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OBJECTIVE: To establish a method for simultaneous determination of 56 kinds of pesticide residues in Radix Ophiopogonis by using gas chromatography tandem mass spectrometry (GC-MS /MS), and apply it to screening of 137 batches of samples. METHODS: The forbidden, restricted and frequently-used pesticides were selected as the detecting indexes. The samples were prepared by QuEChERS, and quantitative analysis was carried out by GC-MS /MS in multiple-reaction monitoring (MRM) mode. There were three supplemental levels for detection recoveries and RSD. RESULTS: All the 56 pesticides had good linearity in certain ranges with correlation coefficients(r) higher than 0.997 8. The recoveries of 96.4% pesticides ranged from 60% to 130% at three supplemental levels (1, 2 and 10 LODs), with the RSDs of 92.9% pesticides less than 15%. The LODs for most of the selected pesticides were below 0.01 mg•kg-1. Twelve pesticides were detected in 137 batches of samples. CONCLUSION: The detecting indexes are meaningful and the developed method is simple, rapid, sensitive and reliable for screening multiple pesticide residues in Radix Ophiopogonis. The test result has certain reference value for the cultivation and distribution supervision of Radix Ophiopogonis.
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The method of UHPLC-LTQ-Orbitrap mass spectrometry coupled with higher energy collision dissociation (HCD) was established to rapidly analyze the constituents absorbed into blood after oral administration of steroidal saponins from Radix Ophiopogonis. A total of 31 constituents, including 13 furostanol steroidal saponins and 18 spirostanol steroidal saponins, were characterized based on the accurate mass measurements, fragmentation patterns, chromatographic retention times, and diagnostic product ions. Among them, 8 compounds were unambiguously identified by comparison with their corresponding standards. The results provide comprehensive insights and guidance for elucidation of material basis of Radix Ophiopogonis activity.
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In the storage of Radix Ophiopogonis, browning often happens to cause potential risk with regard to safety. Previously few reports investigate the browning of Radix Ophiopogonis. In this research, the causes and mechanisms of the browning of Radix Ophiopogonis were preliminarily elucidated. Content determination by high-performance liquid chromatography (HPLC) and spectrophotometry, enzyme activity determination by colorimetry, and morphological observation by electron microscopy were performed in the present study. Uniform design and three-dimensional response surfaces were applied to investigate the relationship between browning and storage factors. The cortex cell wall of browned Radix Ophiopogonis was ruptured. Compared with the normal Radix Ophiopogonis, cellulase and polyphenol oxidase enzymes were activated, the levels of 5-hydroxymethylfurfural (5-HMF), total sugars, and reducing sugars were increased, while the levels of polysaccharides and methylophiopogonanone A were decreased in browned Radix Ophiopogonis. The relationship between the storage factors and degree of browning (Y) could be described by following correlation equation: Y = - 0.625 4 + 0.020 84 × X3 + 0.001 514 × X1 × X2 - 0.000 964 4 × X2 × X3. Accompanied with browning under storage conditions, the chemical composition of Radix Ophiopogonis was altered. Following the activation of cellulase, the rupture of the cortex cell wall and the outflow of cell substances flowed out, which caused the Radix Ophiopogonis tissue to become soft and sticky. The main causes of the browning were the production of 5-HMF, the activation of polyphenol oxidase, Maillard reactions and enzymatic browning. Browning could be effectively prevented when the air relative humidity (HR), temperature, and moisture content were under 25% RH, 12 °C and 18%, respectively.
Subject(s)
Carbohydrates , Catechol Oxidase , Cell Wall , Cellulase , Chromatography, High Pressure Liquid , Food Storage , Methods , Furaldehyde , Humidity , Maillard Reaction , Ophiopogon , Chemistry , TemperatureABSTRACT
A competitive enzyme-linked immunosorbent assay (ELISA) was developed to determine ruscogenin (RUS) by using the monoclonal antibody (McAb). The monoclonal antibody against RUS, secreted from the established hybridoma cell lines, was identified as being of the IgG1 isotype. The McAb exhibited high specificity to RUS, showing a very slight cross reactivity with diosgenin (15.7%), and no cross-reactivity to sarsasapogenin, diammonium glycyrrhizinate, oleanolic acid and notoginsenoside R1. The established ELISA, at an IC50 value of 157.55 ng·mL(-1) and a detection limit (IC20) of 20.57 ng·mL(-1), was compared with HPLC analyses, and a good correlation between ELISA and HPLC-ELSD analyses of RUS in the extract of Radix Ophiopogonis was obtained. The experimental data indicated that the ELISA method exhibits more advantages over HPLC-ELSD, such as low detection limit, high specificity, low background, and no requirement for sample pre-treatment, and is more suitable for the determination of natural components in Chinese traditional medicines and in biological samples for pharmacokinetic studies.
