Multiple reaction monitoring chromatograms of the objective compounds. (1) Eleutheroside B, (2) eleutheroside E, (3) isofraxidin, (4) hyperoside, (5) rutin, and (6) kaempferol.
Secondary mass spectrum of six active pharmaceutical ingredients. (1) Eleutheroside B, (2) eleutheroside E, (3) isofraxidin, (4) hyperoside, (5) rutin, and (6) kaempferol.
Effect of water stress treatments on stem length, leaf number, and leaf area of Eleuthero. The soil moisture of the control (GK), moderate drought stress (W1), and severe drought stress (W2) groups was maintained at 0.8 to 0.9 g/g, 0.5 ± 0.05 g/g, and 0.3 ± 0.05 g/g, respectively. n = 6. **Significant difference between the treatment group and the control group (P < 0.01); *significant difference between the treatment group and the control group (P < 0.05).
Effect of water stress treatments on net photosynthetic rate, transpiration rate, stomatal conductance, and intercellular CO2 concentration of Eleuthero. The soil moisture of control (GK), moderate drought stress (W1), and severe drought stress (W2) groups was maintained at 0.8 to 0.9 g/g, 0.5 ± 0.05 g/g, and 0.3 ± 0.05 g/g respectively. n = 6. **Significant difference between the treatment group and the control group (P < 0.01); *significant difference between the treatment group and the control group (P < 0.05).
Global effects of different water stress conditions on medicinal compounds accumulation in Eleuthero. The soil moisture of control (GK), moderate drought stress (W1), and severe drought stress (W2) groups was maintained at 0.8 to 0.9 g/g, 0.5 ± 0.05 g/g, and 0.3 ± 0.05 g/g respectively. n = 6.
Effects of different water stress on the accumulation of six medicinal compounds in Eleuthero roots. The soil moisture of control (GK), moderate drought stress (W1), and severe drought stress (W2) groups was maintained at 0.8 to 0.9 g/g, 0.5 ± 0.05 g/g, and 0.3 ± 0.05 g/g respectively. Bars indicate standard deviation (n = 6). **Significant difference between the treatment group and the control group (P < 0.01); *significant difference between the treatment group and the control group (P < 0.05).
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In this investigation, changes in growth and photosynthetic parameters were used to explain the effects of drought stress on morphology and photosynthesis of Eleutherococcus senticosus. Liquid chromatography (LC)-mass spectroscopy (MS) was used to determine the content of eleutheroside B, eleutheroside E, isofraxidin, hyperoside, rutin, and kaempferol under different drought stress conditions to explain the effects of drought stress on secondary metabolism of Eleuthero. Growth and photosynthetic physiological parameters showed that drought stress could inhibit the growth and photosynthesis of Eleuthero. The compounds studied showed the same cumulative trend in various organs of Eleuthero under different drought stress conditions, with the highest content in the moderate drought stress group and the lowest in the severe drought stress group. Among them, the content of eleutheroside B was found to be higher in the 5-year-old stem. The content of eleutheroside E was higher in the 3-year root. The content of isofraxidin was highest in the 5-year-old root. The content of hyperoside, rutin, and kaempferol were higher in the 3-year-old leaves. The results show that a wet soil environment was beneficial to growth and photosynthesis of Eleutherococcus senticosus, and moderate drought stress is conducive to the accumulation of its active ingredients.
Eleutherococcus senticosus (Ruper. et Maxim.) Harms is a species of a small woody shrub that belongs to the family Araliaceae (Huang et al., 2011). It is commonly used in China (called ciwujia), Korea, Japanese, and Russia (called Siberian ginseng). The roots and stems of the plant are recognized as a tonic herb that has a ginseng-like effect (Bucci, 2000). Eleuthero root is considered a pharmacopeia raw material in many countries (Europe, United States, Japan). Eleuthero invigorates qi, strengthens the spleen, and nourishes the kidney (Han et al., 2014). Thus, Eleuthero may be used for yang deficiency of the spleen and kidney, body weakness and hyperdynamics, poor appetite, aches of the waist and knee, insomnia (Han et al., 2017). Eleuthero has anti-inflammatory (Fei et al., 2014; Jiang and Wang, 2015), antioxidant (Kim et al., 2015), antifatigue (Huang et al., 2011; Jiang and Wang, 2015), and anticancer properties (Cichello et al., 2015) and encourages immunomodulation; thus, it has been widely used to treat chronic bronchitis, neurasthenia, hypertension, and ischemic heart disease (Sun et al., 2016). The components of phenolic, triterpenoid saponins, lignan, coumarins, flavones, polysaccharides, and volatility have been detected in the Eleuthero (Huang et al., 2011; Jiang et al., 2006; Li et al., 2016).
