Transcriptomic Analysis of Wuzi Yanzongwan on Testicular Spermatogenic Function in Semi-castrated Male Mice / 中国实验方剂学杂志

ObjectiveTo screen out the transcriptomes related to the intervention of Wuzi Yanzongwan on the spermatogenic function of semi-castrated male mice， and to explore its potential mechanism in the intervention of the progress of low spermatogenic function. MethodBalb/c mice were randomly divided into sham-operated group， model group， testosterone propionate group（0.2 mg·kg-1·d-1， intramuscular injection） and Wuzi Yanzongwan group（1.56 g·kg-1·d-1， intragastric administration） according to body weight， with 12 mice in each group. The right testicle and epididymis were extracted from the model group and the drug administration group to construct the semi-castrated model of low spermatogenic function， while the fur and the right scrotum of the sham-operated group were only cut and immediately sterilized and sutured. At the end of the intervention， hematoxylin-eosin（HE） staining was used to observe the histopathology of testis， enzyme-linked immunosorbent assay（ELISA） was used to detect the levels of serum testosterone（T）， luteinizing hormone（LH） and follicle stimulating hormone（FSH）. The sperm count and motility of epididymis were measured by automatic sperm detector of small animal. Transcriptomic microarray technology was used to detect the mRNA expression level of testicular tissue in each group， the transcriptome of genes related to the regulation of Wuzi Yanzongwan was screened， and three mRNAs were selected for Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） to verify the transcriptome data. Through the annotation analysis of Gene Ontology（GO） and the signaling pathway analysis of Kyoto Encyclopedia of Genes and Genomes（KEGG）， the related functions of drugs regulating transcriptome were analyzed. ResultCompared with the sham-operated group， the testicular tissue of mice in the model group showed spermatogenic injury， contraction and vacuolization of the seminiferous tubules， reduction of spermatogenic cells at all levels， widening of the interstitial space， obstruction of spermatogonial cell development and other morphological abnormalities， and serum T significantly decreased， LH significantly increased（P<0.01）， and FSH elevated but no statistically significant difference， the count and vitality of epididymal sperm significantly decreased（P<0.01）. There were 882 differentially expressed mRNAs in the testicular tissues， of which 565 were up-regulated and 317 were down-regulated. Cluster analysis showed that these differentially expressed mRNA could effectively distinguish between the sham-operated group and the model group. Compared with the model group， the damage to testicular tissue in the Wuzi Yanzongwan group was reduced， the structure of the seminiferous tubules was intact， vacuolization was reduced， and the number of spermatogenic cells at all levels was significantly increased and arranged tightly. The serum T significantly increased， LH significantly decreased（P<0.01）， and FSH decreased but the difference was not statistically significant. The count and vitality of sperm in the epididymis were significantly increased（P<0.01）. Moreover， Wuzi Yanzongwan could regulate 159 mRNA levels in the testes of semi-castrated mice， of which 32 were up-regulated and 127 were down-regulated， and the data of the transcriptome assay was verified to be reliable by Real-time PCR. GO and KEGG analysis showed that the transcriptome functions regulated by Wuzi Yanzongwan were involved in the whole cell cycle process of sperm development such as sex hormone production of interstitial cells in testis， renewal， differentiation， metabolism， apoptosis and signal transduction of spermatogenic cells， and were closely related to the biological behaviors of signaling pathways such as spermatogenic stem cell function， endoplasmic reticulum protein processing and metabolic program. ConclusionWuzi Yanzongwan can effectively improve the low spermatogenic function of semi-castrated male mice， and its mechanism may be related to the regulation of testicular transcriptional regulatory network， the synthesis of sex hormones in testicular interstitial cells， the function of spermatogenic stem cells， the whole cell cycle process of spermatogenesis， as well as the expression of endoplasmic reticulum protein processing and metabolic program related genes transcription.

