Rhizomes bark

752.13b

761.10b

882.49a

798.57

Roots

40.30

611.54b

741.77a

698.32a

522.98

Roots bark

1059.19b

1077.07b

1244.73a

1127.00

Shoots without bark

194.62

226.65

233.34

218.20

Shoots bark

733.99b

859.36a

821.54a

804.96

Rosmarinic acid

Rhizomes

tr.

65.12

64.00

88.35

72.48

Rhizomes bark

44.02

38.93

50.43

44.46

Roots

tr.

145.67a

177.14a

241.48a

188.10

Roots bark

111.13

110.33

137.71

119.72

Shoots without bark

5.29

11.23

12.09

9.54

Shoots bark

35.47

30.28

50.14

38.63

Caffeic acid

Rhizomes

tr.

3.83

6.45

8.07

7.63

Rhizomes bark

13.45

11.20

19.35

14.67

Roots

tr.

12.76

10.73

11.69

11.73

Roots bark

7.25b

9.68b

16.98a

11.30

Shoots without bark

4.11b

11.23a

12.09a

9.14

Shoots bark

33.67b

45.66ab

53.27a

44.20

Ferulic acid

Rhizomes

tr.

0.48

3.92

2.30

2.23

Rhizomes bark

21.03b

25.66b

58.07a

34.92

Roots

tr.

3.84

1.89

2.01

2.58

Roots bark

53.15b

58.90b

90.21a

67.42

Shoots without bark

0.92

3.27

3.97

2.72

Shoots bark

2.54

2.89

3.05

2.83

Protocatechuic acid

Rhizomes

tr.

19.18b

25.38ab

31.16a

25.24

Rhizomes bark

52.07b

75.99a

69.80a

65.95

Roots

tr.

23.51b

29.46b

49.38a

34.12

Roots bark

75.83

64.63

78.41

72.96

Shoots without bark

5.20

7.72

7.21

6.71

Shoots bark

38.37b

40.22b

55.46a

44.68

–In the 1st year of vegetation, the mass of these organs was too low to collect raw materials

Values marked in rows with different letters differ at P < 0.05, P < 0.05 (in column), Tukey’s test

1Analysis performed using HPLC, method described in Bączek et al. [50]; tr. trace amount

Fig. 22

Chromatograms of eleuthero rhizomes extracts (a), rhizomes bark extract (b), roots extract (c), roots bark extract (d), shoots without bark extract (e), shoots bark extract (f)

3.2 Secretory Structures of Eleuthero Organs

It can be assumed that the majority of biologically active compounds identified by HPLC (see Sect. 3.1) in eleuthero organs are accumulated in the schizogenic secretory reservoirs. Their presence was found in all organs, i.e., rhizomes, roots, shoots, and leaves of 3-year-old eleuthero plants, as well as in the primary bark and the secondary phloem of hypocotyl of the 6-week-old seedlings (Figs. 23, 24, 25, 26, 27, and 28). These reservoirs differ in the size and number of epithelial cells surrounding them (from a few to even above 20). However, in all the observed reservoirs, there was a secretion characterized by high heterogeneity (Figs. 25 and 28). This secretion had a different staining ability (from light blue to light gray, olive, and dark blue), different ultrastructure (fibrous, moderately electronically dense and electronically dense), and stained black under the influence of osmium tetroxide, which would indicate the phenolic nature of compounds present in it [64]. Most likely, these are phenolics such as eleutherosides B and E and phenolic acids. The phenolic nature of compounds found in the secretion is indirectly confirmed by chemical analyzes using HPLC (see Sect. 3.1). In external tissues (phloem and periderm), in which a much larger number of secretory reservoirs were found (often with significantly higher diameter), eleutherosides and phenolic acids were found in higher amounts compared to the whole organs. However, the occurrence of biologically active compounds of a phenolic nature in the vacuoles of secretory cells and parenchyma of the secondary phloem cannot be excluded, because all these cells contain an electronically thick material. Phenolic compounds are characterized by a strong blue autofluorescence, which was observed in the reservoirs; however, if the secretion was present in significant amounts, the autofluorescence light was almost white (Fig. 29). Nevertheless, neither autofluorescence nor the dyes used allowed the exact determination of the composition of the secretion because of its heterogeneous character. Therefore, histochemical studies of the secretion are necessary.
Fig. 23

Cross-section of the rhizomes obtained from 3-year-old plants. Arrows – secretory reservoirs. C cambium, SX secondary xylem, SPh secondary phloem, PF phloem fibers, R phloem/xylem radius

Fig. 24

Secretory reservoirs in the rhizomes. Arrows – secretory reservoirs. Black rosette – clinched phloem elements

Fig. 25

Secretory reservoir filled with heterogeneous secretion (rhizomes). Double arrowheads – lipid bodies. Black rosette – heterogeneous secretion within the schizogenic reservoir. V vacuole, N nucleus

Fig. 26

Cross-section of 1-year-old shoots. White arrow – phellogen. Black arrows – secretory reservoirs. Black star – undegraded parenchyma cells of primary cortex. PF phloem fibers, Co collenchyma, Pe pericycle, SPh secondary phloem, PF phloem fibers, R phloem/xylem radius, SX secondary xylem

Fig. 27

Schizogenic reservoir in the shoots. Black arrow – secretory reservoir. Black star – undegraded parenchyma cells of primary cortex. Black rosette – heterogeneous secretion within the schizogenic reservoir

Fig. 28

Epithelial cells surrounding schizogenic reservoir in the shoots. Black rosette – heterogeneous secretion within the schizogenic reservoir. Black stars and arrows – exocytotic activity of anticlinal walls of epithelial cells. LB lipid bodies, CW cell wall, V vacuole

Fig. 29

Autofluorescence of the secretory canal. Thin arrows – secretion drops on the section surface. Wide arrows – secretion within the reservoir. Rosette – phellem autofluorescence

4 Conclusions

Eleuthero is a very important source of raw materials with a distinct, documented adaptogenic activity. Strong chemical diversity of wild-growing plants from which raw materials have been obtained and the increasing difficulties associated with their collection are the main reasons for introducing eleuthero into cultivation. However, the primary problem in the cultivation seems to be the reproductive material needed to establish its plantations. According to our results, vegetative propagation using modern in vitro techniques, with application of somatic embryos, seems to be the most efficient method of eleuthero propagation. It makes possible to produce uniform, both in terms of morphological traits and chemical profile, plants useful for the phytopharmaceutical industry. In addition to the standard raw material, included in pharmacopoeial monographs, i.e., Eleuthero radix, the valuable source of biologically active compounds, particularly eleutheroside B, is the shoots bark. The anatomical studies on eleuthero organs indicate that eleutherosides B and E, as well as phenolic acids, are accumulated primarily in schizogenic secretory reservoirs in external tissues (phloem and periderm) of both underground organs and shoots.

Notes

Acknowledgments

The study was supported by Polish Ministry of Agriculture and Rural Development, grant no. N N310 312834

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Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Katarzyna Bączek
    • 1
  • Anna Pawełczak
    • 1
  • Jarosław L. Przybył
    • 1
  • Olga Kosakowska
    • 1
  • Zenon Węglarz
    • 1
    Email author
  1. 1.Laboratory of New Herbal Products, Department of Vegetable and Medicinal PlantsWarsaw University of Life Sciences – SGGWWarsawPoland

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