Rapid screening and purification of potential alkaloid neuraminidase inhibitors from Toddalia asiatica (Linn.) Lam. roots via ultrafiltration liquid chromatography combined with stepwise flow rate counter-current chromatography.

Toddalia asiatica (Linn.) Lam. is a medical plant traditionally used to treat coughs, fevers, and various diseases. Alkaloids are the main active ingredients in Toddalia asiatica (Linn.) Lam., but traditional methods for screening and separation are complex and labor-intensive. In this work, an efficient strategy was developed to rapidly screen, identify, and separate neuraminidase inhibitors from Toddalia asiatica (Linn.) Lam. Ultrafiltration, high performance liquid chromatography, and time-of-flight mass spectrometry were employed for rapid screening and identification of neuraminidase inhibitors. A two-phase solvent system comprising n-hexane/ethyl acetate/methanol/water (5:5:3:7, v/v) was then selected for separation by high-speed counter-current chromatography. A sample loading of 200 mg and a stepwise flow rate were achieved by increasing the flow rate from 2 to 4 mL/min after 4 h. Three main fluoroquinoline alkaloids (haplopine, skimmianine, and 5-methoxydictamnine) along with two coumarins were obtained via one-step separation and their structures were determined by mass spectrometry and nuclear magnetic resonance. In vitro assays revealed skimmianine with half-maximal inhibitory concentration of 16.2 ± 0.7 µmol/L was selected as the potential highest neuraminidase inhibitor. The results suggest that ultrafiltration high performance liquid chromatography-mass spectrometry combined with high-speed counter-current chromatography is efficient for the screening and isolation of neuraminidase inhibitors from complex natural products.

Fetal microtia and FGFR2 polymorphism.

Association of the single-nucleotide polymorphism (SNP) of rs3135718 site in fibroblast growth factor receptor 2 (FGFR2) gene with congenital microtia was investigated. A total of 193 patients with congenital microtia (observation group) and 150 normal and healthy fetuses (control group) treated in Maternity and Child Health Care of Zaozhuang from January 2010 to October 2017 were randomly selected. The gene and genotype of the rs3135718 site of FGFR2 gene SNP were detected via quantitative polymerase chain reaction (qPCR). The association between rs3135718 site SNP and congenital microtia was analyzed. No statistically significant difference in the prevalence of congenital microtia was observed in the rs3135718 genotype (AG) between the observation and control group (P>0.05). The GG and G genotypes in rs3135718 were closely related to fetal microtia (P<0.05). Results revealed that the rs3135718-GG mutation was more correlated with the risk of microtia in male (P<0.05), but not correlated with the risk of microtia in female (P>0.05). Moreover, there was a statistically significant difference in the distribution of rs3135718-G allele frequency in male between the two groups (P<0.05). The rs3135718-G gene in FGFR2 has a certain association with the incidence of congenital microtia with high prevalence and risk.