RESUMO
Ephedra herb (EH), an important medicine prescribed in herbal formulas by Traditional Chinese Medicine practitioners, has been widely used in the treatment of viral pneumonia in China. However, the molecular basis of EH in viral pneumonia remains unclear. In this study, a ternary correlation multi-symptom network strategy was established based on in vivo chemical profile identification and metabolomics to explore the molecular basis of EH against viral pneumonia. Results showed that 143 compounds of EH and 70 prototype components were identified in vivo. EH could reduce alveolar-capillary barrier disruption in rats with viral pneumonia and significantly downregulate the expression of inflammatory factors and bronchoalveolar lavage fluid. Plasma metabolomics revealed that EH may be involved in the regulation of arachidonic acid, tryptophan, tyrosine, nicotinate, and nicotinamide metabolism. The multi-symptom network showed that 12 compounds have an integral function in the treatment of viral pneumonia by intervening in many pathways related to viruses, immunity and inflammation, and lung injury. Further verification demonstrated that sinapic acid and frambinone can regulate the expression of related genes. It has been shown to be a promising representative of the pharmacological constituents of ephedra.
Assuntos
Medicamentos de Ervas Chinesas , Ephedra , Metabolômica , Ratos Sprague-Dawley , Animais , Ratos , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/química , Ephedra/química , Masculino , Pneumonia Viral/tratamento farmacológico , Pneumonia Viral/metabolismo , Pneumonia Viral/virologiaRESUMO
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.
Assuntos
Alcaloides , Antineoplásicos , Camundongos , Animais , Receptores de IgE/metabolismo , Efedrina/metabolismo , Degranulação Celular , Mastócitos/metabolismo , Antineoplásicos/farmacologia , Alcaloides/farmacologia , Alérgenos/metabolismo , Mediadores da Inflamação/metabolismo , Imunoglobulinas , Monoaminoxidase/metabolismo , Monoaminoxidase/farmacologiaRESUMO
The microscopic test method (microscopic examination) used to identify crude drugs is a common method in the identification of the original plant source by determining the characteristics from a small sample quantity. However, in recent years, the number of examiners who are familiar with the microscopic examination technique has decreased. In recent years, high-resolution X-ray computed tomography (HRXCT) has been used to visualize the internal structure of plants. HRXCT scans an object using X-rays and enables visualization of the internal structure of the crude drug using a computer. Therefore, in this report, HRXCT was used to easily observe the internal morphology of crude drugs using the Ephedra Herb as an example. The same internal morphological characteristics were observed using both, microscopic examination and HRXCT methods. Image analysis using HRXCT did not require specific techniques, such as sectioning, and the same tissue could be observed from any orientation using a single scan. It afforded remarkable technical simplification and reduction in time to inspect the organization's characteristics. Therefore, image analysis using HRXCT is a useful method for crude drug identifications.
Assuntos
Tomografia Computadorizada por Raios X , Tomografia Computadorizada por Raios X/métodos , Raios XRESUMO
PURPOSE: Ephedra herb (Mao) exerts potent anti-allergic effects. This study aimed to examine the underlying mechanisms of Mao on allergic inflammation using in vitro cultured mast cells (MCs) and an in vivo model of MC-dependent anaphylaxis. METHODS: Bone marrow-derived MCs (BMMCs) were presensitized with anti-2,4-dinitrophenol (DNP) immunoglobulin E (IgE) and challenged with antigens (Ag; DNP-human serum albumin). Degranulation responses and cell surface high-affinity receptor for IgE (FcεRI) expression were assessed with/without Mao treatment. Passive systemic anaphylaxis (PSA)-treated mice were administered Mao and the pathophysiological responses were evaluated. RESULTS: Mao inhibited Ag-induced BMMC degranulation, but not polyclonal activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, indicating that Mao inhibits IgE-dependent activation of BMMCs. Mao-treated BMMCs exhibited significant reductions in expression of surface IgE and its receptor FcεRI. Analysis of subcellular localization revealed that Mao induces FcεRI internalization in BMMCs without degranulation. In the PSA mouse model, Mao administration prevented antigen-induced hypothermia. Mao administration significantly reduced cell surface expression of IgE-bound FcεRI on peritoneal MCs. CONCLUSIONS: Mao induced FcεRI internalization in MCs, thereby inhibiting Ag-induced IgE-dependent degranulation. The inhibitory effects of Mao on MC degranulation may offer a novel therapeutic approach for allergic diseases.
