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ASHS 2024 Annual Conference

 

Effects of Rare Earth Elements on Growth and Determination of Secondary Metabolites under In Vitro Conditions in Salvia miltiorrhiza

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Zhibin Fan School of Life Sciences, Qufu Normal University, Qufu, Shandong 273165, China

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Kai Zhang Forest Farm of Mountain Culai, Taian, Shandong 271000, China; and College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia 010018, China

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Fengyun Wang School of Life Sciences, Qufu Normal University, Qufu, Shandong 273165, China

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Xiaodan Zhao School of Life Sciences, Qufu Normal University, Qufu, Shandong 273165, China

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Ruiqin Bai College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia 010018, China

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Boling Liu School of Life Sciences, Qufu Normal University, Qufu, Shandong 273165, China

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Abstract

Salvia miltiorrhiza, known as danshen, is one of most valued medicinal plants in China. Although it has been cultivated since ancient times, an optimal culture system needs to be standardized for this important species. Here, we explored the phytochemical properties of S. miltiorrhiza with the treatments of rare earth elements (REEs) to develop an optimal tissue culture system. Four-week-old in vitro-grown S. miltiorrhiza plantlets were used as explants. The experiment was conducted in a randomized block design on a Murashige and Skoog (MS) medium containing 0.2 mg·L−1 naphthaleneacetic acid (NAA) to induce rooting at four different concentrations (50, 100, 200, and 300 μM) of REEs such as cerium (Ce), lanthanum (La), or praseodymium (Pr), respectively. Compared with all REEs at different concentrations, 100 μM Pr induced greater root length than Ce or La at any concentrations. Concomitantly, 0.38 μg tanshinone IIA/mg dry weight (DW) was observed, which was 54.84% higher than in the control. Similarly, chlorophyll content, antioxidant enzyme activity, and secondary metabolite were enhanced in rooting medium supplemented with 100 μM Pr. Therefore, this study showed that 100 μM Pr is an adequate concentration in the optimal culture system for promoting plant growth as well as enhancing secondary metabolite content in S. miltiorrhiza.

Salvia miltiorrhiza, the Lamiaceae family, is a perennial herb that bears large zygomorphous flowers and is known as danshen in China. Medicinal components from danshen are widely used to treat cardiovascular diseases (CVD), particularly angina pectoris and myocardial infarction (Ryu et al., 1996). Similarly, it has been applied for liver dysfunction, renal deficiency, and diabetic vascular complication (Shan et al., 2007). A previous study found that methanolic extracts obtained from danshen had a significant cytotoxicity against cultured human tumor cell lines (Ryu et al., 1996). Dried roots of S. miltiorrhiza have been found to contain relatively large amounts of the important secondary metabolites (tanshinones and diterpenoid pigments, such as tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I). These compounds are relatively abundant in danshen, and they are largely responsible for its therapeutic effects (Wang and Wu, 2010).

In the Global Burden of Diseases (GBD) overview study, 17.8 million (233.1 per 100,000) people reportedly died of CVD in 2017 globally (Ram et al., 2019). Because of danshen’s high medicinal value for treating CVD and increased market demand, over-exploitation of danshen now threatens its wild resource; although the danshen cropping system has been optimized, repeated cropping tends to reduce the content of the medicinal components. Concomitantly, viral infections often reduce the yield under greenhouse cultivation. Therefore, it is highly desirable to develop a standard in vitro growth system to enhance tanshinone production of S. miltiorrhiza.

REEs are a set of 17 elements among which 15 have been included in the periodic table as “lanthanides,” plus scandium and yttrium (Aquino et al., 2009). In recent years, REEs have been widely used for medical, industrial, agricultural, and aquaculture applications because of their properties (Hu et al., 2004). Previous studies have reported that REEs accelerated cell growth, enhanced synthesis of secondary metabolites (Wu et al., 2001; Zhang et al., 2013), and provided tolerance against fungal diseases (Liu et al., 2008). Previous studies have also found that the addition of biotic elicitors and heavy metal salts can stimulate the accumulation of tanshinone and increase its yield (Zhao et al., 2010a, 2010b). However, to the best of our knowledge, the effect of REEs on the metabolism of danshen has not been studied. Although many physiological effects of REEs on plants remain to be studied and discovered, we are interested in the effects of REEs on the secondary metabolites of in vitro-cultured plants. Recent studies have proposed that REEs are good elicitors for secondary metabolites accumulation. Further, it has been shown that REEs have no severe deleterious effects on growth at optimal levels. Indeed, cerium (Ce), lanthanum (La), and praseodymium (Pr) reportedly showed positive effects on plant growth and stimulated secondary metabolites accumulations (Chen et al., 2003; Ting et al., 2016; Wu et al., 2001).

Secondary metabolites are generally believed to be a part of plant stress and defense responses (Bai et al., 1995; Peng et al., 2013). Thus, plant defense processes can be elicited by the enzymatic antioxidant system, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX) (Bowler et al., 1994). In general, secondary metabolites of most medicinal plants are flavonoids or phenols that can counter some diseases of the human body caused by oxidative stress, such as cardiovascular diseases, cancer, neurological diseases, etc. (Kinsella et al., 1993; Reddy et al., 2012). Superoxide anion (O2-), followed by hydrogen peroxide (H2O2) and hydroxy (·OH), are by-products of metabolic reaction, which can be scavenged by enzymatic reactions to maintain redox homeostasis (Kumaran and Karunakaran, 2007). Excess amounts of these oxidative free radicals could denature biological macromolecules such as proteins, DNA, and unsaturated fatty acids, and ultimately lead to plant cell death (Halliwell and Gutteridge, 2007). Enzymatic and nonenzymatic antioxidants can detoxify excess reactive oxygen species (ROS) in plant cells (Angkhana et al., 2019). Therefore, the antioxidant content in plants is also a standard strategy to estimate the value of medicinal plants.

