Comparison of Non-ephedrine Constituents from Ephedra Plants Cultivated in Japan

Abstract

Ephedra plants, the main components of which are ephedrine alkaloids, are used as traditional medicines in Eastern Asian countries. In this study, we isolated non-ephedrine constituents from various Ephedra plant species cultivated in Japan. HPLC analysis suggested that kynurenic acid and its derivatives accumulated in a wide range of Ephedra plant species. Furthermore, a large amount of (2R,3S)-O-benzoyl isocitrate has been isolated from E. intermedia. This study suggests that Ephedra plants have diverse non-ephedrine constituents.

Introduction

Ephedra plants (Ephedraceae) are used as traditional medicines in Eastern Asian countries, including Japan.^1,2) Ephedra herbs have been used as an important medicine for the treatment of asthma, fever, and nasal congestion.^2,3) The major ingredients contained in Ephedra plants are ephedrine alkaloids (EAs), such as ephedrine (Ep) and pseudoephedrine (PEp), which accumulate in herbal stems. The amounts of EAs in Ephedra are one of the criteria for medicinal usage in the Japanese Pharmacopoeia 18th edition (JP18).⁴⁾ However, EAs have side effects, such as elevated blood pressure, agitation, and dysuria.⁵⁾ Attenuation of these side effects is keenly desired for the medicinal use of Ephedra plant extracts.^6,7)

The accumulation of various non-ephedrine constituents has been observed in Ephedra plant species.^8,9) Flavonoids, catechins, polyphenols, and kynurenic acid (KA) has been isolated from various Ephedra plant species.^10–13) Recently, some reports have shown that non-ephedrine constituents of Ephedra herb extracts have potential antiviral activity.^14,15) Thus, non-ephedrine constituents in Ephedra plants could be complementary to EAs for the medicinal use of Ephedra plant extracts without side effects.

In this study, we isolated species-characteristic non-ephedrine constituents from Ephedra plants cultivated in Japan. Significantly, we isolated a novel metabolite, (2R,3S)-O-benzoyl isocitrate, which has never been observed in the plant kingdom except in Lemna paucicostata 151 under benzoic acid-feeding conditions.¹⁶⁾

Results and Discussion

We identified three Ephedra plants that were cultivated at the Nippon Shinyaku Yamashina Botanical Research Institute (Table 1). The first Ephedra plant (5880), introduced from Moscow as E. gerardiana Wall. ex Stpf, was identified as E. intermedia Schrenk et C. A. Mey. based on its morphological features, EAs content and genotypic analysis of the nuclear internal transcribed spacer 1 (ITS1) region. A larger amount of PEp accumulated in Ephedra plant 5880 than in Ep, that is a characteristic feature of E. intermedia.¹⁷⁾ Moreover, sequence analysis of ITS1 region of Ephedra plant 5880 was identical to that of E. intermedia (AY755758). The second Ephedra plant (5879), introduced from Moscow as E. fragilis Desf., was identified as E. foeminea Forssk. based on its morphological features and genotypic analysis of ITS1. Ephedra plant 5879 did not produce EAs and exhibited vine morphology, which are characteristic features of E. foeminea.^9,18) Sequence analysis of ITS1 region of Ephedra plant 5879 was identical to that of E. foeminea (GU968546). The third Ephedra plant (30746), introduced from Peru, was identified as E. americana Humb. Et Bonpl. ex Willd. based on its morphological features and genotypic analysis of ITS1. In some previous studies, E. americana was collected from South America and produce very small amount of EAs.^19,20) Sequence analysis of ITS1 region of Ephedra plant 30746 was identical to that of E. americana (GU968545).

Table 1. Species of Ephedra Plants Cultivated at Nippon Shinyaku Yamashina Botanical Research Institute

Number	Species	Derivation	Ep content (% of dry weight)	PEp content (% of dry weight)	Ep/(EP + PEp)	ITS1
5880	E. intermedia	Moscow (1965)	0.62	1.03	0.37	LC790072
5879	E. foeminea	Moscow (1965)	0.00	0.00	—	LC790074
30746	E. americana	Peru (2003)	0.00	0.00	—	LC790073

To isolate the non-ephedrine constituents, we first analyzed E. intermedia extracts using HPLC (Fig. 1A). A characterized compound (1) was detected at 6.3 min in E. intermedia extracts. Compound 1 was purified from E. intermedia (100 g) and obtained as a colorless solid (70.6 mg). High-resolution MS (HRMS) analysis of 1 showed an ion [M − H]⁻ at m/z 295.0446, which corresponds to the molecular formula C₁₃H₁₁O₈. In the NMR analysis, ¹H-NMR spectrum of 1 was identical to that of O-benzoyl isocitrate¹⁶⁾ (1, Fig. 1B). 1 was not isolated from the plant kingdom, except from Lemna paucicostata 151 under benzoic acid-feeding conditions.¹⁶⁾

Fig. 1. A) Chromatogram of Aqueous 80% MeOH Extract from E. intermedia

A chromatogram was presented with intensity of 254 nm. B) Chemical structure of (2R,3S)-O-benzoyl isocitrate (1). C) Synthetic scheme for tert-butyl isocitrate (2).

