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

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.

A Modified 1H-NMR Quantification Method of Ephedrine Alkaloids in Ephedrae Herba Samples.

A previous 1H-NMR method allowed the quantification of ephedrine alkaloids; however, there were some disadvantages. The cyclized derivatives resulted from the impurities of diethyl ether were identified and benzene was selected as the better extraction solvent. The locations of ephedrine alkaloids were confirmed with 2D NMR. Therefore, a specific 1H-NMR method has been modified for the quantification of ephedrine alkaloids. Accordingly, twenty Ephedrae Herba samples could be classified into three classes: (I) E. sinica-like species; (II) E. intermedia-like species; (III) others (lower alkaloid contents). The results indicated that ephedrine and pseudoephedrine are the major alkaloids in Ephedra plants, but the concentrations vary greatly determined by the plant species and the collection locations.

Ephedrine Alkaloid-Independent High-Affinity Immunoglobulin-E Receptor (FcεRI) Internalization Results in CCL2 Production without Inducing Mast Cell Degranulation.

Mast cells (MCs) play an important role in allergies, leading to the development of MC-targeted therapies. Ephedra herb (Mao) has potent anti-allergic activity, but contains ephedrine alkaloids (EAs); therefore, its hazardous effects are taken into consideration during its clinical use. We previously reported that Mao attenuates robust MC degranulation by an allergen through high-affinity immunoglobulin E (IgE) receptor (FcεRI) internalization, in which an EA-independent mechanism was suggested to be at play. This study aimed to deepen our understanding of the potential of Mao against FcεRI internalization using two strains with different EA contents. Mao extracts were administered to bone marrow-derived MCs (BMMCs), and their cellular responses, including FcεRI internalization, were analyzed. In addition, physiological events were evaluated using a passive cutaneous anaphylactic (PCA) reaction mouse model. BMMCs mediate the production of diverse inflammatory mediators. Among these, the potent chemokine CCL2 is thought to be differentially regulated from other pro-inflammatory mediators. We found that Mao significantly induces CCL2 expression in BMMCs despite suppressing robust degranulation through FcεRI internalization. Importantly, this was a distinctly EAs-independent response. In the PCA reaction, local MC activation following allergen challenge was suppressed by Mao treatment, which strengthened the view that Mao sufficiently decreased the rapid activation of MCs and promoted CCL2 secretion. Collectively, these observations provide additional insights into the mechanism of Mao-induced silent FcεRI internalization in MCs and the complex and heterogeneous secretory responses operating in MCs.

Ecological and pharmacognostical characteristics in Ephedra saxatilis in the Yarlung Zangbo River Valley of southeastern Tibetan Plateau, China.

In the Yarlung Zangbo River Valley of the southeastern Tibetan Plateau, China (29°07'49.5"N, 92°41'11.0"E, 3256 m above sea level), we found an Ephedra saxatilis community in the xeric steppe with shrubland vegetation habitat of the broad alluvial plain of the river with soil having relatively higher water-soluble cation (Ca2+, 8.62; K+, 1.94; Mg2+, 2.38 mmol/100 g dry soil weight) and nitrogen (NO3-, 21.78; NH4+, 1.82 mmol/100 g dry soil weight) content. The ranges of ephedrine and pseudoephedrine in 13 E. saxatilis samples were as follows: ephedrine, not detected-3.03 of dry weight (%DW) and pseudoephedrine, not detected-1.36%DW. The 13 E. saxatilis plants collected in the study area showed intraspecific variability of ephedrine and pseudoephedrine with 6 samples containing ephedrine and pseudoephedrine, 6 samples containing only ephedrine, and 1 sample containing only pseudoephedrine.

Overlooked switch from transient sedation to sustained excitement in the Biphasic effects of Ephedra Herb extract administered orally to mice.

ETHNOPHARMACOLOGICAL RELEVANCE: In our previous study, we reported that Ephedra Herb extract (EHE) increased the locomotor activity of mice in the open-field test and reduced the immobility time in the forced swim test. Ephedrine alkaloids (EAs) are thought to be responsible for the adverse effects of Ephedra Herb. However, there are no reports to verify that the adverse effects of Ephedra Herb are caused by the amount of EAs in the herb. Therefore, we investigated whether these adverse effects of EHE are caused by the amounts of ephedrine (Eph) and pseudoephedrine (Pse) in the herbal extract. In a preliminary study of the time course analysis of the open field test, we newly observed that EHE evoked switching from transient sedation to sustained excitement. AIM OF THE STUDY: We aimed to confirm whether EHE evokes switching from transient sedation to sustained excitement, investigate whether these actions of EHE are caused by the amount of ephedrine (Eph) and pseudoephedrine (Pse) in the herbal extract, and clarify the molecular mechanism of the transient sedative effect. MATERIALS AND METHODS: The locomotor activity of mice was tested using the open-field test. The immobility times were measured using a forced swim test, and the motor dysfunction in mice was tested using the rotarod test. RESULTS: EHE, Eph, and Pse induced transient motionlessness between 0 and 15 min after oral administration, however, they did not induce depression-like behavior and motor dysfunction in mice, suggesting that the motionlessness induced by EHE, Eph, or Pse resulted from sedation. The α2a adrenoceptor inhibitor, atipamezole, decreased their sedative effects. Thus, immediately after EHE administration, the transient sedative effect is mediated through the activation of the α2a adrenoreceptor by Eph and Pse. EHE and Eph increased total locomotor activity for 15-120 min after oral administration; however, Pse had no effect. Therefore, the slow-onset and sustained excitatory effects of EHE are mediated by Eph. CONCLUSIONS: We discovered for the first time that EHE evokes diphasic action by switching from transient sedation to sustained excitement. The transient sedation was evoked by the Eph and Pse in the herbal extract via activation of the α2a adrenoceptor and the sustained excitement was caused by the Eph in the herbal extract.