The chemical compounds of Eleuthero raw materials used in this study are phenolic compounds. Phenols are the most widely distributed metabolites involved in interactions between biology and the environment (Garcia-Calderon et al., 2015). The accumulation of phenols may also affect other secondary metabolite pathways, including alkaloid pathways, because plant defense is a complicated system (Ferreres et al., 2008; Mustafa and Verpoorte, 2007). Phenolic compounds and related pathways can be influenced by exposure of plants to abiotic stresses, such as adverse environmental conditions (Dixon and Paiva 1995; Harb et al., 2010). However, plants have evolved to survive harsh conditions, for example, by using their metabolic capacity to produce a variety of secondary metabolites. Eleutheroside B, eleutheroside E, isofraxidin, hyperoside, rutin, and kaempferol are the main phenols in Eleuthero (Bączek et al., 2017; Yang et al., 2012, 2013). Eleutheroside B and E are lignans (Lee et al., 2004), isofraxidin is a coumarin compound (Yamazaki and Tokiwa, 2010), and hyperoside, rutin, and kaempferol belong to the flavonoid group (Bączek et al., 2017). These compounds are derived from shikimic acid (Gamir et al., 2014; Schafellner et al., 1999), the contents of which also seem to increase from water deficit (Becerra-Moreno et al., 2015).
To the best of our knowledge, there is no comprehensive study on the effects of drought stress on Eleuthero. The present study focused on photosynthetic physiological parameters and targeted analysis of metabolite features of roots, stems, and leaves under different water treatments. Collectively, these data will enable assessment of the difference in growth, photosynthetic physiological responses, and secondary metabolism responses to water restriction throughout the plant.
Eleutheroside B, eleutheroside E, isofraxidin, hyperoside, rutin, and kaempferol were purchased from the Chinese National Institute of Control of Pharmaceutical and Biological Products (Beijing, China). Water used for ultra performance LC-tandem MS (UPLC-MS) analysis was prepared with a Milli-Q water purification system bought from Millipore (Milford, MA). Acetonitrile (J & K Scientific Ltd., Beijing, China) was high-performance LC grade. All other chemicals used in the method were of analytical grade.
Seedlings of 3-year-old Eleuthero were obtained from Qitaihe, Heilongjiang Province, China (geographic coordinates: lat. 45°95′N, long. 131°05′E) and planted in the Heilongjiang University of Chinese Medicine Botanical Garden, Harbin, Heilongjiang Province, China (geographic coordinates: lat. 45°72′N, long. 126°64′E) in February. A month later (March), seedlings were transplanted into 30-cm diameter pots containing a mixture of garden soil and vermicomposting (3:1, w/w); soil pH was 6.50. The experiment was conducted in a glasshouse with temperature ranging from 20 to 30 °C under natural light conditions (day length: 13 h). The climate of Harbin is temperate continental monsoon type. The whole plant was dissected to obtain roots, stems, and leaves for metabolic analysis. The raw materials were dried at 60 °C for 48 h. The experiment was performed in three replications.
One month after transplantation (April), the control (GK), moderate (W1), and severe (W2) drought stress treatment was started. The soil moisture of GK, W1, and W2 was maintained at 0.8 to 0.9 g/g, 0.5 ± 0.05 g/g, and 0.3 g ± 0.05 g/g, respectively. The moisture in pot soil was evaluated regularly by measuring the soil water content percentage: 1 g soil from pots of each treatment, as well as the control, was taken, oven-dried, and weight was taken again. Soil water content percentage was calculated by using the formula:
This measurement was repeated regularly at 1-d intervals, and water (100–400 mL) was supplied to pots of each treatment during plant development. There were three replicates in each experimental group, and six seedlings in each replicate. The experiment was conducted throughout plant developmental stages (from April to June).