Subject(s)
Antibodies, Monoclonal , Drugs, Chinese Herbal , Enzyme-Linked Immunosorbent Assay , Methods , Sensitivity and Specificity , SpirostansABSTRACT
Objective To determine the content of fructose in radix ophiopogonis cropped in Sichuan and Zhejian, and in extracts of radix ophiopogonis. Methods A HPLC-ELSD method was used for determining the content of fructose. Results The content of fructose in radix ophiopogonis cropped in Sichuan and Zhejiang were 11.32%～19.96% and 3.85%～10.19%, respectively. The contents of fructose in extracts of radix ophiopogonis cropped in Sichuan and Zhejiang were 23.48%～25.82% and 21.39%～ 23.29%, respectively. Conclusion The content of fructose in radix ophiopogonis cropped in Sichuan was higher than that of in Zhejiang. The content of fructose in extracts of radix ophiopogonis cropped in Sichuan was almost the same as that of in Zhejiang.
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Objective To detect the content of polysaccharides in Radix and Rootlet ophiopogonins. Methods Phenylhydrate-sulfuric acid method was used. Results The maximum absorption wavelength was 485 nm. Linear range was 0.101～1.012 mg/L (A=0.826 7C+0.016 3, r=0.999 2). The polysaccharide content in Radix ophiopogonis was 0.301 6 mg/g, and in Rootlet ophiopogonis was 0.359 4 mg/g. The average recovery rate was 100.30% and RSD was 3.14%. Conclusion The polysaccharide content in Rootlet Ophiopogonis was not lower than in Radix Ophiopogonis. It deserves to be more developed and researched.
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Objective To determine the contents of methylophiopogonanone A (MOA) and methylophiopogonanone B (MOB) in Radix Ophiopogonis and its extracts. Methods An HPLC-UV method was used for determining the contents of MOA and MOB in all samples. Analytical column was Kromasil C18 (250 mm?4.6 mm, 5 ?m). Mobile phase was acetonitrle-water (55∶45) and detection wavelength was 298 nm. The flow rate of mobile phase was 1 mL/min, and temperature was 30 ℃. Results The contents of MOA in Radix Ophiopogonis cropped in Sichuan and Zhejiang Provinces were 0.004 0%-0.009 6%, 0.006 7%-0.013 4%, and the contents of MOB were 0.002 1%-0.006 2%, 0.015 9%-0.028 2%, respectively. The contents of MOA in the extract of Radix Ophiopogonis cropped in Sichuan and Zhejiang Provinces were 0.007 5%-0.008 8%, 0.011 2%-0.012 6%, and the contents of MOB were 0.003 8%-0.005 1%, 0.020 7%-0.023 8%, respectively. Conclusion The contents of MOA and MOB in Radix Ophiopogonis cropped in Zhejiang Province and its extracts are more than those in Sichuan Province and its extrouts. The method can be used for the purpose of the quality control of Radix Ophiopogonis and its extracts.
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AIM: To establish chromatography fingerprint of Radix Ophiopogonis. METHODS: HPLC was applied on a Kromasil C_(18) column(5 ?m,4.6 mm?250 mm) with CH_3CN-0.01%H_3PO_4 solution by gradient elution,flow-rate of 1.0 mL/min,and the UV absorbance was monitored at 297 nm. RESULTS: 30 common peaks were picked up. CONCLUSION: This method is reliable,simple and provides a reference standard for the quality control of Radix Ophiopogonis.
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AIM: To explore the molecular mechanism of Radix Ophioponis against vascular endothelial cell (VEC) apoptosis induced by LPS. METHODS: The apoptosis of human umbilical vascular endothelial cells(HUVEC) was induced by lipopolysaccharide(LPS). The influence of Radix Ophiopogonis on the expression of bcl-2 and intracellular Ca 2+ was detected by flow cytometry and laser confocal microscope. RESULTS: The serum containing Radix Ophiopogonis suppressed the increase in bcl-2 expression and overloading of Ca 2+ induced by LPS in HUVEC.CONCLUSION: The mechanism of Radix Ophiopogonis against HUVEC apotosis may be related with its regulatory effect on bcl-2 expression and remission of Ca 2+ overloading.