Previously, we have studied the effect of plant growth regulators on seed-derived S. miltiorrhiza under in vitro conditions (Liu et al., 2018). However, to standardize the optimal in vitro system for S. miltiorrhiza culture, different plant sources (in vitro-grown adventitious shoots) were used here and treated with REEs (Ce, La, and Pr) in this study. We aimed to increase an understanding of the effects of REEs as efficient elicitors to improve growth and secondary metabolite biosynthesis in medicinal plants.

Materials and Methods

Plant materials and culture conditions.

S. miltiorrhiza were collected in the town of Shilai, Xintai county, Shandong Province, China. Surface sterilized plants were cultured in vitro on Murashige and Skoog (MS) basal medium with 3% (w/v) sucrose and 0.75% (w/v) agar containing 1.0 mg·L−1 6-benzylaminopurine (6-BA) and 0.1 mg·L−1 NAA. Cultures were maintained under 40 μmol·m−2·s−1 light irradiance and a 12-h photoperiod at 25 ± 1 °C. Four-week-old well-grown shoots were inoculated for rooting on an MS medium containing 0.2 mg·L−1 NAA with different concentrations (50, 100, 200, and 300 μM) of REEs, including Ce, Ce(NO3)3; La, La(NO3)3; or Pr, Pr(NO3)3. Growth and biochemical analysis were carried out after roots were well developed (four weeks).

Assessment of root length and chlorophyll content.

The roots’ lengths were measured with a Vernier caliper. Chlorophyll content was calculated with an ultraviolet-5500 spectrophotometer (Shanghai Metash Instruments, Shanghai, China) (Qiu et al., 2016). Briefly, in vitro leaves (1-mm segments, 50 mg) were soaked with 2 mL of dimethyl sulfoxide (DMSO) and boiled at 65 °C until the chlorophyll extraction was completed. After cooling, total volume was brought to 10 mL with 80% acetone. Optical density (OD) was recorded at 663.6 and 646.6 nm to determine total chlorophyll concentration by this formula: Cchl = 7.35A663.6 + 17.58A646.6.

Enzyme extraction and activity assessment.

To estimate antioxidant enzyme activity, 100 mg of leaves were homogenized in a 1 mL phosphate buffer (50 mm, pH 7.0). Supernatants were centrifuged at 10,000 rpm (D-37520; Heraeus Thermo, Osterode, Germany) for 20 min at 4 °C and used for determining antioxidant enzyme activity. Finally, the antioxidant enzymes activity was calculated based on total protein (by the Bradford method).

Superoxide dismutase (SOD) activity was measured by the change in absorbance at 560 nm during incubation of the extracts (100 μL) at 25 °C with 75 μM nitroblue tetrazolium (NBT), 10 μM ethylene diamine tetraacetic acid (EDTA), 2.0 μM riboflavin in 50 mm phosphate buffer (pH 7.8) in a total volume of 3 mL. SOD activity was determined by the formula (Ack − Ae)/(Ack × 50% × W), where Ack is the OD value of the control, Ae is the OD of extracts, and W is the sample protein content (Siddiqui et al., 2012). CAT activity was assessed by the change in absorbance at 240 nm during incubation of the extracts (100 μL) at 25 °C with 10 mm H2O2 in a 50 mm phosphate buffer (pH 7.0) in a total volume of 3 mL (Wang et al., 2004b). POD activity was detected at 460 nm with 18 mm guaiacol and 5 mm H2O2 in a 50 mm sodium acetate buffer (pH 5.4) in a total volume of 3 mL of enzyme extract (Manivannan et al., 2015a). APX activity was measured in a reaction mixture containing a 50 mm potassium phosphate buffer (pH 7.0), 5 mm ascorbic acid, 0.1 mm H2O2, and 100 μL enzyme extract. The reaction was induced by addition of H2O2, and the absorbance was recorded at 290 nm for 3 min at 30 s intervals. One enzyme unit was defined as a 0.1 change in absorbance in 30 s (Nakano and Asada, 1981).

Estimation of ROS scavenging activity.

In vitro roots samples (100 mg) were lyophilized and extracted overnight with 5 mL of 80% (v/v) methanol at 150 rpm in a rotating shaker. The resulting homogenates were centrifuged at 10,000 rpm for 10 min, and the supernatants were used for the in vitro assays (Manivannan et al., 2015b).

Superoxide (O2-) scavenging activity of the extract was determined by bleaching the superoxide radical produced by nitro-blue tetrazolium salt with riboflavin and light. Extraction (0.1 mL) was added to the 2 mL reaction mixture [0.1 mg NBT, 12 mm EDTA, and 20 μg of riboflavin sodium phosphate in a 50 mm buffer (pH 7.6)], and the light was radiated. Absorbance was measured at 590 nm after 90 s (Kumaran and Karunakaran, 2007).

For the H2O2 scavenging experiment, 0.6 mL H2O2 (2 mm) was mixed with the extract (0.1 mL) and 0.8 mL distilled water, then incubated for 10 min. Absorbance was measured at 240 nm according to the method described by Kumaran and Karunakaran (2007).

The 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability was analyzed by mixing sample extracts (40 μL) with a 0.1-mm methanolic solution of DPPH (total 2 mL) and allowed to stand for 25 min under dark conditions. Absorbance of the sample was measured at 517 nm (Manivannan et al., 2015b).

Radical scavenging percentages for O2-, H2O2, and DPPH were calculated using the formula [(Ac − As)/Ac] × 100, where Ac is the absorbance value of the control (i.e., reaction mixture without extract) and As is the OD value of the extract.