A sampling of plant material was done after 2 months of drought stress treatment. Six plants were randomly selected in each treatment group; measurements of total leaf area (TLA), plant height (PH), and leaf number (LN) were performed. TLA was measured with LI-3100 leaf area meter (LI-COR Biosciences, Lincoln, NE), and PH was measured using digital calipers and a ruler.
Photosynthetic parameters were measured using a portable photosynthesis system (Li-6400XT, LI-COR) in a glasshouse from 10:00 am to 1:00 pm on 30 June 2017. Weather was normal during the investigation. Six plants were randomly selected in each treatment group, and the net photosynthetic rate (Pn), stomatal conductance (gS), transpiration rate (E), and intercellular CO2 concentration (Ci) values were read directly using the photosynthesis system.
Dried leaf, stem, and root of Eleuthero were ground in a mill and passed through a 35-mesh sieve. Approximately 2 g of dry powdered plant material was extracted with 10 mL of methanol (80%) by reflux for 45 min. The extract was repeatedly filtered, and the filtrate was collected. The extract was subjected to centrifugation at 14,000 rpm at 4 °C for 10 min. The supernatant was removed, and the extract was concentrated by evaporation under a vacuum to dryness. Then the precipitate was dissolved with methanol to a volume of 1.0 mL. All samples were filtered through a 0.22-µm diameter micropore filter membrane, which could be directly injected for LC-MS analysis.
The analysis of compounds is described in our previous studies (Wu et al., 2018); roots and stems were pulverized by using a grinding instrument (MM 400, Retsch, Haan, Germany), and 50-mg tissue aliquots were extracted with 1.0 mL 70% aqueous methanol containing 0.1 mg/L lidocaine for water-soluble metabolites at 4 °C overnight and vortexed three times. The extracts were clarified by centrifugation, combined, evaporated, and then filtered through 0.45-µm nylon membranes (SCAA 104; ANPEL http://www.anpel.com.cn) before LC-MS analysis.
UPLC-MS analysis was performed with a Waters ACQUITY UPLC system (Waters Corporation, Tokyo, Japan) coupled to an LC-20AD pump, SIL-20A autosampler (Waters Corporation). The ACQUITY-UPLC BEH C18 column (1.7 μm, 2.1 mm × 50 mm) used for UPLC analysis was held at 25 °C; injection volume was 10.0 μL, and the flow rate was 0.5 mL/min. Mobile phase A comprised methanol, and mobile phase B comprised water. The column was eluted with a linear gradient of 25% A for 0 to 1.5 min, 25% to 50% A for 1.5 to 2.0 min, 50% A for 2.0 to 4.0 min, 50% to 90% A for 4.0 to 4.5 min, 90% A for 4.0 to 4.5 min, 90% to 25% A for 5.5 to 6.0 min, and 25% A for 6.0 to 7.0 min. The chromatograms of six medicinal compounds under a multireaction monitoring mode are shown in Fig. 1.
Citation: HortScience horts 54, 12; 10.21273/HORTSCI14366-19
MS detection was performed using a QTRAP 5500 (AB SCIEX, Boston, MA) equipped with an Electrospray Ionization source with the following operating parameters: cone voltage of 3 kV and ion source atomization temperature of 500 °C; 25 psi atomizing gas and 20 psi air curtain gas. The ion pair, cluster voltage, collision voltage, and collision chamber injection voltage of six active metabolites are shown in Table 1. Both MS and MS/MS data were determined in the positive mode, and data were used for multiple reaction monitoring. Secondary MS of six active pharmaceutical ingredients is shown in Fig. 2.
Citation: HortScience horts 54, 12; 10.21273/HORTSCI14366-19
It subjected all results to the analysis of variance (ANOVA) to determine the significant differences between various levels of salt treatment times. If ANOVA was performed, Duncan’s honestly significant difference (HSD) post hoc tests were conducted to determine the differences between individual treatments (SPSS 22.0; SPSS Inc., Chicago, IL).
