Ephedra Sinica Research Articles

Structural characteristics of a hyperbranched acidic polysaccharide from the stems of Ephedra sinica and its effect on T-cell subsets and their cytokines in DTH mice

An acidic hetero-polysaccharide (ESP-B4) was acquired from the stems of Ephedra sinica. Its structure was characterized by periodate oxidation, Smith degradation, partial acid hydrolysis, methylation analysis combined with HPCE, GC–MS and FT-IR spectra. The results indicated that ESP-B4 contained a repeating unit of [→4)-GalpA-(1→2)-Rhap-(1→] as hairy region, substituted by arabinose, xylose, galactose, glucose, mannose and glucuronic acid. To better understand the mechanism of bioactivity for this compound, the quantities of ESP-B4 was further enriched by ion exchange and gel filtration chromatography. Immunological tests in vivo showed that ESP-B4 exhibits compelling immunosuppressive effects by hematoxylin and eosin staining, enzyme-linked immunosorbent assay and flow cytometric analysis. The mechanism of action might be attributed to its inhibition of IL-2 and IL-4 levels in serum, and also effectively suppressed the ratio of CD4+/CD8+ in serum of T cell mediated delayed-type hypersensitivity mice. The results suggest that ESP-B4 could be a valuable lead material for immunosuppressive drug development.

The structure of plant communities in the dry steppes of Central Mongolia and their response to irrigation

The present state of the dry-steppe vegetation cover is considered on the example of Central Mongolia, including the structure of plant communities and the vitality of dominant species at the time of peak vegetation and in a winter period. Data on the plant communities have revealed the dominance of Ephedra sinica, a small xeromorphic shrub, and a sizeable decrease in the share of the tussocky graminoid Stipa krylovii, of which the majority of specimens was found in a state of inactivity. Due to the intensification of grazing and a prolonged period of soil-moisture deficiency, the response of Stipa krylovii tussocks to an artificial water supply was insignificant.

Cleaner production of ephedrine from Ephedra sinica Stapf by membrane separation technology

A promising cleaner approach, including chemical extraction, separation and purification by membranes separation technology, for producing ephedrine from Ephedra sinica Stapf was introduced. The extraction yield of ephedrine reached 92.45 ± 0.46%, increased by 28.25 ± 0.13% than that of the traditional process, at solid-to-liquid ratio of 1/10, extraction temperature of 80 °C, total extraction time of 20 h and reextraction for 3 times. In microfiltration, the transmissivity for ephedrine was up to 97.88 ± 1.06% and the retention rate of impurities reached 78.56 ± 0.96% when the membranes with pore size of 0.45 μm were employed at inlet and outlet operating pressure of 0.26 MPa and 0.14 MPa, respectively. The surface velocity of membrane channel was 3.5 m s −1 and membrane flux was 207 ± 3.71 l m −2 h −1. Nanofiltration membranes with 160 Da molecular weight cut-off (MWCO) were adopted to separate the ephedrine from microfiltration permeate at a transmembrane pressure of 0.6 MPa wherein the retention rate of ephedrine reached 99.88 ± 0.23% and the membrane flux was 19.88 ± 1.12 l m −2 h −1. For this improved approach, the COD of nanofiltration permeate was only 110 ± 12.56 mg l −1 which could be recycled to the extraction process, causing a decrease by 59.38 ± 1.67% of water consumption and 75.76 ± 1.89% of wastewater generation in comparison with those of the traditional process.

Genetic Diversity of Ephedra Plants in Mongolia Inferred from Internal Transcribed Spacer Sequence of Nuclear Ribosomal DNA