Relationship between ephedrine alkaloid profile in Ephedra gerardiana and soil characteristics of glacial landforms in southeastern Tibetan Plateau, China.

In the Kaluxung River catchment of the southeastern Tibetan Plateau in China, we identified three Ephedra gerardiana communities on different soils and glacial landforms from 4842 to 4899 m above sea level: a moraine community located on constantly collapsing sandy gravel alpine steppe slopes with exposed bedrock on the outer slope of the terminal moraine of the Qiangyong Glacier on Mt. Kaluxung; an outwash plain community located on a gentle alpine steppe slope with exposed bedrock at the terminal end of the outwash plain in the glacial valley of the southeast side of Mt. Noijinkangsang; and a river terrace community located in an alpine meadow on a rock-scattered flat river terrace along a glacier-fed river in the outwash plain in the glacial valley of the southeast side of Mt. Noijinkangsang. Based on the finding of identical DNA sequences of the intergenic spacers of chloroplast trnT-trnF and trnS-trnfM regions for all Ephedra specimens examined in this study, the E. gerardiana in this study were considered to comprise a genetically homogeneous population. Analysis of the relationship between ephedrine alkaloid profiles of these three communities and soil characteristics showed that the river terrace community in wet alpine meadow had significantly lower ephedrine content than did the moraine and outwash plain communities in dry alpine steppe (moraine community, 1.52 ± 0.44; outwash plain community, 1.42 ± 0.68; river terrace community, 0.33 ± 0.65%DW), but pseudoephedrine content showed the reverse pattern (moraine community, 0.86 ± 0.30; outwash plain community, 0.73 ± 0.60; river terrace community, 1.50 ± 0.71%DW). In addition, total alkaloid (ephedrine and pseudoephedrine) content in the river terrace community (1.83 ± 0.24%DW) was significantly lower than that in the moraine community (2.38 ± 0.64%DW) and outwash plain community (2.15 ± 0.55%DW).

Stability and Reproducibility of Ephedra sinica Ephedrine Alkaloid Content and Terrestrial Stem Dry Weight.

Dried terrestrial stems of Ephedra sinica are known as 'Ephedra herb.' The pharmacological effects are mainly related to two major ingredients, (-)-ephedrine and (+)-pseudoephedrine (total alkaloids which are defined in Japanese Pharmacopoeia, TA). In this study, in order to aid in cultivation and breeding, the stability of TA content and stem dry weight of 46 E. sinica genets was evaluated from the first year of transplantation to the sixth year. TA content and composition ratio of these genets were stable after the second year, and dry weight was stable after the fourth year. These traits showed high inter-genet variability but low annual variability for each genet. Additionally, rank correlation coefficients of each trait among the genets were high. There was no significant correlation between these traits. Furthermore, to assess the reproducibility of these traits in clones, we evaluated TA content and dry weight of three clonal lines with high TA contents. TA content and composition ratio of the clonal lines were also stable after the second year of transplantation, and dry weight of the clonal lines was also stable after the fourth year. Moreover, TA content and composition ratio in each clonal line were comparable with those of each original genet after the second year. These results suggested that ephedrine alkaloids content and dry weight of E. sinica plants are stable, and that these traits are highly reproducible in clones. Therefore, selection breeding of E. sinica using vegetative propagation can be effective for high and stable quality of Ephedra herb.

Environmental and soil characteristics in Ephedra habitats of Uzbekistan.

