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.

Correlation between Content of Active Ingredients of Aurantii Fructus and Soil Factor / 中国实验方剂学杂志

ObjectiveTo study the correlation between the content of active ingredients of Aurantii Fructus in different main production areas and soil factors， so as to provide a theoretical basis for implementing ecological regulation of soil， improving the quality of Aurantii Fructus， and revealing the origin of genuine medicinal materials. MethodThe content of naringin， neohesperidin， total flavonoids， volatile oil， total nitrogen， total phosphorus， total potassium， and 17 soil factor-related indicators in 25 batches of Aurantii Fructus from different production areas were determined. The main soil factors affecting the content of active ingredients of Aurantii Fructus were analyzed by Pearson correlation analysis， principal component analysis， and grey correlation analysis. ResultThe pH value of the soil is between 4.83 and 8.21， and the soil is weakly acidic and neutral in general. Soil fertility exceeds the average. Pearson correlation analysis shows that the soil factors most related to the four active ingredients of Aurantii Fructus are total phosphorus， available copper， available zinc， exchangeable magnesium， available sulfur， available phosphorus， and available molybdenum. Principal component analysis shows that total nitrogen， alkali-hydrolyzable nitrogen， organic matter， available phosphorus， and available zinc are the main characteristic factors in soil. Grey correlation analysis shows that the main soil factors affecting the active ingredients of Aurantii Fructus are total phosphorus， total nitrogen， available zinc， available copper， exchangeable magnesium， and pH. ConclusionIn the cultivation of Aurantii Fructus， the medicinal material quality of Aurantii Fructus could be improved by adjusting the level of beneficial factors in the soil and improving the soil texture.

Three-dimensional multi-component analysis of Aurantii Fructus quality and research on influencing factors / 中国中药杂志

The present study explored the consistency of the content proportions of active components of Aurantii Fructus and analyzed the influencing factors based on three-dimensional multi-component analysis. A total of 839 Aurantii Fructus samples in 65 research articles were analyzed using the three-dimensional multi-component analysis mode. The content data of flavonoid components(naringin, hesperidin, neohesperidin, narirutin, and nobiletin), coumarin components(meranzin and gluconolactone), and alkaloid(synephrine) in 386 samples which met the criteria of 2020 edition of the Chinese Pharmacopoeia were extracted and adjusted to percentages, and the content ratios between components were calculated. The influencing factors of Aurantii Fructus quality were analyzed. The results showed content ratios of components as follows: neohesperidin∶naringin in the range of 0.4-1.2; narirutin∶naringin in the range of 0.02-0.16; hesperidin∶naringin in the range of 0.01-0.3; nobiletin∶naringin in the range of 0.000 588 3-0.069 68; synephrine∶naringin in the range of 0.02-0.042; gluconolactone∶naringin in the range of 0.001-0.01; meranzin∶naringin in the range of 0.000 4-0.035. The quality of Aurantii Fructus was closely related to the origin, variety, harvesting time, and processing method of medicinal materials. Harvesting time had a greater impact on the quality of Aurantii Fructus, and the origin and variety had a certain impact on the quality of Aurantii Fructus. The findings of this study indicated that the ratios between flavonoid components, flavonoids and coumarin components, and flavonoids and alkaloids fluctuated. The production base should optimize the varieties, harvesting period, and processing methods of Aurantii Fructus to provide a scientific basis for the production of high-quality Aurantii Fructus.

Dryness-alleviating effect of processed Aurantii Fructus from Lingnan based on correlation of fecal metabolomics and dryness / 中国中药杂志

With the approach of untargeted metabolomics and correlation analysis, this study aimed to explore the mechanism of Aurantii Fructus from Lingnan region in alleviating dryness by analyzing the different effects of raw Aurantii Fructus(RAF) and processed Aurantii Fructus(PAF) on fecal endogenous metabolism in normal rats. Eighteen Sprague-Dawley(SD) rats were randomly divided into a control group(C), an RAF group(10 g·kg~(-1)), and a PAF group(10 g·kg~(-1)). After seven days of administration, the effects of RAF and PAF on dryness-related indexes were compared, including water intake, fecal water content, salivary secretion, the expression of AQP5, VIP, and 5-HT in the submandibular gland, as well as the expression of AQP3, VIP, and 5-HT in the colon. The fecal samples in each group were determined by LC-MS. Multivariate statistical analysis and Pearson correlation coefficient were used for screening the differential metabolites and metabolic pathways in alleviating dryness of RAF. The results indicated that both RAF and PAF showed certain dryness, and the dryness of RAF was more significant. Moreover, PAF could alleviate dryness of RAF to a certain extent by reducing the water intake, fecal water content, and the expression of AQP3, VIP, and 5-HT in the colon and increasing the salivary secretion and the levels of AQP5, VIP, and 5-HT in the submandibular gland. According to the analysis of fecal metabolomics, 99 and 58 metabolites related to dryness were found in RAF and PAF respectively, where 16 of them played an important role in alleviating dryness of RAF. Pathway analysis revealed that the mechanism of PAF in alleviating dryness of RAF was presumably related to the regulation of riboflavin metabolism, purine metabolism, arginine biosynthesis, pyrimidine metabolism, alanine metabolism, aspartate metabolism, glutamate metabolism, and retinol metabolism pathways. This study suggested that PAF might alleviate dryness of RAF by affecting the metabolic levels of the body, which provides a new basis for further clarifying the processing mechanism of PAF.

