[Quality evaluation of Lysimachiae Herba from different habitats based on simultaneous determination of multiple bioactive constituents combined with multivariate statistical analysis].

A method based on ultra-high performance liquid chromatography coupled with triple quadrupole linear ion trap-tandem mass spectrometry(UHPLC-QTRAP-MS/MS) was developed for the simultaneous determination of 41 bioactive constituents of flavonoids, organic acids, nucleosides, and amino acids in Lysimachiae Herba. The content of multiple bioactive constituents was compared among the samples from different habitats. The chromatographic separation was performed in a Waters XBridge®C_(18) column(4.6 mm×100 mm, 3.5 µm) at 30 ℃. The gradient elution was performed with 0.4% methanol(A)-formic acid water(B) as the mobile phase at a flow rate of 0.8 mL·min~(-1), and the multiple-reaction monitoring(MRM) mode was adopted. According to the content of 41 constituents, hierarchical cluster analysis(HCA), orthogonal partial least squares discriminant analysis(OPLS-DA), and gray relational analysis(GRA) were perfomed to comprehensively evaluate the samples from different habitats. The results showed that the 41 constituents exhibited good linear relationship within the tested concentration ranges, with the correlation coefficients(r) greater than 0.999 4. The method featured good precision, repeatability, and stability with the relative standard deviations(RSDs) less than 5.0%. The average recoveries of the 41 constituents ranged from 98.06% to 101.9%, with the RSDs of 0.62%-4.6%. HCA and OPLS-DA separated 48 batches of Lysimachiae Herba samples from different habitats into three categories: the producing areas in Sichuan and Chongqing, the producing areas in Jiangsu, Zhejiang, and Jiangxi, and the producing areas in Guizhou. The content of 41 constituents varied among the Lysimachiae Herba samples from different habitats. The GRA results revealed that the Lysimachiae Herba sample from Nanchong City, Sichuan Province had the best comprehensive quality. The method developed in this study was accurate and reliable and thus can be used for comprehensive evaluation of Lysimachiae Herba quality and provide basic information for the selection of habitats.

[Analysis and evaluation of bioactive constituents from different parts of Epimedium brevicornum].

A comprehensive analytical method based on ultra-fast liquid chromatography coupled with triple quadrupole/linear ion trap tandem mass spectrometry(UFLC-QTRAP-MS/MS) was established for simultaneous determination of the content of 45 bioactive constituents including flavonoids, alkaloids, amino acids, phenolic acids, and nucleosides in Epimedium brevicornum. The multiple bioactive constituents in leaves, petioles, stems and rhizomes of E. brevicornum were analyzed. The gradient elution was performed at 30 ℃ in an XBridge~® C_(18) column(4.6 mm×100 mm, 3.5 µm) with 0.4% formic acid aqueous solution-acetonitrile as the mobile phase at a flow rate of 0.8 mL·min~(-1). Single factor experiment and response surface methodology were employed to optimize the extraction conditions. Multivariate statistical analyses including systematic cluster analysis(SCA), principal component analysis(PCA), partial least squares discriminant analysis(PLS-DA), and one-way analysis of variance(One-way ANOVA) were carried out to classify the samples from different parts and identify different constituents. Grey relation analysis(GRA) and entropy weight-TOPSIS analysis were performed to build a multi-index comprehensive evaluation model for different parts of E. brevicornum. The results showed that there was a good relationship between the mass concentrations of 45 constituents and the corresponding peak areas, with the correlation coefficients(r) not less than 0.999 0. The precision, repeatability, and stability of the established method were good for all the target constituents in this study, with the relative standard deviations(RSDs) less than 5.0%(0.62%-4.9%) and the average recovery of 94.51%-105.7%. The above results indicated that the bioactive constituents varied in different parts of E. brevicornum, and the overall quality followed the trend of leaves > petioles > rhizomes > stems. This study verified the rationality of the Chinese Pharmacopoeia(2020 edition) stipulating that the medicinal part of E. brevicornum is the leaf. Moreover, our study indicated that the rhizome had the potential for medicinal development. The established method was accurate and reliable, which can be used to comprehensive evaluate and control the quality of E. brevicornum. This study provides data reference for clarifying the medicinal parts and rationally utilizing the resources of E. brevicornum.

