Inhibitory effect of endophytic fungi from Dysosma versipellis on HIV-1 IN-LEDGF/p75 interaction / 中国中药杂志

To determine the inhibitory effect of endophytic fungi from Dysosma versipellis on HIV-1 IN-LEDGF/p75 interaction,the protein-protein interaction between human immunodeficiency virus type 1( HIV-1) integrase and lens epithelial growth factor p75 protein( LEDGF/p75) was used as a target. The homogeneous time-resolved fluorescence( HTRF) technique was used in the inhibitory activity assay. The results showed that eight endophytic fungi with anti-IN-LEDGF/p75 interaction activity were screened out from fifty-three strains with different morphological characteristic. Among them,106 strain showed strong inhibitory activity against HIV-1 IN-LEDGF/p75 interaction with IC50 value of 5. 23 mg·L-1,and was identified as a potential novel species of Magnaporthaceae family by the analyses of ITS-rDNA,LSU and RPB2 sequences data. This study demonstrated that potential natural active ingredients against the HIV-1 IN-LEDGF/p75 interaction exist in the endophytic fungi of D. versipellis. These results may provide available candidate strain resources for the research and development of new anti-acquired immunodeficiency syndrome drugs.

Effects of exogenous IBA and fungal elicitor on growth of in vitro roots culture of Dysosma versipellis and production of podophyllotoxin / 中国中药杂志

Using the White as basic medium, the effects of the exogenous IBA and endophytic fungal elicitor on the growth of in vitro roots cultures of Dysosma versipellis and production of podophyllotoxin were investigated in this study. The results showed that the IBA and the endophytic fungus Zasmidium syzygii elicitor could increase the content of podophyllotoxin of in vitro roots of D. versipellis after 3 weeks. The White medium added with 3 mg·L~(-1) IBA induced the highest increase of podophyllotoxin(1 830.86 μg·g~(-1)), which was 2.07 folds greater than the control, and followed by 1.5 mg·L~(-1) IBA, fungal elicitor, 1 mg·L~(-1) IBA, 0.5 mg·L~(-1) IBA and 4.5 mg·L~(-1) IBA, which was 1.82, 1.71, 1.63, 1.43 and 1.1 folds greater than the control, respectively. The results also showed that the growth of roots was certain positively correlated with the change of IBA concentration. Therefore, 3 mg·L~(-1) IBA was the most suitable for the production of podophyllotoxin in the in vitro roots of D. versipellis, and the stimulating effect of Z. syzygii fungal elicitor was between 1.5 mg·L~(-1) and 1 mg·L~(-1) IBA, which was a potential natural elicitor to induce the accumulation of podophyllotoxin in future production.

Identification of chemical constituents in Sinopodophylli Fructus by HPLC-DAD-ESI-IT-TOF-MSn / 中国中药杂志

This experiment was performed to analyze and identify the chemical constituents of Sinopodophylli Fructus by HPLC-DAD-ESI-IT-TOF-MSn. The analysis was performed on an Agilent Zorbax SB-C₁₈ (4.6 mm×250 mm, 5 μm) column.The mobile phase consisted of 0.1% formic acid was used for gradient at a flow rate of 1.0 mL·min⁻¹. Electrospray ionization ion trap time-of-flight multistage mass spectrometry was applied for qualitative analysis under positive and negative ion modes. The results indicated that 54 compounds consisted of 18 lignans and 36 flavonoids from Xiaoyelian had been detected by their HRMS data, the information of literature and reference substance. Among them, 27 compounds were reported in Sinopodophylli Fructus for the first time. In conclusion, an HPLC-DAD-ESI-IT-TOF-MSn method was established to qualitative analysis of Xiaoyelian in this study, which will provide the evidence for evaluating the quality of Xiaoyelian herbs, clarifying the mechanism, and guiding the development of pharmacological active ingredients.

