Research on Material Basis of Homologous and Heterogeneous Effect of Aurantii Fructus Immaturus and Aurantii Fructus Based on Total Statistical Moment Analysis and Molecular Connectivity Index / 中国实验方剂学杂志

ObjectiveTo investigate the material basis of homologous and heterogeneous effect of Aurantii Fructus Immaturus（AFI） and Aurantii Fructus（AF） based on the total statistical moment analysis and molecular connectivity index（MCI）. MethodRelevant literature at home and abroad and Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP） were consulted to establish the chemical composition database of AFI and AF， and set up their fingerprints by ultra-high performance liquid chromatography（UPLC）， and the total statistical moments and similarity parameters of the fingerprint were calculated. According to MCI， all components of AFI and AF were divided into different component groups， the average values of 0-8th order（0χ-8χ） MCI of the common component groups of AFI and AF were calculated. ResultThe values of total zero-order moment（AUCT） of AFI and AF were （10.57±2.45）×106， （5.09±0.89）×106 μV·s， the values of total first-order moment（MCRTT） were （11.57±1.58）， （12.10±1.29） min， the values of total second-order moments（VCRTT） were（24.49±2.30）， （26.49±2.54） min2， respectively. It showed that qualitative and quantitative parameters of AFI and AF were significantly different. The components with high similarity such as neohesperidin， hesperidin and narirutin were screened as the common potential pharmacodynamic components of AFI and AF. The non-common components of AFI， such as alysifolinone and imperatorin， and the non-common components of AF， such as neoeriocitrin and isosakuranin， with high similarity were screened out as potential heterogeneous components of AFI and AF. The composition groups of AFI and AF were classified into six categories， and the similarities between the composition groups of AFI and AF and the total constituents were 0.872-0.979 and 0.918-0.997， the average values of 0χ-8χ MCI of alkaloids in AFI and AF were 3.65 and 3.14， the average values of 0χ-8χ MCI of flavonoids were 8.47 and 8.47， the average values of 0χ-8χ MCI of volatile oils were 2.71 and 3.48， respectively. It showed that there were some differences in MCI of chemical constituents（groups） between AFI and AF. ConclusionThe chemical constituents（groups） of AFI and AF not only differ in content and species， but also in structural characteristics and structure-activity relationship， which can provide a basis for further explaining the scientific connotation of homologous and heterogeneous effect of AFI and AF.

Quality Difference Analysis of Raw and Steamed Products of Citri Reticulatae Pericarpium Based on Electronic Eye Technique and Chemical Fingerprint / 中国实验方剂学杂志

ObjectiveTo explore the quality differences between steamed products and raw products of Citri Reticulatae Pericarpium（CRP）. MethodThe color of steamed products and raw products of CRP was determined from the perspective of appearance by electronic eye technique， and the quality differences between them was objectively characterized by the luminous value（L*）， yellow-blue value（b*）， red-green value（a*） and total chromatic value（E*ab）. Based on this， ultra-high performance liquid chromatography（UPLC） was used to establish a fingerprint evaluation method with the mobile phase of acetonitrile（A）-0.1% formic acid aqueous solution（B） for gradient elution（0-5 min， 5%A； 5-30 min， 5%-20%A； 30-60 min， 20%-52%A）， detection wavelength at 270 nm， flow rate of 0.3 mL·min-1 and column temperature of 30 ℃. The quality differences between steamed products and raw products of CRP were compared from the perspective of chemical composition， and correlation analysis was used to reveal the correlation between the difference in appearance color and the difference in internal chemical composition. ResultAfter being steamed， L*， b* and E*ab of CRP showed an overall decreasing trend， indicating that the color of the steamed products darkened and deepened from yellow to blue but still tended to be yellow， while a* showed an overall increasing trend， indicating that the color of the steamed products tended to red. A total of 24 peaks were identified in the fingerprint profiles of raw products and steamed products of CRP， and 13 of the main peaks were identified. The precision， stability and repeatability studies showed that compared with the reference peak （peak 14， hesperidin）， the relative standard deviations（RSDs） of the relative peak area and relative retention time of the remaining peaks were<3.0%.The results of chemometric statistical analysis showed that there were some differences between raw products and steamed products of CRP， and 7 main differential components were identified， among which 5-hydroxymaltol（peak 1） and 5-hydroxymethylfurfural（peak 2） were the characteristic components of steamed products. The correlation analysis results showed that， in addition to the above two characteristic components， four components of peak 4， peak 10 （vicenin-2）， peak 23 （tangeretin） and peak 24 （5-demethylnobiletin） also correlated significantly with the color change （E*ab） of the samples （P<0.05， P<0.01）. ConclusionBefore and after steaming， not only the chemical composition changes， but also the color. Comparing the characteristic peaks of chemical composition difference and color difference before and after steaming of CRP， it is found that 5-hydroxymaltol， 5-hydroxymethylfurfural and peak 4 are common characteristic difference components， which can provide a reference for establishing the characteristic quality control method of steamed products， and quickly evaluating the quality difference between raw products and steamed products of CRP.

