Development and application of an indirect ELISA to detect antibodies to Neospora caninum in cattle based on a chimeric protein rSRS2-SAG1-GRA7.

Neospora caninum is an important apicomplexan parasite causing neosporosis in cattle. The disease is recognized as one of the most important cause of reproductive problems and abortion in cattle worldwide. In this context, we developed an indirect enzyme-linked immunosorbent assays (ELISA) with chimeric protein rSRS2-SAG1-GRA7 to diagnose antibodies to Neospora-infection. This indirect ELISA was compared to indirect fluorescent antibody test (IFAT) and western blotting (WB), and the sensitivity and specificity results of ELISA were calculated to be 86.7 and 96.1%, respectively. The overall coincidence rate was 92.6% using IFAT and WB. Additionally, 329 aborting dairy cattle serum samples were tested using this ELISA to evaluate the prevalence of N. caninum in Ningxia, China. The positive rate of N. caninum in these farms was from 19.05 to 57.89%, and the mean rate was 41.64% (±11.01%), indicating that infection with N. caninum may be one of the important causes of cattle abortion in this region. This established rSRS2-SAG1-GRA7 indirect ELISA is capable for detecting the antibodies against N. caninum, and it could be a useful screening tool for monitoring the epidemiology of neosporosis in cattle.

A new ionone derivative from the leaves of Picrasma quassioides.

Nigakialcohol A (1), as unusual cyclization ionone derivative, together with eight known ones (2-9), were isolated from the leaves of Picrasma quassioides (D. Don) Benn (Simaroubaceae). Their structures were elucidated by extensive spectroscopic analyses and comparison with literature data. Compound 2 showed a weak inhibitory effect on NO production at non-cytotoxic concentration (100 µM) with inhibitory rate of 59%, and thus it should be regarded as potential anti-inflammatory agents.

[Systematical analysis of multiple components in drainage bear bile powder from different sources].

Bear Bile Powder contains bile acids, protein, amino acids, bilirubin and microelements and other compounds. Among them, the bile acids are the most active components. Currently, there are many studies on bile acids, but few reports on other components. Therefore, the purpose of this study is to carry out a systematical analysis of multiple components in drainage Bear Bile Powder from different sources. Bilirubin and protein were quantified by microplate spectrophotometer. The contents of bile acids and amino acids were determined by liquid chromatography coupled with mass spectrometry (LC-MS). The contents of microelements were determined by inductively coupled plasma mass spectrometry (ICP-MS) The result indicated that among 20 batches of bear bile powder from different sources there is high similarity (0.922-0.977). Tauroursodeoxycholic acid (TUDCA) and taurochenodeoxycholic acid (TCDCA) were the two most abundant components. The total contents of them were 41%-59% and met the current standard for quality control of bear bile powder. However, significant differences were found in their contents among samples from different sources. Besides, bilirubin, protein, amino acids and microelements also contributed to the differentiation of samples from different sources. The main components of bear bile powder from the different sources were with satisfactory similarity. But bile acids, bilirubin, protein, amino acids and microelements all contributed to the different among samples. Our present study provided a systematical approach for the better quality control and evaluation of bear bile powder.

Anti-oral common pathogenic bacterial active acetylenic acids from Thesium chinense Turcz.

Discovery of agents for oral infectious diseases is always encouraged in natural products chemistry. A bioassay-guided isolation led to the isolation of two new acetylenic acids (1, 2) along with seven known ones (3-9) from the ethanol extract of Thesium chinense Turcz, a commonly used oral anti-bacterial and anti-inflammatory herb. Their structures were elucidated on the basis of spectroscopic and chemical evidence. Exocarpic acid (3) demonstrated the most promising activity against three tested oral pathogenic bacterial strains, Porphyromonas gingivalis, Fusobacterium nucleatum, and Streptococcus mutans, with minimum inhibitory concentration values of 0.86, 3.43, and 13.70 µg/mL, respectively. Compounds 1, 2, 4, 5 and 7 also showed potential activities against periodontal bacteria (P. gingivalis, F. nucleatum).

Antifungal Amide Alkaloids from the Aerial Parts of Piper flaviflorum and Piper sarmentosum.