In Uzbekistan, Ephedra distachya L., E. equisetina Bunge, E. foliata Boiss. ex C. A. Mey., E. lomatolepis Schrenk, and E. strobilacea Bunge show species specificity for habitat environments and physical and chemical characteristics of habitat soils. Furthermore, the relationship between soil characteristics and ephedrine and pseudoephedrine contents was examined. E. distachya was found growing from 80 to 200 m above sea level (a.s.l) in the Plateau Ustyurt on the desert steppe of cliffs on soil having relatively higher loss on ignition (19.8-33.8%) and water-soluble cations (Ca2+, 5.14-133.13; Mg2+, 0.85-3.18; and Na+, 2.27-8.33 mmol/100 g dry soil weight) than for other Ephedra habitats. E. strobilacea was found growing on the flat sandy Kyzylkum desert at 94 m a.s.l. and had habitat soil that was the driest with the lowest loss on ignition (2.9%) and highest Na+ (9.05 mmol/100 g dry soil weight) of all the Ephedra habitat soils. On dry steppe from 1054 to 1819 m a.s.l., E. foliata, E. lomatolepis, and E. equisetina formed not only a single community but also a complex community on constantly collapsing sandy gravel slope with relatively higher Ca2+ (3.40-17.44 mmol/100 g dry soil weight) soil content. Notably, E. equisetina grew on the dry steppe of constantly collapsing sandy gravel slopes, in rocky areas, on sandy gravel floodplains of rivers, and on stable humus soil at the base of coniferous trees in a wide range of habitats from dry steppe to coniferous forest zones at altitudes ranging from 1392 to 1819 m a.s.l., as reflected in the greater variability than for other Ephedra habitats in the parameters of loss on ignition (1.4-34.8%), pH (7.1-9.6), NO3- (0.08-35.17 mmol/100 g dry soil weight), Ca2+ (0.24-17.44 mmol/100 g dry soil weight), Mg2+ (not detected-1.25 mmol/100 g dry soil weight), and Na+ (0.13-5.19 mmol/100 g dry soil weight). Ephedrine alkaloids were not detectable in E. strobilacea, E. foliata, and E. lomatolepis. Almost all E. distachya contained only pseudoephedrine (1.25-1.59% of dry weight, %DW), while E. equisetina contained from 1.31 to 2.05%DW ephedrine and from 1.29 to 2.80%DW pseudoephedrine. Ephedrine and pseudoephedrine in E. equisetina showed a statistically significant negative correlation with soil Cl- and Mg2+, respectively.

[The Adverse Effects of Ephedra Herb and the Safety of Ephedrine Alkaloids-free Ephedra Herb Extract (EFE)].

Ephedra Herb is defined in the 17th edition of the Japanese Pharmacopoeia (JP) as the terrestrial stem of Ephedra sinica Stapf., Ephedra intermedia Schrenk et C.A. Meyer, or Ephedra equisetina Bunge (Ephedraceae). The stems of Ephedra Herb contain greater than 0.7% ephedrine alkaloids (ephedrine and pseudoephedrine). Despite its high effectiveness, Ephedra Herb exert several adverse effects, including palpitation, excitation, insomnia, and dysuria. Both the primary and adverse effects of Ephedra Herb have been traditionally believed to be mediated by these ephedrine alkaloids. However, our study found that several pharmacological actions of Ephedra Herb were not associated with ephedrine alkaloids. We prepared an ephedrine alkaloid-free Ephedra Herb extract (EFE) by eliminating ephedrine alkaloids from Ephedra Herb extract (EHE) using ion-exchange column chromatography. EFE exerted analgesic, anti-influenza, and anticancer activities in the same manner as EHE. Moreover, EFE did not induce adverse effects due to ephedrine alkaloids, such as excitation, insomnia, and arrhythmias, and showed no toxicity. Furthermore, we evaluated the safety of EFE in healthy volunteers. The number of adverse event cases was higher in the EHE-treated group than in the EFE-treated group, although the difference was not significant. Our evidence suggested that EFE was safer than EHE.

Ephedrine Alkaloids-Free Ephedra Herb Extract, EFE, Has No Adverse Effects Such as Excitation, Insomnia, and Arrhythmias.

Ephedrine alkaloids-free Ephedra Herb extract (EFE) has been developed to eliminate the adverse effects caused by ephedrine alkaloid-induced sympathetic hyperactivation. Previously, we reported that EFE possesses analgesic, anti-influenza, and cancer metastatic inhibitory effects at comparable levels to that of Ephedra Herb extract (EHE). However, it has not yet been demonstrated that EFE is free from the known side effects of EHE, such as excitation, insomnia, and arrhythmias. In this study, the incidence of these adverse effects was compared between mice administered EHE and those administered EFE. Increased locomotor activity in an open-field test, reduced immobility times in a forced swim test, and reduced sleep times in a pentobarbital-induced sleep test were observed in EHE-treated mice, when compared to the corresponding values in vehicle-treated mice. In contrast, EFE had no obvious effects in these tests. In electrocardiograms, atrial fibrillation (i.e., irregular heart rhythm, absence of P waves, and appearance of f waves) was observed in the EHE-treated mice. It was suggested that this atrial fibrillation was induced by stimulation of adrenaline ß1 receptors, but not by hypokalemia. However, EFE did not affect cardiac electrophysiology. These results suggest that the abovementioned side effects are caused by ephedrine alkaloids in EHE, and that EFE is free from these adverse effects, such as excitation, insomnia, and arrhythmias. Thus, EFE is a promising new botanical drug with few adverse effects.