Evaluation of the Influence of Genetic and Environmental Factors on the Ephedrine Alkaloids Content of Ephedra sinica

Abstract

Ephedra herb, a dried terrestrial stem of Ephedra sinica, is used in traditional Japanese medicine (Kampo) and Chinese medicine to treat the common cold, headaches, bronchial asthma, and nasal inflammation. E. sinica predominantly contains two ephedrine alkaloids—(−)-ephedrine and (+)-pseudoephedrine—which are crucial for its medicinal effects. This study aimed to reveal the influence of genetic and environmental factors on ephedrine alkaloids content using statistical genetic analyses. To evaluate the influence of genetic factors on ephedrine alkaloids content, 25 clonal lines were cultivated in Ibaraki and the broad-sense heritability of the traits was estimated. The heritabilities of (−)-ephedrine, (+)-pseudoephedrine, and “total alkaloids” (TA) content were 0.871, 0.969, and 0.865, respectively. The heritabilities of ephedrine alkaloids content were high. To evaluate the influence of environmental factors on ephedrine alkaloids content, four clonal lines which have different genotypes were cultivated in three locations (Ibaraki, Shizuoka, and Yamanashi prefectures). The effects of genotype (G), location (L), and genotype by environment (G × E) interactions on ephedrine alkaloids content were found to be significant (p < 0.05) by two-way ANOVA, and, in particular, the genotypic effects were found to be the largest. Our results indicate that the ephedrine alkaloids content in E. sinica is under relatively strong genetic control and remains stable under various environments. These findings suggest that E. sinica with a higher and stable ephedrine alkaloids content could be cultivated in different locations through selective breeding.

INTRODUCTION

Ephedra sinica Stapf is a medicinal plant that is distributed in arid areas in northern China and Mongolia.¹⁾ The plant is a dioecious gymnosperm and shrub whose dried terrestrial stems, the crude drug known as Ephedra herb, have long been used as a traditional medicine in eastern Asia. Ephedra herb is an important component of many traditional Japanese (Kampo) and Chinese medicines that have long been employed for treating the common cold, headaches, bronchial asthma, and nasal inflammation. The pharmacological actions of these medicines are thought to be derived from ephedrine alkaloids contained in the Ephedra herb.^2,3) The Japanese Pharmacopoeia⁴⁾ states that Ephedra herb for medicinal use must contain more than 0.7% dry weight (DW) of “total alkaloids” (TA), which is defined as the sum of the contents of two major alkaloids, (−)-ephedrine (Eph) and (+)-pseudoephedrine (PEph). It is important for the pharmacology of Ephedra herb to control the content of ephedrine alkaloids in Ephedra sinica plants.

However, some studies reported that variations in the ephedrine alkaloids of Ephedra sinica plants were large.^5,6) Previous studies have shown that ephedrine alkaloids content is influenced by environmental factors (habitats,^5–7) soil pH⁵⁾ and precipitation⁸⁾) and genetic factors,^9–11) but few studies have examined the effects of genetic and environmental factors (heritability and G × E) from a statistical genetics perspective. Therefore, the comprehensive evaluation of both genetic and environmental factors for ephedrine alkaloids in Ephedra sinica plants is necessary.

This study aimed to evaluate the effects of genetic and environmental factors on the content of ephedrine alkaloids in E. sinica using statistical genetic analyses. To evaluate the effect of genetic factors on ephedrine alkaloids content, we cultivated 25 clonal lines (Lines 1–25) in Ibaraki over years (2020–2021) and evaluated the broad-sense heritability of the traits (Experiment 1). In addition, to evaluate the magnitude of the effect of environmental factors and G × E interaction on ephedrine alkaloids content, we cultivated four selected clonal lines which have different genotypes (Lines 26–29) in three different environmental locations (Ibaraki, Shizuoka, and Yamanashi prefectures) over years (2017–2018) and evaluated ephedrine alkaloids content (Experiment 2).