Aurantii Fructus Immaturus， Atractylodis Macrocephalae Rhizoma， and Their Combination Treat Colonic Motility Disorders in Rats with Slow Transit Constipation via PINK1/Parkin Signaling Pathway-mediated Mitophagy / 中国实验方剂学杂志

ObjectiveTo observe the effects of Aurantii Fructus Immaturus， Atractylodis Macrocephalae Rhizoma， and their combination on slow transit constipation via PTEN-induced putative kinase 1 （PINK1）/Parkin pathway-mediated mitophagy. MethodFifty-six male SD rats were randomly assigned into normal group， model group， natural recovery group， Aurantii Fructus Immaturus group， Atractylodis Macrocephalae Rhizoma group， Aurantii Fructus Immaturus combined with Atractylodis Macrocephalae Rhizoma group， and mosapride group， with 8 rats in each group. Slow transit constipation model was established by gavage with loperamide （3 mg·kg-1·d-1） for 14 days in other groups except the normal group. After successful modeling， except that the model group was continuously induced by loperamide， the normal group and the natural recovery group were administrated with 0.9% normal saline by gavage， and the rats in the Aurantii Fructus Immaturus （1.35 g·kg-1·d-1） group， the Atractylodis Macrocephalae Rhizoma （2.7 g·kg-1·d-1） group， the Aurantii Fructus Immaturus combined with Atractylodis Macrocephalae Rhizoma （4.05 g·kg-1·d-1） group， and the mosapride （1.56 mg·kg-1·d-1） group were administrated with corresponding drugs by gavage for 7 days. The amount of feces， fecal water content， and intestinal propulsion rate of rats were determined. The pathological changes of the colon were evaluated by hematoxylin-eosin （HE） staining and Alcian blue-periodic acid-Schiff （AB-PAS） staining. The activity of respiratory chain complex and the ultrastructure of the colon tissue were determined by ultraviolet spectrophotometry and observed by transmission electron microscopy， respectively. Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was employed to determine the mRNA levels of PINK1， Parkin， and p62， and Western blot to determine the protein levels of microtubule-associated protein 1 light chain 3 （LC3）， PINK1， and Parkin. ResultCompared with the normal group， the model group and the natural recovery group showed decreases in the amount of feces， fecal water content， intestinal propulsion rate （P<0.05，P<0.01）， and activities of mitochondrial respiratory chain complexes Ⅱ， Ⅲ， and Ⅳ in the colon tissue （P<0.05，P<0.01）. Further， the mRNA levels of PINK1 and Parkin and the protein levels of PINK1， Parkin， and LC3 were up-regulated （P<0.01） and the mRNA level of p62 was down-regulated in the model group （P<0.05） and the natural recovery group. Compared with the model group and the natural recovery group， the Aurantii Fructus Immaturus combined with Atractylodis Macrocephalae Rhizoma group showed increased amount of feces， fecal water content， intestinal propulsion rate， and activities of mitochondrial respiratory chain complexes Ⅱ， Ⅲ， and Ⅳ （P<0.05，P<0.01）. Moreover， the combination meliorated the degree of mitochondrial swelling in the colon tissue， down-regulated the mRNA levels of PINK1 and Parkin and the protein levels of PINK1， Parkin， and LC3 （P<0.05，P<0.01）， and up-regulated the mRNA level of p62 （P<0.05）. ConclusionAurantii Fructus Immaturus and Atractylodis Macrocephalae Rhizoma， and their combination may remedy the colonic motility disorders in rats with slow transit constipation by blocking PINK1/Parkin signaling pathway to inhibit the excessive mitophagy in interstitial cells of Cajal in the colon tissue.

