Ephedrine Alkaloids-free Ephedra Herb Extract Research Articles

Antiviral Effect of Ephedrine Alkaloids-Free Ephedra Herb Extract against SARS-CoV-2 In Vitro

We report for the first time that ephedrine alkaloids-free Ephedra Herb extract (EFE) directly inhibits the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro and that the addition of EFE to the culture medium before viral infection reduces virus titers in the culture supernatant of SARS-CoV-2, including those of variant strains, by more than 99%, 24 h after infection. The addition of Ephedra Herb macromolecule condensed-tannin, which is the main active ingredient responsible for the anticancer, pain suppression, and anti-influenza effects of EFE, similarly suppressed virus production in the culture supernatant by 99% before infection and by more than 90% after infection. Since EFE does not have the side effects caused by ephedrine alkaloids, such as hypertension, palpitations, and insomnia, our results showed the potential of EFE as a safe therapeutic agent against coronavirus disease 2019.

Effect of Ephedra Herb on Erlotinib Resistance in c-Met-Overexpressing Non-Small-Cell Lung Cancer Cell Line, H1993, through Promotion of Endocytosis and Degradation of c-Met

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKIs) are used to treat non-small-cell lung cancer (NSCLC), harboring an EGFR-activating mutation. However, acquired resistance to these treatments emerges after a few years. One of causes of resistance to EGFR-TKIs is a high level of c-Met amplification or c-Met protein overexpression/hyperactivation. Therefore, combination therapy with EGFR-TKIs and a c-Met inhibitor is thought to be effective treatment for patients with NSCLC resistance carrying c-Met amplification and/or protein hyperactivation. Ephedra Herb is a crude drug and is used in Japan as a component in many Kampo formulae. We previously reported that Ephedra Herb extract (EHE) inhibits HGF-induced phosphorylation of c-Met by preventing c-Met tyrosine kinase activity. Thus, we investigated the combination effect of EHE and erlotinib, an EGFR-TKI, on growth of H1993 cells, an erlotinib-resistant NSCLC cell line with overexpression of c-Met. The EHE and erlotinib combination proved to be effective in suppression of the growth of H1993 xenograft tumors and on inhibition of proliferation of H1993 cells, suggesting that EHE is effective in rescuing NSCLC cells from erlotinib resistance. Moreover, EHE not only inhibited the phosphorylation of c-Met, but also downregulated the expression of c-Met by facilitating clathrin-mediated endocytosis and lysosomal degradation of c-Met. EHE also promoted downregulation of the expression of EGFR and phosphorylation of EGFR. Ephedrine alkaloids-free Ephedra Herb extract (EFE) had the same effects as EHE, and the 40% MeOH fraction from EFE, which mainly contained the high-molecular mass condensed tannins, decreased the expression levels of c-Met, pMet, EGFR, and pEGFR to almost the same level as EFE. These results suggest that recovery from resistance to erlotinib by EHE is derived from the high-molecular mass condensed tannins and that EHE may be suitable for treatment of c-Met-overexpressing NSCLC with resistance to EGFR-TKIs.

Quality Evaluation and Characterization of Fractions with Biological Activity from Ephedra Herb Extract and Ephedrine Alkaloids-Free Ephedra Herb Extract.

Previously, we reported that the c-Met inhibitory effect of Ephedra Herb extract (EHE) is derived from ingredients besides ephedrine alkaloids. Moreover, analgesic and anti-influenza activities of EHE and ephedrine alkaloids-free Ephedra Herb extract (EFE) have been reported recently. In this study, we examined the fractions containing c-Met kinase inhibitory activity from EHE and the fractions with analgesic and anti-influenza activities from EFE, and elucidated the structural characteristics of the active fractions. Significant c-Met kinase activity was observed in 30, 40, and 50% methanol (MeOH) eluate fractions obtained from water extract of EHE using Diaion HP-20 column chromatography. Similarly, 20 and 40% MeOH, and MeOH eluate fractions obtained from water extract of EFE were found to display analgesic and anti-influenza activities. Reversed phase-HPLC analysis of the active fractions commonly showed broad peaks characteristic of high-molecular mass condensed tannin. The active fractions were analyzed using 13C-NMR and decomposition reactions; the deduced structures of active components were high-molecular mass condensed tannins, which were mainly procyanidin B-type and partly procyanidin A-type, including pyrogallol- and catechol-type flavan 3-ols as extension and terminal units. HPLC and gel permeation chromatography (GPC) analyses estimated that the ratio of pyrogallol- and catechol-type was approximately 9 : 2, and the weight-average molecular weight based on the polystyrene standard was >45000. Furthermore, GPC-based analysis was proposed as the quality evaluation method for high-molecular mass condensed tannin in EHE and EFE.