For the ·OH scavenging experiment, the reaction mixture (4 mL) contained 1 mL of FeSO4 (4 mm), 1 mL of H2O2 (4 mm), 1 mL of sodium salicylic acid (4 mm), and 1 mL of sample or water, as reference. Absorbance at 510 nm was measured after incubation for 1 h at 37 °C. The percentage of ·OH scavenging was calculated as: [1 − (A1 − A2)/A0] × 100, where A1 is the absorbance of the sample; A0 is the absorbance of the reaction without any sample; and A2 is the absorbance of the reagent blank without sodium salicylic acid addition (Smironff and Cumbes, 1989).

Extraction and quantitation of total phytochemical compounds.

Dried samples were ground in a 1 mL methanol solution, followed by extraction by ultrasonication for 30 min and centrifugation at 5000 rpm (D-37520; Heraeus Thermo) for 10 min; the supernatants were employed in phytochemical analysis.

The Folin-Ceiocalteu theory was used to evaluate the total phenolic content of extracts (Singleton et al., 1999). Some extracts (100 μL) were diluted with 2 mL of distilled water and then added to Folin-ciocalteu reagent (1 mL). The reaction mixture was placed in the dark for 5 min and then mixed with 3 mL of 2% (w/v) Na2CO3 and diluted to 10 mL using distilled water. After incubation at 25 °C for 1 h, the reaction mixture absorbance was recorded at 760 nm using an ultraviolet-5500 spectrophotometer. Finally, the equivalent content of gallic acid was used as the content of total phenol.

Total flavonoid content was determined by aluminum chloride calorimetric analysis (Kim et al., 2003). Samples (100 μL) were mixed with methanol (80%) to 4 mL, and a 0.3 mL 5% (w/v) NaNO2 solution. The reaction mixture was placed at room temperature (25 °C) for 5 min and added with 0.3 mL of 10% (w/v) AlCl3. After 6 min, 4 mL of 4% (w/v) NaOH was added. Finally, the reaction mixture was brought to a final volume of 10 mL with distilled water. The absorbance of the reaction mixture was recorded at 510 nm with a ultraviolet-5500 spectrophotometer, and total flavonoid content of each extract was calculated by a standard calibration curve.

Total tanshinone content of the extracts was evaluated following quantitative analysis of multicomponents by single-marker (Li et al., 2012). We selected standards tanshinone IIA to represent total tanshinone content. Samples (100 μL) were added to 3 mL ethanol solution, and absorbance was recorded at 269 nm using an ultraviolet-5500 spectrophotometer; total tanshinone content was calculated using a standard tanshinone IIA calibration curve (Lan et al., 2012; Li et al., 2012).

Statistical analysis.

All experiments were in a completely randomized design with three replications. Additionally, each experiment was repeated three times to verify the reproducibility of the results. The significance test of the differences among treatments was conducted by analysis of variance, and Duncan’s multiple range test was performed at the 5% significance level by the SPSS.19 software package to separate significantly different means (IBM, Armonk, NY).

Results

Effects of REEs on root length and chlorophyll content.

The highest root length (8.85 cm) of S. miltiorrhiza was observed at 100 μM Pr (Table 1). All REEs induced more roots than those in the control at 100 μM. In other words, both 200 μM and 300 μM reduced root elongation relative to 100 μM. Especially, root growth was inhibited at 300 μM in all treatments (Fig. 1, Table 1). Promotion of root growth by low concentration and inhibition at high concentration was consistently observed in all experiments. Further, we found that increased chlorophyll content consistently correlated with greater root length (Table 1).

Table 1.

Effect of Ce(NO3)3, La(NO3)3, and Pr(NO3)3 on root growth and chlorophyll content of S. miltiorrhiza.

Table 1.
Fig. 1.
Fig. 1.

In vitro effect of REEs on root growth of S. miltiorrhiza. (A) Inhibition of root growth. (B) Root in blank treatment. (C) Promotion of root growth. (D) Comparison of root growth of S. miltiorrhiza (bar = 1 cm). Roots (ae) were cultured for 4 weeks from adventitious buds: a, 300μM Pr(NO3)3; b, 300 μM Ce(NO3)3; c, control; d, 200 μM Ce(NO3)3; e, 100 μM Pr(NO3)3.

Citation: HortScience horts 55, 3; 10.21273/HORTSCI14661-19

Effect of REEs on SOD, CAT, POD, and APX activities.

As shown in Fig. 2, the highest SOD activity was observed at 100 μM Pr(NO3)3 (35.80 U/mg protein), which was ≈34.51% higher than SOD activity in controls. However, SOD activity was significantly inhibited by 200 μM La(NO3)3; and when the concentration was up to 300 μM, all REE treatments inhibited SOD activity (Fig. 2A). In turn, Ce promoted CAT activity at 50 μM, but further increase in Ce concentration decreased it. On the other hand, CAT was stimulated more at 100 μM Pr, which was followed by La. Especially, about an 80.48% increase in CAT activity was observed under 100 μM Pr over the control level (Fig. 2B). The POD activity was not much variation in the range between 50 to 200 μM concentration for Ce, Pr, or La. Although POD activity was reduced at 300 μM, no significant difference was detected between REEs at this high concentration. And 50 μM La increased the APX activity to a similar extent relative to other REEs, but an unexpected decrease in APX activity was observed at 100 μM (Fig. 2D).

Fig. 2.
Fig. 2.

Effect of Ce(NO3)3, La(NO3)3, and Pr(NO3)3 on antioxidant enzyme activities of plant tissue cultures. The activity of superoxide dismutase (A); catalase (B); peroxidase (C); and ascorbate peroxidase (D) estimated in the plant tissue culture. Data are means ±se from three replicates. Different letters on the bars corresponding to the different treatments indicate statistically significant difference at P < 0.05 by Duncan’s multiple range test.

