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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.
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ObjectiveTo research the mechanism underlying the effect of raw and processed Aurantii Fructus Immaturus switched to Zhishi Shaoyaosan （ZSS） on constipation-predominant irritable bowel syndrome （C-IBS） rats via the brain-gut-microbiota axis. MethodEighty rats were randomly divided into the blank， model， positive drug （pinaverium bromide， 15.625 mg·kg-1）， raw ZSS， stir-fried ZSS， bran-fried ZSS， charcoal-fried ZSS and finished ZSS groups （3.75 g·kg-1）， with 10 rats in each group. Except for the blank group， which received intragastric administration of 0.9% sodium chloride solution at room temperature， all other groups were administered the ice solution at 0 to 4 ℃ （2 mL·d-1， for a total of 14 d） to establish the C-IBS rat model. The fecal water content and the propulsion rate of small intestine were detected after 14 d of continuous drug administration. The levels of 5-hydroxytryptamine （5-HT）， vasoactive intestinal peptide （VIP）， neuro-peptide Y （NPY）， calcitonin gene-related peptide （CGRP）， substance P （SP）， diamine oxidase （DAO） and D-lactic acid （D-LA） were detected by enzyme linked immunosorbent assay （ELISA）. Hematoxylin-eosin （HE） staining was used to observe the changes in colonic pathological injury in each group. The expression levels of cyclic adenosine monophosphate （cAMP）， protein kinase A （PKA） and aquaporin-3 （AQP3） mRNA in colon tissues were detected by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and the protein expressions of VIP and AQP3 in colon tissues were detected by Western blot. The content of short chain fatty acids （SCFAs） was determined by gas chromatography-mass spectrometry. ResultCompared with the blank group， the fecal water content and intestinal propulsion rate of rat in the model group were significantly decreased （P<0.01）， and the levels of 5-HT， VIP， CGRP and SP in serum were significantly increased. Simultaneously， the NPY levels significantly decreased （P<0.01）， the levels of DAO and D-LA in plasma were significantly increased （P<0.01）， and the mucosal epithelium of colon tissue was slightly damaged， with reduced goblet cells and significantly reduced luminal granules. The mRNA expression levels of AQP3， cAMP and PKA and the protein expression levels of AQP3 and VIP in colon tissue were significantly decreased （P<0.05， P<0.01）. The total amount of SCFAs in feces showed an obvious decreasing trend， with the contents of acetic acid， isobutyric acid， isovaleric acid， valeric acid and caproic acid decreased significantly， while the contents of propionic acid and butyric acid increased significantly （P<0.05， P<0.01）. Compared with the model group， the treatment groups increased the intestinal propulsion rate， improved the intestinal mucosal barrier function， and adjusted the level of serum brain-gut peptide in C-IBS rats （P<0.05， P<0.01）. The expression levels of AQP3， cAMP， PKA mRNA and VIP， AQP3 protein in colon tissue of rats in all treatment groups were increased. All the treatment groups had a significant downregulation of the content of SCFAs except for isobutyric acid in rat feces， and the effect of ZSS prepared by the bran-fried Aurantii Fructus Immaturus was superior than that of other ZSS. ConclusionThe raw and processed Aurantii Fructus Immaturus switched to ZSS could influence the brain-gut-microbiota axis to treat C-IBS rats and it is more reasonable to use bran-fried Aurantii Fructus Immaturus in ZSS.
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The differences in dryness between raw Aurantii Fructus Immaturus(AFI) and bran-fried products were investigated based on a slow-transit constipation(STC) model. Seventy healthy SPF-grade rats were randomly divided into a blank group(K), a positive drug group(Y), a model group(M), low-and high-dose raw AFI groups(SD and SG), and low-and high-dose bran-fried AFI groups(FD and FG). During the experiment, it was found that compared with the K group, the groups with drug treatment had little effect on the daily body weight of the STC rats. The first defecation time of black stool in the M group was significantly higher than that in the K group, and the 24-hour fecal output significantly decreased starting from the 13th day, indicating successful modeling. The SG group showed a significant increase in the first defecation time, fecal water content, urine output, and water intake than other groups with drug treatment. The FG group had the highest fecal output than other groups with drug treatment. The FD group had the highest salivary secretion than other groups with drug treatment. The levels of cAMP/cGMP, VIP, 5-HT, AQP1, and AQP5 were measured in each group with drug treatment, and the expression of c-Kit and SCF mRNA in gastric antrum tissue and AQP3 mRNA in the kidney and colon were detected by RT-PCR. The results showed that the SD and SG groups had a more significant impact on AQP1, AQP5, and other water channel indexes in STC rats than the FD and FG groups. The FD and FG groups had a more significant impact on c-Kit, SCF, VIP, 5-HT, and other gastrointestinal motility indicators than the SD and SG groups. This study, through in vitro biological observations, immunological detection, and gene expression analysis, found that raw AFI had strong dryness property, while bran-fried AFI could alleviate its dryness property.