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.

Analgesic Effects of Ephedra Herb Extract, Ephedrine Alkaloids-Free Ephedra Herb Extract, Ephedrine, and Pseudoephedrine on Formalin-Induced Pain.

The analgesic effect of Ephedra Herb (EH) is believed to be derived from the anti-inflammatory action of pseudoephedrine (Pse). We recently reported that ephedrine alkaloids-free EH extract (EFE) attenuates formalin-induced pain to the same level as that achieved by EH extract (EHE), which suggests that the analgesic effect of EH may not be due to ephedrine alkaloids (EAs). To examine the contribution of EAs to the analgesic effect of EH, mice were injected with formalin to induce a biphasic pain reaction (first phase, 0-5 min; second phase, 10-45 min) at various time points after oral administration of the following test drugs: ephedrine (Eph), Pse, "authentic" EHE from Tsumura & Co. (EHE-Ts), EFE, and EHE that was used as the source of EFE (EHE-To). Biphasic pain was suppressed at 30 min after administration of Eph, EHE-Ts, and EHE-To. At 6 h after administration of EFE, EHE-To, and Pse-and at 4 to 6 h after administration of EHE-Ts-only second-phase pain was suppressed; however, the effect of Pse at 6 h was not significant. These results suggested that EHE has a biphasic analgesic effect against biphasic formalin-induced pain: in the first phase of analgesia (30 min after administration), biphasic pain is suppressed by Eph; in the second phase of analgesia (4-6 h after administration), second-phase pain is alleviated by constituents other than EAs, although Pse may partially contribute to the relief of second-phase pain.

A Double-Blind, Randomized, Crossover Comparative Study for Evaluating the Clinical Safety of Ephedrine Alkaloids-Free Ephedra Herb Extract (EFE).

Ephedra Herb is an important crude drug; it is used in various Traditional Japanese Medicine (Kampo) formulations. Its significant pharmacological effects have been believed to be attributed to ephedrine and pseudoephedrine, which sometimes induce adverse effects. On the other hand, it has been reported that some of these pharmacological effects are not dependent on ephedrine or pseudoephedrine. Ephedrine alkaloids-free Ephedra Herb extract has been newly developed. It has been reported to have analgesic, anti-influenza, and antimetastatic effects. This clinical trial was aimed at verifying the noninferiority of EFE's safety compared to that of Ephedra Herb extract (EHE) in humans. This was a single-institution, double-blinded, randomized, two-drug, two-stage, crossover comparative study. Twelve healthy male subjects were equally and randomly allocated into two groups: prior administration of EFE (EFE-P) and prior administration of EHE (EHE-P). In Stage 1, EFE and EHE were orally administered to the EFE-P and EHE-P groups, respectively, for six days. After a 4-week washout period, Stage 2 was initiated wherein the subjects were given a study drug different from Stage 1 study drug for six days. Eleven adverse events with a causal relationship to the study drugs (EHE: 8; EFE: 3) were noted; all events were mild in severity. With regard to the incidence of adverse events, EHE and EFE administration, respectively, accounted for 4 cases (out of 12 subjects, similarly below) and 1 case of increased pulse rate (p=0.32) and 3 cases and 1 case of insomnia (p=0.59). Further, there was one case of hot flashes (p=1.00) due to EFE administration and one case of dysuria (p=1.00) due to EHE administration. There were no significant differences in the incidences of adverse events between EHE administration and EFE administration. Therefore, we concluded that EFE is not inferior to EHE in terms of safety.

Two flavone C-glycosides as quality control markers for the manufacturing process of ephedrine alkaloids-free Ephedra Herb extract (EFE) as a crude drug preparation.

As part of our continuing study of ephedrine alkaloids-free Ephedra Herb extract (EFE) in pursuit of its approval as a crude drug preparation, we identified two quantitative markers for the quality control of the manufacturing process of EFE and sought to establish cost-effective and simple methods for quantitative analyses. We analysed Ephedra Herb extracts grown in different habitats and collection years by liquid chromatography/high-resolution mass spectrometry (LC/HRMS) and detected two notable peaks common to each extract. These peaks were identified as vicenin-2 (1) and isovitexin 2″-O-rhamnoside (2). Quantitative analyses using the isocratic condition of LC/MS showed that the content percentages of 1 and 2 in EFE were 0.140-0.146% and 0.350-0.411%, respectively. We concluded that 1 and 2 were adequate quality control markers for quantitative analysis of EFE. Furthermore, we quantitatively analysed apigenin (3), an aglycon common to 1 and 2, and found that the conversion factors of 1 to 3 and 2 to 3 were 1.3 and 1.5, respectively. Therefore, we concluded that 3 was a secondary standard for quantifying the contents of 1 and 2 in EFE. A series of results obtained from this study will be valuable for the quality control of EFE.

