ObjectiveHeadspace solid-phase microextraction-gas chromatography-mass spectrometry（HS-SPME-GC-MS） and GC-triple quadrupole MS（GC-QqQ-MS） in combination with non-targeted and targeted metabolomics were employed to systematically analyze the chemical composition differences of Xihuangwan prepared with natural musk and artificial musk， and establish an identification system for them. MethodsThe volatile components of 9 batches of Xihuangwan samples from 8 manufacturers were analyzed by HS-SPME-GC-MS non-targeted metabolomics， and identified by comparing their MS data with the National Institute of Standards and Technology（NIST） spectral library. Orthogonal partial least squares-discriminant analysis（OPLS-DA） was used to identify differential volatile components of Xihuangwan prepared with natural musk and artificial musk. Additionally， GC-QqQ-MS targeted metabolomics was applied to quantify the levels of α-pinene， β-elemene， muscone， dehydroepiandrosterone， bornyl acetate， and octyl acetate in 27 batches of samples from 9 manufacturers. Cluster analysis， principal component analysis（PCA）， and partial least squares-discriminant analysis（PLS-DA） were conducted to further explore the differences in volatile components between Xihuangwan samples prepared with natural musk and artificial musk. ResultsNon-targeted metabolomics identified 291 volatile compounds in Xihuangwan， including alkanes， esters， alkanes， alcohols， ketones， naphthalenes and others. OPLS-DA analysis revealed distinct separation between Xihuangwan samples containing artificial musk（A1， C1， D1， E1， F1， G1， I1） and those containing natural musk（H1， H3）. A total of 30 differential metabolites were identified. The relative contents of these 30 differential metabolites were visualized using a radar chart， revealing significant differences in the levels of octanol， borneol acetate and muscone. Cluster analysis and PCA results from targeted metabolomics indicated that Xihuangwan could be classified into two distinct groups：one composed of natural musk（H1， H3） and the other of artificial musk， sample H2. PLS-DA identified muscone， octyl acetate， and dehydroepiandrosterone as key differential volatile components. Although no significant difference was observed in the content of octyl acetate between the two groups， statistically significant differences were found for muscone and dehydroepiandrosterone（P<0.05）. ConclusionMuscone and dehydroepiandrosterone can be used for the differentiation of Xihuangwan samples containing natural musk from those containing artificial musk. This study systematically and comprehensively analyzed the differences in the types and contents of major volatile components in Xihuangwan prepared with natural musk and artificial musk， providing a scientific basis for quality evaluation and control of Xihuangwan.

ObjectiveHeadspace solid-phase microextraction-gas chromatography-mass spectrometry（HS-SPME-GC-MS） and GC-triple quadrupole MS（GC-QqQ-MS） in combination with non-targeted and targeted metabolomics were employed to systematically analyze the chemical composition differences of Xihuangwan prepared with natural musk and artificial musk， and establish an identification system for them. MethodsThe volatile components of 9 batches of Xihuangwan samples from 8 manufacturers were analyzed by HS-SPME-GC-MS non-targeted metabolomics， and identified by comparing their MS data with the National Institute of Standards and Technology（NIST） spectral library. Orthogonal partial least squares-discriminant analysis（OPLS-DA） was used to identify differential volatile components of Xihuangwan prepared with natural musk and artificial musk. Additionally， GC-QqQ-MS targeted metabolomics was applied to quantify the levels of α-pinene， β-elemene， muscone， dehydroepiandrosterone， bornyl acetate， and octyl acetate in 27 batches of samples from 9 manufacturers. Cluster analysis， principal component analysis（PCA）， and partial least squares-discriminant analysis（PLS-DA） were conducted to further explore the differences in volatile components between Xihuangwan samples prepared with natural musk and artificial musk. ResultsNon-targeted metabolomics identified 291 volatile compounds in Xihuangwan， including alkanes， esters， alkanes， alcohols， ketones， naphthalenes and others. OPLS-DA analysis revealed distinct separation between Xihuangwan samples containing artificial musk（A1， C1， D1， E1， F1， G1， I1） and those containing natural musk（H1， H3）. A total of 30 differential metabolites were identified. The relative contents of these 30 differential metabolites were visualized using a radar chart， revealing significant differences in the levels of octanol， borneol acetate and muscone. Cluster analysis and PCA results from targeted metabolomics indicated that Xihuangwan could be classified into two distinct groups：one composed of natural musk（H1， H3） and the other of artificial musk， sample H2. PLS-DA identified muscone， octyl acetate， and dehydroepiandrosterone as key differential volatile components. Although no significant difference was observed in the content of octyl acetate between the two groups， statistically significant differences were found for muscone and dehydroepiandrosterone（P<0.05）. ConclusionMuscone and dehydroepiandrosterone can be used for the differentiation of Xihuangwan samples containing natural musk from those containing artificial musk. This study systematically and comprehensively analyzed the differences in the types and contents of major volatile components in Xihuangwan prepared with natural musk and artificial musk， providing a scientific basis for quality evaluation and control of Xihuangwan.