Ephedrae herba has been used for treating colds, relieving coughs and asthma from ancient times. We previously reported the distribution of Ephedra sinica, E. equisetina, E. przewalskii, E. regeliana, E. monosperma and Ephedra sp. in Mongolia, and among them E. sinica and E. equisetina were potential new resources of Ephedrae herba of Japanese pharmacopoeia grade, based on our field survey and subsequent molecular and chemical assessments. However, the Ephedra population in southwestern areas showed a high possibility of having hybrid origins. Further field surveys in southwestern areas, and sequence analysis of the partial nuclear internal transcribed spacer 1 (ITS1) region, besides trnK and 18S ribosomal RNA (rRNA) gene regions, were conducted in order to obtain detailed evidence of hybridization status. As a result, the distribution of E. glauca in western area and E. lomatolepis in western-most area was confirmed. The ITS sequences from all 8 Ephedra species collected in Mongolia were roughly divided into 5 types (types I-V). Type II sequence, having several additive nucleotides, was found in Ephedra sp., E. glauca, E. regeliana and E. sinica, which provided useful information for tracing hybrid origins. Morphological, genetic and distribution evidence suggested that the hybridization of Ephedra species occurred widely in southwestern Mongolia, and several Ephedra species including E. przewalkskii and E. intermedia were involved in these events. Integrated with our previous report, trnK-, 18S- and ITS-types from pure lines of each species are proposed. In addition, we propose a practicable method for detecting additive peaks on a direct sequencing electropherogram.

Development and application of a rapid and efficient CZE method coupled with correction factors for determination of monosaccharide composition of acidic hetero‐polysaccharides from Ephedra sinica

Ephedrine alkaloids cannot account for all the effects of Ephedra sinica and the polysaccharides are also demonstrated to be one of the main bioactive constituents of E. sinica. However, no work has been reported on the analysis of monosaccharide composition of purified polysaccharides isolated from the stem of E. sinica. To develop a rapid and efficient capillary zone electrophoresis (CZE) method based on pre-column derivatisation with 1-phenyl-3-methyl-5-pyrazolone for the simultaneous determination of neutral and acidic sugars of purified polysaccharides from E. sinica. Three polysaccharides (ESP-A3, ESP-A4 and ESP-B4) were isolated and purified by ion exchange and gel-filtration chromatography from the stem of E. sinica. The effects of background electrolyte pH and concentration, applied voltage and temperature on the separation were investigated. Meanwhile, factors affecting the hydrolysis of ESP-B4 with sulphuric acid were investigated by changing the hydrolysis time, acid concentration and hydrolytic temperature to achieve complete hydrolysis. The standard curves coupled with correction factors were used to calculate molar ratios. The optimal CZE method coupled with correction factors was successfully applied to the determination of molar ratios of three purified polysaccharides and their corresponding partial acid hydrolysis products. ESP-A3, ESP-A4 and ESP-B4 were all typical acidic hetero-polysaccharides and consisted of xylose, arabinose, glucose, rhamnose, mannose, galactose, glucuronic acid and galacturonic acid, and their corresponding molar ratios were 6.8:7.5:1.0:14.0:13.7:22.3:10.2:3.8 for ESP-A3, 1.2:4.1:1.0:5.1:1.6:17.3:3.1:2.2 for ESP-A4, and 1.0:4.5:1.0:2.0:1.0:5.5:1.5:50.0 for ESP-B4. The results provided scientific evidence for the further study of the structure and bioactivity of complex acidic E. sinica polysaccharides.

Optimum extraction of acidic polysaccharides from the stems of Ephedra sinica Stapf by Box–Behnken statistical design and its anti-complement activity

The response surface methodology was employed to study the extraction of acidic polysaccharides from the stem of Ephedra sinica Stapf. The quantitative effects of extraction temperature, time, number and ratio of water to raw material on yield of ephedra acidic polysaccharides were investigated with Box–Behnken design. The experimental data were fitted to three second-order polynomial equations using multiple regression analysis and also analyzed using the appropriate statistical methods. By solving the regression equation and analyzing 3-D plots, the optimum condition was at extraction temperature 100°C, time 3.5h, numbers 4 and ratio of water to raw material 13.6. Under these conditions, the experimental extraction yield, polysaccharide yield and uronic acid yield were 49.47mg/g, 33.25mg/g, and 22.08mg/g, which were in good agreement with the predicted values. Ephedra acidic polysaccharide exhibited excellent anti-complement activity in vitro, indicating it could be a potential anti-complement therapeutic agent.