Flavonoids isolated from Sinopodophylli Fructus and their bioactivities against human breast cancer cells / 中国天然药物

Four prenylated flavonoids compounds 1-4, named sinopodophyllines A-D, and a flavonoid glycoside (compound 13), sinopodophylliside A, together with 19 known compounds (compounds 5-12 and 14-24) were isolated from the fruits of Sinopodophyllum hexandrum. The structures of new compounds were elucidated by extensive spectroscopic analysis, including HRESIMS, 1D and 2D NMR. Compounds 1-6, 9-11, and 14-17 were tested for their cytotoxicity against human breast-cancer T47D, MCF-7 and MDA-MB-231 cells in vitro, and compounds 2, 5, 6, 10 and 11 showed significant cytotoxicity (IC values < 10 μmol·L) against T47D cells.

Correlation research on contents of podophyllotoxin and total lignans in Sinopodophyllum hexandrum and ecological factors / 中国中药杂志

To explore the correlation between the ecological factors and the contents of podophyllotoxin and total lignans in root and rhizome of Sinopodophyllum hexandrum, podophyllotoxin in 87 samples (from 5 provinces) was determined by HPLC and total lignans by UV. A correlation and regression analysis was made by software SPSS 16.0 in combination with ecological factors (terrain, soil and climate). The content determination results showed a great difference between podophyllotoxin and total lignans, attaining 1.001%-6.230% and 5.350%-16.34%, respective. The correlation and regression analysis by SPSS showed a positive linear correlation between their contents, strong positive correlation between their contents, latitude and annual average rainfall within the sampling area, weak negative correlation with pH value and organic material in soil, weaker and stronger positive correlations with soil potassium, weak negative correlation with slope and annual average temperature and weaker positive correlation between the podophyllotoxin content and soil potassium.

Dynamic study on two lignans contents in different parts of Sinopodophyllum hexandrum / 中国中药杂志

The contents of two lignans, namely 4'-demethylpodophyllotoxin and podophyllotoxin in cultivated and wild Sinopodophyllum hexandrum plants were extracted by ultrasonicaction and determined by HPLC. According to the result showed, the order of parts of cultivated plants containing 4'-demethylpodophyllotoxin from high to low is as follows: stem > root, no 4'-demethypodophyllotoxin was detected in leaves of cultivated plants; The order of parts of wild plants 4'-demethylpodophyllotoxin from high to low is as follows: lateral root > petiole > rhizome > leaf, no 4'-demethypodophyllotoxin was detected in fruit. The order of parts of cultivated and wild S. hexandrum containing podophyllotoxin from high to low is as follows: root > petiole > leaf ( > fruit). Both of the lignan contents in different parts of cultivated plant varied in a " W" curve with the changes in seasons, with the highest content in July.

Effect of topographical factors on podophyllotoxin content in Sinopodophyllum hexandrum and study on ecological suitability / 中国中药杂志

In order to find the optimal topographical factor for regionslization, the content of cimetidine in 116 Sinopodophyllum hexandrum sample collected from Sichuan, Qinghai, Gansu, Tibet, Yunnan and Shaanxi provinces, was determined. Using mathematical statistics and geographical spatial analysis of GIS analysis, the relationship between content of podophyllotoxin and influencing factors including altitude gradient and gradient position was analyzed. It is found that the optimal altitude was 2 800 m to 3 600 m, the aspect of slope north or northeast and northwest and the slope 12 degrees to 65 degrees with a high suitability degree. Considering the artificial planting, the suitable planting area for S. hexandrum is comfirmed. The topographical factor is important for S. hexandrum regionalization, but has hardly effect on podophyllotoxin content. The results of the study provide an important scientific basis for S. hexandrum production development. But there are many factors which affect suitability index and podophyllotoxin content of S. hexandrum, it is necessary to consider other factors like climate and soil while exploitation and protection of S. hexandrum.