Sixty-three amide alkaloids, including three new, piperflaviflorine A (1), piperflaviflorine B (2), and sarmentamide D (4), and two previously synthesized ones, (1E,3S)-1-cinnamoyl-3- hydroxypyrrolidine (3) and N-[7'-(4'-methoxyphenyl)ethyl]-2-methoxybenzamide (5), were isolated from the aerial parts of Piper flaviflorum and Piper sarmentosum. Their structures were elucidated by detailed spectroscopic analysis and, in case of 3, by single-crystal X-ray diffraction. Most of the isolates were tested for their antifungal and antibacterial activities. Ten amides (6-15) showed antifungal activity against Cryptococcus neoformans ATCC 90 113 with IC50 values in the range between 4.7 and 20.0 µg/mL.

Two New Oleanane-type Triterpenoids from Methanolyzed Saponins of Momordica cochinchinensis.

Two new oleanane-type triterpenoid saponins (1 and 2) were isolated from the methanolyzed total saponins of the seeds of Momordica cochinchinensis (Lour.) Spreng, together with 16 known compounds (3-18). Their structures were elucidated on the basis of detailed spectroscopic, including 1D and 2D NMR, mass spectrometric, methanolysis and LC-MS analysis. All the isolates were tested for their cytotoxic activities against five human cancer cell lines (HL-60, SMMC-7721, PANC-1, A-549, and SW-480) and the glucose uptake activity. The known compound 6 exhibited toxic effects against HL-60 with an IC50 value of 18.1 µM, while 10 showed cytotoxicity against SMMC-7721 and A-549 cell lines, with IC50 values of 34.4 and 32.8 µM, respectively. In addition, the new compound 2 showed glucose uptake activity with a glucose consumption value of 0.29 µM at 10 µM concentration.

Antioxidant and hyaluronidase inhibitory activities of diverse phenolics in Phyllanthus emblica.

Fifty-eight phenolic compounds isolated from Phyllanthus emblica were screened and compared for their in vitro and in vivo antioxidant properties, as well as hyaluronidase (HAase) inhibitory activities. Among them, 20 compounds showed to be promising antioxidants due to the stronger scavenging activity in both DPPH radical and Danio rerio reactive oxygen species assays, while nine compounds were potential HAase inhibitors with 100-fold stronger activities than that of the positive control, DSCG. The structure activity relationship was discussed.

Anti-viral and cytotoxic norbisabolane sesquiterpenoid glycosides from Phyllanthus emblica and their absolute configurations.

In an effort to identify anti-viral and cytotoxic compounds from Phyllanthus spp., 14 highly oxygenated norbisabolane sesquiterpenoids, phyllaemblicins H1-H14, were isolated from the roots of Phyllanthus emblica Linn, along with phyllaemblicins B and C and glochicoccinoside D. Their structures were determined on the basis of detailed spectroscopic analysis and chemical methods. Determination of absolute configurations of these compounds was facilitated by theoretical calculations of electronic circular dichroism (ECD) spectra using time-dependent density functional theory (TDDFT) for the aglycone components, and pre-column derivative/chiral HPLC analysis for the monosaccharides. The known glochicoccinoside D displayed potent activity against influenza A virus strain H3N2 and hand, foot and mouth virus EV71, with IC50 values of 4.5±0.6 and 2.6±0.7 µg/ml, respectively. Phyllaemblicin H1 showed moderate cytotoxicity against human cancer cell lines A-549 and SMMC-7721, with IC50 values of 4.7±0.7 and 9.9±1.3 µM, respectively.

Lignans and aromatic glycosides from Piper wallichii and their antithrombotic activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Piper wallichii (Miq.) Hand.-Mazz. is a medicinal plant used widely for the treatment of rheumatoid arthritis, inflammatory diseases, cerebral infarction and angina in China. Previous study showed that lignans and neolignans from Piper spp. had potential inhibitory activities on platelet aggregation. In the present study, we investigated the chemical constituents of Piper wallichii and their antithrombotic activities, to support its traditional uses. MATERIALS AND METHODS: The methanolic extract of the air-dried stems of Piper wallichii was separated and purified using various chromatographic methods, including semi-preparative HPLC. The chemical structures of the isolates were determined by detailed spectroscopic analysis, and acidic hydrolysis in case of the new glycoside 2. Determination of absolute configurations of the new compound 1 was facilitated by calculated electronic circular dichroism using time-dependent density-functional theory. All compounds were tested for their inhibitory effects on platelet aggregation induced by platelet activating factor (PAF) in rabbits׳ blood model, from which the active ones were further evaluated the in vivo antithrombotic activity in zebrafish model. RESULTS: A new neolignan, piperwalliol A (1), and four new aromatic glycosides, piperwalliosides A-D (2-5) were isolated from the stems of Piper wallichii, along with 25 known compounds, including 13 lignans, six aromatic glycosides, two phenylpropyl aldehydes, and four biphenyls. Five known compounds (6-10) showed in vitro antiplatelet aggregation activities. Among them, (-)-syringaresinol (6) was the most active compound with an IC50 value of 0.52 mM. It is noted that in zebrafish model, the known lignan 6 showed good in vivo antithrombotic effect with a value of 37% at a concentration of 30 µM, compared with the positive control aspirin with the inhibitory value of 74% at a concentration of 125µM. CONCLUSION: This study demonstrated that lignans, phenylpropanoid and biphenyl found in Piper wallichii may be responsible for antithrombotic effect of the titled plant.