Citation: HortScience horts 55, 3; 10.21273/HORTSCI14661-19

Effect of chemical elicitors on the free radical scavenging potential.

Treatment with REEs treatments significantly enhanced the free radical scavenging potential of the danshen root extracts in comparison with the control (Fig. 3). The best O2 scavenging potential of an extract (85.58%) was recorded in the 100 µM Pr treatment, followed by La (Fig. 3A). However, H2O2 was higher at 100 μM Pr than at any other concentration, and no significant difference was observed between REEs treatments (Fig. 3B). Free radical scavenging activity of danshen extracts elicited by treatment with 100 µM Pr resulted in the best ·OH scavenging potential of 92.99%, followed by the 50 µM La(NO3)3 treatment (Fig. 3C). When the concentration of La went to 50 µM, the ·OH scavenging activity was inhibited. DPPH activity was increased at 50 μM in all REEs treatments. Optimal DPPH scavenging percent (83.71%) was observed in the 50 µM Ce treatment, followed by the 100 µM Pr(NO3)3 treatment (Fig. 3D). Overall, low concentrations (50 and 100 μM) improved the scavenging ability of oxidized free radicals, whereas high concentrations inhibited the ability of the scavenging enzyme activities.

Fig. 3.
Fig. 3.

Effect of Ce(NO3)3, La(NO3)3, and Pr(NO3)3 treatments on free radical scavenging activities potentials of danshen plant tissue cultures. Superoxide radical scavenging potential (A); Hydrogen peroxide scavenging percentage (B); Hydroxyl radical scavenging capacity (C); DPPH radical scavenging potential (D). Data are the means ±se from three replicates. Different letters on bars of the different treatments indicate statistically significant difference at P < 0.05 by Duncan’s multiple range test.

Citation: HortScience horts 55, 3; 10.21273/HORTSCI14661-19

Effect of REEs on the accumulation of bioactive compounds.

Rare earth elements had significant effects on the synthesis of bioactive compounds, such as total flavonoids, total phenols, and total tanshinone (Fig. 4). Like the antioxidant enzyme activities, the highest concentration of REEs (300 μM) significantly reduced the production of the total flavonoids. The greatest content of total flavonoids was registered in the danshen extracts treated with 100 μM Ce and Pr (Fig. 4A). Similarly, the synthesis of total phenols was significantly enhanced by the treatment with 100 μM REEs. The most content of total phenol was recorded in the danshen extracts treated with the 100 μM Pr, namely 62.69 μg gallic acid·mg−1, i.e., 73.48% better than total phenol content measured in the controls (Fig. 4B). Similarly, accumulation of total tanshinone was triggered by the increasing concentrations of REEs. A decrease in tanshione content at higher concentrations (200 and 300 μM) was correlated with a decrease in total flavonoids and total phenol contents. Most importantly, the improvement of tanshinone content was achieved at 100 μM Pr, which caused a 54.84% increase more than control content (Fig. 4C). Similarly, 100 μM Ce and La resulted in a 41.21% and 51.89% increase in tanshinone content relative to control levels, respectively.

Fig. 4.
Fig. 4.

Effect of Ce(NO3)3, La(NO3)3, and Pr(NO3)3 treatments on the secondary metabolites of S. miltiorrhiza. Total flavonoid content (A); total phenol content (B); and total tanshinone content (C) of plant tissue cultures. Data are the mean ±se from three replicates. Different letters (a, b, c) on bars of the different treatments indicate statistically significant difference at P < 0.05 by Duncan’s multiple range test.

Citation: HortScience horts 55, 3; 10.21273/HORTSCI14661-19

Discussion

Plant-derived secondary metabolites are important medicinal resources. Danshen extracts reportedly show important secondary metabolites with pharmaceutical activities (Ryu et al., 1996). However, increasing market demand and difficulties in cultivation of danshen pose a challenge that increases content of tanshinones, which are important active medicinal compounds (Shan et al., 2007). Plant tissue culture is one of the techniques that could be used for year-round production of danshen. Furthermore, micropropagation might improve the medical properties and tanshinone content of in vitro cultures (Liu et al., 2018). Therefore, the present study was conducted to promote the phytochemical properties of in vitro-grown danshen. Additionally, the effects of REEs, such as Ce(NO3)3, La(NO3)3, and Pr(NO3)3 as supplements to induce medicinal compound accumulation were also explored. The induction of adventitious buds and roots was achieved using leaf explants cultured with a combination of 1.0 mg·L−1 BA, 0.1 mg·L−1 NAA, and 0.2 mg·L−1 NAA in the growth medium (Liu et al., 2018). At low concentrations, especially at 100 μM Pr, three REEs could improve root elongation and chlorophyll content (Figs. 1 and 2). Similar results were observed in studies that reported that the appropriate concentration of REEs improved the root elongation of radish, tomato, and peach (Philippe et al., 2014; Song et al., 2003). The enhancement of photosynthetic rate was observed in spinach with treatments of REEs (Brown et al., 1990; Hong et al., 2002). And the inhibition of root elongation at high REE concentrations in danshen correlated with the decreased root growth in Chinese cabbage at high Pr concentration (Tang and Tong, 1988).