Since endogenous (2R,3S)-isocitrate derived from the tricarboxylic acid cycle (TCA cycle) was used as a precursor of isocitrate-derivatives, some isocitrate-conjugated natural products in plants and fungi also had the same configuration.^21–23) To confirm the absolute stereochemistry of 1, we performed derivatization of 1 to a reported (2R,3S)-tert-butyl isocitrate (2, Fig. 1C) and following the comparison of NMR spectra and optical rotation.²⁴⁾ Esterification of 1 by O-tert-butyl-N,N′-diisopropylisourea and hydrolysis under basic conditions afforded 2²⁴⁾ (Fig. 1C). The NMR spectra and specific rotation of 2 were identical to those reported (2R,3S)-tert-butyl isocitrate (Table 2). Therefore, 1 was identified as (2R,3S)-O-benzoyl isocitrate.

Table 2. Comparison of NMR Spectra and Specific Rotation Data for 2

	Synthetic 2	Reported 2*
1H-NMR (δH)	4.16 (dd, 1H)	4.14 (d, 1H)
	3.27 (ddd, 1H)	3.26 (ddd, 1H)
	3.15 (d, 1H)	3.14 (d, 1H)
	2.71 (dd, 1H)	2.70 (dd, 1H)
	2.47 (dd, 1H)	2.46 (dd, 1H)
	1.48 (s, 9H)	1.50 (s, 9H)
	1.43 (s, 9H)	1.45 (s, 9H)
	1.42 (s, 9H)	1.43 (s, 9H)
13C-NMR (δC)	172.4	172.5
	171.2	171.2
	170.3	170.3
	83.2	83.2
	81.8	81.8
	80.9	80.9
	70.9	71
	45.9	45.9
	34.0	34.0
	28.1 (9C)	28.2
[α]D	+7.3 (c 0.86, CH2Cl2)	+7.6 (c 0.67, CH2Cl2)

*S. Tsegay et al., Aust. J. Chem. (2009)

Next, we performed HPLC analyses of E. foeminea and E. americana extracts to isolate non-ephedrine constituents (Figs. 2B, C). The HPLC chromatogram of E. foeminea showed two characterized compounds (3 and 4), and E. americana extracts contained one characterized compound (5). Compound 3 was identified to be KA (Fig. 2I) by direct comparison with an authentic standard. To identify 4 and 5, we purified these compounds from water extracts of the dried herbal stems of E. foeminea and E. americana. The structures of isolated 4 and 5 were determined by comparison with reported values for 6-hydroxykynurenic acid and 6-methoxykynurenic acid, respectively^6,12,25) (Fig. 2I). KA derivatives are abundant in Ephedra plants.⁹⁾

Fig. 2. A–H) Chromatogram Comparison of Aqueous 80% MeOH Extracts from Ephedra Plants

Chromatograms were presented at the intensity of 254 nm. I) Chemical structures of kynurenic acid (3), 6-hydoxykynurenic acid (4), and 6-methoxykynurenic acid (5).

Finally, we quantified the characterized compounds (1, 3, 4, and 5) in Ephedra plants cultivated in Japan and commercially available E. sinica (Figs. 2A–H, Table 3). 11E08-1 and 12E37-1, which are progenies of wild E. intermedia and E. equisetina Bunge collected from the Zarashan Mountains of Tajikistan, respectively.¹⁷⁾ 1 was mainly accumulated in E. intermedia and E. equisetina. 3 and 4 were mainly accumulated in E. intermedia and E. foeminea. Large amounts of 5 were detected in E. americana. In contrast, very small amounts of these compounds were detected in E. sinica used in this study.