[Analysis and evaluation of dynamic accumulation of multiple active constituents in Taxilli Herba in Guangxi].

A method based on ultra-high performance liquid chromatography with triple quadrupole/linear ion trap mass spectrometry(UHPLC-QTRAP-MS/MS) was developed for the simultaneous determination of 33 active constituents, including flavonoids, organic acids, nucleosides, and amino acids in Taxilli Herba to analyze and evaluate the dynamic accumulation of their multiple active constituents. The separation was performed at 30 ℃ on an XBridge~® C_(18) column(4.6 mm×100 mm, 3.5 µm) with gradient elution using 0.1% formic acid aqueous solution-methanol as the mobile phase at a flow rate of 0.5 mL·min~(-1), and the injection volume was 2 µL. The constituents were ionized in the electrospray ionization source(ESI) and quantitated by the multiple reaction monitoring(MRM) mode. The entropy weight TOPSIS method was used to objectively assign weights to the target constituents and rank them according to their relative closeness coefficient(C_i) to construct a multi-index comprehensive evaluation model of Taxilli Herba. The results showed that the concentrations and peak areas of 33 target constituents had good linearity in their respective linear ranges, and the correlation coefficients(r) were not less than 0.999 0. The RSD of precision, reproducibility, and stability were not higher than 4.7%. The average recoveries were 98.03%-101.3% with RSD less than 4.0%. There were differences in the content of 33 active constituents in Taxilli Herba at different harvest periods. The overall quality of Taxilli Herba harvested from mid-February to early March was better, which was consistent with the traditional harvest period. This study provides basic information for revealing the rule of dynamic accumulation of multiple active constituents in Taxilli Herba and determining the suitable harvesting period. Meanwhile, it also provides a new methodological reference for the comprehensive evaluation of the intrinsic quality of Taxilli Herba.

Quality Evaluation of Taxilli Herba from Different Hosts Based on Simultaneous Determination of Multiple Bioactive Constituents Combined with Multivariate Statistical Analysis.

Taxilli Herba (TAXH) is an important traditional Chinese medicine with a long history, dating from the Eastern Han Dynasty to the present times. However, the active constituents in it that parasitize different hosts vary, affecting its clinical efficacy. Given the complexity of the host origins, evaluating the quality of TAXH is critical to ensure the safety and effectiveness of clinical medication. In the present study, a quantitative method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was established, which simultaneously determined the content of 33 active constituents, including 12 flavonoids, 4 organic acids, 12 amino acids, and 5 nucleosides in 45 samples. Orthogonal partial least squares discriminant analysis (OPLS-DA) was employed to classify and distinguish between TAXH and its adulterants, Tolypanthi Herba (TOLH). A hierarchical clustering analysis (HCA) was conducted combined with a heatmap to visually observe the distribution regularity of 33 constituents in each sample. Furthermore, gray relational analysis (GRA) was applied to evaluate the quality of samples to get the optimal host. The results demonstrated that TAXH excelled TOLH in quality as a whole. The quality of TAXH parasitizing Morus alba was also better, while those that were parasitic on Cinnamomum camphora and Glyptostrobus pensilis had relatively poor quality. This study may provide comprehensive information that is necessary for quality control and supply a scientific basis for further exploring the quality formation mechanism of TAXH.

[Thoughts of omics research on quality formation in Dao-di herbs].