Absolute configuration of podophyllotoxone and its inhibitory activity against human prostate cancer cells / 中国天然药物

Podophyllotoxone (1) was isolated from the roots of Dysosma versipellis. The structure was determined by spectroscopic analysis in combination with single-crystal X-ray analysis. The absolute configuration of compound 1 was assigned based on the Flack parameter. It showed significant inhibitory activities against human prostate cancer cells PC3 and DU145 with IC50 values being 14.7 and 20.6 μmol·L(-1), respectively. It also arrested the cells at G2/M phase. Tubulin polymerization assay showed that it inhibited the tubulin polymerization in a dose-dependent manner, and molecular docking analysis revealed a different binding mode with tubulin as compared with those known tubulin inhibitors.

A 14-Day Repeated Dose Toxicity of Epimedii Herba Aqueous Extract Administered by Oral Gavage in F344 Rats

The objective of this study is to characterize a toxicity of Epimedii Herba (EH) in F344 rats and to find a dose levels for the 13 weeks toxicity study. EH is well known as medicinal herb in many Asian countries for traditional medicines of antibacterial and antiviral effects, estrogenic and antiestrogenic effects, and for treatment of osteoporosis, hypotensives, fatigue, kidney disorders, and related complications. However, the indispensable and basic information of toxicological evaluation of EH extract is insufficient to support its safe use. Therefore, we conducted toxicological evaluation of this drug in compliance with OECD and MFDS guideline in this study. The extract of EH was administered orally to F344 rats at dose levels of 0, 500, 1000, 2000, 3500, and 5000 mg/kg/day for 2 weeks. Each group was composed of 5 male and female rats. In this study, there were no treatment of EH-related adverse changes in clinical observations, mortality, body weights, food consumption, urinalysis, gross finding at necropsy, and organ weight examination. Total red blood cell count, hematocrit, mean corpuscular hemoglobin concentration, total cholesterol, and phospholipid were decreased in males and females at 5000 mg/kg/day compared to the control animals. Mean corpuscular volume and reticulocyte counts were increased in males and females at 5000 mg/kg/day compared to control animals. Therefore, we recommend that dose level of 5000 mg/kg/day is a highest treatment group in 13-week EH extract exposure study for further toxicity assessment.

Meroterpenoids and isoberkedienolactone from endophytic fungus Penicillium sp. associated with Dysosma versipellis / 药学学报

Seven meroterpenoids and five small-molecular precursors were isolated from Penicillium sp., an endophytic fungus from Dysosma versipellis. The structures of new compounds, 11beta-acetoxyisoaustinone (1) and isoberkedienolactone (2) were elucidated based on analysis of the spectral data, and the absolute configuration of 2 was established by TDDFT ECD calculation with satisfactory match to its experimental ECD data. Meroterpenoids originated tetraketide and pentaketide precursors, resepectively, were found to be simultaneously produced in specific fungus of Penicillium species. These compounds showed weak cytotoxicity in vitro against HCT-116, HepG2, BGC-823, NCI-H1650, and A2780 cell lines with IC 50 > 10 micromol x L(-1).

Studies on main interspecific association of rare and endangered medicinal plant Sinopodophyllum hexandrum community in Kangding Zheduo mountain of Sichuan province / 中国中药杂志

Based on the 2 x 2 contingency table, by using multi-species relevance (variance ratio, VR), chi2-test, Ochiai index, Dice index, Jaccard index, t-test of v/x and F-test of Morisita, s index, the interspecific relationships and the spatial distribution pattern between 20 dominants in Kangding Zheduo Mountain of Sichuan province were studied. The results indicated that the interspecific association between dominants of Sinopodophyllum hexandrum community in this area did not show significant association, which suggested that the S. hexandrum community was in mature stage, and showed stronger independency, among total 190 pairs in 20 dominant species, 2 species pairs exhibited extremely significantly positive association, 12 species pairs showed significantly positive association, 6 species pairs exhibited significantly negative association and there were no pairs showed extremely significantly negative association. S. hexandrum in community did not show significant association, which indicates they are independent in community, the spatial distribution pattern of S. hexandrum is characterized by random distribution.