The interaction between polyphyllin I and SQLE protein induces hepatotoxicity through SREBP-2/HMGCR/SQLE/LSS pathway / 药物分析学报

Polyphyllin Ⅰ(PPⅠ)and polyphyllin Ⅱ(PⅡ)are the main active substances in the Paris polyphylla.However,liver toxicity of these compounds has impeded their clinical application and the potential hepatotoxicity mechanisms remain to be elucidated.In this work,we found that PPⅠ and PⅡ exposure could induce significant hepatotoxicity in human liver cell line L-02 and zebrafish in a dose-dependent manner.The results of the proteomic analysis in L-02 cells and transcriptome in zebrafish indicated that the hepa-totoxicity of PPⅡ and PⅡwas associated with the cholesterol biosynthetic pathway disorders,which were alleviated by the cholesterol biosynthesis inhibitor lovastatin.Additionally,3-hydroxy-3-methy-lglutaryl CoA reductase(HMGCR)and squalene epoxidase(SQLE),the two rate-limiting enzymes in the choles-terol synthesis,selected as the potential targets,were confirmed by the molecular docking,the over-expression,and knockdown of HMGCR or SQLE with siRNA.Finally,the pull-down and surface plasmon resonance technology revealed that PPⅠ could directly bind with SQLE but not with HMGCR.Collectively,these data demonstrated that PPⅠ-induced hepatotoxicity resulted from the direct binding with SQLE protein and impaired the sterol-regulatory element binding protein 2/HMGCR/SQLE/lanosterol synthase pathways,thus disturbing the cholesterol biosynthesis pathway.The findings of this research can contribute to a better understanding of the key role of SQLE as a potential target in drug-induced hepatotoxicity and provide a therapeutic strategy for the prevention of drug toxic effects with similar structures in the future.

Comparison of the Effects on Angiogenesis Activity between Whitmania Pigra Whitman and Hirudinaria Manillensis Lesson Based on a Zebrafish Model / 中国药师

Objective:To investigate and compare the effects of water extracts of Whitmania pigra Whitman and Hirudinaria ma-nillensis Lesson on the angiogenesis of Tg (kdrl:mCherry) zebrafish. Methods:The zebrafish embryos 6-8 hours after fertilization (6-8hpf) were transferred to the culture medium containing Whitmania pigra Whitman or Hirudinaria manillensis Lesson extract at different concentrations, and the culture medium containing the drugs was replaced every 24 h. And then, at 72 hpf, the larvalmorphology and intersegmental vessels were observed under a microscope. The hatchability of 48-and 72-hpf embryos, and the number of intersegmen-tal vessels and the heart rate of 72-hpf juveniles were measured. Results:Compared with the control group, when the concentration of Whitmania pigra Whitmanis was higher than 30μg· ml-1 , and the concentration of Hirudinaria manillensis Lesson was higher than 20μg· ml-1, the number of intersegmental vessels was significantly reduced (P<0. 01). Compared with the control group, at 48 hpf, when the concentration of the drug groups was higher than 40 μg· ml-1 , the hatchability of the two groups significantly decreased ( P<0. 01);at 72 hpf, the hatchability of Whitmania pigra Whitman decreased significantly at the concentration of 100 μg·ml-1 (P<0.01), while the hatchability of Hirudinaria manillensis Lesson decreased significantly at the concentration of 80 μg· ml-1(P <0. 01). There was no obvious yolk sac edema, pericardial edema and spine curvature in the two groups. The heart rate decreased sig-nificantly (P<0. 01), while was still within the normal range. Conclusion:Both Whitmania pigra Whitman and Hirudinaria manillen-sis Lesson have notable anti-angiogenic activity, and the anti-angiogenesis activity of Hirudinaria manillensis Lesson is stronger. They both have effects on the development of zebrafish embryos, while the toxicity is not obvious.