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Ratos , Animais , Serotonina , Constipação Intestinal/tratamento farmacológico , Medicamentos de Ervas Chinesas , RNA MensageiroRESUMO
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.
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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.
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Cromatografia Líquida de Alta Pressão , Citrus , Medicamentos de Ervas Chinesas , SinefrinaRESUMO
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.
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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.
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The grade classification of Chinese herbal medicines and their decoction pieces has always been a hot issue in the industry. In the trading history of Chinese herbal medicine,buyers and sellers often determine the price according to quality,so the naturally formed specifications and grades are changing with the change of technology and the increasing demand of the public for medical care.The planting,processing and circulation methods of Chinese medicinal materials have also changed greatly. Under the mode of pursuing yield and short-term benefit,the traditional rating evaluation method has been difficult to be used in the current quality status of prepared slices. Aurantii Fructus Immaturus is a genuine medicinal material of Jiangxi province,which is widely used,but has different quality levels on the market. In this paper,the quality levels were evaluated by the mass constant method. A set of grading method of Aurantii Fructus Immaturus slices was established,laying a foundation for " high quality and high price" Aurantii Fructus Immaturus slices. The grading method of Chinese herbal slices with quality constant was applied to 16 batches of Aurantii Fructus Immaturus slices collected form the market,and the final assessment results were as follows for different piece shapes( hemispherical type and thin type)respectively. The hemispherical Aurantii Fructus Immaturus pieces: quality constant ≥1. 11 for the first grade,< 1. 11 but ≥0. 69 for the second grade,and <0. 69 for the third grade. The thin type Aurantii Fructus Immaturus pieces: quality constant ≥0. 024 for the first grade,<0. 024 but ≥0. 015 for the second grade,and <0. 015 for the third grade. This work indicated that the quality constants grading method can be used to objectively classify the grades of traditional Chinese medicine,providing basis for standardizing market behavior and providing useful reference for grading of hemispherical or thin Aurantii Fructus Immaturus pieces.
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Citrus , Química , Medicamentos de Ervas Chinesas , Padrões de Referência , Medicina Tradicional Chinesa , Controle de QualidadeRESUMO
Objective: To establish a quality control method of standard decoction of Aurantti Fructus Immaturus(AFI),and to provide reference for quality evaluation of AFI dispensing granules and other related products of AFI. Method: A total of 16 batches of AFI pieces with different quality were collected from the market,including 13 batches of Citrus aurantium and 3 batches of C. sinensis,and the standard decoction of AFI was prepared according to the standard decoction process.Transfer rate of synephrine,dry extract rate and others of the standard decoction were regarded as evaluation indicators and relative assessment are conducted. Result: Transfer rates of synephrine in 13 batches of standard decoction of AFI(C. aurantium) were ranged from 35.7% to 92.7% with the average value was 61.9%;dry extract rates were varied from 20.7% to 43.8% and the average value was 28.4%;pH values were 4.48-5.32 with the average value was 4.99;the HPLC fingerprint similarities were >0.9 by comparing with the corresponding control fingerprint,6 common peaks were found and 3 of them were identified as naringin,hesperidin and neohesperidin.Transfer rates of synephrine in 3 batches of standard decoction of AFI(C. sinensis) were changed from 53.1% to 84.4%,and the average value was 73.2%;dry extract rates were shifted from 13.8% to 17.6% and the average value was 15.4%;pH values were 4.77-5.38 with the average value was 5.06;the HPLC fingerprint similarities were >0.9 by comparing with the corresponding control fingerprint,2 common peaks were found and one of them were identified as hesperidin. Conclusion: From the HPLC fingerprint of standard decoction of AFI,we can easily understand that the number of peaks in C. aurantium is obviously more than that of C. sinensis.This method has good precision,reproducibility and stability,it is suitable for quality evaluation for related products of AFI.Simultaneously,the research provides a good reference for identifying sources of AFI.
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Objective: To compare the pharmacokinetics of naringin and neohesperidin after oral administration of Zhishi total flavonoid glycosides (ZSTFG) extracted from Aurantii Fructus Immaturus in normal and gastrointestinal motility disorders (GMD) mice. Methods: ZSTFG was orally given to normal and GMD mice induced by atropine or dopamine. The plasma samples were incubated with β-glucuronidase/sulfatase, the total (free + conjugated) naringenin and hesperitin were extracted with acetonitrile. The validated HPLC-MS/MS method was successfully applied to the pharmacokinetic study. Results: The results showed that, compared with the normal group, AUC0–∞, AUC0–t and Cmax for total naringenin and hesperitin were significantly higher (P < 0.01 or P < 0.05), while CLZ/F for total naringenin and hesperitin was significantly lower (P < 0.01) in the GMD group. Tmax, t1/2z, MRT0-t, and MRT0-∞ for naringenin were longer (P < 0.01) in the GMD group than those in the normal group. Conclusion: The results showed that there were significant differences in pharmacokinetic parameters of naringenin and hesperitin between normal and GMD groups. It was suggested that the absorption of naringenin and hesperitin was increased, and the elimination processes of naringenin and hesperitin were slower in the GMD group than the normal group. The data are of value for further pharmacological studies of ZSTFG and would be useful to provide a reference for improving the therapeutic regimen of ZSTFG in clinical trials.