To further explore the effects of REE on the growth of danshen, we measured the antioxidant enzymes activities including SOD, CAT, POD, and GPX. As Fig. 2 shows, the different treatments distinctly affected the activities of antioxidant enzymes. In particular, SOD, CAT, and APX activities were significantly increased under Pr at 100 μM. Our results were similar to those in a study reporting that low concentrations of REEs had a positive effects on plant growth and simultaneously increase yield, metabolite production, and quality; whereas excessive concentrations of REEs inhibited plant growth by negatively altering photosynthesis and antioxidant enzyme activity (Chen et al., 2015). According to Feng et al. and Wu et al. (Feng et al., 1999; Wu et al., 2001), the induction of ROS could increase plasma membrane permeability to a proper concentration in long-term REEs-treated cells than in control cells, which may lead to the enhancement of nutrient uptaking and promotion of plant growth (Diatloff et al., 2008; Hong et al., 1999, 2000; Huang et al., 2012; Wu et al., 2001). Oxidative stress induced by the elicitors could stimulate antioxidant enzymes (such as SOD, CAT, POD, and GPX) to protect the plant cell from the harm of free radicals. During the process of redox homeostasis, the primary reaction is catalyzed by SOD, which provides the first line of defense against the toxic effects of ROS by catalyzing the dismutation of O2 into H2O2 and O2; subsequently H2O2 is catalyzed into H2O and O2 by CAT or peroxidases such as POD or APX (Ali et al., 2006).

According to previous reports, exogenous application of chemical elicitors, such REEs, might stimulate plant secondary metabolism to improve the medicinal value of plants by interfering with their signaling process (Chen et al., 2003; Ting et al., 2016; Wu et al., 2001). We speculated that the accumulation of endogenous ROS such as O2, H2O2, ·OH, and DPPH under REEs treatment might induce secondary metabolites to resist oxidative stress (Kumaran and Karunakaran, 2007). The induction of ROS production upon exogenous application of REEs has been evidenced in cell suspension cultures of Vicia faba (Wang et al., 2012). In addition, REEs might promote the synthesis of flavonoids through enhanced PAL activities (Liu et al., 2008). Although the effect of the elicitor molecules varied among plant species (Wyttenbach et al., 1998), Pr(NO3)3 significantly enhanced the production of total flavonoids and total phenols in danshen. Like our study, lower concentration of REEs reportedly increased the production of secondary metabolites, whereas high concentrations had negative effects in Tetrastigma hemsleyanum, Arnebia euchroma, and Saussurea medusa (Ge et al., 2006; Wu et al., 2001; Yuan et al., 2002). Flavonoids and phenols are nonenzymatic antioxidants and aromatic substitutions involved in various processes such as pigmentation, root nodule formation, and cell signaling pathways. Higher concentration of metabolite contents, which was observed in the plants with the treatments of REEs, may associate with increasing expression of biosynthetic genes (Wang et al., 2004a). Therefore, further study is needed explore biosynthetic pathway(s) involving REEs in the production of tanshinone in danshen.

Overall, our results showed that the highest phytochemical content, antioxidant enzyme activity (SOD, CAT, and APX) and oxidation radical scavenging activity (O2, H2O2, and ·OH) in danshen were caused by 100 μM Pr(NO3)3.

Conclusion

Our study clearly demonstrated the effects of Ce(NO3)3, La(NO3)3, and Pr(NO3)3 on danshen root elongation and photosynthetic efficiency. Highest enhancement effects on in vitro danshen cultures were achieved at 100 μM Pr(NO3)3; this concentration promoted growth of in vitro danshen plants, and increased antioxidant activities and synthesis of bioactive compounds. Further studies are needed to elucidate the regulatory mechanism of Ce(NO3)3, La(NO3)3, and Pr(NO3)3 underlying the promotion of plant growth and the accumulation of secondary metabolites of pharmaceutical interest.

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  • Lan, T.F., Wang, X., Wang, D.J., Wang, L., Liu, Q. & Yu, Z.Y. 2012 Determination of four tanshinones in Salvia miltiorrhiza Radix et Rhizoma with quantitative analysis of multi-components by single-marker Chin. Tradit. Herbal Drugs 43 2420 2423 (in Chinese)

    • Search Google Scholar
    • Export Citation
  • Li, Q., Liu, W., Luo, Z.L. & Yang, M.H. 2012 Simultaneous determination of four tanshinones in Salvia miltiorrhiza by QAMS Chin. J. Chinese Mater. Med. 37 824 828 (in Chinese)

    • Search Google Scholar
    • Export Citation
  • Liu, B.L., Fan, Z.B., Liu, Z.Q., Qiu, X.H. & Jiang, Y.H. 2018 Comparison of phytochemical and antioxidant activities in micropropagated and seed-derived Salvia miltiorrhiza plants HortScience 53 1038 1044

    • Search Google Scholar
    • Export Citation
  • Liu, Y.J., Wang, Y., Wang, F.B., Liu, Y.M., Cui, J.Y., Hu, L. & Mu, K.G. 2008 Control effect of lanthanum against plant disease J. Rare Earths 26 115 120

  • Manivannan, A., Prabhakaran, S., Laras, S.A., Chung, H.K., Muneer, S. & Jeong, B.R. 2015a Silicon-mediated enhancement of physiological and biochemical characteristics of Zinnia elegans “Draml and Yellow” grown under salinity stress Hort. Environ. Biotechnol. 56 721 731

    • Search Google Scholar
    • Export Citation
  • Manivannan, A., Soundararajan, P., Park, Y.G. & Jeong, B.R. 2015b In vitro propagation, phytochemical analysis, and evaluation of free radical scavenging property of Scrophularia kakudensis Franch tissue extracts BioMed Res. Intl. 2015 480564

    • Search Google Scholar
    • Export Citation
  • Nakano, Y. & Asada, K. 1981 Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts Plant Cell Physiol. 22 867 880

  • Peng, X., Zhou, S.L., He, J.Y. & Li, D. 2013 Influence of rare earth elements on metabolism and related enzyme activity and isozyme expression in Tetrastigma hemsleyanum cell suspension cultures Biol. Trace Elem. Res. 152 82 90

    • Search Google Scholar
    • Export Citation
  • Philippe, J.T., Carpenter, D., Boutin, C. & Allison, J.E. 2014 Rare earth elements (REEs): Effects on germination and growth of selected crop and native plant species Chemosphere 96 57 66