Applicability of Zebra Fish Thrombosis Model in Antithrombotic Activity Screening of Chinese Materia Medica / 中国中医药信息杂志

Objective To investigate the applicability of zebra fish thrombosis model in antithrombotic activity screening of Chinese materia medica.Methods The living zebra fish thrombosis model was induced by adrenaline hydrochloride. Zebra fish were randomly divided into blank control group, model group, positive medicine group and medication group. Each group was given the corresponding medicine or embryo culture water. O-anisidine staining solution was used to stain and calculate the staining intensity of erythrocytes in zebra fish heart, and quantitative analysis was carried out. The platelet aggregation of transgenic zebra fish was observed and under qualitative analysis. Results Compared with the model group, 100μg/mL salvianolic acid B, 300, 900μg/mL aqueous extract of Salvia miltiorrhiza, 45μg/mL 95% ethanol extract and 400, 1200μg/mL hypothalamus could significantly inhibited the formation of zebra fish thrombosis (P<0.01).ConclusionZebra fish thrombosis model has good applicability in antithrombotic activity screening of Chinese materia medica.

Determination of triterpenes in Melastoma dodecandrum Lour. from different producing areas by HPLC / 国际中医中药杂志

Objective To develop a HPLC method for the simultaneous content determination of three terpenoids inMelastoma dodecandrum Lour. (asiatic acid, betulinic acid, oleanolic acid).Methods The chromatographic column was set with waters SunFire C18 column (4.6 mm×150 mm, 5μm); the moving phase was acetonitrile -0.1%H3PO4; the column temperature was 30℃; the detection wavelength was 200 nm; the flow rate was 0.6 ml/min; and the sample volume was 25μl.Results A good linear relationship was observed in the range of 0.310-6.200μg (r=0.9999), 0.405-8.100μg (r=0.9999), 0.169-3.375μg (r=0.9998) for asiatic acid, betulinic acid, oleanolic acid respectively, with the average recovery rates of 102.08%, 101.81%, 102.22%. Conclusions The established method is convenient and sensitive, repeatability and stability, quality evaluation for Melastoma dodecandrum Lour.

Three Determination Methods for Residual Sulfite in Traditional Chinese Medicinal Materials and Pieces / 中国药师

Objective:To determine residual sulfite in traditional Chinese medicinal materials or pieces processed by sulfur fumi-gation respectively by iodine titration, acid-base titration and ion chromatography and compare the results. Methods:The three meth-ods were used to determine four kinds of Chinese herbal medicines including Codonopsis radix, Dioscoreae rhizoma, Achyranthis bident-atae Radix and Atractulodis Macrocephalae Rhizoma, the recovery tests were also performed and the results were analyzed and compared to summarize the characteristics and quality control requirements of each method. Results:Iodine titration and acid-base titration had the advantages of simple operation process and low cost. However, there were many interference factors in the two methods, and due to different principles, they were suitable for the determination of different varieties of herbal medicines. Ion chromatography method had the advantages of high sensitivity and strong specificity, while the cost was high. Conclusion: It is suggested that proper methods should be chosen for the determination of sulfur dioxide residues according to actual situations.

HPLC Simultaneous Determination of Vitexin and Isovitexin inMelastoma Dodecandrum / 世界科学技术-中医药现代化

This study was aimed to develop HPLC for determination of vitexin and isovitexin inM. dodecandrum. The HPLC column was SunFireTM C18 (4.6 mm× 150 mm, 5μm). The detection wavelength was 365 nm. The mobile phase was methanol-0.2% formic acid aqueous solution. The column temperature was 40℃. The flow rate was 1.0 mL·min-1. The results showed that the regression equations of vitexin and isovitexin wereY = 1× 106X– 14 396, Y = 1× 106X– 13 900, respectively. The linear ranges were 0.210μg - 1.050μg (r = 0.999) and 0.186μg - 0.930μg (r = 1.000), respectively. The recovery rates were 97.48% and 104.64%, respectively. The RSD were 2.32%and 1.51%, respectively. The sample contents of vitexin and isovitexin were 1.25 and 1.86 mg·g1, respectively. It was concluded that the method was simple, feasible and reproducible for the content determination of vitexin and isovitexin inM. dodecandrum.