Assuntos
Anafilaxia/tratamento farmacológico , Antialérgicos/farmacologia , Ephedra/química , Mastócitos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Anafilaxia/imunologia , Animais , Antialérgicos/uso terapêutico , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Células Cultivadas , Modelos Animais de Doenças , Feminino , Humanos , Imunoglobulina E/metabolismo , Ionomicina/imunologia , Mastócitos/imunologia , Medicina Kampo/métodos , Camundongos , Extratos Vegetais/uso terapêutico , Caules de Planta/química , Cultura Primária de Células , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Acetato de Tetradecanoilforbol/imunologiaRESUMO
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.
Assuntos
Ephedra/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Alcaloides/química , Alcaloides/farmacologia , Analgésicos/química , Analgésicos/farmacologia , Animais , Antivirais/química , Antivirais/farmacologia , Biflavonoides/química , Biflavonoides/farmacologia , Catequina/química , Catequina/farmacologia , Linhagem Celular Tumoral , Cães , Efedrina/química , Efedrina/farmacologia , Humanos , Células Madin Darby de Rim Canino , Masculino , Camundongos , Proantocianidinas/química , Proantocianidinas/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidoresRESUMO
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.
Assuntos
Analgésicos/uso terapêutico , Ephedra/química , Efedrina/uso terapêutico , Dor/tratamento farmacológico , Extratos Vegetais/uso terapêutico , Pseudoefedrina/uso terapêutico , Administração Oral , Analgésicos/isolamento & purificação , Animais , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos , Medição da Dor , Extratos Vegetais/isolamento & purificação , Teste de Desempenho do Rota-Rod , Fatores de TempoRESUMO
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.
Assuntos
Ansiedade/prevenção & controle , Arritmias Cardíacas/prevenção & controle , Suplementos Nutricionais/efeitos adversos , Ephedra/química , Efedrina/efeitos adversos , Extratos Vegetais/efeitos adversos , Distúrbios do Início e da Manutenção do Sono/prevenção & controle , Alcaloides/análise , Alcaloides/toxicidade , Analgésicos não Narcóticos/administração & dosagem , Analgésicos não Narcóticos/efeitos adversos , Analgésicos não Narcóticos/química , Animais , Animais não Endogâmicos , Ansiedade/sangue , Ansiedade/induzido quimicamente , Ansiedade/etiologia , Arritmias Cardíacas/sangue , Arritmias Cardíacas/induzido quimicamente , Arritmias Cardíacas/etiologia , Comportamento Animal , Cafeína/intoxicação , Estimulantes do Sistema Nervoso Central/intoxicação , Suplementos Nutricionais/análise , Efedrina/administração & dosagem , Efedrina/química , Contaminação de Alimentos , Hipnóticos e Sedativos/farmacologia , Japão , Masculino , Camundongos , Pentobarbital/farmacologia , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Caules de Planta/química , Potássio/sangue , Distúrbios do Início e da Manutenção do Sono/sangue , Distúrbios do Início e da Manutenção do Sono/induzido quimicamente , Distúrbios do Início e da Manutenção do Sono/etiologiaRESUMO
Respiratory syncytial virus (RSV) is a major cause of respiratory tract infection in children. Despite decades of efforts, no effective therapies are available. We recently reported that extracts of Ephedra Herb and Cinnamon Bark interacted with the G attachment protein of RSV to inhibit infectivity. The present in vitro study aimed to investigate the antiviral effect of ephedrine alkaloids-free Ephedra Herb extract (EFE), which is characterized by free of harmful effects of ephedrine alkaloids in Ephedra Herb, on experimental RSV infection. Infection of RSV into A549 cells simultaneously with EFE resulted the significant reduction of RSV RNA, viral protein, and viral titers after the incubation of the cells. We found that RSV attachment to the cell surface was inhibited both in the presence of EFE and when RSV particles were pre-treated with EFE. We also found that EFE specifically interacted with the central conserved domain of RSV G protein by surface plasmon resonance, demonstrating that specific binding of G protein to the cellular receptor was inhibited by EFE. Another mechanism was found in which a higher concentration of EFE inhibited the viral load immediately after the viral entry into host cells, suggesting the inhibition of viral RNA replication. These results demonstrate that EFE worked against RSV infection through multiple antiviral mechanisms, a unique feature of this crude drug extract.