Clinical Research Testing the Usefulness of Ephedrine Alkaloids-free Ephedra Herb Extract.

We aimed to test the clinical usefulness of ephedrine alkaloids-free Ephedra Herb extract (EFE), which has been proven to be effective and safe in animal studies. First, we performed a safety trial with 7 healthy volunteers, and increased white blood cell counts were observed in 2. However, it was unknown whether this observation was a result of EFE administration. Then, we implemented a controlled, double-blind, randomized crossover trial as the second safety trial for EFE. The trial was conducted under strict conditions, and the participants were managed in the hospital and monitored using an increased number of endpoints. Twelve healthy volunteers were randomly allocated to group 1, who were administered EFE first, or group 2, who were administered Ephedra Herb first. In both groups, the drug was administered for 6 d. After a 4-week washout period with no drug administration, each group was administered the alternate drug for 6 d. In the third stage, a dose-response trial is planned to test the effectiveness and safety of EFE on chronic joint pain. Patients with chronic knee joint pain will be randomly allocated to three groups, and each group will be administered EFE prepared from different amounts of Ephedra Herb. Until this stage, the studies will be conducted as investigator-initiated clinical trials. In the fourth stage, industry-sponsored clinical trials will be planned. This multicenter trial will involve 300 patients with joint pain, neuralgia, and/or muscle pain.

Analgesic Effects and Side Effects of Ephedra Herb Extract and Ephedrine Alkaloids-free Ephedra Herb Extract.

Ephedra Herb is classified "pungent, slightly bitter, and warm" in tastes and natures, and is used to provide warmth to the body, dispel coldness, remove dampness, and reduce pain. Similar herbs are "pungent and hot" chili peppers, "pungent and hot" evodia fruit," "pungent and warm" ginger, "pungent and hot" processed ginger, "pungent and hot" Zanthoxylum fruit, etc. These herbs are prescribed to provide heat to the outer or inner body. Some pungent components such as capsaicin, evodiamine, gingerol, and shogaol are known to be activators of transient receptor potential vanilloid 1 (TRPV1). TRPV1, a pain receptor, is activated in response to irritant chemicals such as capsaicin and high heat (>43℃) and strongly acidic conditions (pH<6). The typical TRPV1 activator capsaicin has various effects such as improvement of peripheral circulation, enhancement of thermogenesis, and pain relief. These effects are commonly observed for the "pungent and hot/warm" herbs, suggesting that TRPV1 stimulation plays an important part in their pharmacological action. In this study, we demonstrated that Ephedra Herb extract (EHE) shows strong TRPV1 activation, although ephedrine didn't show such effects. Both EHE and ephedrine alkaloids-free EHE (EFE) expressed similar analgesic action following oral administration, suggesting the presence of active components other than ephedrine alkaloids. Furthermore, EFE did not show side effects such as loss of sleep and irregular heartbeat in mice. Caution needs to be exercised while prescribing Ephedra Herb because it contains ephedrine. The application of EFE in Kampo medicine might be a better alternative in some cases.

Efficient Preparation of Ephedrine Alkaloids-free Ephedra Herb Extract and Its Antitumor Effect and Putative Marker Compound.

Ephedra Herb is a crude drug for the treatment of headache, bronchial asthma, nasal inflammation, and the common cold. Although it has been considered that ephedrine alkaloids (EAs) are the principal active ingredients of Ephedra Herb, EAs are known to induce palpitations, hypertension, insomnia, and dysuria as major side effects. Therefore, the administration of EAs-containing drugs to patients with cardiovascular-related diseases is strongly contraindicated. Previously, we isolated herbacetin 7-O-neohesperidoside from Ephedra Herb. In addition, we found that herbacetin, a flavonoid aglycone in Ephedra Herb, had antiproliferative and analgesic effects. Therefore, the prospect of preparing safer natural medicines without the adverse effects associated with EAs was appealing. In this symposium review, to achieve the aim of producting a clinically useful Ephedra Herb extract with none of the adverse effects associated with EAs, I present an efficient preparation method of EAs-free Ephedra Herb extract, together with its chemical composition, antiproliferative effects, and a putative marker for quality control.