Research Progress in Toxicity Assessment Using Zebrafish / 中国药师

Zebrafish toxicity assessment system is one of the important vertebrate model systems. Zebrafish is playing an increas-ingly important role in the field of toxicology studies because of its small size, short generation cycle, the transparent embryo and high reproductive rate. Now it is widely used in the embryonic derelopmental toxicology, pathological toxicology, environmental toxicology and other areas of toxicology studies with its unique advantages.

Molecular Identification of Metacordyceps Liangshanensis, Its Adulterants and Its Relative Species Based on DNA Barcode / 世界科学技术-中医药现代化

This study was aimed to identify and distinguish Metacordyceps liangshanensis recorded by the Sichuan Province Traditional Chinese Medicine Standard from its adulterants and its relative species by combining ITS and COI barcode sequences in order to study the feasibility of this new method. After extracting DNA of 28 species of Cordyceps samples, DNA were amplified and sequenced. And then, ITS and COI sequences were received. Codon-Code Aligner V3.7.1 and Mega 5.0 were used to analyze the variable site and construct the N-J tree. The results showed that the minimum ITS inter-specific K-2P distance was relatively higher than the maximum intra-specific K-2P distance. The inter-specific sequence divergence between M. liangshanensis and its adulterants exhibited high while intra-specific sequence divergence exhibited low. And COI one was the same case. N-J tree of both ITS and COI indicated that same genus belonged together and each species belonged to relatively independent branch. It was concluded that based on the ITS and COI gene, the technology of DNA barcode can be an excellent identification of M. liangshanensis, its adulterants and its relative species. It provided technical support for the further research on species molecular identification and phylogenetics of Cordyceps .

Macroscopic and microscopic characteristics of Chinese caterpillar fungus / 药学学报

Chinese Caterpillar Fungus (CCF) is one of the rare Chinese traditional drugs. As the resource is reducing sharply, the price is rising higher and higher, and there have been much more adulterants in the markets, but until now we don't have a scientific and accurate research on the identification study for this drug. On the basis of resource investigation, during the study of the samples collected by ourselves and the specimens stored in the museum, using the macroscopic and microscopic methods, referring to the literatures of entomology, emphasizing on the characteristics of polypide part, we have studied this species in detail of the macroscopic characters such as the insertion position of the stroma part, the annulations and segments of the caterpillar, the abdominal leg, the pinaculum, and the microscopic characters of the body wall; firstly added the microscopic character of the crotchets on the planta of abdominal leg. The result turned out that the characters which we have studied are regular and stable, and it have laid the foundation for the powder products and patent medicines which have used the crude drug of CCF.

Effect of Euphorbia kansui on urination and kidney AQP2, IL-1beta and TNF-alpha mRNA expression of mice injected with normal saline / 中国中药杂志

<p><b>OBJECTIVE</b>To observe the effect of Euphorbia kansui (E. KS) alcohol extracts on urination and kidney-related expressions of mice injected with normal saline and to discuss its impact on kidney.</p><p><b>METHOD</b>Mice intraperitoneally injected with normal saline were observed for urination and changes in kidney-related histiocytic factors of after intragastrical administration of E. KS and compared with normal mice.</p><p><b>RESULT</b>E. KS alcohol extracts can promote urination of mice injected with normal saline and enhance peripheral serum creatinine, with no obvious pathological change showed in tissue sections. It had a certain effect on reducing AQP2 expression and enhancing TNF-alpha expression.</p><p><b>CONCLUSION</b>Euphorbia kansui in large dose has a remarkable effect on kidney but may be accompanied with pathological reactions to some extent, especially the dose of 1.2 g x kg(-1). The pathological reactions may be related with increased serum creatinine and TNF-alpha expression.</p>

Rapid detection of alkaloids in Ipecac by direct analysis in real time tandem mass spectrometry (DART-MS/MS) / 中国中药杂志