Table 3. Quantification of the Characterized Compounds in Ephedra Plants Cultivated in Japan and Commercially Available E. sinica

Species	Content (% of dry weight)
	1	3	4	5
E. intermedia 5880	2.5	0.023	0.075	n.i.
E. foeminea 5879	n.d.	0.20	0.10	n.i.
E. americana 30746	n.d.	n.i.	n.d.	0.31
E. sinica IMU-ESI*	n.i.	n.i.	n.i.	n.i.
E. intermedia 11E08-1*	3.2	n.i.	n.i.	n.d.
E. equisetina 12E37-1*	1.3	n.d.	<0.01	n.d.
Ephedra sinica (Takasago) commercially available	<0.01	n.d.	<0.01	<0.01
Ephedra sinica (Tochimoto) commercially available	n.d.	<0.01	n.i.	<0.01
Wave length for quantification	275 nm	330 nm	350 nm	350 nm

Each value was calculated by three independent experiments. *H. Hayashi et al., Biol. Pharm. Bull. (2019); n.d.: Not Detected; n.i.: Not Identified.

The bioactivity of KA and its derivatives (3–5) has been suggested as a neuroactive compound that plays important roles in various brain diseases such as Alzheimer’s and Huntington’s.^26,27) Recently, some reports showed that 3–5 from Ephedra plants have an impact on antiviral activity against some viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2).¹⁵⁾ Therefore, 3–5 might be important non-EAs components in Ephedra plants. In the plant kingdom, O-caffeoyl-, p-coumaroyl-, and feruloyl-isocitrate have been identified in various plant species.^28–30) In particular, O-caffeoylisocitrate activates nuclear factor erythroid 2-related factor 2 (Nrf2), which is an important transcription factor for diabetic nephropathy therapy.³¹⁾ Since 1 and O-caffeoylisocitrate have similar structures, 1 could be a potential bioactive compound.

Conclusion

In this study, we identified a novel isocitrate derivative from E. intermedia. Our results suggest the diversity of non-ephedrine constituents derived from species and other conditions.

Experimental

General Information

NMR spectra were recorded on a JNM-ECZL-500 spectrometers (JEOL, Tokyo, Japan). HRMS analyses were conducted using a LCMS-IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan).

Plant Material

Ephedra plants (5880, 5879, and 30746) were collected at the Nippon Shinyaku Yamashina Botanical Research Institute, and were identified by Hiroaki Hayashi. Ephedra plants cultivated in the Herbal Garden of Iwate Medical University (IMU-ESI, 11E08-1, and 12E37-1), presented in Table 3, were grown outdoors for >3 years.¹⁷⁾ Commercially available Ephedra were purchased from Takasago Yakugyo Inc. (Lot No. 102212; Osaka, Japan) and Tochimoto Tenkaido Co., Ltd. (Lot No. 011721003; Osaka, Japan). Voucher specimens (RIN-240101, RIN-240102, and RIN-240103) of Ephedra plants used in this study were deposited at the Museum of Materia Medica, College of Pharmaceutical Sciences, Ritsumeikan University (Shiga, Japan). Quantification of EAs and ITS1 genotyping were performed as previously reported.¹⁷⁾ The nucleotide sequence data reported in this study are deposited in the GenBank under the accession numbers: LC790072 (E. intermedia), LC790073 (E. americana), and LC790074 (E. foeminea).

Metabolite Analyses of Ephedra Plant Extracts

Dried herbaceous stems from each Ephedra plant were powdered using grinding. Each powder (10.0 mg) was added to 1 mL of aqueous 80% MeOH and incubated at 60 °C for 2 h with shaking. Debris were removed by centrifugation (15000 × g, 4 °C, 10 min), and each supernatant was subjected to HPLC analysis. Operation of the HPLC systems and analysis of the output data files were performed using LabSolutions (Version 5.111, Shimadzu). The degasser, pump, photo diode array (PDA) detector, and system controller used were DGU-20A, LC-20AD, SPD-M40, and CBM-20A (Shimadzu), respectively. We used an Inertsil ODS-SP 3 µm (φ 2.1 × 100 mm; GL Sciences, Tokyo, Japan) for the analysis of compounds (the mobile phases: A, H₂O with 0.1% (v/v) formic acid; B, MeCN with 0.1% (v/v) formic acid: the gradient program: 0 to 30.0 min, linear gradient 10 to 100% B, with a flow rate of 0.2 mL/min).

Quantification of the Characterized Metabolites

The accumulated metabolites were quantified by HPLC analysis. The extraction and HPLC conditions were the same as described above. An authentic sample of kynurenic acid (3) was purchased from TCI Co. Ltd. (Tokyo, Japan). Isolated 1 was used as an authentic standard. Authentic standards of 4 and 5 were prepared as previously reported.^6,32)

Acknowledgments

We thank Dr. Yuto Nishidono (Ritsumeikan University) for his support in measuring the physical data of the isolated compounds.

Conflict of Interest

The authors declare no conflict of interest.

Supplementary Materials

This article contains supplementary materials.

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