Quality Evaluation of Aurantii Fructus from Sichuan by Specific Chromatogram Combined with Chemometrics and Grey Correlation Analysis / 中国实验方剂学杂志

ObjectiveHigh performance liquid chromatography （HPLC） was used to establish the specific chromatograms of Aurantii Fructus from different origins， and the quality variability of Aurantii Fructus from Sichuan was analyzed and evaluated by combining entropy weighting method and grey correlation method. MethodHPLC was performed on an Agilent Eclipse Plus C18 column （4.6 mm×250 mm， 5 μm） with a gradient elution of methanol （A）-0.1% phosphoric acid aqueous solution as the mobile phase （0-12 min， 25%-33%A； 12-21 min， 33%-41%A； 21-30 min， 41%-42%A； 30-40 min， 42%-59%A； 40-53 min， 59%-72%A； 53-60 min， 72%A； 60-65 min， 72%-100%A； 65-70 min， 100%A； 70~71 min， 100%-25%A； 71-80 min， 25% A） at a flow rate of 1.0 mL·min-1， the injection volume was 10 μL and the detection wavelength was 330 nm. Fifty batches of Aurantii Fructus samples from different origins （Sichuan， Chongqing， Jiangxi and Hunan） were tested， and the similarity evaluation software is used to generate characteristic profiles and compare them with control profile for peak identification， and then to evaluate the similarity of the samples. IBM SPSS 19.0 and SIMCA 14.1 were used to perform multivariate statistical analysis on the results of the samples， and then the entropy weighting method and grey correlation were used to calculate the overall quality score of samples from Sichuan. ResultHPLC specific chromatogram of Aurantii Fructus was established， and 14 common peaks were identified as eriocitrin， neoeriocitrin， narirutin， naringin， hesperidin， neohesperidin， meranzin hydrate， poncirin， meranzin， marmin， nobiletin， 3，3′，4′，5，6，7，8-heptamethoxyflavone， tangeretin and auraptene. And the similarities between the samples from Sichuan and the control chromatogram were all above 0.980. The samples could be classified into four categories according to their main origins by chemical pattern recognition， and the results of cluster analysis， principal component analysis and orthogonal partial least squares-discriminant analysis were all able to discriminate the samples of different main origins effectively. The comprehensive evaluation results of entropy weighting method combined with grey correlation showed that the quality of Aurantii Fructus from Sichuan varied greatly among different origins， and the quality of Aurantii Fructus from Sichuan was ranked as Bazhong>Luzhou>Chongqing>Neijiang. ConclusionIn this study， the characteristic mapping of Aurantii Fructus from Sichuan is established， and combined with the analytical methods of chemometrics and grey correlation， the quality of samples from different origins can be effectively differentiated， which can provide a reference for the comprehensive evaluation and control of the quality of Aurantii Fructus from Sichuan.

Predictive Analysis of Quality Markers of Jiangxi Aurantii Fructus Based on Fingerprint and Network Pharmacology / 中国实验方剂学杂志

ObjectiveTo analyze and predict the potential quality markers （Q-Marker） in the Genuine medicinal materials Jiangxi Aurantii Fructus based on fingerprints and network pharmacology. MethodUltra-high performance liquid chromatography （UPLC） and gas chromatography-mass spectrometry （GC-MS） fingerprints were established for 18 batches of Jiangxi Aurantii Fructus ，combined with chemometric methods to screen out candidate Q-Marker components.Use network pharmacology to construct a "core component-target-pathway" network to predict the Q-Marker and core targets of Jiangxi Aurantii Fructus，and then verify the biological activity of Jiangxi Aurantii Fructus Q-Marker by molecular docking method. ResultThe 18 batches of Jiangxi Aurantii Fructus use UPLC，GC-MS fingerprints combined with chemometric analysis，a total of 9 Q-Marker candidate components were screened out.Through network pharmacological analysis，it is predicted that nobiletin，neohesperidin，meranzin，naringin and D-limonene are the Q-Marker of Jiangxi Aurantii Fructus，acting on the core targets transforming protein p21/H-Ras-1（HRAS），cellular tumor antigen p53 （TP53），mitogen-activated protein kinase 8 （MAPK8），transcription factor AP-1（JUN），glycogen synthase kinase-3 beta（GSK3B），tumor necrosis factor（TNF），cyclin-dependent kinase inhibitor 1（CDKN1A），cAMP-dependent protein kinase catalytic subunit alpha（PRKACA），cysteine aspartate-specific protease-9（Caspase-9），cyclic AMP-responsive element-binding protein 1（CREB1），exerting gastrointestinal motility and antidepressant，anti-inflammatory，anti-tumor，etc.； molecular docking shows that nobiletin，neohesperidin，meranzin，naringin and D-limonene and the selected 10 core targets have good binding ability，reflecting the better biological activity of the Q-Marker of Jiangxi Aurantii Fructus. ConclusionThe Q-Marker of Jiangxi Aurantii Fructus can be comprehensively predicted from the two aspects of volatile and non-volatile components，providing a reference for the quality control of Jiangxi Aurantii Fructus and the further study of its pharmacodynamic mechanism.