MATERIALS AND METHODS

Experiment 1. Evaluation of Heritability on Ephedrine Alkaloids Content

Plant Materials and Clonal Propagation

In Experiment 1, to evaluate the influence of genetic factors on ephedrine alkaloids content, we obtained clones from 25 E. sinica genets that were preserved in the experimental cultivation field of Tsumura & Co., in Ibaraki prefecture. Twenty-five clonal lines (Lines 1–25) were propagated using stolons of each genet on March 25, 2020 (Fig. 1). The replicates for each clonal line were as follows; Lines 1, 3–6, 11–13, 18, 21, 23–24 (n = 3), Lines 2, 10, 16–17, 25 (n = 4), Lines 7–9, 15, 19, 22 (n = 5), Line 14 (n = 7) and Line 20 (n = 13).

Fig. 1. Method of Vegetative Propagation Using Stolons

In this study, the clones were propagated from stolons of genets. (A): Selection of plants. Whole E. sinica plant. White dotted lines: stolons, black dotted lines: roots. Modified from Hiyama¹⁸⁾ (B): Harvesting strains derived from stolon of E. sinica plant (C): Transplantation of clones derived from stolon.

Experimental Design

To evaluate the effect of genetic factor on the content of ephedrine alkaloids, we estimated broad-sense heritability. In order to remove the influence of macro-environments (locations), we cultivated the 25 clonal lines (Lines 1–25) in Ibaraki prefecture (35°99′N; 140°20′E) from March 24, 2020. Clones in each clonal line were transplanted into rows with a spacing of 0.3 m between plants and a spacing of 1.0 m between rows. Farmyard manure (2000 g·m⁻²) was used as a basal fertilizer. A chemical fertilizer was applied to supply 2 g·m⁻² of N, 2 g·m⁻² of P₂O₅, 2 g·m⁻² of K₂O, and 0.2 g·m⁻² of Mg.

Harvest and Processing

The terrestrial stems of the E. sinica plants were harvested by cutting them at a height of 5 cm above the ground when the growth of terrestrial stems in these plants had ceased. The terrestrial stems of the 25 clonal lines (Lines 1–25) were harvested on August 2, 2021 (the second year of transplantation). In this study, the ephedrine alkaloids content in clones was evaluated after the second year of transplantation because the ephedrine alkaloids content in E. sinica plants became stable after the second year of transplantation in our previous study.¹⁰⁾ In the first year of transplantation, terrestrial stems were cut and not harvested to avoid mixing with woody terrestrial stems of the plants.

The harvested terrestrial stems were dried using a food dryer (DSK-30-3; Shizuoka Seiki Co., Ltd., Japan) at 35 °C for 3 d, followed by 50 °C for 6 h. Subsequently, the dry weight of the terrestrial stems (stems-DW) was measured.

Determination of the Content of Ephedrine Alkaloids

Quantitative analysis of the ephedrine alkaloids [(−)-Eph and (+)-PEph] was performed using a HPLC system (LC-20; Shimadzu Corp., Kyoto, Japan) equipped with an Inertsil ODS-3 column (5 µm, 4.6 × 150 mm; GL Sciences Inc., Japan) according to previously reported method.¹⁰⁾ The TA content was the sum of the contents of both (−)-Eph and (+)-PEph. The standards for (−)-Eph and (+)-PEph were provided by Tsumura & Co. (Japan). The herbal samples were pulverized using a vibrating rod mill (TI-100; CMT Co., Ltd., Japan).

Statistical Analysis

One-way ANOVA was performed for the contents of Eph, PEph, and TA using R software (version 4.1.0).

To assess the magnitude of the effect of genetic factor on ephedrine alkaloids content in a macro-environment, broad-sense heritability (h²) was estimated from variance components using one-way ANOVA as follows:

  

where σ_g² and σ_e² are the components of the variance for the genotype (G) and environment (E) variances, respectively.^12,13) The number of effective replications (r) for the estimation of σ_g² and h² was calculated as described by Yoshida¹⁴⁾ as follows:

  

where a and r_i are the number of levels and replications at each level, respectively. These variance components were estimated using mean squares. We used the calculation model for vegetatively propagated crops, such as tall fescue.¹³⁾

Experiment 2. Evaluation of Effects of Macro-Environmental Factors on Ephedrine Alkaloids Content under Different Cultivation Locations