Optimization of Extraction and Purification Process of Total Iridoids and Total Crocins in Gardeniae Fructus / 中国实验方剂学杂志

ObjectiveTo optimize the extraction and purification process of Gardeniae Fructus for industrial production， and to obtain the total iridoid and total crocin extracts. MethodOrthogonal test was used to optimize the water extraction process by taking contents of geniposide， genipin gentiobioside， gardenoside， crocin-1 and crocin-2 as indicators and the decocting time， decocting times and water amount as factors. The purification process was optimized by single factor test， and four different types of macroporous adsorption resins were screened. The process conditions such as resin type， maximum loading amount， water washing amount， ethanol concentration， ethanol dosage， and flow rate of sample loading were mainly investigated. In addition， the drying methods （vacuum drying and spray drying） of the extract were investigated， and a pilot scale-up verification test was carried out. ResultThe optimal water extraction process of Gardeniae Fructus was to add 15， 10 times the amount of water for decocting twice， 1 h each time. The optimal purification process was as follows：the water extract through SP825L macroporous resin column， the amount of crude drug-the amount of resin （1∶1.5）， the sample loading flow rate of 3 BV h-1， adding 2 BV of water to remove impurities， adding 4 BV of 30% ethanol to obtain the iridoid part， then adding 3 BV of 70% ethanol to obtain the crocin part， collecting the ethanol lotion， and drying at 70 ℃. Under these conditions， the extraction amount of total iridoids was 590.75 mg·g-1 with the transfer rate of 70.48%， and the yield of dry extract was 8.89%. The extraction amount of total crocins was 83.37 mg·g-1 with the transfer rate of 22.20%， and the dry extract yield was 2.60%. ConclusionThe optimized extraction and purification process is stable and feasible with high extraction rate of active components， which is suitable for the industrial extraction and purification of active parts of Gardeniae Fructus.

Analysis and Application of Seven Common New Psychoactive Substances of Synthetic Cannabinoids in Hair / 法医学杂志

Objective To establish a detection method for common new psychoactive substances of synthetic cannabinoids in hair with ultra-high performance liquid chromatography-tandem mass spectrometry （UPLC-MS/MS）. Methods In the 1 mL of internal standard methanol solution, 20 mg hair was added. After cryogenic grinding and ultrasonic extraction, the extract was separated by ACQUITY UPLC HSS T3 column （100 mm×2.1 mm, 1.8 μm）. The mobile phase A was aqueous solution that composed of 20 mmol/L ammonium acetate, 0.1% formic acid, and 5% acetonitrile. The mobile phase B was acetonitrile. Electrospray ionization source in positive ion mode was used for data acquisition in multi-reaction monitoring （MRM） mode. Results The seven common new psychoactive substances of synthetic cannabinoids in hair had a good linear relationship within their respective linear ranges （r>0.99）, the limits of detection were 0.5-2 pg/mg, the limits of quantification were 1-5 pg/mg, the intra-day and inter-day precisions were 0.1%-12.6%, the intra-day and inter-day accuracies were 89.2%-110.7%, the recovery rates were 52.3%-93.3%, and the matrix effects were 19.1%-95.2%. Conclusion The established method has a simple sample preparation process and high sensitivity. It is suitable for qualitative and quantitative analysis of common new psychoactive substances of synthetic cannabinoids in hair.

Metabolomics Changes of Serum and Tissues in Mice Died of Acute Tetracaine Poisoning / 法医学杂志