Quality Evaluation of Aurantii Fructus Based on Fingerprint Qualitative Analysis， Multi-component Quantitative Analysis and Chemometrics / 中国实验方剂学杂志

ObjectiveTo establish an integrated method of fingerprint qualitative， multi-component quantitative analysis and chemometrics， and to evaluate the quality attributes and differences of Aurantii Fructus from different production areas and origins. MethodAnalysis was performed on COSMOSIL 5C18-MS-Ⅱ column （4.6 mm×250 mm， 5 μm） with the mobile phase of acetonitrile-0.2% phosphoric acid solution for gradient elution （0-4 min， 19%A； 4-5 min， 19%-21%A； 5-18 min， 21%A； 18-19 min， 21%-28%A； 19-27 min， 28%A； 27-28 min， 28%-40%A； 28-36 min， 40%A； 36-37 min， 40%-50%A； 37-42 min， 50%-60%A； 42-46 min， 60%-95%A； 46-55 min， 95%-100%A）， the flow rate was 1 mL·min-1， the column temperature was 30 ℃， the detection wavelength was set at 320 nm， and the injection volume was 10 μL. High performance liquid chromatography （HPLC） fingerprints of Aurantii Fructus from different production areas and origins were established. Then， the quality of 26 batches of samples was evaluated by cluster analysis （CA）， principal component analysis （PCA） and orthogonal partial least squares discriminant analysis （OPLS-DA）. A method for the determination of 12 components was developed and verified， and a thermal map-based CA of Aurantii Fructus from different production areas and origins was carried out based on the content difference of samples. ResultThe fingerprint and determination methods were well verified. The similarity of HPLC fingerprint of 12 batches of Aurantii Fructus was 0.85-0.996， 20 common peaks were calibrated and 14 of them were assigned. The resolution and linear relationship of 12 components in quantitative analysis were good. The recovery rates were 99.2%-101.0% with RSD≤2.0%. The results of CA， PCA and OPLS-DA indicated that the differentiation of Aurantii Fructus in different production areas was great， and there were differences among different cultivars. ConclusionThe qualitative analysis of fingerprint and quantitative analysis of multiple indexes based on the same chromatographic analysis conditions are convenient， accurate and reliable， and combined with chemometrics， the identification and quality analysis of Aurantii Fructus from different production areas and origins can be realized， which can provide reference for quality control and evaluation of Aurantii Fructus.

Comparison between peel and pulp of Aurantii Fructus Immaturus by UPLC fingerprint and multicomponent quantitative analysis / 中国中药杂志

Twenty batches of Aurantii Fructus Immaturus(AFI) were collected, with their peel and pulp taken as research objects. Ultra-high performance liquid chromatography(UPLC) fingerprints of peel and pulp of AFI were established with 17 common peaks in peel and 10 in pulp. Six kinds of flavonoids were identified, i.e., narirutin, naringin, rhoifolin, hesperidin, neohesperidin and nobiletin. The Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine was employed for similarity analysis, which showed that the chromatographic peaks of peel and pulp were basically similar to their respective reference fingerprints, with all similarities greater than 0.90. The similarity between peel and pulp of the same batch of AFI ranged from 0.850 to 0.983. Cluster analysis(CA), principal component analysis(PCA), and orthogonal partial least squares discriminant analysis(OPLS-DA) were conducted on the common peaks of peel and pulp of AFI with SPSS 17.0 and SIMCA 14.1. Combined with the reference fingerprints, these analyses revealed 12 differential components regarding peel and pulp. Further, the content of the 6 flavonoids and synephrine was determined. The proposed method integrating UPLC fingerprint and multicomponent quantitative analysis is applicable to the quality evaluation of AFI. The results provide a certain basis for the scientific connotation about the appearance characteristic of AFI.

Dryness comparison of different fractions of Aurantii Fructus extract on normal mice and gastrointestinal motility disorder rats and spectrum-dryness study / 中国中药杂志

Aurantii Fructus is a commonly used qi-regulating medicinal herb in China. Both traditional Chinese medicine theory and modern experimental research demonstrate that Aurantii Fructus has dryness effect, the material basis of which remains unclear. In recent years, spectrum-effect relationship has been widely employed in the study of active ingredients in Chinese medicinal herbs, the research ideas and methods of which have been constantly improved. Based on the idea of spectrum-effect study, the ultra-high perfor-mance liquid chromatography-quadrupole-time of flight mass spectrometry(UHPLC-Q-TOF-MS) fingerprints of different fractions of Aurantii Fructus extract were established for the identification of total components. Then, the dryness effects of the fractions on normal mice and gastrointestinal motility disorder(GMD) rats were systematically compared. Finally, principal component analysis(PCA), Pearson bivariate correlation analysis and orthogonal partial least squares analysis(OPLS) were integrated to identify the dryness components of Aurantii Fructusextract. The results showed that narirutin, naringin, naringenin, poncirin, oxypeucedanin, and eriodictyol-7-O-glucoside had significant correlations with and contributed to the expression of AQP2 in kidney, AQP3 in colon, and AQP5 in submandibular gland, which were the main dryness components in Aurantii Fructus.