Liquid Chromatography-Particle Beam Electron Ionization Mass Spectrometry Method for Analysis of Botanical Extracts: Evaluation of Ephedrine Alkaloids in Standard Reference Materials

The preliminary validation of a high-performance liquid chromatography particle beam mass spectrometry method (HPLC-PB/MS) with electron impact ionization source for analysis of botanical extracts is presented. The LC-PB/MS system was evaluated for the analysis of ephedrine alkaloids using ephedra-containing National Institute of Standards and Technology dietary supplement standard reference materials (SRMs) 3241 Ephedra Sinica Stapf Native Extract and 3242 Ephedra Sinica Stapf Commercial Extract. The ephedrine alkaloids were separated by reversed-phase chromatography using a phenyl column at room temperature. A linear gradient method with a mobile phase composition varying from 5:95 [MeOH:0.1% trifluoroacetic acid (TFA) in water] to 20:80 (MeOH:0.1% TFA in water) at a flow rate of 1.0 ml/min, with an analysis time of less than 20 min, was used. The source block temperature was evaluated to determine the optimal operating conditions by monitoring the intensities and fragmentation patterns of the ephedrine alkaloids. Ephedrine and N-methylephedrine were taken as a representative of the test alkaloids. The LODs on the sub-nanogram level were achieved, with ephedrine, pseudoephedrine, and methylephedrine in the SRMs quantified by a standard addition method with recoveries of > or = 86% and RSDs of < or = 14% (n = 3).

Ephedrannin A and B from roots of Ephedra sinica inhibit lipopolysaccharide-induced inflammatory mediators by suppressing nuclear factor-κB activation in RAW 264.7 macrophages

Ephedra sinica is a traditional Chinese medicinal herb and has pharmacological functions including anti-inflammatory effects. However, the active ingredients from Ephedra roots have not been characterized. Here, two active constituents were isolated and their structures and mechanisms of action were defined. Active constituents from Ephedra roots were isolated by continuous solvent-extractions and column chromatography. Their structures were determined by use of multiple types of spectrometry. The mechanisms of action were examined using lipopolysaccharide (LPS)-stimulated RAW 264.7 cells through PCR, ELISA, electrophoretic mobility shift assays, and immunocytochemistry. Two active constituents, ephedrannin A and B, belonging to the A-type proanthocyanidin family were identified. Both ephedrannin A and B effectively suppressed the transcription of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). These compounds exerted their anti-inflammatory actions on LPS-stimulated macrophages by suppressing the translocation of nuclear factor-kappa B (NF-κB) and the phosphorylation of p38 mitogen-activated protein (MAP) kinase. Ephedrannin A and B both exhibited strong anti-inflammatory effects, however, the optimal dose of ephedrannin B was 10 times lower than that of ephedrannin A. This is the first report describing effective anti-inflammatory activity for ephedrannin A and B isolated from Ephedra roots. Ephedrannin B may be a good candidate for delaying the progression of human inflammatory diseases and warrants further studies.

Effect of the oral administration of Ephedra Sinica extract on suppression of body weight gains

Ephedra Sinica (Ma-hwang, ES) is a medicinal extract used in Oriental Medicine. ES is used clinically to release exterior symptoms, disperse cold, control wheezing, promote urination and reduce edema. Historically, ES have suppressive effect on body weight gains. Using diet-induced obesity C57BL/6 mouse model, anti-obesity effect and cytotoxicity of the longterm oral administration of these herbal extracts were investigated. Herbal extract treated groups were arrested in weight increment only when they were lodged together. Such effects were abolished when they kept individually. ES fed mice behaved very rudely and violently. On the basis of histological studies of liver tissues and also in vitro cytotoxicity tests of the liver and kidney cell lines, no significant toxicity was found by 14 weeks of ES treatments. However, we found significant changes in gene expression profile between ES treated group and SA treated group by micro-array analysis. In case of ES group, up-regulated genes were 113 and down-regulated were 120. Some of lipid metabolism related genes also significantly changed in both treatment groups. In conclusion, ES had effects on increasing the basal metabolic rate by stimulating the sympathetic nervous systems.