Anti-hepatitis B virus activities and absolute configurations of sesquiterpenoid glycosides from Phyllanthus emblica.

During the process exploring anti-viral compounds from Phyllanthus species, eight new highly oxygenated bisabolane sesquiterpenoid glycoside phyllaemblicins G1­G8 (1­8) were isolated from Phyllanthus emblica, along with three known compounds, phyllaemblicin F (9), phyllaemblic acid (10) and glochicoccin D (11). Phyllaemblicin G2 (2), bearing a tricyclo [3.1.1.1] oxygen bridge ring system, is an unusual sesquiterpenoid glycoside, while phyllaemblicins G6­G8 (6­8) are dimeric sesquiterpenoid glycosides with two norbisabolane units connecting through a disaccharide. All the structures were elucidated by the extensive analysis of HRMS and NMR data. The relative configuration of phyllaemblicin G2 was constructed based on heteronuclear coupling constants measurement, and the absolute configurations for all new compounds were established by calculated electronic circular dichroism (ECD) using time dependent density functional theory. The sesquiterpenoid glycoside dimers 6­9 displayed potential anti-hepatitis B virus (HBV) activities, especially for the new compound 6 with IC50 of 8.53 ± 0.97 and 5.68 ± 1.75 µM towards the HBV surface antigen (HBsAg) and HBV excreted antigen (HBeAg) secretion, respectively.

New phenylpropanoid-substituted flavan-3-ols from Pu-er ripe tea.

Pu-er ripe tea is a special microbial post-fermented tea made from Pu-er raw tea, a kind of green tea produced from the leaves of Camellia sinensis var. assamica. It is one of the most consumed teas in the past two decades in China, due to its special flavor, taste, and beneficial effects. This work aimed to obtain diverse catechin structures from Pu-er ripe tea, which led to the isolation of four new phenylpropanoid-substituted flavan-3-ols, puerins C-F (1-4), together with four known flavan-3-ols, (+)-catechin (5), (-)-epicatechin (6), (+/-)-gallocatechin (7), and (-)-epigallocatechin (8). Their structures were elucidated on the basis of detailed spectroscopic analysis, including 1D and 2D NMR, mass and CD spectra. Compounds 1-4, which could be formed in the post-fermentative process of Pu-er tea, were isolated for the first time from tea and Theaceae plant.

Highly Oxygenated Limonoids and Lignans from Phyllanthus flexuosus.

ABSTRACT: Two new highly oxygenated limonoids, flexuosoids A (1) and B (2), and three new arylnaphthalene lignan glycosides, phyllanthusmins D-F (3-5), were isolated from the roots of Phyllanthus flexuosus, in addition to three known lignans, phyllanthusmin C, arabelline, and (+)-diasyringaresinol. Their structures were elucidated on the basis of detailed spectroscopic analysis and chemical methods. Compounds 1 and 2, two new decaoxygenated limonoids with a C-19/29 lactol bridge and heptaoxygenated substituents at C-1, C-2, C-3, C-7, C-11, C-17, and C-30, represent the second example of limonoids in the Euphorbiaceae family. Most of the isolates were tested for their antifeedant, anti-herpes simplex virus 1, and cytotoxic activities. The new limonoids 1 and 2 showed promising antifeedant activity against the beet army worm (Spodoptera exigua) with EC50 values of 25.1 and 17.3 µg/cm(2), respectively. In addition, both of them displayed moderate cytotoxicity against the ECA109 human esophagus cancer cell line, along with the known lignan glycoside, phyllanthusmin C, with the IC50 values of 11.5 (1), 8.5 (2), and 7.8 (phyllanthusmin C) µM, respectively. GRAPHICAL ABSTRACT: Two new highly oxygenated limonoids, flexuosoids A (1) and B (2), and three new arylnaphthalene lignan glycosides, phyllanthusmins D-F (3-5), were isolated from the roots of Phyllanthus flexuosus, along with three known lignans. Flexuosoids A (1) and B (2) showed promising antifeedant activity against the beet army worm (Spodoptera exigua) with EC50 values of 25.1 and 17.3 µg/cm(2), respectively. In addition, both of them and the known lignan glycoside, phyllanthusmin C, displayed moderate cytotoxicity against the ECA109 human esophagus cancer cell line, with the IC50 values of 11.5, 8.5, and 7.8 µM, respectively.