Changes in plant growth parameters are planted responses to drought stress on external morphology (Pellegrino et al., 2010). Changes in PH showed in Fig. 3A. The moderate stress group (W1) and the severe stress group (W2) showed apparent differences from the control group (GK). The plant height in W1 was 57% lower than GK, and in the W2 group it was 67% lower than GK. The results of LN count is shown in Fig. 3B. Higher LN was found in GK. LN in W1 was 43.84% lower than GK, and in W2 it was 32.61% lower than GK. The change of LA was opposite that of LN (Fig. 3C), it was the highest in W1 and the lowest in the GK.
Citation: HortScience horts 54, 12; 10.21273/HORTSCI14366-19
The results showed that drought stress could significantly inhibit PH and LN of Eleuthero and increase its leaf area. Soil water shortage also affects plant growth in many ways. On one hand, plant somatic cell division and differentiation require water participation. When water is absent, the speed of division and differentiation slows down or even stops (Van der Weele et al., 2000). On the other hand, it affects the transport of substances in plants, and many substances in plants need water as a carrier (Walton et al., 1976). Under drought stress, the transport rate of substances slowed, and the assimilation products could not be readily distributed (Chaves, 1991). Also, drought affects plants’ water absorption (Farooq et al., 2009). In this study, drought stress increased leaf area to a certain extent, which may be a response to adapt to drought conditions.
When Eleuthero was subjected to different drought stress treatments, significant differences in all photosynthetic parameters were observed. The Pn and gS decreased with the increase of drought stress (Fig. 4A and B). There was no significant difference between the GK and W1. Pn and gS in W2 were significantly lower than GK and W1. The transpiration rate (E) was lower in the two drought stress groups (Fig. 4C), and it was lowest in W2. The concentration of intercellular carbon dioxide (Ci) showed the opposite trend (Fig. 4D). With worsening of drought stress, the two treatments were significantly higher than the control group, and the highest was in W2. This result showed that photosynthetic capability was best in leaves of Eleuthero cultivated under GK.
Citation: HortScience horts 54, 12; 10.21273/HORTSCI14366-19
The factors leading to decline in photosynthetic rate include stomatal and nonstomatal limitations. The stomatal limiting factor was the decrease of Ci because of the decrease of the stomatal opening of leaves, whereas the nonstomatal factor was the decrease and accumulation of CO2 solubility, which resulted in the decrease of mesophyll photosynthetic capacity. Drought stress can inhibit photosynthesis in these two means (Berry and Downton, 1982). Previous studies (Colom and Vazzana, 2001; Rajendrudu et al., 2000) have shown that soil moisture is the main limiting factor for plant photosynthetic parameters. Moderate water deficit does not affect leaves’ stomata opening and therefore does not have a significant effect on Pn, gS, Ci, and E. In the present study, Pn and gS were less affected under W1, and there was no significant difference with the GK. Severe drought stress significantly inhibited the photosynthesis of Eleuthero by limiting CO2 and causing photodamage in medicinal plants (Cornic and Massacci, 1996).
In the present study, the UPLC-MS method was successfully applied for the quantitative analysis of six medicinal compounds in Eleuthero. The comprehensive score (Q value) of principal component analysis was used to illustrate the overall trend of the contents of the compounds studied in roots, stems, and leaves under different drought stress conditions (Fig. 5). The compounds studied showed the same accumulation trend in different organs. Higher content of the compounds studied was observed in W1. In the roots, the higher content of eleutheroside B, isofraxidin, kaempferol, and hyperoside accumulated in W1 (P < 0.05). Among them, the contents of eleutheroside B and isofraxidin in GK were higher than those in W2, and the contents of kaempferol and hyperoside in W2 were higher than those in GK. The contents of rutin and eleutheroside E were higher in GK and the lower in W2. In stems, the contents of eleutheroside B, isofraxidin, hyperoside, kaempferol, and rutin in W1 were significantly higher than those in other treatments (P < 0.05). Among them, the contents of eleutheroside B, isofraxidin, hyperoside, and kaempferol in GK were higher than those in W2, and the contents of rutin in W2 were higher than those in GK. The content of eleutheroside E in GK was higher than that in other treatment groups (P < 0.05). In the leaves, the contents of eleutheroside B, isofraxidin, and eleutheroside E in W1 were significantly higher than those in other treatments (P < 0.05), and the contents of eleutheroside B and eleutheroside E in GK were higher than those in W2. Isofraxidin was not detected in GK. The contents of rutin, kaempferol, and hyperoside were the highest in GK and the lowest in W2 (Fig. 6).
