Tissue culture of safflower and analysis of secondary metabolites in suspension cells / 中国中药杂志

Safflower(Carthamus tinctorius), a valuable traditional Chinese medicinal plant, has attracted much attention in recent years. This study established a stable tissue culture system of safflower and analyzed the chromatogram of its secondary metabolites, providing high-quality experimental materials for further research on natural products in safflower. The calluses were established from the safflower seeds germinated in a sterile environment, and then they were differentiated into the aseptic seedlings, or cultured to obtain suspension cells in liquid medium. The ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry(UPLC-Q-TOF-MS), Progenesis QI, and principal component analysis(PCA) were used to detect and analyze the secondary metabolites in the suspension cells before and after induction with different elicitors(methyl jasmonate, silver nitrate, salicylic acid and yeast extract). A total of 23 secondary metabolites including flavonoids, phenylpropanoids, alkaloids, fatty acids and aromatic glycosides were detected in safflower suspension cells. In response to the four elicitors, 11 compounds showed increased or decreased relative content. The results indicate that different elicitors have various effects on the accumulation of secondary metabolites in safflower suspension cells, and yeast extract shows more obvious positive induction. Therefore, different elicitors may play a role in the expression of related genes in the biosynthetic pathway of specific secondary metabolites. The results facilitate the discovery of targeted elicitors and the large-scale production of valuable secondary metabolites in the future.

Discussion on research idea of quality marker of Salvia miltiorrhiza based on biosynthetic pathway of tanshinone compounds / 中国中药杂志

Based on the theory of Q-marker, the hairy root of Salvia miltiorrhiza and S. miltiorrhiza in many provinces were studied. The relative expressions of SmCPS, SmKSL and CYP76AH1 genes in hairy roots were detected by real-time fluorescence quantitative PCR and the contents of tanshinoneⅡ_A, cryptotanshinone, tanshinoneⅠ, 1,2-dihydrotanshinone, ferruginol and miltiradiene were detected by UPLC and GC-MS, respectively. Statistical analysis shows as fllows: in the hairy root of S. miltiorrhiza, the content of miltiradiene and ferruginol is positively correlated with the content of tanshinone compounds in the downstream, and the relative expression of important genes in the biosynthetic pathway of tanshinone can reflect the content of tanshinone compounds to a certain extent; in many provinces of S. miltiorrhiza, the content of ferruginol and tanshinone compounds can also be found that there is a positive correlation between the contents. Based on the biosynthetic pathway of tanshinone compounds, which is a special index component in S. miltiorrhiza, this study focused on the important relationship between the upstream gene, the middle intermediate compound and the downstream tanshinone compound content of the biosynthetic pathway, and explored the possible research ideas of improving the quality marker system of S. miltiorrhiza, and then provided the possible research ideas for understanding and studying the quality marker of traditional Chinese medicine from the biosynthetic pathway.

Molecular cloning and functional characterization of multiple ApOSCs from Andrographis paniculata / 中国天然药物

Triterpenoids have been described in Andrographis paniculata. Oleanolic acid exhibits high biological activity and is widely used in the clinic, and β-sitosterol not only has good biological activity but also plays an important physiological role in plants. However, analysis of the biosynthetic pathway of triterpenoids in Andrographis paniculata has not been reported. Here, we provide the first report of the isolation and identification of nine 2, 3-oxidosqualene cyclases (ApOSC3 to ApOSC11) from A. paniculata. The results showed that ApOSC4 represented a monofunctional synthase that could convert 2, 3-oxidosqualene to β-amyrin. ApOSC5 as a bifunctional 2, 3-oxidosqualene cyclases, could transfer 2, 3-oxidosqualene to β-amyrin and α-amyrin. ApOSC6 to ApOSC8 composed the multifunctional 2, 3-oxidosqualene cyclases that could convert 2, 3-oxidosqualene to β-amyrin, α-amyrin and one or two undetermined triterpenoids. This study provides a better understanding of the biosynthetic pathway of triterpenoids in A. paniculata, and the discovery of multifunctional 2, 3-oxidosqualene cyclases ApOSC5 to ApOSC8 of the facilitates knowledge of the compounds diversity in A. paniculata.