<p><b>OBJECTIVE</b>To detect alkaloids in Ipecac by direct analysis in real time tandem mass spectrometry (DART-MS) without pre-treatment and chromatographic separation.</p><p><b>METHOD</b>Under the optimum conditions, DART-MS characteristic spectra were collected for tablet of Ipecac powder, Ipecac stems and leaves by full scanning, and secondary spectra were adopted for identifying alkaloids. The multiple reaction monitoring mode was adopted to determine the mass spectrum peak intensity of determinands on the surface of determined samples, in order to calculate their average content in samples.</p><p><b>RESULT</b>Spectra of tablet of Ipecac powder and Ipecac stems showed remarkable ionized ion peaks of emetine and cephaeline at m/z 481 and 467, while spectra of leaves showed ionized ion peaks of other alkaloids at m/z 479 and 465. Furthermore, the quantitative analysis was also demonstrated with good reproducibility and linear relationship.</p><p><b>CONCLUSION</b>The mode can play a role in rapid determination of medicinal materials and prepared herbal medicines and real-time rapid quantitative analysis on intermediates and preparations.</p>

Study on water-soluble iron, heavy metals and harmful elements of Magnetitum / 中国中药杂志

<p><b>OBJECTIVE</b>To measure the contents of the water-soluble iron, five heavy metals and harmful elements in Magnetiturn and provide a basis for the quality control and safety evaluation of Magnetitum.</p><p><b>METHOD</b>Iron (Fe), lead (Pb), cadmium (Cd) and copper (Cu) were determined by atomic absorption spectrometry (AAS); arsenic (As) and mercury (Hg) were determined by atomic fluorescence spectrometry (AFS).</p><p><b>RESULT</b>The mean content of element iron is 764.30 mg x kg(-1). The contents of five water-soluble heavy metals and harmful elements in Magnetitum were within the safety range. The recovery of the standard addition was in the range of 93.7% - 110.6%, and the RSD was less than 5.0%.</p><p><b>CONCLUSION</b>Analyzing the water-soluble iron, heavy metals and harmful elements in Magnetitum is effective to the quality control and the safety evaluation of magnetitum.</p>

Determination of total content of ten ginsenosides in yiqifumai lyophilized injection by near infrared spectroscopy / 中国中药杂志

<p><b>OBJECTIVE</b>To determine the total content of 10 ginsenosides in Yiqifumai lyophilized injection by near infrared spectroscopy.</p><p><b>METHOD</b>Sixty samples were collected and determined of the total contents of ten ginsenosides by HPLC. The optimal calibration model was established by the contents of 10 ginsenosides in fifty samples and their NIR spectroscopy using the PLS. And the contents of 10 samples were successfully predicted.</p><p><b>RESULT</b>When using the pretreatment of the first derivative and MSC in the range of 4 246.8 - 4 602.2, 5 446.8 - 61 02.6 cm(-1), the best dimension was 9, and the quantitative model was accurate. The R2 was 94.2, and the RMSECV was 0.186. The RMSEP of ten samples was 0.234.</p><p><b>CONCLUSION</b>This method is easy, rapaid and precise, and can be used to determine the content of 10 ginsenosides in Yiqifumai lyophilized injection.</p>

Simultaneous determination of 4 nortriterpenoids in Schisandra chinensis extract by HPLC / 中国中药杂志

<p><b>OBJECTIVE</b>To determine 4 nortriterpenoids (de-hydroxy arisanlactone D, 25-hydroxy schindilactone, schindilachone A, lancifodilactone D) in Schisandra chinensis extract by HPLC.</p><p><b>METHOD</b>The analysis was performed on a waters symmetry column (4.6 mm x 250 mm, 5 microm) with the mobile phase of acetonitrile-water (33:67) at a flow rate of 1 ml x min(-1). The column temperature was set at 37 degrees C, and the detector wavelength was 264 nm.</p><p><b>RESULT</b>The linear ranges of de-hydroxy arisanlactone D, 25-hydroxy schindilactone, schindilachone A, and lancifodilactone D are 0.075-1.800, 0.098-0.980; 0.095-0.950, and 0.053-0.530 microg, respectively, and the average recoveries were 98.57%, 96.44%, 97.96%, and 97.27%, respectively.</p><p><b>CONCLUSION</b>The four nortriterpenoids were well separated by this method, and it could be used to determine the four nortriterpenoids in Schisandra chinensis extract.</p>