Plant Materials and Clonal Propagation

In Experiment 2, to evaluate the influence of environmental factors and G × E interactions on ephedrine alkaloids content, we obtained clones from four E. sinica genets that have different genotypes were preserved in the experimental cultivation field of Tsumura & Co., in Ibaraki prefecture (35°99′N; 140°20′E) (Supplementary Table 1). We have selected the four genets out of 45 genets, which were preserved in the experimental cultivation field of Tsumura & Co., in Ibaraki prefecture (35°99′N; 140°20′E), based on the genetic distance using EST-SSR markers¹⁵⁾ and having an adequate number of stolons (Supplementary Fig. 1). Four clonal lines (Lines 26–29) of the plants were propagated using the stolon of each genet. The voucher specimens were deposited at the Herbarium of the Tsumura Botanical Raw Material Research Laboratories (Tsumura & Co., Ibaraki, Japan).

Experimental Design

To evaluate the influence of the macro-environmental factors on the content of ephedrine alkaloids in E. sinica under different cultivation locations, we cultivated the four clonal lines (Lines 26–29) in three different environmental cultivation fields: Ibaraki (35°99′N; 140°20′E), Shizuoka (34°64′N; 138°11′E), and Yamanashi (35°78′N, 138°32′E) prefectures in Japan. Based on the result of previous study,¹⁶⁾ we selected these various cultivation locations in which the plants were able to be well grown. The characteristics of the environmental conditions of these cultivation fields are listed in Supplementary Table 2. Climatic data were obtained from the records of the Japan Meteorological Agency.¹⁷⁾ Clones in each clonal line were transplanted into rows with a spacing of 0.3 m between plants and a spacing of 1.0 m between rows at all cultivation locations. The clones were transplanted to Ibaraki on June 7, 2017, Shizuoka on June 9, 2017, and Yamanashi on June 23, 2017. The replicates for each clonal line were as follows; Line 26 (n = 8), Line 27 (n = 7), Line 28 (n = 6) and Line 29 (n = 3) in Ibaraki, Line 26 (n = 5), Line 27 (n = 3), Line 28 (n = 5) and Line 29 (n = 1) in Shizuoka, Line 26 (n = 1), Line 27 (n = 4), Line 28 (n = 2) and Line 29 (n = 2) in Yamanashi. Basal and chemical fertilizers were used in the same manner as described in Experiment 1.

We partially reutilized our unpublished data (TA content and stems-DW of four clonal lines, and genotypes of forty-five genets)¹⁸⁾ to assess the G × E interactions for TA content, and to investigate the relationships between ephedrine alkaloids content and stems-DW.

Harvest and Processing

Harvest and processing methods were also performed as described in Experiment 1. The terrestrial stems of the four clonal lines (Lines 26–29) were harvested on July 11, 2018 at Ibaraki, July 3, 2018 at Shizuoka, and July 18, 2018 at Yamanashi.

Determination of the Content of Ephedrine Alkaloids

Quantitative analysis of the ephedrine alkaloids [(−)-Eph and (+)-PEph] was performed using an HPLC system (LC-10ADvp; Shimadzu Corp., Kyoto, Japan) as described in Experiment 1.

Statistical Analysis

Two-way ANOVA was performed for the contents of Eph, PEph, and TA using R software (version 4.1.0).

The magnitude of the effect of genotype, locations, and G × E interactions on ephedrine alkaloids content were estimated using two-way ANOVA. The factors of the G × E interactions were genotype (G), and location (L) with their interaction effects. σ_g², σ_l², and σ_gl² are the components of variance for genotype (G), location (L), and G × L.¹²⁾ The number of effective replications for the estimation of σ_g², σ_l² and σ_gl² was calculated as described by Experiment 1. These variance components were estimated using mean squares.

RESULTS

Experiment 1. Evaluation of Heritability on Ephedrine Alkaloids Content

Figure 2 illustrated the variations in the ephedrine alkaloids content in 25 clonal lines. Table 1 showed the results of the one-way ANOVA for the content of ephedrine alkaloids, and the results of the components of variance and the broad-sense heritability, which were used to evaluate the selective effects on the content of ephedrine alkaloids in the plants.