    • Search Google Scholar
    • Export Citation
  • Qiu, N.W., Wang, X.S., Yang, F.B., Yang, X.G., Yang, W.R., Diao, J., Wang, X., Cui, J. & Zhou, F. 2016 Fast extraction and precise determination of chlorophyll Chinese Bul. Bot. 51 667 678 (in Chinese)

    • Search Google Scholar
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  • Ram, J., Shivani, A.P., Mohammed, K.A. & Venkat Narayan, K.M. 2019 Global updates on cardiovascular disease mortality trends and attribution of traditional risk factors Curr. Diab. Rep. 19 44

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  • Reddy, N.S., Navanesan, S., Sinniah, S.K., Wahab, N.A. & Sim, K.S. 2012 Phenolic content, antioxidant effect and cytotoxic activity of Leea indica leaves BMC Comp. Alter. Med. 12 128 134

    • Search Google Scholar
    • Export Citation
  • Ryu, S.Y., Lee, C.O. & Choi, S.U. 1996 In vitro cytotoxicity of tanshinones from Salvia miltiorrhiza Planta Med. 63 339 342

  • Shan, C.G., Wang, Z.F., Su, X.H., Yan, S.L. & Sun, H.C. 2007 Study advance in Salvia miltiorrhiza tissue culture Res. Prac. Chin. Med. 22 54 57 (in Chinese)

  • Siddiqui, M.H., Al-Whaibi, M.H., Sakran, A.M., Basalah, M.O. & Ali, H.M. 2012 Effect of calcium and potassium on antioxidant system of Vicia faba L. under cadmium stress Intl. J. Mol. Sci. 13 6604 6619

    • Search Google Scholar
    • Export Citation
  • Singleton, V.L., Orthofer, R. & Lamuela-Raventos, R.M. 1999 Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent Methods Enzymol. 14 152 178

    • Search Google Scholar
    • Export Citation
  • Smironff, N. & Cumbes, Q.J. 1989 Hyroxyl radical scavenging activity of compatible solutes Phytochemistry 28 1057 1060

  • Song, W.P., Hong, F.S., Wan, Z.G., Zhou, Y.Z., Gu, F.G., Xu, H.G., Yu, M.L., Chang, Y.H., Zhao, M.Z. & Su, J.L. 2003 Effects of cerium on nitrogen metabolism of peach plantlet in vitro Biol. Trace Elem. Res. 95 259 268

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    • Export Citation
  • Tang, X.K. & Tong, Z. 1988 Effects of rare earth elements on plant root growth and activity Chin. Rare Metal. 5 22 24 (in Chinese)

  • Ting, X., Su, C.L., Dan, H., Li, F.F., Lu, Q.Q., Zhang, T.T. & Xu, Q.S. 2016 Molecular distribution and toxicity assessment of praseodymium by Spirodela polyrrhiza J. Hazard. Mater. 312 132 140

    • Search Google Scholar
    • Export Citation
  • Wang, C., Luo, X., Tian, Y., Xie, Y., Wang, S., Li, Y., Tian, L. & Wang, X. 2012 Biphasic effects of lanthanum on Vicia faba L. seedlings under cadmium stress, implicating finite antioxidation and potential ecological risk Chemosphere 86 530 537

    • Search Google Scholar
    • Export Citation
  • Wang, H.Y., Luo, H. & Sun, M. 2004a Application of elicitor to cell culture of medicinal plants Chin. Tradit. Herb Drugs. 35 1426 1430 (in Chinese)

  • Wang, J.W. & Wu, J.Y. 2010 Tanshinone biosynthesis in Salvia miltiorrhiza and production in plant tissue cultures Appl. Microbiol. Biotechnol. 88 437 449

    • Search Google Scholar
    • Export Citation
  • Wang, Y.S., Tian, S.P., Xu, Y., Qin, G.Z. & Yao, H.J. 2004b Changes in the activities of pro- and anti-oxidant enzymes in peach fruit inoculated with Cryptococcus laurentii or Penicillium expansum at 0 or 20 °C Postharvest Biol. Technol. 34 21 28

    • Search Google Scholar
    • Export Citation
  • Wu, J.Y., Wang, C.G. & Mei, X.G. 2001 Stimulation of taxol production and excretion in Taxus spp cell cultures by rare earth chemical lanthanum J. Biotechnol. 85 67 73

    • Search Google Scholar
    • Export Citation
  • Wyttenbach, A., Furrer, V., Schleppi, P. & Tobler, L. 1998 Rare earth elements in soil and in soil-grown plants Plant Soil 199 267 273

  • Yuan, X.F., Wang, Q., Zhao, B. & Wang, Y.C. 2002 Improved cell growth and total flavonoids of Saussurea medusa on solid culture medium supplemented with rare earth elements Biotechnol. Lett. 24 1889 1892

    • Search Google Scholar
    • Export Citation
  • Zhang, C.H., Li, Q.Q., Zhang, M.X., Zhang, N. & Li, M.H. 2013 Effects of rare earth elements on growth and metabolism of medicinal plants Acta Pharm. Sin. B 3 20 24

    • Search Google Scholar
    • Export Citation
  • Zhao, J.L., Zhou, L.G. & Wu, J.Y. 2010a Effects of biotic and abiotic elicitors on cell growth and tanshinone accumulation in Salvia miltiorrhiza cell cultures Appl. Microbiol. Biotechnol. 87 137 144

    • Search Google Scholar
    • Export Citation
  • Zhao, J.L., Zhou, L.G. & Wu, J.Y. 2010b Promotion of Salvia miltiorrhiza hairy root growth and tanshinone production by polysaccharide protein fractions of plant growth-promoting rhizobacterium Bacillus cereus Process Biochem. 45 1517 1522

    • Search Google Scholar
    • Export Citation
  • Fig. 1.