Research on chemical fingerprint chromatograms of Sinopodophyllum hexandrum / 中国中药杂志

<p><b>OBJECTIVE</b>To study the HPLC fingerprint of Sinopodophylli Fructus, compare the major chemical differences in fruit of Sinopodophyllum hexandrum as well as the roots and rhizomas of S. hexandrum, and provide scientific evidence for clinical application and quality control.</p><p><b>METHOD</b>HPLC fingerprint method was used to analyze 12 fruits of S. hexandrum. A total of 20 common peaks were confirmed, and 12 peaks in the HPLC fingerprint were identified. Furthermore, similarity evaluation method and clustering analysis method were introduced to compare HPLC chromatograms, between fruits and underground parts.</p><p><b>RESULT</b>In this paper, quercetin-3-O-beta-D-glucopyranosid, kaempferol-3-O-beta-D-glucopyranoside, 8-prenylquercetin and 8-prenylquercetin 3-methyl ether were firstly reported in Sinopodophylli Fructus. Among the existing fingerprint research, a total of 11 peaks were identified for the first time, containing 9 flavonoids and 2 lignans. The chemical constituents differed significantly in different medicinal parts of S. hexandrum. Prenylflavonoid compounds were the main constituents of Sinopodophylli Fructus. However, podophyllotoxin, flavonoids with simple substituent groups and flavonoid glycosides were the major ingredients in the roots and rhizomas of S. hexandrum.</p><p><b>CONCLUSION</b>This method can be used for the quality control of Sinopodophylli Fructus and the roots and rhizomas of S. hexandrum. It has provided a reference for the pharmacodynamic differences of the two different parts.</p>

Microscopic observation on mycorrhiza of rare herb Dysosma versipellis / 中国中药杂志

Endophytic fungi played an important role in the growth of its host plant. To investigate the mycorrhizal characteristics and the distribution of fungi in the root, an endangered wild plant-Dysosma versipellis was collected and observed by electron microscope. The results showed that the host was closely associated with endophytic fungi. The fungi were mainly distributed in the epidermis and cortex. The aseptate and septate fungi with swollen hyphae were observed in some cell of the cortex. The result provides a reference for the study of mycorrhizal structure of Dysosma genus and the interaction between the fungi and its host.

Pathological changes in rats with acute Dysosma versipellis poisoning / 法医学杂志

OBJECTIVE@#To observe the pathological changes of major organs in rats with acute Dysosma versipellis poisoning and investigate the toxic mechanism and the injuries of target tissues and organs.@*METHODS@#Forty Sprague-Dawley (SD) rats were randomly divided into three experimental groups, which were given the gavage with 0.5, 1.0 and 2.0 LDo doses of Dysosma versipellis decoction, and one control group, which was given the gavage with 1.0 LD0 dose of normal saline. The rats were sacrificed 14 days after Dysosma versipellis poisoning and samples including brain, heart, liver, lung, and kidney were taken. After pathological process, the pathological changes of the major organs and tissues were observed by light microscope and electron microscope. The experimental data were statistical analyzed by chi2 test.@*RESULTS@#The observations of light microscopy: loose cytoplasm of neurons with loss of most Nissl bodies; swelling of myocardial cells with disappearance of intercalated disk and striations; hepatocellular edema with ballooning degeneration; and swelling epithelial cells of renal proximal convoluted tubule with red light coloring protein-like substances in the tube. The observations of electron microscopy: the structures of cell membrane and nuclear membrane of neurons were destroyed; cytoplasm of neurons, obvious edema; and most organelles, destroyed and disappeared. The mortalities of rats after acute poisoning of the four groups increased with doses (P < 0.05).@*CONCLUSION@#Acute Dysosma versipellis poisoning can cause multi-organ pathological changes. There is a positive correlation between the toxic effect and the dosage. The target tissues and organs are brain (neurons), heart, liver and kidney.