Effect of extract of Quzhou Aurantii Fructus on hepatic inflammation and NF-κB/NLRP3 inflammasome pathway in CCl_4-induced liver fibrosis mice / 中国中药杂志

To study the effect and mechanism of extract of Quzhou Aurantii Fructus(QAF) on liver inflammation in CCl_4-induced liver fibrosis mice. Totally 60 C57 BL/6 male mice were randomly divided into control group(distilled water, oral), model group(distilled water, oral), colchicines group(Col, colchicines 2 mg·kg~(-1)·d~(-1), oral), low-dose QAF group(QAF-L, QAF 100 mg·kg~(-1)·d~(-1), oral) and high-dose QAF group(QAF-H, QAF 300 mg·kg~(-1)·d~(-1), oral) by random number table method. The model group and each administration group were injected with carbon tetrachloride(CCl_4) 1 mL·kg~(-1)(CCl_4-olive oil 1∶4), twice a week, totally 6 weeks. After the last administration, the mice were sacrificed, and serum and liver tissue were collected. Serum ALT and AST levels were measured in each group to observe the liver function of mice. The pathological changes and inflammatory cell infiltration in liver were observed by HE staining and F4/80 immunohistochemical staining. The mRNA expressions of TNF-α, IL-18 and IL-1β were detected by RT-PCR. The protein expressions of IκBα, p-IKKα/β, p-p65, NLRP3, caspase-1 and cleaved caspase-1 were analyzed by Western blot. The results showed that QAF significantly reduced serum ALT and AST levels, and alleviated the degree of liver damage.The results of immunohistochemistry showed that QAF significantly reduced liver inflammatory cell infiltration in liver fibrosis mice. The results of RT-PCR and Western blot showed that QAF significantly inhibited mRNA expressions of TNF-α, IL-18 and IL-1β in liver of fibrosis mice. QAF also suppressed the degradation of IκBα protein and reduced p-IKKα/β, p-p65, NLRP3 and cleaved caspase-1 protein expressions. In conclusion, QAF improves CCl_4-induced liver fibrosis in mice. The mechanism may be related to the inhibition of NF-κB/NLRP3 inflammasome-mediated inflammation signaling pathway.

Exploration of Active Ingredients and Anti-breast Cancer Mechanism of Glycyrrhizae Radix Et Rhizome and Aurantii Fructus Based on Network Pharmacology / 中国实验方剂学杂志

Objective::To explore the potential active ingredients and possible anti-breast cancer mechanism of Glycyrrhizae Radix et Rhizome and Aurantii Fructus based on the method of network pharmacology. Method::The main potential targets of Glycyrrhizae Radix et Rhizome and Aurantii Fructus on breast cancer were summarized by comparing the Glycyrrhizae Radix et Rhizome-Aurantii Fructus active ingredients screened from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and breast cancer targets searched in Therapeutic Target Database (TTD). Cytoscape 3.7.1 software was used to establish a Glycyrrhizae Radix et Rhizome-Aurantii Fructus active ingredients-target-disease network and perform topology analysis based on the network. Result::According to related conditions of drug-like (DL) and oral bioavailability (OB), the network of Glycyrrhizae Radix et Rhizome-Aurantii Fructus active ingredients-breast cancer target was obtained, covering a total of 133 nodes, 116 chemical components and 17 breast cancer drug targets, 109 active components of Glycyrrhizae Radix et Rhizome interacting with breast cancer drug target, 6 active ingredients of Aurantii Fructus interacting with breast cancer drug targets, and 1 common active ingredient of Aurantii Fructus and Glycyrrhizae Radix et Rhizome interacting with breast cancer targets. There were 400 breast cancer target-interaction target pairs in the network diagram. Conclusion::The anti-breast cancer effect of Glycyrrhizae Radix et Rhizome and Aurantii Fructus is based on the overall pharmacodynamic effect of multi-component, multi-pathway and multi-target, the investigation of its potential anti-breast cancer mechanism provides theoretical basis for further experimental research.