EFEの薬理作用と臨床応用へ向けた取り組み

Ephedra Herb is defined in the 17th edition of the Japanese Pharmacopoeia as the terrestrial stem of Ephedra sinica STAPF., Ephedra intermedia SCHRENK et C.A. MEYER, or Ephedra equisetina BUNGE (Ephedraceae) which contains more than 0.7% ephedrine alkaloids (ephedrine and pseudoephedrine). The primary effects and adverse effects of Ephedra Herb are traditionally believed to be mediated by ephedrine alkaloids. We recently reported that Ephedra Herb extract (EHE) exhibits antimetastatic and antitumor effects by suppressing the hepatocyte growth factor-c-Met signaling pathway through the inhibition of c-Met tyrosine kinase activity. We confirmed that the non-alkaloidal fraction of EHE had c-Met-inhibitory activity. Moreover, we discovered herbacetin glycosides in EHE and demonstrated that herbacetin, the aglycone of the glycosides, shows c-Met-inhibitory activity and analgesic action. These findings suggest that some pharmacological actions of EHE may be produced by its non-alkaloidal fraction, which does not cause the adverse effects of ephedrine alkaloids. Therefore, we prepared ephedrine alkaloids-free EHE (EFE) by removing ephedrine alkaloids from EHE using ion-exchange column chromatography. EFE had c-Met-inhibitory action, analgesic effects, and antiinfluenza activity similar to EHE but had no toxicity. Now, we are evaluating the safety of EFE in healthy volunteers and its efficacy in patients to obtain licensing approval for its therapeutic use in the future.

Efficiently prepared ephedrine alkaloids-free Ephedra Herb extract: a putative marker and antiproliferative effects.

Ephedrine alkaloids (EAs) have been considered the main pharmacologically active substances in Ephedra Herb (麻黄, Mao; EH) since they were first identified by Prof. N. Nagai, and are known to induce palpitation, hypertension, insomnia, and dysuria as side effects. Therefore, the administration of drugs containing EH to patients with cardiovascular-related diseases is severely contraindicated. While our previous studies suggest that some of the effects of EH may not be due to EAs, considering their side effects would be expedient to develop a new EAs-free EH extract (EFE). Here, we established a preparation method for EFE and revealed its chemical composition, including the content of herbacetin, a flavonoid aglycon present in EH and a potential putative marker for EFE quality control. In addition, we showed the antiproliferative effects of EFE against the H1975 non-small cell lung cancer (NSCLC) cell line. EFE was prepared from EH extract using the ion exchange resin SK-1B. LC/Orbitrap MS analysis revealed the removal of EAs, 6-methoxykynurenic acid, and 6-hydroxykynurenic acid from the original extract. Quantitative analysis of herbacetin using LC/MS in acid-hydrolyzed EFE showed that its content was 0.104 %. Although several alkaloidal constituents were removed from EH extract, the antiproliferative effect of EFE against H1975 cells was comparable to that of EH extract. These results indicate that EFE retained the anticancer effect of EH and demonstrated its potential for future development as a new herbal medicine with reduced side effects.Electronic supplementary materialThe online version of this article (doi:10.1007/s11418-016-0977-1) contains supplementary material, which is available to authorized users.

Ephedrine alkaloids-free Ephedra Herb extract: a safer alternative to ephedra with comparable analgesic, anticancer, and anti-influenza activities.

It is generally accepted that the primary pharmacological activities and adverse effects of Ephedra Herb are caused by ephedrine alkaloids. Interestingly, our research shows that Ephedra Herb also has ephedrine alkaloid-independent pharmacological actions, such as c-MET inhibitory activity. This study describes the preparation of an ephedrine alkaloids-free Ephedra Herb extract (EFE) by ion-exchange column chromatography, as well as in vitro and in vivo evaluation of its pharmacological actions and toxicity. We confirmed that EFE suppressed hepatocyte growth factor (HGF)-induced cancer cell motility by preventing both HGF-induced phosphorylation of c-Met and its tyrosine kinase activity. We also investigated the analgesic effect of EFE. Although the analgesic effect of Ephedra Herb has traditionally been attributed to pseudoephedrine, oral administration of EFE reduced formalin-induced pain in a dose-dependent manner in mice. Furthermore, we confirmed the anti-influenza virus activity of EFE by showing inhibition of MDCK cell infection in a concentration-dependent manner. All assessments of toxicity, even after repeated oral administration, suggest that EFE would be a safer alternative to Ephedra Herb. The findings described here suggest that EFE has c-Met inhibitory action, analgesic effect, and anti-influenza activity, and that it is safer than Ephedra Herb extract itself. Therefore, EFE could be a useful pharmacological agent.Electronic supplementary materialThe online version of this article (doi:10.1007/s11418-016-0979-z) contains supplementary material, which is available to authorized users.