Genetic diversity and genetic structure of endangered wild Sinopodophyllum emodi by start codon targeted polymorphism / 中国中药杂志

<p><b>OBJECTIVE</b>Revealed the genetic diversity level and genetic structure characteristics in Sinopodophyllum emodi, a rare and endangered species in China.</p><p><b>METHOD</b>We detected the genetic polymorphism within and among six wild populations (45 individuals) by the approach of Start Codon Targeted (SCoT) Polymorphism. The associated genetic parameters were calculated by POP-GENE1.31 and the relationship was constructed based on UPGMA method.</p><p><b>RESULT</b>A total of 350 bands were scored by 27 primers and 284 bands of them were polymorphic. The average polymorphic bands of each primer were 10.52. At species level, there was a high level of genetic diversity among six populations (PPB = 79.27%, N(e) = 1.332 7, H = 0.210 9 and H(sp) = 0.328 6). At population level, the genetic diversity level was low (PPB = 10.48% (4.00% -23.71%), N(e) = 1.048 7 (1.020 7-1.103 7), H = 0.029 7 (0.012 9-0.063 1), H(pop) = 0.046 2 (0.019 9-0.098 6). The Nei's coefficient of genetic differentiation was 0.841 1, which was consistent with the Shannon's coefficient of genetic differentiation (0.849 4). Two calculated methods all showed that most of the genetic variation existed among populations. The gene flow (N(m) = 0.094 4) was less among populations, indicating that the degree of genetic differentiation was higher. Genetic similarity coefficient were changed from 0.570 8 to 0.978 7. By clustering analysis, the tested populations were divided into two classes and had a tendency that the same geographical origin or material of similar habitats clustered into one group.</p><p><b>CONCLUSION</b>The genetic diversity of samples of S. emodi is high,which laid a certain foundation for effective protection and improvement of germplasm resources.</p>

Determination of podophyllotoxin and total lignans in Sinopodophyllum emodi / 中国中药杂志

<p><b>OBJECTIVE</b>To determine the content of podophyllotoxin and total lignans in Sinopodophyllum emodi Ying from different areas and evaluate the resource utilization of this endangered medicinal plant.</p><p><b>METHOD</b>HPLC and UV spectrophotometry were used to determine the content of podophyllotoxin and total lignans in 126 samples from different habitats and the total lignans content was determined by the reference wavelength method.</p><p><b>RESULT</b>According to the results, the highest content was determined from the samples from Yongdeng Nature Reserve in Gansu province, and the lowest ones was found in the samples from Tibet. The former's podophyllotoxin and total lignans contents were 7.40% and 20.66%, respectively, which were 19 times and 4 times more than those of the latter. The content of podophyllotoxin and total lignans in S. emodi were significantly positively related, meanwhile, samples from both low altitude and high latitude showed the higher content.</p><p><b>CONCLUSION</b>The two determination methods are simple, rapid, accurate and repeatable. It is more scientific and rational to evaluate the resource utilization of S. emodi with two indicators, those are the content of podophyllotoxin and the content of total lignans. This paper is instructive to the collection of wild resources and the establishment of production bases.</p>

Resources of survey in rare and endangered medicinal plant Sinopodophyllum emodi / 中国中药杂志

The resource of Sinopodophyllum emodi in Tibet, Qinghai, Gansu, Sichuan and Yunnan province were surveyed by the ways of documents, interview, quadrat and market investigation. The results indicated that S. emodi were mainly distributed in central part from Taibai Shan of Shanxi province along the middle and high mountain on either side of Hengduan mountain which from north to south and in Tibetan plateau of China, the suitable eco-environment of S. emodi was dominated by valleys, wet forests and low bush-woods vegetation which comparatively dry on plateau with the altitude focused on 1 5004 500 meters, and the distribution density of simple alpine meadow was relatively lower. Illumination, water, soil, temperature and altitude had significant influences on the growth, distribution and reserves of S. emodi from different angles. In recent years, there was sharp increase of market requirement in Xiaoyelian (fruit) which were used in Tibetan medicine and the underground part which was used for the extraction of podophyllotoxin, excess collection of the underground part was the primary cause of the rapid decreasing resource of S. emodi. It is suggested that the management of rational collection should be strengthen and the development of culturing and production should be meanwhile accelerated.