Structures Characteristics and Bioactivity of Polysaccharide CALB-2 from Aurantii Fructus / 中国实验方剂学杂志

Objective:To isolate and purify a polysaccharide CALB-2 fraction from Aurantii Fructus，and analyze its basic chemical structure, morphological characteristics and bioactivity. Method:A refined CALB-2 was obtained from Aurantii Fructus by hot water extraction，then separated and purified by ion exchange resin，ion exchange agarose gel and propylene dextran gel to obtain homogeneous polysaccharide CALB-2. The molecular mass of CALB-2 was determined by high performance liquid chromatography （HPLC）. Monosaccharide composition analysis of CALB-2 was conducted by methylation analysis and Smith degradation. Structural analysis and morphological characterization were conducted by infrared scanning （IR） and scanning electron microscopy （SEM） analysis. Antioxidant activity of CALB-2 was studied by using H2O2-induced cardiomyocyte oxidative damage model. Result:CALB-2 was a homogeneous polysaccharide and the molecular weight of CALB-2 was estimated to be 3.57×107 Da，which was proved to be a kind of highly branched acidic polysaccharides in IR analysis, methylation analysis and Smith degradation, mainly present in form of 1→3，4 bonds. Through SEM observations，we indicated that the molecular morphology of CALB-2 was amorphous solid. The in vitro activity test showed that CALB-2 had obvious protective effects on injury of H9c2 myocardial cells induced by H2O2. Conclusion:CALB-2 is a kind of homogeneous polysaccharide extracted from Aurantii Fructus， with an anti-cardiomyocyte oxidative damage effect, laying a theoretical foundation for further study of Aurantii Fructus polysaccharides．

Analysis of Influence of Jiangxi Characteristic Processing Technology on Chemical Compositions of Aurantii Fructus Based on UPLC-Q/TOF-MS / 中国实验方剂学杂志

Objective:To compare the effects of different processing techniques on the chemical constituents of Aurantii Fructus for screening the dominant decoction pieces. Method:UPLC-Q/TOF-MS was used to detect the chemical constituents of Aurantii Fructus, chromatography separation was achieved on an ACQUITY UPLC BEH C18 column (2.1 mm×100 mm, 1.7 μm), and gradient elution was performed with 0.1% formic acid aqueous solution (A)-0.1% formic acid acetonitrile solution (B) as mobile phase (0-10 min, 5%-35%B; 10-18 min, 35%-75%B; 18-21 min, 75%-100%B; 21-24 min, 100%B; 24-24.1 min, 100%-5%B; 24.1-28 min, 5%B). Data acquisition was carried out in electrospray ionization (ESI) under the positive ion mode, the scanning range was m/z 50-1 200. The chemical constituents in methanol extract of Aurantii Fructus were identified according to reference substance, relative molecular weight, mass spectrometric cleavage rule and literature information. SIMCA-P 13.0 software was used to establish principal component analysis (PCA) model and partial least squares-discriminant analysis (PLS-DA) model of Aurantii Fructus processed products, PCA score plot, PLS-DA loading plot and variable importance in the protection (VIP) values were obtained to screen the material basis for the main differences before and after processing of Aurantii Fructus. Result:A total of 54 chemical components were identified by UPLC-Q/TOF-MS. PCA indicated that there were significant differences among different groups of Aurantii Fructus processed by different methods. A total of 14 chemical components with VIP value >1 were screened by PLS-DA as the main chemical markers for the differences before and after processing, including hesperidin, poncirin, narirutin, etc. The comprehensive weighted score showed that the content of effective components in Aurantii Fructus processed with honey bran was the highest. Conclusion:The contents of chemical constituents in Aurantii Fructus before and after processing are significantly changed. Flavonoids are the most important compounds to distinguish different processed products of Aurantii Fructus. Aurantii Fructus processed with honey bran is the dominant variety.

Changes of HPLC Fingerprint and Main Flavonoids Content of Aurantii Fructus Before and After Processing / 中国实验方剂学杂志

Objective::To establish HPLC fingerprints of Aurantii Fructus and its processed products, and to quantitatively analyze the contents of four flavonoids in these products. Method::HPLC was employed with Inertsil ODS-3 C18 column (4.6 mm×250 mm, 5 μm), the mobile phase of acetonitrile-0.1%phosphoric acid aqueous solution for gradient elution, the detection wavelength of 283 nm, and the flow rate of 1.0 mL·min-1. HPLC fingerprints of raw products, stir-fried bran products and processing products of Aurantii Fructus were established. Similarity evaluation and cluster analysis were used to analyze the chromatographic data. At the same time, the contents of narirutin, naringin, hesperidin and neohesperidin were determined. Result::HPLC fingerprints of Aurantii Fructus and its processed products were established, taking naringin as the reference peak, 8, 15, 11 common peaks were demarcated for raw products, stir-fried bran products, processing products, respectively, the similarities of fingerprints were >0.95.Contents of the above four flavonoids in raw products were 0.574 7%, 5.986 3%, 0.302 2%and 3.574 7%, respectively. After processing, the contents of these four components in stir-fried bran products turned into 0.948 4%, 5.103 4%, 0.549 3%and 3.533 7%, their contents in processing products turned into 0.605 3%, 4.762 3%, 0.404 7%and 3.264 9%, respectively. Conclusion::The HPLC fingerprint of Aurantii Fructus changes significantly before and after processing. The contents of four flavonoids change to a certain extent before and after processing. The order of contents of narirutin and hesperidin in samples was stir-fried bran products>processing products>raw products, while the order of contents of naringin and neohesperidin was raw products>stir-fried bran products>processing products.