Effect of GR24 on accumulation of diterpenoids in Tripterygium wilfordii suspension cells / 中国中药杂志

Terpenoids are main bioactive components in Tripterygium wilfordii,but the contents of some terpenoids are relatively low. In order to provide scientific evidence for the regulation of terpenoids in T. wilfordii,this research explored the effect of GR24 on accumulations of four diterpenoids( triptolide,tripterifordin,triptophenolide,and triptinin B) in T. wilfordii suspension cells by biological technology and UPLC-QQQ-MS/MS. The results indicated that 100 μmol·L-1 GR24 inhibited the accumulations of triptolide,tripterifordin,triptophenolide,and triptinin B to different degrees. Compared with the control group,the contents of 4 diterpenoids( in the induced group) were down to 96.59%,63.80%,61.02% and 33.59% in 240 h,respectively. Among them,the accumulation of triptinin B iswas significantly inhibited. In addition,the key time point of inhibitory effect was 120 h after induction with GR24 in some diterpenoids. This is the first systematic study focusing on the effect of GR24 on the accumulations of diterpenoids in T. wilfordii suspension cells. The dynamic accumulation ruleregularity of four diterpenoids after induced by GR24 was summarized,which laid a foundation for further study on the chemical response mechanism of terpenoids to GR24.

Bioinformatics and tissue distribution analysis of Tripterygium wilfordii CYP450 / 中国中药杂志

Cytochrome P450 family is a kind of biocatalyst widely existing in nature. It has many functions such as catalyzing the biosynthesis of plant secondary metabolites and regulating phytoremediation. Based on the analysis of proteome data of Tripterygium wilfordii,the CYP450 gene of T. wilfordii was preliminarily analyzed and predicted by various bioinformatics methods. The results showed that after the expression of T. wilfordii suspension cells was induced by methyl jasmonate,the proteomic data of T. wilfordii were obtained and analyzed,and 10 CYP450 proteins of T. wilfordii were finally screened out. By analyzing the phylogenetic tree constructed with CYP450 gene of Arabidopsis family,the 10 CYP450 proteins were clustered into 6 different CYP450 families. The physical and chemical properties of CYP450 proteins in different families were different. The secondary structure of CYP450 proteins was mainly composed of irregular curls. Eight subcellular localization results of CYP450 proteins were chloroplasts and the rest were plastids. Subsequently,the conserved domains( heme active sites) shared by CYP450 genes were found by analyzing the results of multiple sequence alignment. Finally,by analyzing the transcriptome data of T. wilfordii,the expression distribution of T. wilfordii in different tissues was preliminarily confirmed,which verified its correlation with the biosynthesis of active components of T. wilfordii,and provided important genetic resources for the analysis of biosynthesis pathway of active components of T. wilfordii.

Analysis of diagnosis and treatment for cervical cancer of stage Ⅰ A1 / 复旦学报（医学版）

Objective To evaluate the evolution of the effective and safe diagnostic and surgical approaches for patients with cervical cancer of stage Ⅰ A1 during ten years.Methods A retrospective cohort study of 721 cases diagnosed with cervical cancer of stage Ⅰ A1 from 2005 to 2014 was conducted by reviewing the archived medical records.Results Of patients with cervical cancer of stage Ⅰ A1,396 cases (54.9％) had no symptoms and follow-up materials.The transformation of treatment and curative effect were evaluated with Pearson chi square test,Spearman correlation analysis as well as Fisher's exact probability.Pathology of biopsy clarified the diagnosis for 82 cases (11.4 ％),and pathologic results of conization made the definite diagnosis for 640 cases (89.9 ％) (P＜0.01).Laparoscopy grew to be the main trend of surgical approach for cervical cancer of stage Ⅰ A1 instead of laparotomy from 2010 to 2014 compared with 2005 to 2009 (P＜0.01).Non-radical surgery became more popular to the cohort from 2010 to 2014 (P＜0.01).Thirty-five cases (4.9％) were lost to follow-up.There was no dead case and a case of recurrence.Conclusions Conization was very important to clarify the diagnosis for cervical cancer of stage Ⅰ A1.Laparoscopic hysterectomy was a safe and effective treatment for cervical cancer of stage Ⅰ A1 patients.