Objective To study the changes of metabolites in serum and tissues （kidney, liver and heart） of mice died of acute tetracaine poisoning by metabolomics, to search for potential biomarkers and related metabolic pathways, and to provide new ideas for the identification of cause of death and research on toxicological mechanism of acute tetracaine poisoning. Methods Forty ICR mice were randomly divided into control group and acute tetracaine poisoning death group. The model of death from acute poisoning was established by intraperitoneal injection of tetracaine, and the metabolic profile of serum and tissues of mice was obtained by ultra-high performance liquid chromatography-electrostatic field orbitrap high resolution mass spectrometry （UPLC-Orbitrap HRMS）. Multivariate statistical principal component analysis （PCA） and orthogonal partial least square-discriminant analysis （OPLS-DA） were used, combined with t-test and fold change to identify the differential metabolites associated with death from acute tetracaine poisoning. Results Compared with the control group, the metabolic profiles of serum and tissues in the mice from acute tetracaine poisoning death group were significantly different. Eleven differential metabolites were identified in serum, including xanthine, spermine, 3-hydroxybutylamine, etc.; twenty-five differential metabolites were identified in liver, including adenylate, adenosine, citric acid, etc.; twelve differential metabolites were identified in heart, including hypoxanthine, guanine, guanosine, etc; four differential metabolites were identified in kidney, including taurochenodeoxycholic acid, 11, 12-epoxyeicosatrienoic acid, dimethylethanolamine and indole. Acute tetracaine poisoning mainly affected purine metabolism, tricarboxylic acid cycle, as well as metabolism of alanine, aspartic acid and glutamic acid. Conclusion The differential metabolites in serum and tissues of mice died of acute tetracaine poisoning are expected to be candidate biomarkers for this cause of death. The results can provide research basis for the mechanism and identification of acute tetracaine poisoning.

Simultaneous Determination of Six Flavonoids in Astragali Radix Directional Processed with Four Enzymes by UPLC / 中国实验方剂学杂志

Objective::To establish an UPLC method for the simultaneous determination of 6 flavonoids, and to research for the effect of Astragali Radix directional processed with four enzymes (complex enzyme, plant cellulase, snail enzyme, and β-glucosidase) on the contents of flavonoid glycosides and their aglycones in this herb. Method::Chromatographic separation was carried out on ACQUITY UPLC HSS T3 column (2.1 mm×100 mm, 1.8 μm) with the mobile phase of 0.1 mol·L-1 formic acid solution-0.1 mol·L-1 formic acid acetonitrile solution for gradient elution. The detection wavelength was set at 260 nm, the flow rate was 0.3 mL·min-1, the column temperature was 30 ℃, and the injection volume was 2 μL. Result::Calycosin-glucoside, calycosin, ononin, formononetin, 9, 10-dimethoxy-pterocarpan-3-O-β-D-glucoside and 3-hydroxy-9, 10-dimethoxy-pterocarpan showed good linear relationships within their own ranges (R2≥0.998 5), the relative standard deviations (RSDs) of precision, stability and repeatability were all <5.0%, and the average recovery was 97.62%-101.13% with RSDs of 1.4%-2.7%. In 0.5 g·L-1 level of enzyme solution, the contents of calycosin-glucoside, calycosin, ononin, formononetin, 9, 10-dimethoxy-pterocarpan-3-O-β-D-glucoside and 3-hydroxy-9, 10-dimethoxy-pterocarpan in Astragali Radix processed with complex enzyme were 0.082 0, 0.335 9, 0.055 9, 0.104 9, 0.015 0, 0.009 7 mg·g-1, the contents of them in Astragali Radix processed with plant cellulase were 0.105 7, 0.364 2, 0.070 2, 0.117 4, 0.020 8, 0.012 5 mg·g-1, their contents in Astragali Radix processed with snail enzyme were 0.031 4, 0.510 0, 0.043 5, 0.210 9, 0.013 0, 0.013 0 mg·g-1, and their contents in Astragali Radix processed with β-glucosidase were 0.085 3, 0.312 4, 0.061 5, 0.110 8, 0.005 8, 0.009 6 mg·g-1, respectively. Conclusion::After the processing of Astragali Radix by four enzymes, in addition to 9, 10-dimethoxy-pterocarpan-3-O-β-D-glucoside, the contents of calycosin-glucoside and ononin are reduced, but the contents of their three corresponding aglycones are significantly increased. The established method is simple, accurate and reproducible, and is suitable for the simultaneous determination of 6 flavonoids in Astragali Radix, which can provide a reference for this herb directional processed with enzymes.

Application and Research Progress of UPLC in Analysis of Traditional Chinese Medicine and Its Compound / 中国实验方剂学杂志

Ultra-high performance liquid chromatography(UPLC) is a new liquid chromatography technology,which developed on the basis of high performance liquid chromatography(HPLC).It has the characteristics of super high efficiency,high separation degree and high sensitivity,and has been widely used in the analysis of components in traditional Chinese medicine,the studies on metabolomics,establishment of fingerprint chromatogram,determination of unknown compounds combined with mass spectrometry and other detectors,etc.In this paper,the authors reviewed the application of UPLC in the field of traditional Chinese medicine through a review of the literature in the past 5 years,and summed up its relative advantages and development prospects,in order to provide scientific guidance and basis for pharmaceutical workers to make better use of UPLC technology.