Identification of Prototype Compounds and Their Metabolites in Rat Plasma After Oral Administration of Aurantii Fructus Extract by UPLC-Q-TOF/MS / 中国实验方剂学杂志

Objective:To study the serum pharmacochemistry of Aurantii Fructus （AF）， and to investigate the pharmacological material basis of AF extract in rats. Method:Rapid identification and speculation of the prototype constituents and their metabolites in vivo were carried out according to the relative retention time， accurate relative molecular mass， cleavage fragments of MS/MS and neutral loss of metabolites with ultrahigh performance liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry （UPLC-Q-TOF/MS） technique by comparing the differences between different samples such as AF extracts， blank plasma， and administered plasma under the same chromatographic and mass spectrometric conditions. Result:After oral administration of the AF extract， 74 transitional constituents absorbed into the blood were detected in serum， in which 49 compounds were prototype constituents and the other 25 were metabolites. The prototype constituents could be divided into dihydroflavones， polymethoxyflavonoids， limonins， coumarins and alkaloids. The identified metabolites included glucuronic acid conjugates， sulfuric acid conjugates， hydroxylated products of flavonoid glycosides and polymethoxyflavonoids， as well as the simultaneous glucuronidation and sulfation products. Conclusion:The constituents absorbed into the blood and their metabolites may be the pharmacodynamic components of AF. Among them， alkaloids， polymethoxyflavonoids and coumarins are mainly introduced into the blood in the prototype form， while naringin and neohesperidin （the index components） exert effect mainly through hydrolysis into aglycones. This work will help to further elucidate the material basis of AF.

Analysis of Differential Chemical Compositions of Aurantii Fructus Immaturus Before and After Stir-frying with Bran and Chemical Compositions of Wheat Bran After Processing by UPLC-Q-TOF-MSE / 中国实验方剂学杂志

Objective:To quickly analyze and identify the differential chemical compositions of Aurantii Fructus Immaturus before and after stir-frying with bran and chemical compositions of wheat bran after processing by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MSE） combined with UNIFI database screening method. Method:ACQUITY UPLC BEH C18 column （2.1 mm×100 mm， 1.7 µm） was used for chromatographic separation with 0.1% formic acid solution （A）-acetonitrile （B） as the mobile phase for gradient elution （0-11 min， 98%-70%B； 11-15 min， 70%-55%B； 15-16 min， 55%-35%B； 16-20 min， 35%-5%B； 20-20.5 min， 5%-98%B； 20.5-22 min， 98%B） at the flow rate of 0.3 mL·min-1 and the injection volume of 2 µL. The analytes were determined in positive ion mode with electrospray ionization （ESI） and data collection range of m/z 50-1 500. Principal component analysis （PCA） and orthogonal partial least squares discriminant analysis （OPLS-DA） were used to find the component differences between raw and processed products of Aurantii Fructus Immaturus， and the chemical compositions of wheat bran after processing were determined. Result:There were 64 compounds in raw products， 58 compounds in bran-fried products， and 18 compounds in wheat bran.There were 19 different components between raw and processed products of Aurantii Fructus Immaturus， mainly volatile oil， flavonoids， phenolic acid， coumarins and saponins. Conclusion:Based on the analysis of these different components before and after stir-frying with bran and the chemical compositions carried by wheat bran， the stir-frying with bran can alleviate the intensity of Aurantii Fructus Immaturus， which proves the necessity of stir-frying with bran for the processing technology of this herb， and provides a comprehensive experimental basis for research on processing mechanism of Aurantii Fructus Immaturus.