Fig. 2. The Content of Ephedrine (Eph), Pseudoephedrine (PEph), and Total Alkaloids (TA) in 25 Clonal Lines (Lines 1–25) Cultivated in Ibaraki

The clonal lines were arranged by high rank of each mean value. The arrows indicate the differences in the content of each component within a clonal line.

Table 1. One-Way ANOVA for the Content of Ephedrine Alkaloids in 25 Clonal Lines (Lines 1–25) Cultivated in Ibaraki

Traits	Factors	Df	Mean Sq	F value	P (>F)	σg2	σe2	r	h2
Eph	Geneotype (G)	24	0.18	29.9	<2.0E-16**	0.040	0.006	4.28	0.871
	Residuals	83	0.0059
PEph	Geneotype (G)	24	0.089	136.9	<2.0E-16**	0.021	0.001	4.28	0.969
	Residuals	83	0.0007
TA	Geneotype (G)	24	0.24	28.4	<2.0E-16**	0.055	0.009	4.28	0.865
	Residuals	83	0.0086

Df: degree of freedom; Mean Sq: mean square; σ_g²: the component of variance for genotype; σ_e²: the component of variance for environment. r: effective replication; h²: broad-sense heritability. Eph: ephedrine content; PEph: pseudoephedrine content; TA: total alkaloids (sum of Eph and PEph content). G: genotype. ** Probability value for the test of significance <0.01, respectively.

Figure 2 showed that the variations in the contents of Eph, PEph, and TA among clonal lines were large. The content of each component (Eph, PEph, and TA) in clones ranged from 0.49 to 1.70% DW, 0.00 to 0.49% DW and 0.64 to 1.67% DW, respectively. In contrast, the variations in the content of the components among clones in each clonal line were small (Fig. 2); that is, the differences in the content of each component (Eph, PEph, and TA) within each clonal line ranged from 0.02 to 0.30, 0.00 to 0.14, and 0.02 to 0.40, respectively.

The results of one-way ANOVA for the Eph, PEph, and TA contents showed that significant effects of G (Table 1). The component of variance for G (σ_g²) was the higher than that for E (σ_e²) in Eph, PEph, and TA contents (Table 1). The broad-sense heritability for the Eph, PEph, and TA contents was estimated to be high at 0.871, 0.969, and 0.865, respectively (Table 1).

Figure 3 illustrates the relationship between the ephedrine alkaloids content and stems-DW of clones in Ibaraki. The correlation coefficients between the stems-DW and the contents of Eph, PEph, and TA were −0.16, −0.01 and −0.16, respectively (Fig. 3). These correlation coefficients were low, and there were no significant differences.

Fig. 3. Relationship between the Content of Ephedrine Alkaloids and Stems Dry Weight of Clones in 25 Clonal Lines (Lines 1–25) Cultivated in Ibaraki

Eph: ephedrine content; PEph: pseudoephedrine content; TA: total alkaloids (sum of Eph and PEph content); DW: stems dry weight. Data are presented as mean value.

Experiment 2. Evaluation of Effects of Macro-Environmental Factors on Ephedrine Alkaloids Content under Different Cultivation Locations

Table 2 showed the results of the two-way ANOVA for the contents of ephedrine alkaloids (Eph, PEph, and TA), as well as the results of the components of variance. Figure 4 showed that the interaction plots between genotypes and locations for contents of Eph, PEph and TA.