Dao-di herbs have been recognized as "quality models" with a firmly stable status. The formation of Dao-di herbs quality is involved from the genetic inheritance on the molecular level to the metabolic phenotype of final products, and the full material-based biosynthetic pathway remains unknown. In recent years, an increasing variety of omics technologies has provided new methods and ideas for the analysis of complex life systems and are suitable for explanation of quality formation in Dao-di herbs as well. In order to alleviate the scarcity of natural resources and offer scientific guidance of transplanting varieties, achievements of omics in the aspects of Dao-di herbs from genetics to phenotyping, the biosynthetic pathway of secondary metabolites, the interaction with human body and the new methods of quality evaluation have been summarized. It will be a fundamental work for protection and utilization of Chinese medicine resources.

Site-specific accumulation and dynamic change of flavonoids in Apocyni Veneti Folium.

Site-specific accumulation of flavonoids in Apocyni Veneti Folium was determined by laser scanning confocal microscope (LSCM) and the localization of catechins also was observed via vanillin-HCl staining under the conventional optical microscope. The contents of five flavonoids in Apocyni Veneti Folium from different harvest times and growth parts were measured using HPLC method. LSCM observation showed that flavonoids are accumulated in cuticle of epidermal cells and vessel walls, especially in protoplasts and nucleolus of the collenchyma cells and the epidermal cells. Catechins are localized in the palisade parenchyma cells and vessel walls, particularly in the laticifers found in the phloem. On the basis of the difference of the maximal emission wavelength between quercetin and kaempferol derivatives which have fluorescence behavior by appropriate treatment, kaempferol and its derivatives are localized exclusively in the cuticle. Results showed that the content of astragalin in Apocyni Veneti Folium from different parts revealed the decreasing trend, while hyperin and isoquercitrin were higher in June and July analyzed by HPLC. In summary, the site-specific accumulation of flavonoids in Apocyni Veneti Folium can be determined by LSCM and vanillin-HCl staining. The contents of flavonoids in Apocyni Veneti Folium are correlated with harvest times and growth parts.

[Correlation analysis of fetal middle cerebral artery peak systolic velocity, cardiothoracic ratio and crown-rump length in early pregnancy].

OBJECTIVE: To explore the pattern of variations in middle cerebral artery peak systolic velocity (MCA PSV) and cardiothoracic ratio (CTR) during early pregnancy, establish their reference ranges and explore their correlation with the crown-rump length (CRL). METHODS: A total of 522 pregnant women with normal findings in antenatal examinations underwent routine color Doppler ultrasound examination to collect the data of MCA PSV, CTR and CRL. The reference ranges of MCA PSV and CTR for different CRL levels were established, and the correlation of MCA PSV and CTR with CRL was analyzed. RESULTS: During the first trimester, MCA PSV and CRL showed a moderate positive correlation with a correlation coefficient of 0.426 (P<0.001), while CTR and CRL showed no significant correlation (0.168, P<0.001). The reference range of MCA PSV was 14.35 (14.08-14.62) cm/s and that of CTR was 0.34 (0.33-0.34) during early pregnancy. CONCLUSION: Color Doppler ultrasound is a safe and feasible modality to assess fetal MCA PSV and CTR for detecting fetal growth abnormalities in early pregnancy. The established reference ranges of MCA PSV and CTR offer a clinical theoretical basis for detecting α-thalassemia in early pregnancy.

Expression of FOXJ1 in hepatocellular carcinoma: correlation with patients' prognosis and tumor cell proliferation.