Isolation and Identification of Phenols Compounds in Aurantii Fructus Immaturus / 中国药房

OBJECTIVE：To sepa rate and identify the phenols compounds in Aurantii Fructus Immaturus ，and to provide reference for basic research of its effective substances. METHODS ：The phenols compounds were isolated and separated by silica gel，HW-40F gel column ，ODS reversed column and preparation HPLC. Their physicochemical properties and spectral data were used to identify the structures. RESULTS & CONCLUSIONS ：Totally 12 compounds were isolated from Aurantii Fructus Immaturus，identified as 6′-O-trans- cinnamoyl- 3，5-dihydroxyphenyl β-D-glucopyranoside（compound 1），methyl 3-（2′，4′- dihydroxy phenyl ）propionate（compound 2），phloroglucinol（compound 3），aurantiside A （compound 4），5，7，4′-trihydroxy-8， 3′-dimethoxyflavone-3-O-6″-（3-hydroxyl-3-methylglutaroyl） β-D-glucopyranoside （compound 5）， aromadendrin-7-O-β-D- glucopyranside（compound 6），hesperidin-7-O-β-D-glucoside（compound 7），naringin（compound 8），hesperidin（compound 9）， narirutin （compound 10），neoeriocitrin （compound 11），eriocitrin （compound 12）. Among them ，compound 1 is a new compound，and compounds 2 and 3 are isolated from Critus for the first time.

Principal component analysis and discriminant analysis of 12 effective chemical constituents in Aurantii Fructus from different areas / 中草药

Objective: To analyze and compare the contents of 12 effective chemical constituents in Aurantii Fructus from different areas, and identify their source of origin by using principal component analysis and discriminant analysis. Methods: Using isonaringin, narigin, hesperidin, neohesperidin, meranzin hydrate, naringin, hesperidin, meranzin, marmin, nobiletin, tangeretin, and auraptene as index, HPLC method was used to determine the content, and the average percentage content was analyzed by principal component analysis and discriminant analysis. Results: Principal component analysis results showed that Aurantii Fructus from different areas had certain similarities, and the content of Aurantii Fructus from Jiangxi Province was relatively stable. Discriminant analysis results showed that the samples from Zhangshu, Yichun, Yuanjiang, Huaihua, Jinhua, and Lanxi could be distinguished, while the samples from Xin’gan in Jiangxi, Jinhua, and Lanxi in Zhejiang overlapped. Conclusion: This method is simple, convenient and reliable, which can be used to screen out the suitable areas for the growth of Aurantii Fructus, so as to lay a foundation for explaining the authenticity of Aurantii Fructus.

Predictive analysis of Q-marker composition library of Zhangbang Aurantii Fructus pieces with honeyed bran associated with efficacy of “regulating vital energy and eliminating flatulence” / 中草药

Objective: Based on the idea of Q-marker, UPLC-Q-TOF-MS, GC-MS and network pharmacology techniques were used to establish a potential Q-Marker component library associated with the core efficacy of Zhangbang Aurantii Fructus with honeyed bran, in order to lay the foundation for exploring the establishment of pieces quality standards. Methods: Chromatography separation was achieved on an Acquity UPLC BEH C18 column (100 mm × 2.1 mm, 1.7 μm), and eluted with a gradient of methanol-water containing 0.1% formic acid. Data acquisition was carried out in electrospray ionization (ESI) under the positive ion mode, the scanning range was 50-1 200 m/z. Ingredients in alcohol extract of Aurantii Fructus with honeyed bran were identified according to reference substance, relative molecular weight, mass spectrometric cleavage rule and literature information; The volatile oil and aromatic water was extracted by steam distillation and analyzed by gas chromatography-mass spectrometry (GC-MS). The inlet temperature was 260 ℃, the transmission line temperature was 250 ℃, the carrier gas was helium, the flow rate was 1.0 mL/min, the split ratio was 10:1, the injection volume was 1 μL; The program temperature was 70 ℃ for the starting column, 2 ℃/min to 150 ℃ for 2 min, 6 ℃/min to 240 ℃ for 3 min, and then 25 ℃/min to 300 ℃ for 2 min. Mass spectrometry was identified by NIST08 standard mass spectrometry library to identify chemical constituents; Cytoscape 3.7.1 was used to construct a “pieces-compounds-diseases-targets” network of chemical constituents and functional dyspepsia in Aurantii Fructus with honeyed bran. Results: A total of 55 chemical components were identified by UPLC-Q-TOF-MS, and 63 chemical components were identified by GC-MS. Naringin, neohesperidin, eriodictyol-7-O-glucoside, naringenin, limonin, nobiletin, hesperitin, 5-hydroxy- 6,7,8,4’-tetramethoxyflavone, didymin, D-limonene, cymene, γ-terpinene nomilin, (1R)-(+)-α-pinene, caffeic acid and synephrine can be used as a Q-marker ingredient library for Aurantii Fructus with honeyed bran. Conclusion: UPLC-Q-TOF-MS and GC-MS can quickly and qualitatively analyze the chemical constituents in the pieces. The network pharmacology can predict the potential Q-marker library related to efficacy of regulating vital energy and eliminating flatulence, and provide the basis for the establishment of quality standards for the pieces.