RESUMO
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.
Assuntos
Alcaloides , Ephedra , Camundongos , Animais , Ephedra/química , Efedrina/farmacologia , Pseudoefedrina , Alcaloides/química , Extratos Vegetais/química , Hipnóticos e Sedativos/farmacologia , Receptores AdrenérgicosRESUMO
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.
RESUMO
This study aimed to investigate the effect and mechanisms of Ephedra Herb (EH) extract on adriamycin-induced nephrotic syndrome (NS), providing an experimental basis for the clinical treatment of NS. Hematoxylin and eosin staining, creatinine, urea nitrogen, and kidn injury molecule-1 were used to evaluate the activities of EH extract on renal function. The levels of inflammatory factors and oxidative stress were detected by kits. The levels of reactive oxygen species, immune cells, and apoptosis were measured by flow cytometry. A network pharmacological approach was used to predict the potential targets and mechanisms of EH extract in the treatment of NS. The protein levels of apoptosis-related proteins and CAMKK2, p-CAMKK2, AMPK, p-AMPK, mTOR and p-mTOR in the kidneys were detected by Western blot. The effective material basis of EH extract was screened by MTT assay. The AMPK pathway inhibitor (compound C, CC) was added to investigate the effect of the potent material basis on adriamycin-induced cell injury. EH extract significantly improved renal injury and relieve inflammation, oxidative stress, and apoptosis in rats. Network pharmacology and Western blot results showed that the effect of EH extract on NS may be associated with the CAMKK2/AMPK/mTOR signaling pathway. Moreover, methylephedrine significantly ameliorated adriamycin-induced NRK-52e cell injury. Methylephedrine also significantly improved the phosphorylation of AMPK and mTOR, which were blocked by CC. In sum, EH extract may ameliorate renal injury via the CAMKK2/AMPK/mTOR signaling pathway. Moreover, methylephedrine may be one of the material bases of EH extract.
Assuntos
Doxorrubicina , Síndrome Nefrótica , Ratos , Animais , Doxorrubicina/efeitos adversos , Proteínas Quinases Ativadas por AMP/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , ApoptoseRESUMO
We previously reported that the combined application of Ephedra Herb extract (EHE) and epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI), erlotinib, is effective in suppressing the growth of erlotinib-resistant non-small-cell lung cancer (NSCLC) cell line, H1993, xenograft tumor, and cell proliferation, and that EHE downregulates c-Met and wild-type EGFR in H1993 cells. However, it was unclear whether EHE could affect EGFR with active mutations. Clinically, advanced NSCLC patients who are eligible for EGFR-TKI treatment are those with detected EGFR with activating mutations. Therefore, it is important to clarify the effect of EHE on EGFR with activating mutations. H1975 cells express EGFR with activating mutations, L858R and T790M, and c-Met; this NSCLC cell line was used in the present study. EHE downregulated the expression of EGFR with activating mutations and c-Met, and inhibited autophosphorylation of c-Met. Proliferation of H1975 cells was suppressed by EHE in a concentration-dependent manner. These results suggest that EHE may be effective against NSCLC harboring EGFR with activating mutations. Considering the fact that advanced NSCLC patients, with an EGFR T790M mutation, are currently widely treated with the third-generation EGFR-TKI, osimertinib, we examined the combined effects of osimertinib and EHE on H1975 cells. The osimertinib and EHE combination downregulated the expression of these receptors and suppressed the proliferation of H1975 cells more effectively than did osimertinib alone, suggesting that this combination may be effective in treating patients with advanced NSCLC with the L858R + T790M EGFR mutation and c-Met. Graphical Abstract was created with BioRender.com.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Cloridrato de Erlotinib , Receptores ErbB/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Inibidores de Proteínas Quinases/farmacologia , Mutação , Linhagem Celular , Linhagem Celular TumoralRESUMO