FOXJ1 is a member of the forkhead box (FOX) family of transcription factors. Recent studies suggested that FOXJ1 may function as a tumor suppressor gene in breast cancer. To investigate the potential roles of FOXJ1 in hepatocellular carcinoma (HCC), expression of FOXJ1 was first examined in eight paired frozen HCC and adjacent noncancerous liver tissues by Western blot, and we found that FOXJ1 was upregulated in HCC specimens. In addition, immunohistochemistry was performed to confirm our results in 108 HCC samples. Moreover, we also evaluated its relation with clinicopathological variables and the prognostic significance. The data showed that high expression of FOXJ1 was associated with histological grade (P < 0.001), and FOXJ1 was positively correlated with proliferation marker Ki-67 (P < 0.01). Univariate analysis suggested that FOXJ1 expression was associated with poor prognosis (P < 0.001). Multivariate analysis indicated that tumor grade (P < 0.0001), metastasis (P = 0.0451), tumor size (P = 0.0459), FOXJ1 (P = 0.0011), and Ki-67 (P = 0.0006) were independent prognostic markers for HCC. Furthermore, we noted that there existed the change of the level of FOXJ1 subcellular localization during cell-cycle transition in HepG2 cells by immunofluorescence and cell fractionation. Besides, we employed FOXJ1 overexpression/knockdown approaches to investigate the effects of FOXJ1 on HCC cell proliferation and cell-cycle distribution and found that overexpression of FOXJ1 can promote tumor cell proliferation and cell-cycle transition. Our results suggested that FOXJ1 was overexpressed in HCCs and associated with histological grade and poor prognosis. Overexpression of FOXJ1 was also involved in tumor cell proliferation and cell-cycle progression in HCC cell lines.

Graphene oxide-hairpin probe nanocomposite as a homogeneous assay platform for DNA base excision repair screening.

Uracil-DNA glycosylase (UDG) as one of the most important base excision repair enzymes plays a crucial role in protecting the genome from endogenous DNA damage and sustaining the genome integrity. Quantitative activity analysis of UDG is a central challenge and of fundamental importance in bioanalysis. Here, we proposed a novel biosensor constituted by adsorbing a fluorophore-labeled hairpin probe onto the surface of graphene oxide (GO) as a homogeneous assay platform for sensitive UDG activity assay. Active UDG could excise the uracil base in the hairpin probe, and further hydrolysis of the leaving abasic site gave rise to high fluorescence. Thus, it provided a convenient approach for UDG activity quantification. Because of the unique ability of GO in universal fluorescence quenching, a low background fluorescence signal can be obtained for the efficient fluorescence resonant energy transfer from the fluorophore-labeled on the hairpin probe to GO sheet. A quite wide dynamic range from 0.0017 U/mL to 0.8 U/mL was achieved for UDG assay and the detection limit was estimated to be 0.0008 U/mL. The results indicated that this strategy offers a simple, cost-effective, highly sensitive and selective homogeneous detection platform for UDG activity assay related biochemical studies.

[Prenatal diagnosis of agenesis of corpus callosum and its relationship with fetal chromosomal abnormalities].

OBJECTIVES: To evaluate prenatal imaging diagnosis of agenesis of corpus callosum and to investigate the relationship between ACC and chromosomal abnormalities. METHODS: Forty singleton pregnancies diagnosed ACC prenatally in Southern Medical University,Nanfang Hospital,General Hospital of Guangzhou Military Command of PLA and Shenzhen Maternity and Children Health Care Hospital from 2007 to 2012 were recruited. The correlation between ACC and chromosomal abnormalities, the consistence of sonographic characteristics and MRI diagnosis were analyzed retrospectively. RESULTS: (1) Among the 40 cases, 15 (38%, 15/40) were diagnosed isolated ACC, while 25 (63%, 25/40) were non-isolated ACC.In the non-isolated ACC cases, 18 (72%) had central nervous system abnormalities, including cerebellar vermis hypoplasia,Dandy-Walker syndrome, cerebellar cyst, holoprosencephaly, etc.Extra-CNS abnormalities were identified in 16 cases, including 5 cardiac abnormalities, 3 facial abnormalities, 2 congenital anomalies of urinary system, 1 limb skeletal abnormality and 5 other congenital anomalies.(2) In the 40 cases, 3 were chromosomal polymorphisms, including 2 cases of 46,XX, 1qh+ and 1 case of 46,XY, 13cenh+. Chromosomal abnormalities were identified in 4 cases, including trisomy13, trisomy18, trisomy 21 and 47,XYY.(3) 36 cases(90%, 36/40) diagnosed by ultrasound were consistent with MRI, while 4 cases were different with MRI.37 pregnancies were terminated, in which 28 cases were confirmed by fetal autopsy.3 cases continued pregnancy and ACC was confirmed by postnatal MRI.(4) 25 non-isolated ACC and 12 isolated ACC pregnancies were terminated. Among the 3 isolated ACC cases that continued pregnancy, 2 were term delivery and 1 was premature delivery. All of them were confirmed by postnatal MRI.No mental or growth retardation was found during follow-up. CONCLUSION: MRI was prior to detect cases with non-isolated ACC and could be a supplementary method in the diagnosis and classification of ACC. Compared with isolated ACC, non-isolated ACC had a higher incidence of chromosomal abnormalities.