    In vitro effect of REEs on root growth of S. miltiorrhiza. (A) Inhibition of root growth. (B) Root in blank treatment. (C) Promotion of root growth. (D) Comparison of root growth of S. miltiorrhiza (bar = 1 cm). Roots (ae) were cultured for 4 weeks from adventitious buds: a, 300μM Pr(NO3)3; b, 300 μM Ce(NO3)3; c, control; d, 200 μM Ce(NO3)3; e, 100 μM Pr(NO3)3.

  • Fig. 2.

    Effect of Ce(NO3)3, La(NO3)3, and Pr(NO3)3 on antioxidant enzyme activities of plant tissue cultures. The activity of superoxide dismutase (A); catalase (B); peroxidase (C); and ascorbate peroxidase (D) estimated in the plant tissue culture. Data are means ±se from three replicates. Different letters on the bars corresponding to the different treatments indicate statistically significant difference at P < 0.05 by Duncan’s multiple range test.

  • Fig. 3.

    Effect of Ce(NO3)3, La(NO3)3, and Pr(NO3)3 treatments on free radical scavenging activities potentials of danshen plant tissue cultures. Superoxide radical scavenging potential (A); Hydrogen peroxide scavenging percentage (B); Hydroxyl radical scavenging capacity (C); DPPH radical scavenging potential (D). Data are the means ±se from three replicates. Different letters on bars of the different treatments indicate statistically significant difference at P < 0.05 by Duncan’s multiple range test.

  • Fig. 4.

    Effect of Ce(NO3)3, La(NO3)3, and Pr(NO3)3 treatments on the secondary metabolites of S. miltiorrhiza. Total flavonoid content (A); total phenol content (B); and total tanshinone content (C) of plant tissue cultures. Data are the mean ±se from three replicates. Different letters (a, b, c) on bars of the different treatments indicate statistically significant difference at P < 0.05 by Duncan’s multiple range test.

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  • Lan, T.F., Wang, X., Wang, D.J., Wang, L., Liu, Q. & Yu, Z.Y. 2012 Determination of four tanshinones in Salvia miltiorrhiza Radix et Rhizoma with quantitative analysis of multi-components by single-marker Chin. Tradit. Herbal Drugs 43 2420 2423 (in Chinese)

    • Search Google Scholar
    • Export Citation
  • Li, Q., Liu, W., Luo, Z.L. & Yang, M.H. 2012 Simultaneous determination of four tanshinones in Salvia miltiorrhiza by QAMS Chin. J. Chinese Mater. Med. 37 824 828 (in Chinese)

    • Search Google Scholar
    • Export Citation
  • Liu, B.L., Fan, Z.B., Liu, Z.Q., Qiu, X.H. & Jiang, Y.H. 2018 Comparison of phytochemical and antioxidant activities in micropropagated and seed-derived Salvia miltiorrhiza plants HortScience 53 1038 1044

    • Search Google Scholar
    • Export Citation
  • Liu, Y.J., Wang, Y., Wang, F.B., Liu, Y.M., Cui, J.Y., Hu, L. & Mu, K.G. 2008 Control effect of lanthanum against plant disease J. Rare Earths 26 115 120

  • Manivannan, A., Prabhakaran, S., Laras, S.A., Chung, H.K., Muneer, S. & Jeong, B.R. 2015a Silicon-mediated enhancement of physiological and biochemical characteristics of Zinnia elegans “Draml and Yellow” grown under salinity stress Hort. Environ. Biotechnol. 56 721 731

    • Search Google Scholar
    • Export Citation
  • Manivannan, A., Soundararajan, P., Park, Y.G. & Jeong, B.R. 2015b In vitro propagation, phytochemical analysis, and evaluation of free radical scavenging property of Scrophularia kakudensis Franch tissue extracts BioMed Res. Intl. 2015 480564

    • Search Google Scholar
    • Export Citation
  • Nakano, Y. & Asada, K. 1981 Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts Plant Cell Physiol. 22 867 880

  • Peng, X., Zhou, S.L., He, J.Y. & Li, D. 2013 Influence of rare earth elements on metabolism and related enzyme activity and isozyme expression in Tetrastigma hemsleyanum cell suspension cultures Biol. Trace Elem. Res. 152 82 90

    • Search Google Scholar
    • Export Citation
  • Philippe, J.T., Carpenter, D., Boutin, C. & Allison, J.E. 2014 Rare earth elements (REEs): Effects on germination and growth of selected crop and native plant species Chemosphere 96 57 66

    • Search Google Scholar
    • Export Citation
  • Qiu, N.W., Wang, X.S., Yang, F.B., Yang, X.G., Yang, W.R., Diao, J., Wang, X., Cui, J. & Zhou, F. 2016 Fast extraction and precise determination of chlorophyll Chinese Bul. Bot. 51 667 678 (in Chinese)

    • Search Google Scholar
    • Export Citation
  • Ram, J., Shivani, A.P., Mohammed, K.A. & Venkat Narayan, K.M. 2019 Global updates on cardiovascular disease mortality trends and attribution of traditional risk factors Curr. Diab. Rep. 19 44

    • Search Google Scholar
    • Export Citation
  • Reddy, N.S., Navanesan, S., Sinniah, S.K., Wahab, N.A. & Sim, K.S. 2012 Phenolic content, antioxidant effect and cytotoxic activity of Leea indica leaves BMC Comp. Alter. Med. 12 128 134

    • Search Google Scholar
    • Export Citation
  • Ryu, S.Y., Lee, C.O. & Choi, S.U. 1996 In vitro cytotoxicity of tanshinones from Salvia miltiorrhiza Planta Med. 63 339 342

  • Shan, C.G., Wang, Z.F., Su, X.H., Yan, S.L. & Sun, H.C. 2007 Study advance in Salvia miltiorrhiza tissue culture Res. Prac. Chin. Med. 22 54 57 (in Chinese)

