Development of a reporting guideline for systematic reviews of animal experiments in the field of traditional Chinese medicine.

OBJECTIVE: In recent years, there are several systematic reviews published on animal experiments of Traditional Chinese medicine (TCM). PRISMA (preferred reporting items for systematic reviews and meta-analysis) guidelines provide a guarantee for significantly improving the reporting quality of systematic reviews (SRs) and meta-analysis (MAs) to a certain extent; however, there are still certain defects found in the quality of SRs/MAs of animal experiments of TCM. It has been found that especially, the descriptions of the rationale and animal characteristics of TCM interventions are inadequate. As a result, we have developed a novel reporting guideline for SRs/MAs of animal experimental in the field of TCM (PRISMA-ATCM) to overcome these problems. METHODS: PRISMA-ATCM reporting guidelines were formed by analyzing both the status and quality of published SRs/MAs of animal experiments and consulting experts in the related fields, and then by Delphi consultation, consensus meeting and revision. RESULTS: Among the 27 items on the PRISMA checklist, Title (1), Structured summary (2), Rationale (3), Objectives (4), Protocol and registration (5), Eligibility criteria (6), Data items (11), Planned methods of analysis (14), Study characteristics (18), Summary of evidence (24), Limitations (25), and Funding (27) have been extensively revised and expanded, to specifically include the details about TCM intervention and animal characteristics. In addition, illustrative examples and explanations have been provided for each item. CONCLUSION: PRISMA-ATCM could markedly improve the quality SRs/MAs of animal experiments in the field of TCM.

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.

Tyrosol Reduces Amyloid-ß Oligomer Neurotoxicity and Alleviates Synaptic, Oxidative, and Cognitive Disturbances in Alzheimer's Disease Model Mice.

Soluble amyloid-ß (Aß) oligomers (AßOs), which elicit neurotoxicity and synaptotoxicity, are thought to play an initiating role in the pathology of Alzheimer's disease (AD). Since AßOs are a key therapeutic target, we attempted to identify natural agents that reduce AßO neurotoxicity. Using an assay system in which primary cultured neurons are treated with AßOs, we found that Rhodiola rosea extracts and one of its main constituents, tyrosol, significantly inhibited AßO-induced caspase-3 activation. We then assessed the in vivo efficacy of tyrosol by oral administration of the compound into AD model (5XFAD) transgenic and non-transgenic mice from either 2 or 4 to 7 months of age. In both paradigms, tyrosol treatment did not affect body weights of mice. Immunohistochemical analysis revealed that the immunoreactivity of spinophilin, a dendritic synaptic protein, was significantly reduced in three hippocampal subregions of vehicle-treated AD mice compared with non-transgenic mice, which was reversed in tyrosol-treated AD mice. Tyrosol treatment also prevented the enhancement of 4-hydroxy-2-nonenal immunoreactivity in the hippocampal CA3 region of AD mice. By contrast, tyrosol administration did not affect Aß accumulation, as evaluated by immunohistochemical and biochemical analyses. Moreover, the Barnes maze test showed that tyrosol administration modestly mitigated spatial memory impairment in AD mice. These findings collectively indicate that the natural agent tyrosol protects neurons against AßO neurotoxicity in vitro and ameliorates synaptic disturbance, oxidative stress responses, and cognitive impairment in vivo. We thus suggest that tyrosol is potentially an effective, safe, and unique drug candidate for AD.

Non-alkaloidal composition of Ephedra Herb is influenced by differences in habitats.

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.

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.

Quantification of terpene trilactones in Ginkgo biloba with a 1H NMR method.

Ginkgo biloba L. has been used as a herbal medicine in the traditional treatment of insufficient blood flow, memory deficits, and cerebral insufficiency. The terpene trilactone components, the bioactive agents of Ginkgo biloba L., have also been reported to exhibit useful functionality such as anti-inflammatory and neuroprotective effects. Therefore, in the present research, we attempted to analyze quantitatively the terpene trilactone components in Ginkgo biloba leaf extract, with quantitative 1H NMR (qNMR) and obtained almost identical results to data reported using HPLC. Application of the qNMR method for the analysis of the terpene trilactone contents in commercial Ginkgo extract products, such as soft gel capsules and tablets, produced the same levels noted in package labels. Thus, qNMR is an alternative method for quantification of the terpene trilactone components in commercial Ginkgo extract products.

The Improvement of Sleep by Oral Intake of GABA and Apocynum venetum Leaf Extract.

The effects of two food materials, γ-aminobutyric acid (GABA) produced by natural fermentation and Apocynum venetum leaf extract (AVLE), on the improvement of sleep were investigated in humans. The electroencephalogram (EEG) test revealed that oral administration of GABA (100 mg) and AVLE (50 mg) had beneficial effects on sleep. GABA shortened sleep latency by 5.3 min and AVLE increased non-rapid eye movement (REM) sleep time by 7.6%. Simultaneous intake of GABA and AVLE shortened sleep latency by 4.3 min and increased non-REM sleep time by 5.1%. The result of questionnaires showed that GABA and AVLE enabled subjects to realize the effects on sleep. These results mean that GABA can help people to fall asleep quickly, AVLE induces deep sleep, and they function complementarily with simultaneous intake. Since both GABA and AVLE are materials of foods and have been ingested for a long time, they can be regarded as safe and appropriate for daily intake in order to improve the quality of sleep.

Potent SIRT1 enzyme-stimulating and anti-glycation activities of polymethoxyflavonoids from Kaempferia parviflora.

The SIRT1 enzyme-stimulating and anti-glycation activities of Kaempferia parviflora extract and its main polymethoxyflavonoids were evaluated in vitro. K. parviflora extract elevated SIRT1 catalytic activity by eight- and 17-fold at 20 µg/mL and 100 µg/mL, respectively, compared with vehicle only. Two major polymethoxyflavonoids, 3,5,7,3',4'-pentamethoxyflavone (4) and 5,7,4'-trimethoxyflavone (5), were isolated from this extract and are four- and fivefold more potent than resveratrol, hitherto the strongest known natural SIRT1 activator. In addition, the anti-glycation activity of K. parviflora extract was observed to be seven times more effective than aminoguanidine, a clinical anti-diabetes drug. 3,5,7,3',4'-Pentamethoxyflavone (4) and 5,7,4'-trimethoxyflavone (5) showed the strongest anti-glycation activity among the tested polymethoxyflavonoids. Further comparison of the activity of these structurally related polymethoxyflavonoids revealed a possible structure-activity relationship, in particular, for the contribution of methoxy moieties.

A new benzopyran derivative from Pseuduvaria indochinensis Merr.

From the twigs and leaves of Pseuduvaria indochinensis Merr., a new benzopyran derivative, pseudindochin (1), was isolated together with three known compounds, oligandrol (2), (6E,10E)-isopolycerasoidol (3) and polycerasoidol (4). The structure of compound 1 was elucidated on the basis of extensive spectroscopic data interpretation, including 1D, 2D NMR, HR-ESI-MS, UV and IR. Moreover, compounds 1-4 were evaluated in vitro for their cytotoxic activities against HL-60 and SMMC-7721 cell lines, but these compounds were essentially non-cytotoxic (IC50>30 µg/mL).