[Correlation of fetal chromosomal abnormalities to prenatal ultrasound features].

OBJECTIVE: To investigate the correlation between fetal chromosomal abnormalities and the characteristic features of prenatal ultrasound findings. METHODS: A total of 510 cases were underwent chromosome examination by amniotic fluid or cord blood analysis to identify fetal chromosomal abnormalities. The correlation between the abnormalities and the characteristics of the prenatal ultrasound findings was analyzed. RESULTS: Fifty-three cases of abnormal karyotypes were detected with a positivity rate of 10.2%. Of these cases, 32 cases had chromosome number abnormalities, including 15 with 21-trisomy, 11 with 18-trisomy, 2 with 13-trisomy, 2 with 45, XO monomer and 2 with 92, XXXX tetraploid. Chromosome structural abnormalities were found in 21 cases, including 4 with translocation, 3 with insertion, 6 with inversion, 4 with deletion and 4 with derivation. Prenatal ultrasound showed obvious structural abnormalities in 22 cases (41.5%), structural malformation with ultrasonographic soft markers in 18 cases (34.0%), and separate ultrasonographic soft markers in 8 cases (15.1%). CONCLUSION: Prenatal ultrasound fetal abnormalities and chromosome abnormalities are closely related. Prenatal ultrasound of fetal chromosomal abnormalities usually presents with a variety of significant structural abnormalities. A greater number of malformations is associated with a greater risk of chromosomal abnormalities and increased occurrence of ultrasonographic soft markers.

[Lower limb deep vein ultrasonography and blood biochemistry in women after full-term delivery].

OBJECTIVE: To observe the postpartum changes in lower limb deep vein ultrasonography and blood biochemistry in women 2-5 days after full-term delivery. METHODS: A total of 212 women at high risk of thrombosis underwent high-resolution color Doppler ultrasound (CDU) of the lower limb deep veins 2-5 days after full-term delivery (Group A). Sixty-one healthy women 2-5 days after full-term delivery (Group B) and 42 healthy non-pregnant women (Group C) were recruited as the controls. The hematological indexes including the D-dimers (D-D), platelet (PLT), hemoglobin (HGB), hematocrit (HCT), thrombin time (TT), activated partial thromboplastin time (APTT), prothrombin time (PT) and fibrinogen (Fbg) were also determined in these 3 groups. RESULTS: Compared to Group B, the women in group A showed significantly increased diameters and D-D value of the common femoral veins (FV), common superficial femoral veins (SFV) and common popliteal veins (POV) (P<0.01), with decreased peak blood flow in the bilateral POVs (P<0.05). Compared to those in Groups C, the diameters of the bilateral FVs, SFVs, POVs, and posterior tibial veins (PTVs) were significantly increased, but the peak blood flow in the bilateral FVs, SFVs, and POVs were significantly reduced in groups A and B; the PLT, HGB, HCT, DD, TT, APTT, PT, and Fbg also showed significant changes in groups A and B (P<0.01). CONCLUSIONS: The women 2-5 days after full-term delivery are at high risk of prethrombotic state in comparison with non-pregnant women, and the presence of high-risk factors for thrombosis increases the likeliness of prothrombotic state in these postpartum women. CDU examination of the lower limb deep veins can be of value in the diagnosis of prethrombotic state.

[Ultrasonic study of deep-vein diameter and blood flow spectrum changes in full-term pregnant women].