Analysis of the preferred conformations and determination of the concentrations of ephedrine and pseudoephedrine based on hollow fiber liquid-phase microextraction

The preferred conformations of the ephedrine and pseudoephedrine in Ephedra sinica Stapf and rat urine were analyzed by the hollow fiber liquid-phase microextraction (HF-LPME) and their extraction mechanisms were illuminated. The method of the separation of the ephedrine and pseudoephedrine and the determination of their concentrations with high performance liquid chromatography (HPLC) were established. The optimal experimental conditions were as follows: the organic phase carrier was the hollow fiber of polyvinylidene fluoride (MOF-503), organic solvent was n-hexanol, the extraction time was 35 min, the stirring rate was 1200 r/min, the sample phase was the NaOH solution (5 mol/L) of the analyte, the acceptor was 0.01 mol/L H2SO4 solution. The extracts were analyzed by HPLC. Under the optimal conditions, the method is convenient and highly sensitive. In Ephedra sinica Stapf, the linear ranges of ephedrine and pseudoephedrine were 5-100 microg/L, the detection limits were 1.9 microg/L and 1.2 microg/L and the enrichment factors were 38 and 61, respectively. The average recoveries of ephedrine and pseudoephedrine were 100.6% +/- 1.2% and 103.2% +/- 3.5%, respectively. In rat urine, their linear ranges were 100 - 5 x 10(4) microg/L, the detection limits were 30 microg/L and 42 microg/L and the enrichment factors were 20 and 17, respectively. In rat urine, their average recoveries were 108.4% +/- 4.4% and 106. 1% +/- 5.4%, respectively. The obtained results indicated that the method can be successfully applied for the extraction and determination of the ephedrine and pseudoephedrine in Ephedra sinica Stapf and rat urine.

Screening and comparison of the immunosuppressive activities of polysaccharides from the stems of Ephedra sinica Stapf

Three polysaccharide fractions A, B and C (PA, PB and PC) were acquired from the stems of Ephedra sinica Stapf by sequential extraction with cold water, hot water, and 1.0 M NaOH, respectively. Immunosuppressive activities of PA, PB and PC were investigated by carbon clearance test, delayed-type hypersensitivity reaction and humoral immune response in vivo. Then PB was further isolated to afford four purified polysaccharides (ESP-B1, ESP-B2, ESP-B3 and ESP-B4) by various ion exchange and gel-filtration chromatography. Meanwhile, the purified polysaccharides were subjected to composition analysis and valuated for mice splenocyte proliferation activity in vitro. Among four purified polysaccharides, ESP-B4 has the highest inhibitory effect on splenocyte proliferation and its branches are extremely important for the expression of the inhibitory effect. The results in our study indicate that PB and ESP-B4 have therapeutic potential for the treatment of autoimmune and atopic diseases due to their immunosuppressive effects.