Objective:To analyze the effects of processed Epimedii Folium on endogenous metabolites of mouse melanoma cells (B16 cells) before and after processing based on cell metabolomics; To investigate the changes of processed Epimedii Folium before and after processing.Methods:Ultra performance liquid chromatography tandem four-stage orbital trap mass spectrometry (UPLC-Q-Exactive Orbitrap-MS) technology was used, and the endogenous small molecules of B16 cells treated with Epimedii Folium and processed Epimedii Folium were analyzed by metabolomics. The differential metabolites between groups were obtained, and relevant metabolic pathways were analyzed based on the MetaboAnalyst 5.0 database.Results:Significant changes were observed in 13 kinds of endogenous metabolites, including alanine, carnitine C3∶0, glutamic acid-1, lactic acid, isoleucine, choline, phosphatidylcholine (34∶2, 36∶2), free fatty acids, citric acid, carnitine C4∶0, lysophosphatidylcholine 16∶0 and malic acid after the intervention of Epimedii Folium and processed Epimedii Folium. And the impact of processed products on differential metabolites was stronger than that of raw products. The main pathways involved were Warburg effect, pyruvate metabolism, malate-aspartic acid shuttle, pyruvaldehyde degradation and so on.Conclusions:Epimedii Folium and processed Epimedii Folium would have certain effects on cellular metabolic pathways. The results may be related to the pharmacological effects and changes in cold and hot properties of Epimedii Folium before and after processing.