OBJECTIVE: We investigated the protective effects of ephedra herb (HEPH) on adriamycin-induced testicular toxicity in rats and explored the potential mechanisms underlying these effects. METHODS: A rat model of adriamycin injury was established, and sperm motility-related indicator and oxidative stress levels in the testis were evaluated. Serum levels of sex hormones and levels of testicular cell apoptosis were detected by enzyme-linked immunosorbent assay and flow cytometry, respectively. Western blotting (WB), immunofluorescence analyses, and reverse transcription-polymerase chain reaction (RT-PCR) were performed to evaluate the gonadotropin-releasing hormone (GnRH) signalling pathway- and meiosis-related genes and proteins. In subsequent in vitro experiments, adriamycin was used to stimulate GC-1 cells, which were treated with HEPH, ephedrine, or pseudoephedrine. Cell viability was assessed using flow cytometry to detect apoptosis and reactive oxygen species, whereas the GnRH signalling pathway and levels of meiosis-related genes and proteins were evaluated by InCell WB, a high-content imaging system, and RT-PCR. RESULTS: Per in vivo experiments, HEPH restored testicular weight and function, sperm characteristics, serum and tissue hormonal levels, and antioxidant defences and significantly activated the GnRH signalling pathway- and meiosis-related protein levels. All protective effects of HEPH against adriamycin-induced injury were antagonised by the GnRH antagonist cetrorelix. In vitro, HEPH, ephedrine, and pseudoephedrine significantly reduced adriamycin-induced GC-1 cell apoptosis and reactive oxygen species levels and increased the expression of GnRH signalling pathway- and meiosis-related proteins. The effect of pseudoephedrine was greater than that of ephedrine, and these findings may be an important basis for understanding the effects of HEPH.
Assuntos
Ephedra , Testículo , Animais , Doxorrubicina/farmacologia , Efedrina/metabolismo , Efedrina/farmacologia , Hormônio Liberador de Gonadotropina/farmacologia , Masculino , Pseudoefedrina/metabolismo , Pseudoefedrina/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Motilidade dos EspermatozoidesRESUMO
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.
Assuntos
Medicamentos de Ervas Chinesas/efeitos adversos , Medicamentos de Ervas Chinesas/química , Ephedra/química , Idoso , Analgésicos , Antineoplásicos Fitogênicos , Antivirais , Cromatografia por Troca Iônica , Medicamentos de Ervas Chinesas/farmacologia , Efedrina/efeitos adversos , Efedrina/isolamento & purificação , Feminino , Humanos , Masculino , Pseudoefedrina/efeitos adversos , Pseudoefedrina/isolamento & purificação , SegurançaRESUMO
Ephedra Herb is a crude drug defined as the terrestrial stem of Ephedra sinica, E. intermedia, or E. equisetina. It is often used to treat headaches, bronchial asthma, nasal inflammation, and the common cold. In this study, we isolated characteristic non-alkaloidal constituents from the extracts and identified them in relation to the habitat of Ephedra Herb. Extracts were prepared from Ephedra Herb collected from Inner Mongolia and Gansu. High-performance liquid chromatography was performed to quantitatively analyse the amount of ephedrine alkaloids in each extract. We compared the chemical compositions of the extracts by thin layer chromatography (TLC) to find spot characteristics depending on the habitat. 1H-NMR, 13C-NMR, and 2D-NMR spectra of the samples were also examined. The ephedrine content of all extracts satisfied the quality standard stated in the Japanese Pharmacopoeia. Nonetheless, we found each notable constituent characteristic to the Ephedra Herbs from both habitats. In order to identify them, Ephedra Herb extracts were separated by column chromatography, resulting in the isolation of (±)-α-terpineol-ß-D-O-glucopyranoside (1) and (E)-7-hydroxy-3,7-dimethyloct-2-en-1-yl-ß-D-O-glucopyranoside (2) as the characteristic constituents in Ephedra Herb from Inner Mongolia. Epheganoside (3), a new eudesmane-type sesquiterpene glycoside, and scopoletin (4) were found to be the characteristic constituents in Ephedra Herb from Gansu. The results obtained from this study can be used to distinguish between the habitats of Ephedra Herb.
Assuntos
Ephedra/química , Extratos Vegetais/química , Meio Ambiente , HumanosRESUMO
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 2â³-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.