생약복합물의 지방세포형성억제 기전규명을 위한 전사체 분석

생약복합물인 SH21B는 황금(Scutellaria baicalensis Georgi), 행인(Prunus armeniaca Maxim), 마황(Ephedra sinica Stapf), 석창포(Acorus gramineus Soland), 포황(Typha orientalis Presl), 원지(Polygala tenuifolia Willd), 하엽(Nelumbo nucifera Gaertner)의 혼합(비율 3:3:3:3:3:2:2)으로 이루어졌다. SH21B는 예로부터 한의학에서 비만의 치료에 사용되어 왔으나 자세한 분자적 메커니즘과 효능에 대한 연구는 이루어지지 않았다. 본 연구진은 선행연구를 통해 SH21B가 지방세포의 분화에서 adipogenesis (지방세포형성)와 관련된 유전자를 조절하여 중성지방의 축적을 억제함을 밝혔다. 본 연구에서는, microarray 기술을 이용하여 adipogenesis의 in vitro 모델인, 3T3-L1 세포에서 SH21B에 의한 지방세포형성 억제의 분자적 기작을 보다 상세하게 연구하고자 하였다. 전지방세포, 분화된 세포 그리고 SH21B에 의해 분화가 억제된 세포의 각각의 유전자 발현을 분석하기 위해 각 시료들에서 total RNA를 분리하여 cDNA를 합성한 후 microarray에 적용시켰다. 그 결과, 각각의 시료들의 비교에서 2배 이상의 유의한 발현 변화를 가지는 2,568개의 유전자를 확보하였다. 이 유전자들에 대해 Hierarchical clustering과 K-means clustering 분석을 진행하였고 서로 다른 양상을 가지는 9개의 군집(cluster)들을 분류하였다. 그 중, SH21B의 첨가에 의해 뚜렷하게 감소(cluster 4, cluster 6 및 cluster 9)하거나 반대로 뚜렷하게 증가(cluster 7와 cluster 8)하는 양상을 보이는 군집들을 따로 선별하여 그 군집들에 포함되어 있는 유전자들을 분석하였다. 선택 된 5개의 군집에는 지방세포형성과 세포증식에 관련된 유전자가 다수 포함되어 있었다. Cluster 4, cluster 6 그리고 cluster 9에는 peroxisome proliferator activated receptor gamma <TEX>$\gamma$</TEX> (<TEX>$PPAR{\gamma}$</TEX>), CCAAT/enhancer binding protein <TEX>$\alpha$</TEX> (C/<TEX>$EBP{\alpha}$</TEX>), sterol regulatory element binding transcription factor 1 (SREBF1), adiponectin (ADIPOQ), fatty acid synthase (FASN), lipoprotein lipase (LPL) 등의 지방세포형성 유도 및 관련 인자와 B-cell leukemia/lymphoma6 (BCL6), retinoblastoma 1 (RB1), cyclin-dependent kinase inhibitor 2C (CDKN2c), ras homolog gene family, member B (RHOB) 등의 많은 세포증식 억제 유전자가 포함되었다. 이와는 반대로, cluster 7과 cluster 8에는 <TEX>$\beta$</TEX>-catenin, cyclin D1 (CCND1), WNT1 inducible signaling pathway protein 2 (WISP2) 등과 같은 지방 세포형성 억제 조절자와 MARCKS-like1 (MARCKSL1), colony stimulating factor 1 (CSF1), discoidin domain receptor family, member 2 (DDR2), leukemia inhibitory factor receptor (LIFR) 등의 세포증식을 유도하는 조절자가 다수 포함되었다. 결론적으로, 이러한 결과들은 SH21B가 지방세포형성과 관련된 조절자 및 세포증식과 관련 된 조절자들의 유전자 발현을 조절하여 지방세포형성을 억제함을 제시한다. SH21B is a natural composition composed of seven herbs: Scutellaria baicalensis Georgi, Prunus armeniaca Maxim, Ephedra sinica Stapf, Acorus gramineus Soland, Typha orientalis Presl, Polygala tenuifolia Willd and Nelumbo nucifera Gaertner (Ratio 3:3:3:3:3:2:2). In our previous study, we reported that SH21B inhibited adipogenesis and fat accumulation in 3T3-L1 cells through modulation of various regulators in the adipogenesis pathway. The aim of this study was to analyze the transcriptome profiles for the anti-adipogenic effects of SH21B in 3T3-L1 cells. Total RNAs from SH21B-treated 3T3-L1 cells were reverse-transcribed into cDNAs and hybridized to Affymetrix Mouse Gene 1.0 ST array. From microarray analyses, we identified 2,568 genes of which expressions were changed more than two-fold by SH21B, and the clustering analyses of these genes resulted in 9 clusters. Three clusters among the 9 showed down-regulation by SH21B (cluster 4, cluster 6 and cluster 9), and two clusters showed up-regulation by SH21B (cluster 7 and cluster 8) during the adipogenesis of 3T3-L1 cells. It was found that many genes related to cell proliferation and adipogenesis were included in these clusters. Clusters 4, 6 and 9 included genes which were related with adipogenesis induction and cell cycle arrest. Clusters 7 and 8 included genes related to cell proliferation as well as adipogenesis inhibition. These results suggest that the mechanisms of the anti-adipogenic effects of SH21B may be the modulation of genes involved in cell proliferation and adipogenesis.

Total Phenolic Contents and Antioxidant Capacities of Selected Chinese Medicinal Plants

Antioxidant capacities of 56 selected Chinese medicinal plants were evaluated using the Trolox equivalent antioxidant capacity (TEAC) and ferric reducing antioxidant power (FRAP) assays, and their total phenolic content was measured by the Folin-Ciocalteu method. The strong correlation between TEAC value and FRAP value suggested that the antioxidants in these plants possess free radical scavenging activity and oxidant reducing power, and the high positive correlation between antioxidant capacities and total phenolic content implied that phenolic compounds are a major contributor to the antioxidant activity of these plants. The results showed that Dioscorea bulbifera, Eriobotrya japonica, Tussilago farfara and Ephedra sinica could be potential rich sources of natural antioxidants.