Effect of ear-acupoint pressing and Ear Apex (HX6,7) bloodletting on haemorheology in chloasma patients with Gan depression pattern / 中国结合医学杂志

<p><b>OBJECTIVE</b>To explore the therapeutical effect of ear-acupoint pressing combined with Ear Apex (HX6,7) bloodletting on haemorheology in chloasma patients with Gan (Liver) depression pattern.</p><p><b>METHODS</b>A total of 180 chloasma patients were randomly assigned to three groups, 60 cases in each. Patients in the earacupuncture (EA) group were treated with ear-acupoint pressing combined with Ear Apex (HX6,7) bloodletting; vitamins C and E were put into practice in the Western medicine (WM) group together with 0.025% tretinoin cream for local external application; patients in the placebo group were treated with urea-cream by external use, while 30 healthy volunteers were in the control group. After a treatment course of 2 months, the changes of haemorheology, injury skin area, colour score and symptom score before and after the treatment were observed.</p><p><b>RESULTS</b>There was no significant difference on whole blood reduced viscosity (high shear, medium shear, and low shear), erythrocyte aggregation index, hematocrit, plasma viscosity among the four groups (F =2.65, P>0.05). Compared with those before treatment, the whole blood viscosity (high shear) and whole blood reduced viscosity (high shear) after treatment in the EA group, the WM group and the placebo group were with no statistical significance (P>0.05). The injury skin area and colour score after treatment were significantly lower than those before treatment in the EA group and the WM group (P<0.05), while there was no significant difference in placebo group (P>0.05). Clinical symptoms of the EA group were obviously improved after the 2-month treatment, which was significantly different compared with those before treatment (P<0.05), there was significant difference compared with those of WM group and placebo group (P<0.05).</p><p><b>CONCLUSION</b>There was no significant difference on haemorheology index between healthy people and chloasma patients without angionosis, cerebrovascular disease, hematopathy, metabolic disease or any other organic disease. Ear-acupoint pressing combined with Ear Apex (HX6,7) bloodletting can effectively improve concurrent symptoms, lighten chloasma and lower chloasma area in patients accompanied by Gan depression.</p>

The anesthesiologic value of transcutaneous acupoint electrical stimulation combined with general intravenous anesthesia in endoscopic thyroidectomy patients: a clinical study / 中国中西医结合杂志

<p><b>OBJECTIVE</b>To explore the clinical anesthesia value of transcutaneous acupoint electrical stimulation (TAES) combined with general intravenous anesthesia in endoscopic bilateral thyroidectomy patients.</p><p><b>METHODS</b>Totally 60 patients who underwent endoscopic bilateral thyroidectomy were equally randomly assigned to 2 groups, the treatment group and the control group, 30 in each group. Patients in the treatment group received TAES combined general intravenous anesthesia, while those in the control group received total intravenous anesthesia. Anesthesia was maintained by target controlled infusion of propofolum combined constant speed infusion of remifentanil in the two groups. TAES was maintained from 30 min before anesthesia induction to the end of endoscopic thyroidectomy at bilateral Hegu (L14) and Neiguan (PC6). The mean artery pressure (MAP) and heart rate (HR) were recorded at different time points of anesthesia, i.e., immediately after entry into the operating room (TO), immediately after intubation (T1), 5 min after intubation (T2), 5 min before incision (T3), 5 min after incision (T4), 30 min after inflation (T5), at the end of surgery (T6), 5 min before extubation (T7), immediately after extubation 0 (T8), and 5 min after extubation (T9). The concentration of IL-6 and TNF-alpha were measured at TO, T3, T5, and T6. The target concentration of propofol was also recorded at T3, T4, and T5.</p><p><b>RESULTS</b>There was no statistical difference in the operation time between the two groups (P >0.05). Compared with TO in the same group, HR at T3-T4 decreased and increased at T8-T9, and MAP increased at T7-T9 in the treatment group; HR decreased at T3 and increased at T7-T9, MAP increased at T1, T5, T7-T9, and MAP decreased at T2-T3 in the control group. IL-6 increased at T5-T6, while TNF-alpha decreased at T6 in the two groups (P <0.01,P <0.05). Compared with the control group at the same time point, HR decreased at T6-T9, MAP decreased at T1, T4, T5, T7-T9, MAP increased at T3, and IL-6 decreased at T5-T6 in the treatment group (P <0. 05). The concentration and the total amount of propofol were significantly lower in the treatment group than in the control group (P <0.01,P <0.05).</p><p><b>CONCLUSIONS</b>TAES could maintain the hemodynamics more stably and inhibit the stress response in endoscopic thyroidectomy. It also reduce the dosage of anesthetics and improve the safety of anesthesia.</p>