Citation: HortScience horts 54, 12; 10.21273/HORTSCI14366-19
Citation: HortScience horts 54, 12; 10.21273/HORTSCI14366-19
The results showed that the content of eleutheroside B, eleutheroside E, and isofraxidin was higher in roots and stems (Fig. 4A–C), similar to previous research (Kang et al., 2001; Lee et al., 2005), and further confirmed the correctness of the Chinese Pharmacopoeia's roots and stems as the legal medicinal organs of Eleuthero. Eleutheroside B and isofraxidin showed the same accumulation trend in different organs under different drought stress conditions, and their content was higher in the W1 group (Fig. 4A and C), which showed that W1 is suitable for the accumulation of two compounds. Drought stress inhibited the accumulation of eleutheroside E in roots and stems. The content of eleutheroside E was higher in GK, indicating that the moist soil environment is suitable for the accumulation of this component. It should be pointed out that the content of eleutheroside B is the highest among the six compounds, which verified the validity of eleutheroside B as the quality control index of Eleuthero in the Chinese Pharmacopoeia. It has been shown that eleutheroside E possesses an antistress and antifatigue effect (Kimura and Sumiyoshi, 2004; Weng et al., 2007). The results of the present study indicate that the accumulation of eleutheroside E in different organs of Eleuthero was also high, which is in agreement with results of previous studies (Lee et al., 2005; Zhang and Xue, 2008). Eleutheroside E thus has the potential to become a quality control indicator for Eleuthero.
The results of the present study show a significant effect of hyperoside, rutin, and kaempferol (all flavonoids) in different parts of Eleuthero. The content of the three flavonoids was higher in the leaves of Eleuthero, and the content decreased with the soil drought stress. The preceding results indicate that a wet soil environment is suitable for the accumulation of flavonoids in Eleuthero. This is inconsistent with previous research on St. John’s wort and Hypericum pruinatum (Caliskan et al., 2017; Gray et al., 2003). This could be explained by the fact that phenylpropanoids and shikimate pathway enzymes reduce their activity under drought stress, as has been demonstrated in other plants (Mewis et al., 2012; Sanchez-Rodriguez et al., 2011). Previous studies (He et al., 2019; Liu et al., 2019; Qian et al., 2019) have shown that these compounds have antiinfective, immunomodulatory, and other pharmacological effects. Our results show that Eleuthero leaves have good medicinal value. In this study, the content of hyperoside and kaempferol was low, and whether this relates to the growth period remains to be studied.
This investigation provided data on the impact of drought stress on the morphological and photosynthetic characteristics and the medicinal compounds of Eleuthero. The growth parameters results show that drought stress may inhibit plant growth, and moderate drought stress may significantly inhibit photosynthesis. The medicinal compounds were significantly affected in different parts of Eleuthero under various water treatments. Moderate drought stress is the most suitable condition for the accumulation of medicinal compounds in Eleuthero. This study may provide the experimental basis for further study on the metabolic regulation mechanisms of the medicinal components of Eleutherococcus senticosus under drought stress.
Multiple reaction monitoring chromatograms of the objective compounds. (1) Eleutheroside B, (2) eleutheroside E, (3) isofraxidin, (4) hyperoside, (5) rutin, and (6) kaempferol.
Secondary mass spectrum of six active pharmaceutical ingredients. (1) Eleutheroside B, (2) eleutheroside E, (3) isofraxidin, (4) hyperoside, (5) rutin, and (6) kaempferol.
Effect of water stress treatments on stem length, leaf number, and leaf area of Eleuthero. The soil moisture of the control (GK), moderate drought stress (W1), and severe drought stress (W2) groups was maintained at 0.8 to 0.9 g/g, 0.5 ± 0.05 g/g, and 0.3 ± 0.05 g/g, respectively. n = 6. **Significant difference between the treatment group and the control group (P < 0.01); *significant difference between the treatment group and the control group (P < 0.05).