생약복합물에 의한 지방세포형성 조절자의 유전자 발현 연구

본 연구의 목적은 생약복합제제인 SH21B의 adipogenesis 억제 효능에 대한 상세한 분자적 메커니즘을 3T3-L1 지방세포를 이용하여 밝히는 데 있다. 실험에 사용된 SH21B는 7가지 생약성 천연물질인, 황금, 행인, 마황, 석창포, 포황, 원지 및 하엽으로 이루어졌다. 최근, 본 연구진에 의해 3T3-L1을 이용한 in vitro 연구와 마우스를 이용한 in vivo 연구에서 SH21B의 adipogenesis 억제효능이 밝혀진 바 있다. 본 연구에서는 3T3-L1 지방세포가 분화될 때 작용하는 다양한 지방세포형성 조절자들의 유전자 발현이 SH21B에 의해 어떻게 변하는지 살펴보고자 하였다. 실시간중합효소반응(real time PCR) 기술을 이용하여 SH21B를 처리한 지방세포와 그렇지 않은 지방세포를 비교한 결과, 최종마커인 ADIPOQ와 SLC2A4의 유전자 발현이 SH21B에 의해 급격하게 감소함을 알 수 있었다. 최종마커의 발현을 유도하는 핵심전사인자인 <TEX>$PPAR{\gamma}$</TEX>와 C/<TEX>$EBP{\alpha}$</TEX>의 유전자 발현 역시 SH21B의 처리 시 유의하게 억제되었다. 좀 더 상세한 분자적 메커니즘을 규명하기 위해, 핵심전사인자의 상위에 위치한 다양한 조절자들의 유전자 발현을 분석하였다. 그 결과, 여러 지방세포형성 유도조절자 중, Krox20과 KLF15의 유전자 발현이 SH21B 처리에 의해 유의하게 감소된 반면, C/<TEX>$EBP{\beta}$</TEX>와 KLF5의 유전자 발현은 SH21B 처리에 영향을 받지 않았다. 그리고 지방세포형성 억제조절자인 KLF2와 CHOP의 유전자 발현은 SH21B 처리에 의해 유의하게 증가되었다. 이러한 결과들은 SH21B의 지방세포형성 억제효능이 지방세포의 분화에 작용하는 다양한 상위조절자 중 지방세포형성 유도조절자인 Krox20과 KLF15 그리고 지방세포형성 억제조절자인 KLF2와 CHOP 등의 유전자 발현이 변화되면서 일어나는 복합적인 반응의 결과임을 제시한다. In our previous study, it was reported that an herbal mixture, SH21B, inhibits fat accumulation and adipogenesis both in vitro and in vivo models of obesity. SH21B is a mixture composed of seven herbs: Scutellaria baicalensis Georgi, Prunus armeniaca Maxim, Ephedra sinica Stapf, Acorus gramineus Soland, Typha orientalis Presl, Polygala tenuifolia Willd, and Nelumbo nucifera Gaertner (Ratio 3:3:3:3:3:2:2). The aim of this study was to investigate the detailed molecular mechanisms of the effects of SH21B on various regulators of the adipogenesis pathway. During the adipogenesis of 3T3-L1 cells, SH21B significantly decreased the expression levels of central transcription factors of adipogenesis, such as peroxisome proliferator-activated receptor (PPAR)<TEX>$\gamma$</TEX> and CCAAT/enhancer binding protein (C/EBP)<TEX>$\alpha$</TEX>. To elucidate the detailed molecular mechanism of the anti-adipogenic effects of SH21B, we examined the expression levels of the various pro-adipogenic or anti-adipogenic regulators of adipogenesis upstream of <TEX>$PPAR{\gamma}$</TEX> and C/<TEX>$EBP{\alpha}$</TEX>. The mRNA levels of Krox20 and Kruppel-like factor (KLF) 15, which are pro-adipogenic regulators, were significantly down-regulated by SH21B treatment, whereas the mRNA levels of C/<TEX>$EBP{\gamma}$</TEX> and KLF5 were not changed. KLF2 and C/EBP homologous protein (CHOP), which are anti-adipogenic regulators, were significantly up-regulated by SH21B treatment. These results suggest that the molecular mechanism of the anti-adipogenic effect of SH21B involves both the down-regulations of pro-adipogenic regulators, such as Krox20 and KLF15, and the up-regulations of anti-adipogenic regulators, such as KLF2 and CHOP, which results in the suppression of central transcription factors of adipogenesis including <TEX>$PPAR{\gamma}$</TEX> and C/<TEX>$EBP{\alpha}$</TEX>.