Table 2. Two-Way ANOVA for the Content of Ephedrine Alkaloids in E. sinica Clonal Lines (Lines 26–29) Cultivated in Ibaraki, Shizuoka, and Yamanashi Prefectures

Traits	Factors	Df	Mean Sq	F value	P (> F)	σg2	σl2	σgl2	r
Eph	G	3	1.344	77.3	1.6-E15**	0.170	0.000	0.009	3.80
	L	2	0.028	1.6	0.212
	G × L	6	0.051	2.9	0.020*
	Residuals	35	0.017
PEph	G	3	0.489	109.8	<2.0E-16**	0.064	0.006	0.000	3.80
	L	2	0.069	15.4	1.6-E05**
	G × L	6	0.003	0.7	0.627
	Residuals	35	0.005
TA	G	3	2.579	75.2	2.5E-15**	0.330	0.005	0.009	3.80
	L	2	0.125	3.7	0.036*
	G × L	6	0.070	2.1	0.085
	Residuals	35	0.034

Df: degree of freedom; Mean Sq: mean square; r: effective replication. Eph: ephedrine content; PEph: pseudoephedrine content; TA: total alkaloids (sum of the Eph and PEph contents). G: genotype; L: location. *, ** Probability value for the test of significance <0.05 and <0.01, respectively.

Fig. 4. G × L Interaction Plot for the Content of Ephedrine Alkaloids in Four Clonal Lines Cultivated in 3 Locations (Ibaraki, Shizuoka, and Yamanashi Prefectures)

Circle, square, triangle, and rhombus indicate clonal lines, Lines 26, 27, 28, and 29, respectively.

In Eph-content, the results of the two-way ANOVA showed that although there were significant effects of genotype (G), G × location (L) interaction (p < 0.05), the component of variance for G (σ_g²) (0.170) was the higher than that for L (σ_l²) (0.000) and G × L (σ_gl²) (0.009) (Table 2). In particular, the clonal lines with higher Eph-content were common among all three locations, e.g., Line 27 (Fig. 4). Similarly, the clonal lines with lower Eph-content were common among all three locations, e.g., Line 29.

In PEph-content, the results of two-way ANOVA showed that although there were significant effects of G, and L (p < 0.05), the component of variance for G (σ_g²) (0.064) was higher than that for L (σ_l²) (0.006) and G × L (σ_gl²) (0.000) (Table 2). In particular, the clonal lines with higher PEph-content were common among all three locations, e.g., Line 27 (Fig. 4). Similarly, the clonal line with lower PEph-content were common among all three locations, e.g., Lines 26 and 28.

In the TA content, the results of the two-way ANOVA results showed that although there were significant effects of G, and L (p < 0.05), the component of variance for G (σ_g²) (0.330) was also higher than that for L (σ_l²) (0.005) and G × L (σ_gl²) (0.009) (Table 2). In particular, the clonal line with higher TA content was common among all three locations, e.g., Line 27 (Fig. 4). Similarly, the clonal line with lower TA content was common among all three locations, e.g., Lines 28 and 29.

DISCUSSION

In this study, we evaluated the influence of genetic and environmental factors on the ephedrine alkaloids content in E. sinica plants by performing two experiments (Experiment 1 and Experiment 2) using clonal lines.

In Experiment 1, the range of ephedrine alkaloids content between clonal lines was large, whereas the range of ephedrine alkaloids content among clones in each line was small (Fig. 2). The broad-sense heritabilities for the contents of Eph, PEph, and TA in E. sinica were also high (Table 1), suggesting that the contents of the ephedrine alkaloids in the plants are highly influenced by genetic factors. Our previous transplantation experiment¹⁴⁾ showed that the selection of high ephedrine alkaloids content in E. sinica grown in different locations in Japan is valid and supports the results of the present study. Additionally, our result indicated that it is possible to select E. sinica plants with high contents of both Eph and PEph (i.e., Lines 18 and 24) (Fig. 2). Moreover, the correlations between the ephedrine alkaloids content and stems-DW were low (Fig. 3). Therefore, E. sinica plants with high ephedrine alkaloids content could be selected irrespective of stems-DW.

In general, for the breeding of clonal plants, such as E. sinica, the magnitude of their genetic variation must be estimated, and selection breeding strategies for clonal plants require the exclusion of environmental variance in individual selection. Thus, effective breeding can be achieved by selecting genets with a higher ephedrine alkaloids content, even if the selected genets carry both dominant and additional genetic components. For example, in grass and sugarcane, the broad-sense heritability of breeding target traits estimated in these plants showed that effective breeding can be achieved by selecting the genets of these plants.^19,20) In addition, the broad-sense heritability of alkaloid content in many plant species has commonly been shown to be high.^21–24) This suggests that selection breeding could be an effective way to achieve stable production of Ephedra herb with a high ephedrine alkaloids content that is suitable for medicinal use.