ObjectiveTo determine the pharmacodynamic substance basis of Epimedii Folium（EF） and Epimedii Wushanensis Folium（EWF） in promoting osteogenic differentiation， and to establish a method to analyze the material basis of Chinese materia medica based on the correlation between chemical fingerprint and cellular metabolomics. MethodThe chemical fingerprints of 15 batches of EF with 4 species and 3 batches of EWF were analyzed by ultra performance liquid chromatography-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry（UPLC-Q-Exactive Orbitrap-MS）， and partial least squares-discriminant analysis（PLS-DA） was used to analyze the peak areas of chemical fingerprints of samples. The effects of different samples on proliferative activity of MC3T3-E1 osteoblast precursors， as well as the activity of alkaline phosphatase（ALP） in osteoblasts were detected by cell counting kit-8（CCK-8） and enzyme-linked immunosorbent assay（ELISA）. At the same time， UPLC-quadrupole-time-of-flight mass spectrometry（UPLC-Q-TOF-MS/MS） was used to analyze the effects of different samples on the metabolomics of MC3T3-E1 cells， then metabolic peak table of osteogenic differentiation cells was constructed， and pharmacodynamic index mean Y₀ was introduced into the peak table. PLS was used to calculate mean Y₀ of each group， and the mean Y₀ was added to the peak table of chemical fingerprint to construct the correlation between chemical fingerprint and cell metabolome， the pharmacodynamic components of EF and EWF that promote bone differentiation were screened according to variable importance in the projection（VIP） value>1. The pharmacodynamic effects of EF and EWF were evaluated according to the mean Y₀ of each group. ResultThe chemical fingerprints of EF with different origins and EWF were completely separated. Compared with the blank group， the activity of MC3T3-E1 cells in EF and EWF groups was significantly increased， the activity of ALP in the Epimedium brevicornu（Gansu province）， E. koreanum and E. pubescens groups was significantly increased（P<0.05）. The results of cell metabolomics showed that the blank group and the model group had an obvious trend of separation. EF with different origins and EWF had different distance from the model group， indicating that EF with different origins and EWF had different effect on promoting osteogenic differentiation. Chemical fingerprint-cell metabolomics integration analysis screened 9 components closely related to the efficacy of EF and EWF， including diphylloside B， epimedin C， icariin， baohuoside Ⅰ， yinyanghuo B， β-anhydroicaritin， magnoflorine， cryptochlorogenic acid and quercetin. E. koreanum had the strongest effect on promoting osteogenic differentiation. ConclusionThis study determined that the material basis of EF and EWF promoting osteogenic differentiation were mostly flavonoids， alkaloids and organic acids， which provided ideas and methods for the screening of pharmacodynamic components and the prediction of therapeutic effect of Chinese materia medica.

Objective:To explore the effects of different processed products of Epimedii Folium on cytotoxicity and the material basis of toxicity. Methods:By using the SRB method to investigate the effects of different processed products of Epimedii Folium on the proliferation of HaCaT cells; and based on the grey correlation analysis method to establish the data spectrum effect relationship of HPLC fingerprint spectrum-toxicity so as to determine the toxic components and processing methods. Results:The value of cytotoxicity IC 50 of different processed products of Epimedii Folium is as follow: vinegar fried> oil fried > original > single fried > salt fried > wine fried. Among the 12 characteristic chromatographic peaks, Peak No.3 (magnoflorine, correlation value: 0.870) and Peak No.6 (epimedin C, correlation value: 0.851) are highly correlated with the toxicity value. Conclusions:Both vinegar fried and oil fried Epimedii Folium have the effect of reducing toxicity. Magnoflorine and epimedin C may be the main toxic components in Epimedii Folium. The study provides scientific basis for the research on the process optimization of Epimedii Folium concocting to reduce toxicity.

Objective:To study the curative effect of platelet-rich plasma (PRP) combined with internal fixation with hollow screws on femoral neck fractures.Methods:The clinical data of 160 patients with femoral neck fracture were retrospectively analyzed who had been treated by internal fixation with hollow screws at Orthopedic Department Ⅲ, The Second Affiliated Hospital to Luohe Medical College from May 2012 to May 2018. According to whether PRP was used or not to assist their internal fixation, they were divided into a PRP group ( n=80) and a control group ( n=80). In the PRP group, there were 46 males and 34 females with an age of 52.3 years±7.6 years, and one case of type Ⅰ, 5 cases of type Ⅱ, 57 cases of type Ⅲ and 17 cases of type Ⅳ by the Garden classification. In the control group, there were 41 males and 39 females with an age of 50.6 years ± 7.3 years, and 2 cases of type Ⅰ, 7 cases of type Ⅱ, 51 cases of type Ⅲ and 20 cases of type Ⅳ by the Garden classification. The 2 groups were compared in terms of fracture healing time, nonunion, femoral head necrosis and Harris hip scores. Results:The 2 groups were comparable because their preoperative general data showed no significant differences ( P>0.05). The 160 patients obtained follow-up for 12 to 36 months. The PRP group showed significantly shorter fracture healing time (4.3 months ± 1.0 months), significantly lower incidences of nonunion [0% (0/80)] and avascular necrosis of femoral head [3.8% (3/80)] than the control group [7.3 months ± 1.3 months, 7.5% (6/80) and 15.0% (12/80), respectively] (all P< 0.05). The Harris scores at 6 and 12 months after operation for the PRP group (88.7±5.3 and 94.2±4.8) were significantly higher than those for the control group (81.4±4.6 and 84.2±5.2) (both P<0.05). Conclusion:In the treatment of femoral neck fractures, compared with internal fixation with hollow screws alone, platelet rich plasma combined with internal fixation with hollow screws can significantly shorten fracture healing time, reduce incidence of avascular necrosis of the femoral head and improve functional recovery of the hip joint.