Composition and stereochemistry of ephedrine alkaloids accumulation in Ephedra sinica Stapf

Ephedra sinica Stapf (Ephedraceae) is a widely used Chinese medicinal plant (Chinese name: Ma Huang). The main active constituents of E. sinica are the unique and taxonomically restricted adrenergic agonists phenylpropylamino alkaloids, also known as ephedrine alkaloids: (1 R,2 S)-norephedrine (1 S,2 S)-norpseudoephedrine, (1 R,2 S)-ephedrine, (1 S,2 S)-pseudoephedrine, (1 R,2 S)- N-methylephedrine and (1 S,2 S)- N-methylpseudoephedrine. GC–MS analysis of freshly picked young E. sinica stems enabled the detection of 1-phenylpropane-1,2-dione and ( S)-cathinone, the first two putative committed biosynthetic precursors to the ephedrine alkaloids. These metabolites are only present in young E. sinica stems and not in mature stems or roots. The related Ephedra foemina and Ephedra foliata also lack ephedrine alkaloids and their metabolic precursors in their aerial parts. A marked diversity in the ephedrine alkaloids content and stereochemical composition in 16 different E. sinica accessions growing under the same environmental conditions was revealed, indicating genetic control of these traits. The accessions can be classified into two groups according to the stereochemistry of the products accumulated: a group that displayed only 1 R stereoisomers, and a group that displayed both 1 S and 1 R stereoisomers. ( S)-cathinone reductase activities were detected in E. sinica stems capable of reducing ( S)-cathinone to (1 R,2 S)-norephedrine and (1 S,2 S)-norpseudoephedrine in the presence of NADH. The proportion of the diastereoisomers formed varied according to the accession tested. A (1 R,2 S)-norephedrine N-methyltransferase capable of converting (1 R,2 S)-norephedrine to (1 R,2 S)-ephedrine in the presence of S-adenosylmethionine (SAM) was also detected in E. sinica stems. Our studies further support the notion that 1-phenylpropane-1,2-dione and ( S)-cathinone are biosynthetic precursors of the ephedrine alkaloids in E. sinica stems and that the activity of ( S)-cathinone reductases directs and determines the stereochemical branching of the pathway. Further methylations are likely due to N-methyltransferase activities.

Ephedrine Controls Heart Rhythms by Activating Cardiac Iks Currents

Ephedrine (Eph) is an alkaloid extracted from the Chinese traditional medicine plant Ephedra Sinica or Ma huang, which has been known for effects on the central nervous system, cardiovascular system, and smooth muscles. However, the corresponding molecular mechanism of these effects remains unknown. In this study, we investigated the influences of Eph on heart rate, QTc interval in vivo, and the slowly activated K channels (IKs) that were composed of both KCNQ1 and KCNE1 subunits in vitro. Results demonstrated that Eph, but not pseudoephedrine, could increase the heart rate and shorten QTc interval of BALB/c mouse. Besides, Eph markedly activated cardiac IKs currents with EC50 = 50 nM and shifted G-V curves to left. But pseudoephedrine had no effects on Iks currents. The onset and offset time constants of IKs currents activated by Eph at 1 M were tauon = 49 seconds and tauoff = 400 seconds. A pair of binding sites of Eph on KCNQ1/KCNE1 channel was also shown to occur at F296 and Y299 in the S5-S6 P-loop of the KCNQ1 channel. As both amino acids are highly conserved in the KCNQ family, Eph can possibly activate other members of the KCNQ family. The mechanism of Iks activated by Eph may provide a clue for drug design in the future.

Identification of Two Cold Water-Soluble Polysaccharides from the Stems of Ephedra sinica Stapf

Two polysaccharides (ESP-A1 and ESP-A2) were isolated from the cold water extract of Ephedra sinica Stapf and purified through ethanol precipitation, deproteinization and by ion exchange and gel-filtration chromatography. Their molecular weight was determined using high performance size exclusion chromatog-raphy and evaporative light scattering detector (HPSEC-ELSD) and their monosaccharide composition was analyzed by high performance capillary electrophoresis (HPCE) based on pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP). It was shown that ESP-A1 consisted of xylose, arabinose, glucose, mannose and galactose and ESP-A2 consisted of xylose, arabinose, rhamnose and galactose, in a molar ratio (%) of 3.2: 61.1: 11.1: 12.9: 11.6 and 20.6: 67.7: 5.0: 6.7, respectively. The molecular weights (Mw) of ESP-A1 and ESP-A2 were 5.83 × 104 Da and more than 200 × 104 Da, respectively. To the best of our knowledge, two neutral polysaccharides are now being reported for the first time in this study.