  • Siddiqui, M.H., Al-Whaibi, M.H., Sakran, A.M., Basalah, M.O. & Ali, H.M. 2012 Effect of calcium and potassium on antioxidant system of Vicia faba L. under cadmium stress Intl. J. Mol. Sci. 13 6604 6619

    • Search Google Scholar
    • Export Citation
  • Singleton, V.L., Orthofer, R. & Lamuela-Raventos, R.M. 1999 Analysis of total phenols and other oxidation substrates and antioxidants by means of folin-ciocalteu reagent Methods Enzymol. 14 152 178

    • Search Google Scholar
    • Export Citation
  • Smironff, N. & Cumbes, Q.J. 1989 Hyroxyl radical scavenging activity of compatible solutes Phytochemistry 28 1057 1060

  • Song, W.P., Hong, F.S., Wan, Z.G., Zhou, Y.Z., Gu, F.G., Xu, H.G., Yu, M.L., Chang, Y.H., Zhao, M.Z. & Su, J.L. 2003 Effects of cerium on nitrogen metabolism of peach plantlet in vitro Biol. Trace Elem. Res. 95 259 268

    • Search Google Scholar
    • Export Citation
  • Tang, X.K. & Tong, Z. 1988 Effects of rare earth elements on plant root growth and activity Chin. Rare Metal. 5 22 24 (in Chinese)

  • Ting, X., Su, C.L., Dan, H., Li, F.F., Lu, Q.Q., Zhang, T.T. & Xu, Q.S. 2016 Molecular distribution and toxicity assessment of praseodymium by Spirodela polyrrhiza J. Hazard. Mater. 312 132 140

    • Search Google Scholar
    • Export Citation
  • Wang, C., Luo, X., Tian, Y., Xie, Y., Wang, S., Li, Y., Tian, L. & Wang, X. 2012 Biphasic effects of lanthanum on Vicia faba L. seedlings under cadmium stress, implicating finite antioxidation and potential ecological risk Chemosphere 86 530 537

    • Search Google Scholar
    • Export Citation
  • Wang, H.Y., Luo, H. & Sun, M. 2004a Application of elicitor to cell culture of medicinal plants Chin. Tradit. Herb Drugs. 35 1426 1430 (in Chinese)

  • Wang, J.W. & Wu, J.Y. 2010 Tanshinone biosynthesis in Salvia miltiorrhiza and production in plant tissue cultures Appl. Microbiol. Biotechnol. 88 437 449

    • Search Google Scholar
    • Export Citation
  • Wang, Y.S., Tian, S.P., Xu, Y., Qin, G.Z. & Yao, H.J. 2004b Changes in the activities of pro- and anti-oxidant enzymes in peach fruit inoculated with Cryptococcus laurentii or Penicillium expansum at 0 or 20 °C Postharvest Biol. Technol. 34 21 28

    • Search Google Scholar
    • Export Citation
  • Wu, J.Y., Wang, C.G. & Mei, X.G. 2001 Stimulation of taxol production and excretion in Taxus spp cell cultures by rare earth chemical lanthanum J. Biotechnol. 85 67 73

    • Search Google Scholar
    • Export Citation
  • Wyttenbach, A., Furrer, V., Schleppi, P. & Tobler, L. 1998 Rare earth elements in soil and in soil-grown plants Plant Soil 199 267 273

  • Yuan, X.F., Wang, Q., Zhao, B. & Wang, Y.C. 2002 Improved cell growth and total flavonoids of Saussurea medusa on solid culture medium supplemented with rare earth elements Biotechnol. Lett. 24 1889 1892

    • Search Google Scholar
    • Export Citation
  • Zhang, C.H., Li, Q.Q., Zhang, M.X., Zhang, N. & Li, M.H. 2013 Effects of rare earth elements on growth and metabolism of medicinal plants Acta Pharm. Sin. B 3 20 24

    • Search Google Scholar
    • Export Citation
  • Zhao, J.L., Zhou, L.G. & Wu, J.Y. 2010a Effects of biotic and abiotic elicitors on cell growth and tanshinone accumulation in Salvia miltiorrhiza cell cultures Appl. Microbiol. Biotechnol. 87 137 144

    • Search Google Scholar
    • Export Citation
  • Zhao, J.L., Zhou, L.G. & Wu, J.Y. 2010b Promotion of Salvia miltiorrhiza hairy root growth and tanshinone production by polysaccharide protein fractions of plant growth-promoting rhizobacterium Bacillus cereus Process Biochem. 45 1517 1522

    • Search Google Scholar
    • Export Citation
Zhibin Fan School of Life Sciences, Qufu Normal University, Qufu, Shandong 273165, China

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Kai Zhang Forest Farm of Mountain Culai, Taian, Shandong 271000, China; and College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia 010018, China

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Fengyun Wang School of Life Sciences, Qufu Normal University, Qufu, Shandong 273165, China

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Xiaodan Zhao School of Life Sciences, Qufu Normal University, Qufu, Shandong 273165, China

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Ruiqin Bai College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia 010018, China

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Boling Liu School of Life Sciences, Qufu Normal University, Qufu, Shandong 273165, China

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Contributor Notes

This work was funded by the National Natural Science Foundation of China (Grant 31700624) and the Doctoral Scientific Fund Project of Qufu Normal University (bsqd20130136).

We appreciate Soundararajan Prabhakaran [Department of Agriculture Biotechnology, National Academy of Agricultural Science (NAAS), Rural Development Administration (RDA), Wansan-gu, Jeonju, Korea] for revising this manuscript.

Z.F. and K.Z. contributed equally to this work.

B.L. is the corresponding author. E-mail: lblzzkk@126.com.

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