Qualitative and quantitative research on sulfur fumigation of Angelicae Dahuricae Radix (Baizhi) by near-infrared spectroscopy / 中国中药杂志

The contents of coumarins in the sulfur fumigated Angelicae Dahuricae Radix (Baizhi, ADR) were reduced significantly. To achieve the quality control of ADR, the qualitative identification of sulfur fumigated ADR and quantitative model of imperatorin content should be established. The near-infrared (NIR) spectrograms of non-sulfur and sulfur fumigated ADR were collected by NIR diffuse reflectance spectroscopy technology and pretreated by the method of first derivative derivation and vector normalization. The Ward's Algorithm method was used for the cluster analysis. The non-sulfur and sulfur fumigated ADR can be quickly identified in the range of 8,806. 0-3 811.0 cm(-1) based on the cluster analysis. The NIR quantitative model of imperatorin was established by the contents of imperatorin determined by HPLC in combination with partial least squares regression analysis. According to the calibration model established in this study, correlation coefficients (R2), the root-mean-square error of cross-validation (RMSECV), and the root-mean-square error of prediction (RMSEP) for imperatorin were 0.982 8, 0.006 8, 0.011 8, respectively. The quantitative model of imperatorin can be applied to determine the content of imperatorin in ADR accurately.

Chemical constituents contained in fatty oil from seeds of Cucumis sativus / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical constituents contained in the seeds of Cucumis sativus.</p><p><b>METHOD</b>The fatty oil was extracted by heating and refluxing with petroleum ether. Potassium hydroxide-methanol solution was used for saponification. An unsaponifiable matter was extracted by EtOAc and separated with various chromatographic methods. Its structure was identified on the basis of their physicochemical properties and spectral data. The fatty acid fraction was methyl-esterified and determined by GC. The composition and relative content of fatty acid were determined with normalization method of peak area.</p><p><b>RESULT</b>24-ethylcholesta-7, 22, 25-trienol (1), 24-ethylcholesta-7, 25-dienol (2) ,avenasterol (3), spinasterol (4), karounidiol (5) and isokarounidiol (6) were separated and identified from the unsaponifiable matter. Myristic acid (7, 0.12%), palmitic acid (8, 12.04%), palmitoleic acid (9, 0.09%), heptadecanoic acid (10, 0.06%), stearic acid (11, 5.64%), oleic acid (12, 6.95%), linoleic acid (13, 74.40%), arachidic acid (14, 0.19%), and alpha-linolenic acid (15, 0.51%) were identified from the fatty acids part.</p><p><b>CONCLUSION</b>Compounds 5, 6, 9, 10, 14,and 15 were first reported in C. sativus.</p>

Effect of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry on identifing biomarkers of endometriosis / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Endometriosis is a common gynecological disease. This study aimed to screen proteins that were expressed differently in patients with endometriosis versus normal controls using proteomic techniques, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS).</p><p><b>METHODS</b>Protein chip SELDI-TOF-MS combines the advantages of microarray and mass spectrometry, and can screen latent markers in sera of patients with endometriosis. Serum samples from patients and normal volunteers were analyzed by SELDI-TOF-MS.</p><p><b>RESULTS</b>After comparing the serum protein spectra of 36 patients with 24 normal controls, 24 differently expressed potential biomarkers (P < 0.01) were identified. Using Biomarker Pattern software, we established a tree model of the 60 serum protein spectra. When using the three biomarkers to classify the samples, the sensitivity for diagnosing endometriosis was 91.7%, specificity was 95.8%, and coincidence rate was 93.3%. Then we used serum samples from 12 patients and 8 normal controls to validate the tree model and report the sensitivity for diagnosing endometriosis was 91.7%, specificity was 75%, and coincidence rate was 85%.</p><p><b>CONCLUSIONS</b>SELDI-TOF-MS may be a useful tool in high-risk population screening for endometriosis. The identification and application of the biomarkers need to further study.</p>