SH21B, an anti-obesity herbal composition, inhibits fat accumulation in 3T3-L1 adipocytes and high fat diet-induced obese mice through the modulation of the adipogenesis pathway

Ethnopharmacological relevance SH21B is an anti-obesity composition composed of seven herbs: Scutellaria baicalensis Georgi, Prunus armeniaca Maxim, Ephedra sinica Stapf, Acorus gramineus Soland, Typha orientalis Presl, Polygala tenuifolia Willd and Nelumbo nucifera Gaertner. It has been used for the treatment of obesity in traditional medical clinics in Korea, but its anti-obesity effects and mechanism of action have not been studied until now. Aim of the study This study was conducted to confirm the anti-obesity effects of SH21B and to elucidate its mechanism of action. Materials and methods 3T3-L1 adipocytes and high fat diet-induced obese mice were treated with SH21B, and its effect on gene expression was analyzed using microarray technology, real-time PCR and western blotting experiments. Results SH21B significantly inhibited fat accumulation in 3T3-L1 adipocytes and reduced adipose tissue and serum triglyceride levels in high fat diet-induced obese mice. Microarray analyses showed that SH21B affected more genes in the adipogenesis pathway than any other pathway studied. SH21B significantly decreased the expression of major transcription factors of the adipogenesis pathway and resulted in the down-regulation of lipid metabolizing enzymes involved in the transport, uptake and synthesis of lipids. Conclusions SH21B inhibits fat accumulation by down-regulating the expression of genes involved in the adipogenesis pathway.

Studies of Ephedra plants in Asia. Part 6: Geographical changes of anatomical features and alkaloids content of Ephedra sinica

Ephedra sinica Stapf is the main botanical origin of the Chinese herbal drug Mahuang, Ephedra Herb. Eighty-five samples of E. sinica, collected across eastern China, Mongolia, and Buryatia (Russia), were studied anatomically and chemically to elucidate the local variations and the relation between environmental factors and the variations. The results showed that samples grown in more arid conditions tended to have a more sinuous epidermis, more cuticular tubers, and more subepidermal fiber bundles anatomically, and contained more total ephedrine alkaloids. These samples also had a high pseudoephedrine content. These results imply that Ephedra Herb with good quality should be collected from arid fields, and the chemical quality can be estimated by observing the anatomical characteristics.

Comparison of the contents of kampo decoctions containing ephedra herb when prepared simply or by re-boiling according to the traditional theory

Herbal formulas containing ephedra herb (mao-zai) are among the most important medicinal prescriptions in Japanese traditional kampo medicine to treat cold symptoms, bronchial asthma, arthralgia, and rheumatism. Shokan-zatsubyo-ron (Shanghan zabing lun in Chinese), a classical textbook of kampo medicine published in 220 A.D., describes that when herbal formulas containing ephedra herb (stem of Ephedra sinica) such as maoto (mahuang-tang) and kakkonto (gegen-tang) are prepared, ephedra herb is first boiled alone, the scum is removed, and then other crude drugs are added and decocted. In the present study, we evaluated evidence for the benefit of boiling ephedra herb prior to other crude drugs by analyzing the contents of the extract and four ephedrine alkaloids (ephedrine, methylephedrine, pseudoephedrine, and norephedrine), which are considered the main active ingredients in ephedra herb. We prepared two different decoctions of maoto and kakkonto: one decoction was prepared by boiling all the crude drugs at the same time and the other decoction was prepared according to the classical textbook. In both decoctions of maoto and kakkonto, neither alkaloid contents in the extract nor extracting ratio of four ephedrine alkaloids exhibited significant difference. As far as the ephedrine alkaloid content goes, there is no evidence for the benefit of the boiling method for ephedra herb described in the classical textbook of kampo medicine.