OBJECTIVE: To explore the changes in lower limb deep vein diameters, blood flow velocity and blood biochemistry in full-term pregnant women for early diagnosis and treatment of prothrombotic state. METHODS: One hundred and twenty-eight full-term pregnant women at high risk of thrombosis (Group A), 61 healthy full-term pregnant women (Group B), and 42 healthy non-pregnant women (Group C) underwent high-resolution color Doppler ultrasound (CDU) for examining the deep veins of the lower limbs. The hematological indexes such as D-D, PLT, HGB, HCT, TT, APTT, PT, and FbgC were also observed in these 3 groups. RESULTS: Compared to Group B, the women in group A showed significantly increased diameters of the common femoral veins (CFV) and left superficial femoral vein (SFV), HCT and DD, but with significantly decreased peak blood flow in the bilateral popliteal veins (POPV) (P<0.01) and increased left POPV diameter (P=0.034). Compared to those in group C, the diameters of the bilateral CFVs, SFVs, POPV, and posterior tibial veins (PTVs) were significantly increased, but the peak blood flow in the bilateral CFVs and POPVs were significantly reduced in groups A and B; the PLT, HGB, HCT, DD, TT, APTT, PT, and FbgC also showed significant changes in groups A and B (P<0.01). CONCLUSION: The full-term pregnant women are at higher risk of prothrombotic state than non-pregnant women, and the full-term pregnant women with the high risk factors for thrombosis are more likely to have prothrombotic state than healthy full-term pregnant women. CDU examination of the lower limb deep veins can be of value in the diagnosis of prothrombotic state.

[Clinical study of Osaka formula and improved multiparameter ultrasonic measurement for fetal weight estimation].

OBJECTIVE: To analyze the factors affecting the accuracy of Osaka formula multiparameter ultrasound-based fetal mass estimation, thereby establishing new formulas to improve the accuracy of the estimation. METHODS: A retrospective review was conducted among 519 healthy women with singleton pregnancy. Three days before the delivery (between 37 and 42 weeks' gestation), ultrasonic measurement of the fetal weight and other indices of the fetus was routinely performed. Correlation and multiple linear stepwise regression analysis were used to correct the 3 equations, which, along with Osaka University formula, were used to predict another 219 fetuses' birth weight. The coincidence rate of the predicted value and with the actual birth weight, and the absolute error and relative error were compared between the equations. RESULTS: The fetal abdominal area (AA) and abdominal circumference (AC) showed the most conspicuous influence on the estimated fetal birth weight, and fetal humerus length (HL) was more sensitive than femur length (FL) for the estimation. Three new regression equations were established, among which the equation 2 (fetal birth weight=1082.859+4.116xAAxHL) showed the best accuracy in clinical prediction. CONCLUSION: AA,AC and HL are more sensitive indices for estimation of the fetal birth weight, and the equation 2 established in this study still awaits further verification for its clinical value.

[Chorionic villus sampling for prenatal diagnosis: clinical analysis of 36 cases].

OBJECTIVE: To evaluate the safety and clinical value of chorionic villus sampling via the cervix for prenatal diagnosis of early pregnancy. METHOD: Thirty-six women with early pregnancy who required prenatal diagnosis received chorionic villus sampling via the cervix under the guidance by ultrasonography for laboratory examination of the samples. RESULTS: Villus sampling was performed successfully in 27 of the 36 cases in a single operation, whereas a second operation for sampling was needed in another 7 cases, with a total operation success rate of 94.4%;. The side effects included abdominal pain and vaginal bleeding in small quantity. In the 22 women who chose to continue the pregnancy, one woman experienced miscarriage at the 25th gestational week due to premature rupture of the membrane, and all other women gave birth to normal full-term infants. CONCLUSION: Chorionic villus sampling via the cervix under the guidance by ultrasonography is effective and safe for prenatal diagnosis in early pregnancy to help avoid induced abortion in mid-pregnancy and the occurrence of fetal defects.