In Experiment 2, the results of the two-way ANOVA showed that although G, L, and G × E interactions had significant effects on the content of each ephedrine alkaloids (Eph, PEph, and TA) (p < 0.05), the components of variance for G (σ_g²) were much higher than the other variances (Table 2). These results indicate that the environmental effects on the ephedrine alkaloids content are relatively small and that genotypic effects strongly affect the ephedrine alkaloids content under various macro-environments. Previous studies reported that in habitats, both a higher soil pH and lower precipitation tended to increase the ephedrine alkaloids content of Ephedra plants,^5,8) while Mikage et al.²⁵⁾ recently showed that there were no correlations between the amount of precipitation or soil pH and the content of ephedrine alkaloids of E. sinica clonal lines in Wagner pot cultivation experiments. In our study, the ephedrine alkaloids content did not tend to be higher at higher soil pH or under lower annual rainfall (Supplementary Table 2, Fig. 4). Our results consistent with the recent study.²⁵⁾ Additionally, our results indicate that, regardless of the cultivation location, the content of ephedrine alkaloids remains stable. Therefore, our results suggest that the ephedrine alkaloids content in selected E. sinica clonal lines can remain stable under different macro-environments. In contrast, the variations in stems-DW of each clonal line were large among the three cultivation locations (Supplementary Fig. 2). Moreover, the correlations between the ephedrine alkaloids content and stems-DW were low among the three locations (Supplementary Fig. 2). These results suggest that the ability to produce ephedrine alkaloids in E. sinica plants was not affected by their growth conditions.

From our findings, selection and clonal propagation could be an effective strategy for breeding of E. sinica with higher and stable ephedrine alkaloids content. However, our selection breeding framework is time consuming, with considerable time being required to grow the plant material for selection, select the plants, and propagate a large number of clones. Recent studies have revealed the presence of genes that encode biosynthetic enzymes for ephedrine alkaloids,^26–28) and transcriptome analyses of terrestrial stems and roots of E. sinica have also been performed.^29,30) Considering our genetic analysis in this study, these results suggest that molecular marker selection could be an alternative and effective approach for shortening the time required to select plants with a high content of ephedrine alkaloids. Moreover, although vegetative propagation using stolons is an easy method for clone propagation (Fig. 1), the number of clones obtained from the selected plants via stolon propagation is small. In Experiment 1, the number of biological replicates of clonal lines was partially limited and small. Recently, several other vegetative propagation methods for Ephedra plants have been reported, including cuttings^31–34) and in vitro propagation.³⁵⁾ These propagation methods are intended for application to the large-scale propagation of Ephedra plants. Effective breeding can be performed more rapidly if large-scale propagation and molecular marker selection are established.

In conclusion, our study demonstrates that the ephedrine alkaloids content in E. sinica plants is predominantly governed by genetic factors, and that the content of ephedrine alkaloids remains stable even under different environmental conditions. Therefore, it is possible to develop cultivars of E. sinica with high and stable ephedrine alkaloids content by selection breeding.

Acknowledgments

We are grateful to Dr. Bunsho Makino for technical support in analysis and his helpful suggestions. We thank Ms. Terue Kurosawa, Ms. Akiko Uetake, and Mr. Mikio Sakai for their assistance with the sampling and sample processing. We also thank Mr. Hideyuki Akahori and Mr. Hisashi Sonehara for their assistance with cultivation. We also thank Ms. Miki Sakurai and Dr. Takahiro Tsusaka for their helpful suggestions.

Conflict of Interest

This study was funded by Tsumura & Co. (https://www.tsumura.co.jp/english/). The funder provided support in the form of a salary for Hajime Hiyama. Yosuke Yoshioka and Ryo Ohsawa are supervisors at the University of Tsukuba, to which Hajime Hiyama belongs, and there was no funding from Tsumura & Co.

Supplementary Materials

This article contains supplementary materials.

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