Anti-hepatitis B virus norbisabolane sesquiterpenoids from Phyllanthus acidus and the establishment of their absolute configurations using theoretical calculations.

Nineteen new highly oxygenated norbisabolane sesquiterpenoids, phyllanthacidoid acid methyl ester (1), and C-T (4-21), were isolated from Phyllanthus acidus Skeels, together with two known ones, phyllanthusols A (2) and B (3), whose sugar moiety was revised as glucosamine-N-acetate, rather than the previously assigned mannosamine-N-acetate. Compounds 2 and 3 were renamed respectively as phyllanthacidoids A (2) and B (3) to avoid confusion. All of the isolates except for 1 are glycosides, whose saccharide moieties possess a pentaoxy cyclohexane (scyllo quercitol) connecting with glucosamine-N-acetate or glucosyl moieties, which are first examples in natural products. Phyllanthacidoids N-R (15-19) with 8R configurations and/or 5,8-diketal skeleton, are unprecedented structures among norbisabolane sesquiterpenoids. Phyllanthacidoids S (20) and T (21) have the unusual tricyclo [3.1.1.1] oxygen bridge skeleton formed by a diketal system, of which the relative configurations of the aliphatic chain were assigned on the basis of heteronuclear coupling constants. The absolute configurations of compounds (1-21) were established by means of calculated electronic circular dichroism (ECD) and coupling constants. Compounds 1-5, 7-9, 10, and 14 displayed potential anti-hepatitis B virus (HBV) activities, with IC50 values of 0.8-36 µM against HBV surface antigen (HBsAg) and HBV excreted antigen (HBeAg), and the results indicated that the 5-ketal group and sugar moieties had contributions to the selectivity of HBsAg and HBeAg.

[Value of narrow band imaging endoscopy in the detection of unknown primary site with cervical lymph node metastasis of squamous cell carcinoma].

OBJECTIVE: To investigate the value of narrow band imaging (NBI) endoscopy in the detection of unknown primary tumor site with cervical lymph node metastases of squamous cell carcinoma. METHODS: Fifty-three patients with cervical lymph node metastases of squamous cell carcinoma treated in our department between June 2009 and December 2011 were enrolled in this study. Their primary tumor site was not detected by routine computed tomography, magnetic resonance imaging and laryngoscopy. The nasopharyngolarynx was examined by NBI endoscopy to explore the primary tumor site. RESULTS: A total of 53 cases with cervical lymph node metastasis of squamous cell carcinoma from an unknown primary were examined under NBI endoscopy. The primary tumor site was confirmed by NBI examination in 47.2% (25/53) of patients, significantly better than routine radiology and endoscopy (0, P < 0.001). These primary tumors were small and superficial, with characteristic mucosal vascular morphologies. The superficial nasopharyngeal carcinomas under NBI examination showed the superficial thin branch-like or torturous line microvessels. The notable characteristics of the squamous cell carcinoma of oropharynx, hypopharynx and larynx was the well demarcated brownish area and scattered brown dots. CONCLUSION: The NBI endoscopy can provide better visualization of the morphology of superficial mucosal vasculature and improve the ability to detect possible primary cancer in patients with primary unknown cervical lymph node metastasis.

[Display of esophageal entrance by injecting gas through the laryngoscopic biopsy channel in hypopharyngeal cancer].