Assuntos
Composição de Medicamentos/métodos , Ephedra/química , Efedrina/química , Flavonas/química , Glicosídeos/metabolismo , Efedrina/análise , Controle de QualidadeRESUMO
Kampo medicines containing Ephedra Herb (EH) such as eppikajutsubuto and makyoyokukanto are used to treat myalgia, arthralgia, and rheumatism. The analgesic effects of these Kampo medicines are attributed to the anti-inflammatory action of EH. However, the molecular mechanism of the analgesic effect of EH remains to be clarified. In this study, the effects of EH extract (EHE) on transient receptor potential vanilloid 1 (TRPV1), a nonselective ligand-gated cation channel, which plays an essential role in nociception on sensory neurons, were investigated using mTRPV1/Flp-In293 cells (stable mouse TRPV1-expressing transfectants). Administration of EHE increased the intracellular Ca2+ concentration in these cells, which was inhibited by the TRPV1 antagonist, N-(4-tert-butylphenyl)-1,2-dihydro-4-(3-chloropyridine-2-yl) tetrahydropyrazine-1-carboxamide (BCTC), indicating that EHE activated TRPV1. Examination of EHE-induced nociceptive pain in vivo revealed that an intradermal (i.d.) injection of EHE into the hind paw of mice induced paw licking, a pain-related behavior, and that the extract increased paw licking times in a dose-dependent manner. The EHE-induced paw licking was also inhibited by BCTC. An i.d. injection of EHE 30 min before administration of capsaicin decreased capsaicin-induced paw licking times. Similarly, oral administration of the extract also suppressed capsaicin-induced paw licking, without affecting the physical performance of the mice. These results suggest that EHE suppresses capsaicin-induced paw licking by regulating TRPV1 activity. Thus, the antinociceptive effects of EHE seem to be produced by its direct action on sensory neurons through TRPV1.
Assuntos
Anti-Inflamatórios/farmacologia , Capsaicina/efeitos adversos , Ephedra/química , Dor/induzido quimicamente , Animais , Humanos , Masculino , Camundongos , Medição da Dor , Canais de Cátion TRPV , TransfecçãoRESUMO
Ephedrine alkaloids (EAs) have been considered the main pharmacologically active substances in Ephedra Herb (, 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.
Assuntos
Alcaloides/química , Medicamentos de Ervas Chinesas/química , Ephedra/química , Efedrina/química , Extratos Vegetais/química , Efedrina/análise , HumanosRESUMO
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.
Assuntos
Analgésicos/uso terapêutico , Antineoplásicos/uso terapêutico , Ephedra/química , Efedrina/química , Influenza Humana/tratamento farmacológico , Alcaloides/química , Efedrina/análise , HumanosRESUMO
Chemokine receptors CCR3 and CCR4 are preferentially expressed by TH2 cells, mast cells, and/or eosinophils, all of which are involved in the pathogenesis of allergic diseases. Therefore, CCR3 and CCR4 have long been highlighted as potent therapeutic targets for allergic diseases. Japanese traditional herbal medicine Kampo consists of multiple crude drugs/herbs, which further consist of numerous chemical substances. Recent studies have demonstrated that such chemical substances appear to promising sources in the development of novel therapeutic agents. Based on these findings, we hypothesize that Kampo-related crude drugs/herbs would contain chemical substances that inhibit the cell migration mediated by CCR3 and/or CCR4. To test this hypothesis, we screened 80 crude drugs/herbs to identify candidate substances using chemotaxis assay. Among those tested, Ephedra Herb inhibited the chemotaxis mediated by both CCR3 and CCR4, Cornus Fruit inhibited that mediated by CCR3, and Rhubarb inhibited that mediated by CCR4. Furthermore, Ephedra Herb specifically inhibited the chemotaxis mediated by not only CCR3 and CCR4 but CCR8, all of which are selectively expressed by TH2 cells. This result led us to speculate that ephedrine, a major component of Ephedra Herb, would play a central role in the inhibitory effects on the chemotaxis mediated by CCR3, CCR4, and CCR8. However, ephedrine exhibited little effects on the chemotaxis. Therefore, we fractionated Ephedra Herb into four subfractions and examined the inhibitory effects of each subfraction. As the results, ethyl acetate-insoluble fraction exhibited the inhibitory effects on chemotaxis and calcium mobilization mediated by CCR3 and CCR4 most significantly. In contrast, chloroform-soluble fraction exhibited a weak inhibitory effect on the chemotaxis mediated by CCR8. Furthermore, maoto, one of the Kampo formulations containing Ephedra Herb, exhibited the inhibitory effects on the chemotaxis mediated by CCR3, CCR4, and CCR8. Taken together, our data suggest that these crude drugs/herbs might be useful sources to develop new drugs targeting TH2-mediated allergic diseases.