Screening of some ethnobotanically important plants of Nagar area Gilgit-Baltistan, Pakistan

Certain species of ethnobotanical importance belonging to families Asteraceae, Berberidaceae, Cupressaceae, Elaeagnaceae, Gentianaceae, Salicaceae and Tamaricaceae, were collected from Gilgit during the months of June and July 2008 and were investigated for the presence of alkaloids, amino acids, anthraquinone [free and as glycosides] ascorbic acid, carbohydrates, coumarins, flavonoids, phenolics, proteins, saponins and steroids in their aqueous, ethanol and benzene extracts. Artemisia maritime L. [leaves] showed positive results for carbohydrates, coumarins, phenolics and proteins. Ephedra gerardiana Wall ex. Stapf. [stem] tested positive for alkaloids, ascorbic acid, coumarins, phenolics, proteins, saponins and steroids. Tamarix gallica L. tested positive for alkaloids, amino acids, anthraquinone as glycoside, ascorbic acid, carbohydrates, flavonoids, phenolics, proteins and steroids in stems, roots and leaves. Salix acmophylla Boiss. showed positive results for alkaloids, amino acids, anthraquinone [free and as glycosides] ascorbic acid, carbohydrates, flavonoids, phenolics, proteins, saponins and natural steroids. Hippophae rhamnoides L. showed positive results for alkaloids, amino acids, anthraquinone [free as glycosides] ascorbic acid, carbohydrates, coumarins, flavonoids, phenolics, proteins, saponins and steroids. Berberis glycocarpa Stapf. showed positive results for alkaloids, amino acids, ascorbic acid, carbohydrates, flavonoids, phenolics, proteins and steroids in stem, roots and leaves. Similarly Juniperus excelsa Wall ex. C.A. Meyer showed positive result for anthraquinone [both free and as glycosides], carbohydrates, phenolics, proteins, saponins and natural steroids

Determination of quercetin and kaempferol in Dysosma plants by RP-HPLC / 中国中药杂志

<p><b>OBJECTIVE</b>To determine quercetin and kaempferol in the plant of genus Dysosma that come from different species, different plant parts or different growing areas, which provide the basis of rational utilization of Dysosma plants.</p><p><b>METHOD</b>The analysis was performed on a Diamonsil C18 column (4.6 mm x 150 mm, 5 microm) eluted with the mobile phase of methanol-water containing 0.1% phosphoric acid (60:40). The flow rate was 1 mL x min(-1), the detection wavelength was 360 nm; and the column temperature was set at 25 degrees C.</p><p><b>RESULT</b>The linear ranges of quercetin and kaempferol are 0.22-1.1 microg and 0.42-2.1 microg. The average recoveries of quercetin and kaempferol are 97.1% (RSD 1.4%) and 99.6% (RSD 2.4%); respectively.</p><p><b>CONCLUSION</b>The contents of flavones in different species of Dysosma are significantly different.</p>

Inhibitory effect of taspine on mouse S180 sarcoma and its mechanism / 中国中药杂志

<p><b>OBJECTIVE</b>To study the inhibition effect of taspine on mouse S180 sarcoma and its mechanism.</p><p><b>METHOD</b>The mouse S180 sarcoma model was established and used to observe the antitumor activity of taspine. The microvessel density and protein expressing of the VEGF, bFGF, Bcl-2 and Bax in the tumor were measured by immunohistochemistry.</p><p><b>RESULT</b>Taspine showed antitumor activity on the mouse S180 sarcoma in a good dose-dependent manner. The inhibition rates on tumor of taspine at low, middle and high concentrations were 39.08% , 43.99% and 48.60%, respectively. The microvessel density and protein expressing of the VEGF, bFGF, Bcl-2 and Bax in the tumor were decreased compared with the negative control. The ratio of Bax to Bcl-2 was increased.</p><p><b>CONCLUSION</b>Taspine has antitumor effect on the S180 sarcoma, and the mechanism may be through the way of decreasing the expressing of the VEGF, bFGF, Bcl-2 and Bax and inducing the vascular endothelial cell apoptosis.</p>