Effect of water stress treatments on net photosynthetic rate, transpiration rate, stomatal conductance, and intercellular CO2 concentration of Eleuthero. The soil moisture of control (GK), moderate drought stress (W1), and severe drought stress (W2) groups was maintained at 0.8 to 0.9 g/g, 0.5 ± 0.05 g/g, and 0.3 ± 0.05 g/g respectively. n = 6. **Significant difference between the treatment group and the control group (P < 0.01); *significant difference between the treatment group and the control group (P < 0.05).
Global effects of different water stress conditions on medicinal compounds accumulation in Eleuthero. The soil moisture of control (GK), moderate drought stress (W1), and severe drought stress (W2) groups was maintained at 0.8 to 0.9 g/g, 0.5 ± 0.05 g/g, and 0.3 ± 0.05 g/g respectively. n = 6.
Effects of different water stress on the accumulation of six medicinal compounds in Eleuthero roots. The soil moisture of control (GK), moderate drought stress (W1), and severe drought stress (W2) groups was maintained at 0.8 to 0.9 g/g, 0.5 ± 0.05 g/g, and 0.3 ± 0.05 g/g respectively. Bars indicate standard deviation (n = 6). **Significant difference between the treatment group and the control group (P < 0.01); *significant difference between the treatment group and the control group (P < 0.05).
Contributor Notes
We are grateful for the financial support of the National Key Research and Development Program of China (No. 2016YFC0500303); Heilongjiang Province Foundation for the National Key Research and Development Program of China (No. GX17C006); the Post-Doctoral Foundation of Heilongjiang Province, China (LBH-Z17208); and the Key Project of Heilongjiang Provincial Administration of Traditional Chinese Medicine (No. 2018-009).
Z.-Y.W. is the corresponding author. E-mail: Wangzhen_yue@163.com.
Multiple reaction monitoring chromatograms of the objective compounds. (1) Eleutheroside B, (2) eleutheroside E, (3) isofraxidin, (4) hyperoside, (5) rutin, and (6) kaempferol.
Secondary mass spectrum of six active pharmaceutical ingredients. (1) Eleutheroside B, (2) eleutheroside E, (3) isofraxidin, (4) hyperoside, (5) rutin, and (6) kaempferol.
Effect of water stress treatments on stem length, leaf number, and leaf area of Eleuthero. The soil moisture of the control (GK), moderate drought stress (W1), and severe drought stress (W2) groups was maintained at 0.8 to 0.9 g/g, 0.5 ± 0.05 g/g, and 0.3 ± 0.05 g/g, respectively. n = 6. **Significant difference between the treatment group and the control group (P < 0.01); *significant difference between the treatment group and the control group (P < 0.05).
Effect of water stress treatments on net photosynthetic rate, transpiration rate, stomatal conductance, and intercellular CO2 concentration of Eleuthero. The soil moisture of control (GK), moderate drought stress (W1), and severe drought stress (W2) groups was maintained at 0.8 to 0.9 g/g, 0.5 ± 0.05 g/g, and 0.3 ± 0.05 g/g respectively. n = 6. **Significant difference between the treatment group and the control group (P < 0.01); *significant difference between the treatment group and the control group (P < 0.05).
Global effects of different water stress conditions on medicinal compounds accumulation in Eleuthero. The soil moisture of control (GK), moderate drought stress (W1), and severe drought stress (W2) groups was maintained at 0.8 to 0.9 g/g, 0.5 ± 0.05 g/g, and 0.3 ± 0.05 g/g respectively. n = 6.
Effects of different water stress on the accumulation of six medicinal compounds in Eleuthero roots. The soil moisture of control (GK), moderate drought stress (W1), and severe drought stress (W2) groups was maintained at 0.8 to 0.9 g/g, 0.5 ± 0.05 g/g, and 0.3 ± 0.05 g/g respectively. Bars indicate standard deviation (n = 6). **Significant difference between the treatment group and the control group (P < 0.01); *significant difference between the treatment group and the control group (P < 0.05).