OBJECTIVE: To investigate the method displaying the hypopharynx and esophageal entrance under laryngoscopy to determine the invasion range of hypopharyngeal cancer. METHODS: A total of 113 patients with hypopharyngeal cancer was enrolled in this study and they previously underwent conventional laryngoscopic and radiologic examinations. The esophageal entrance was exposed by injecting gas through the biopsy channel of laryngoscope. The invasion of esophageal entrance in hypopharyngeal cancers was evaluated by this method and compared with that evaluated with radiology. RESULTS: Conventional laryngoscopy was unable to evaluate appreciably esophageal entrance involvement in the 113 patients with hypopharyngeal cancer. After injecting gas through the laryngoscopic biopsy channel, the esophageal entrance was clearly displayed in 96.5% (109/113) patients, showing the presence (33 cases) and absence (76 cases) of esophageal entrance invasion. Imaging examinations only showed the invasion of the esophageal entrance in 25 patients of these patients,but did not in other 8 patients. Laryngoscopy with injecting gas through the biopsy channel was superior to radiological imaging examinations in determining the invasion of the esophageal entrance (χ² = 9.103, P = 0.003). CONCLUSION: Laryngoscopy with injecting gas through the biopsy channel is a useful method for determining the presence or absence of esophageal entrance invasion in hypopharyngeal cancer prior to surgery.

[Value of endobronchial ultrasound-guided transbronchial needle aspiration in hilar and mediastinal lesions].

OBJECTIVE: To evaluate the diagnostic yield of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in cases of undiagnosed intrapulmonary lesions. METHODS: A total of 89 patients with intrathoracic lesions underwent EBUS-TBNA, including 56 pulmonary lesions, 7 lymph node staging in lung cancer patients, 21 unknown hilar and/or mediastinal lymphadenopathies and 5 mediastinal tumors. All samples were evaluated for cytological and pathological examinations. RESULTS: No complication of EBUS-TBNA was observed. Among 89 cases, 76 had positive results, 5 negative and 5 cases excluded as unsatisfied samples. In 56 patients with pulmonary lesions, EBUS-TBNA demonstrated 45 malignant tumors, 5 benign diseases, 3 suspicious cancers, 1 negative and 2 unsatisfied samples. In 7 lung cancer patients staged by EBUS-TBNA, 5 showed metastasis and 2 showed no metastasis. In 21 cases with mediastinal and/or hilar lymphadenopathy, EBUS-TBNA demonstrated 3 malignant tumors, 13 benign diseases, 2 negative and 3 unsatisfied samples. All 5 mediastinal lesions were malignant. Three suspicious cancers were confirmed, 1 by CT-guided percutaneous transthoracic needle biopsy and 2 by clinical follow-ups. In 2 lung cancer patients EBUS-TBNA showed negative, 1 surgical sample showed metastasis and another no metastasis by PET-CT. Three negative cases were diagnosed as benign by clinical follow-ups. The diagnostic sensitivity, specificity, positive predictive value and negative predictive value of EBUS-TBNA were 95%, 100%, 100% and 20% respectively. CONCLUSION: EBUS-TBNA is both effective and safe in making a diagnosis of intrathoracic lesions.

[Diagnostic value of transbronchial needle aspiration combined with transesophageal endoscopic ultrasound-guided fine needle aspiration in mediastinal and pulmonary hilar lesions].

OBJECTIVE: To evaluate the value of transbronchial needle aspiration (TBNA) combined with transesophageal endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) in the diagnosis of mediastinal and pulmonary hilar lesions as well as in the lymph node staging (N staging) of lung cancer. METHODS: 129 patients with mediastinal and pulmonary hilar lesions underwent either TBNA or EUS-FNA with cytological needle aspiration. The samples obtained from TBNA or EUS-FNA were examined by both cytologiy and histopathology. RESULTS: Of the 129 patients, 59 underwent TBNA and 70 EUS-FNA. The diagnostic rate were 84.7% (50/59) by TBNA and 94.3% (66/70) by EUS-FNA, resepectively. The diagnosis of 116 (89.9%) patients were confirmed by either TBNA or EUS-FNA. The pathological and cytological diagnostic rates were 92.2% (107/116) and 88.0% (102/116), resepectively. The diagnostic rate was elevated by 8.4% (9/107) through pathological examination. The histological classification rates by cytological and pathological examination were 73.8% (76/116) and 89.3% (92/103), respectively. The diagnostic rate of histological classification was elevated by 35.5% (27/76) through pathological examination. CONCLUSION: The combination of TBNA and EUS-FNA can improve the diagnostic rate for wider mediastinal and pulmlonary hilar lesions. Pathological examination of the samples obtained from the TBNA and EUS-FNA can elevate not only the rate of diagnosis but also the rate of histological classification.