Herbalism, botany and components analysis study on original plants of frankincense / 中国中药杂志

In order to clarify original plants of traditional Chinese medicine (TCM) frankincense, a GC method for determination essential oils and a HPLC method for determination boswellic acids were carried out together with analysis of herbalism, botany, components and pharmacology papers of frankincense. It was concluded that original plants of TCM frankincense include at least Boswellia sacra, B. papyrifera and B. serrata.

X-ray diffraction Fourier fingerprint of mineral Chinese medicine Chloriti Lapis / 中国中药杂志

The technology of powder X-ray diffraction (XRD) was used for analysis Chloriti Lapis and the XRD Fourier fingerprints were established. The dates were analyzed by fuzzy cluster and fingerprint similarity evaluation software to compare the similarity of samples. XRD fingerprint with 10 common peaks of 14 batches of Chloriti Lapis were established. The average, median coefficients of crystal lattice spacing d (A), peak position 2 theta, relative intensity value I/I0 (%) were all more than 0.95. And similarity( angle cosine value) were all more than 0. 97. There were small number samples differed from others. And obvious differences between the pre-and post-processing samples. This paper shows the powder XRD Fourier fingerprint can be used for appraisal and study of the Chloriti Lapis.

Two iridoid glycoside from roots of Phlomis medicinalis / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical constituents of the roots of Phlomis medicinalis.</p><p><b>METHOD</b>The compounds were isolated and repeatedly purified by macroporous resin, silica gel column chromatography, TLC and PREP-HPLC. Their structures were elucidated by physical and chemical properties and NMR spectra.</p><p><b>RESULT</b>Two iridoid glucosides were obtained and elucidated as 7-epilamalbide (1), chlorotuberoside (2).</p><p><b>CONCLUSION</b>Compound 1 was a new compound, compound 2 was isolated from the plant for the first time.</p>

Application of near infrared spectroscopy in quality control of excipients of traditional Chinese medicine / 中国中药杂志

<p><b>OBJECTIVE</b>To apply the near infrared spectroscopy in quality control for excipients of traditional Chinese medicine (TCM).</p><p><b>METHOD</b>A new type of transmittance and reflectance integration sphere accessory and a transmittance accessory were used to collect the spectra of processed honey and rice wine, respectively. Determination method of reducing sugar and ethanol in processed honey were established with partial least squares (PLS).</p><p><b>RESULT</b>The correlation coefficients (r), the root mean square error of calibration (RMSEC) and the root mean square error of prediction (RMSEP) of the proposed models were 0.9593, 1.00% and 1.19% for reducing sugar; 0.9529, 0.17% and 0.24% for ethanol.</p><p><b>CONCLUSION</b>The proposed method is fast, non-destructive, simple and accurate, and can be applied to at-spot detection,</p>

Chemical constituents of Viola tianshanica / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical constituents of Viola tianshanica.</p><p><b>METHOD</b>Compounds were isolated by silica column, pharmadex LH-20 column and polyamide column, and their structures were elucidated by UV, IR, ESI-MS and NMR.</p><p><b>RESULT</b>Six compounds were isolated and identified as daucosterol (1), kaempferol-7-O-beta-D-glucopyranoside (2), kaempferol- 3-O-beta-D-glucopyranoside (3), isorhamnetin-3-O-beta-glucoside (4), kaempferol (5) and quercetin (6).</p><p><b>CONCLUSION</b>Compounds 2-5 were isolated from this plant for the first time.</p>