Objective To optimize purification technology of total lignans in Myristicae Semen; To study its anti-inflammatory effects. Methods Macroporous adsorption resin was used for enrichment and purification of total lignans in Myristicae Semen, with adsorption rate, desorption rate and content of total lignans as indexes. The resin type, concentration of eluent, sample weight and dosage of eluen were investigated. Anti-inflammatory effects were studied by determining swelling degree and inhibition rate of mice with ear swelling induced by xylene. Results Purification technology of total lignans was processed on type of AB-8 resin. 1 g crude extract was dissolved as sample; 40 mL preprocessed resin including 16 g resin was added and static adsorpted for 12 hours; 30% ethanol was used firstly to elute to colorless and then 300 mL 70% ethanol was used to elute; the eluent of 70% ethanol was collected. The purity of total lignans was 45.8%. There was statistical significance in ear swelling degree of total lignans in Myristicae Semen high-, medium-, and low-dosage groups compared with model group (P<0.05). Conclusion The purification technology of total lignans is stable and reproducible, and total lignans in Myristicae Semen has significant anti-inflammatory activity.

Objective To establish a RP-HPLC method for the determination of encapsulation efficiency (EE) and medicine loading (ML) in β-elemene-loaded Nano Polymeric Micelles; To study its release characteristic in vitro. Methods DSPE-PEG2000 was used as carrier to prepare medicine-loaded micelles. EE and ML were determined by petroleum ether extraction method, and its release characteristic in vitro was studied by dialysis method. Results The average EE and ML ofβ-elemene-loaded Nano Polymeric Micelles were 89.47%and 8.33%, respectively. Its release characteristic was slow. Conclusion The method for EE and ML determination is simple and accurate, and the prepared micelles have the property of sustained release.

Objective To optimize the processing technology of sliced Myristicae Semen. Methods Roasting temperature, roasting time and the amount of bran were set as factors, and the content of total lignans, volatile oil, fatty oil were set as evaluation indicators. The processing technology of sliced Myristicae Semen was optimized by L9(34) orthogonal test. Results The optimal processing technology was as following: 40 g bran plus 100 g sliced Myristicae Semen, roasting for 20 minutes at 110-120 ℃. Conclusion The process is reasonable and reliable, which can provide references for new processing technology of Myristicae Semen.

Objective To establish QAMS method to determine the contents of three alkaloids in bile processed Coptidis Rhizoma; To compare the results of QAMS with those from external standard method; To prove the feasibility of QAMS.Methods An HPLC method was developed. Berberine hydrochloride was selected as the internal reference substance. 2 relative correction factors (RCF) of berberine hydrochloride to palmatine hydrochloride and to jatrorrhizine hydrochloride were established. Obtained RCFs were used to conduct content calculation (calculated value) to complete QAMS method. At the same time, the contents (measured value) of the three components were also determined by external standard method. Calculated value and measured value were compared.Results The analysis results showed that there was no significant difference between the calculated values and the measured values of the three alkaloids in 10 batches of bile processed Coptidis Rhizoma.Conclusion The QAMS method can be applied in the determination of alkaloids in bile processed Coptidis Rhizoma.