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Citri Reticulatae Pericarpium, Aurantii Fructus Immaturus, and Aurantii Fructus belong to the traditional Chinese medicine derived from citrus and are commonly used for regulating vital (Qi in Chinese). But these three are different in the function, efficacy and therapeutic. Based on the concept of Q-marker and the combination of pharmacodynamics, pharmacological and pharmacokinetic studies, their quality markers were predicted. The prediction study provides a theoretical basis for establishing and improving the quality standard of the medicinal materials.
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OBJECTIVE:To study the mechanism of Aurantii fructus immaturus(AFI)and its main active ingredients in pro-moting gastrointestinal motility of model rats with spleen deficiency. METHODS:170 rats were randomly divided into blank group (10 rats) and modeling group (160 rats),rats in modeling group was induced models with spleen deficiency by bitter cold diar-rhea+irregular diet. After modeling, rats were randomly divided into model group, naringin (NA) low-dose, medium-lose, high-dose groups(3.267,6.535,13.070 mg/mL),neohesperidin(NE)low-dose,medium-lose,high-dose groups(3.865,7.730, 15.460 mg/mL),synephrine(SY)low-dose,medium-lose,high-dose groups(0.252,0.504,1.008 mg/mL),compatibility groups with 3 monomer ingredients (NA-NE-SY) low-dose,medium-lose,high-dose and AFI water decoction low-dose,medium-lose, high-dose groups(0.104,0.208,0.416 g/mL,calculated by crude drug),ig,once a day,10 mL/kg,for 7 d. After the last admin-istration,gastrin (GAS) in serum,and acetylcholine (ACh),motilin (MTL),substance P (SP),vasoactive intestinal peptide (VIP)levels in plasma were detected. RESULTS:Compared with blank group,GAS level in serum and ACh,MTL,SP levels in plasma in model group were reduced(P<0.01),VIP level in plasma was increased(P<0.05). Compared with model group,ex-cept for the GAS level in serum showed no obvious change in NA high-dose group and SY doses groups,other medicine groups were obviously increased (P<0.05 or P<0.01);the ACh levels in serum were obviouly increased in NE high-dose group,SY high-dose group and AFI water decoction low-dose group(P<0.01). MTL levels in plasma were obviously increased in NE medi-um-dose,high-dose groups,SY high-dose group,compatibility low-dose,medium-dose groups and AFI water decoction medi-um-dose,high-lose groups (P<0.05);SP levels in plasma were obviously increased in NA low-dose,medium-dose groups and NE doses groups(P<0.05 or P<0.01);VIP levels were reduced in NA low-dose group,SY high-dose group and AFI water decoc-tion low-dose,medium-lose groups(P<0.05). CONCLUSIONS:AFI may promote the gastrointestinal motility of model rats with spleen deficiency by promoting the secretion of GAS,ACh,MTL,and inhibiting the secretion of VIP;there are differences be-tween AFI and the 3 monomer ingredients in regulation of gastrointestinal hormones.
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The present study was designed to investigate the chemical constituents of the fruit of Citrus aurantium L.. The compounds were isolated and purified by various chromatographic techniques, and their structures were elucidated on the basis of physicochemical properties and spectral data. Two new phenolic glycosides (compounds 1 and 2) were obtained and identified as 1-O-3, 5-dihydroxyphenyl-(6-O-4-hydroxybenzoyl)-β-D-glucopyranoside (1) and 1-O-3, 5-dihydroxyphenyl-(6-O-3-methoxy-4-hydroxy benzoyl)-β-D-glucopyranoside (2), respectively.
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Citrus , Química , Frutas , Química , Glucosídeos , Química , Glicosídeos , Química , Estrutura Molecular , Fenóis , Química , Extratos Vegetais , QuímicaRESUMO
Objective: To study the effects of various compatible herbs and pH value conditions on the change rule of anthraquinones in Rhei Radix et Rhizoma (RRR). Methods: The pH value of the extracted solution from seven compatible herbs[vinegar Kansui Radix (vKR), Moutan Cortex (MC), Scutellariae Radix (SR), Coptidis Rhizoma (CR), Aconiti Lateralis Radix Praeparata (ALRP), Aurantii Fructus Immaturus (AFI), and Magnoliae Officinalis Cortex (MOC)] were determined, then RRR was added into hydrochloric acid solution with the same pH value as the above solutions and boiled, in which the contents of anthraquinones were determined by UV-Vis and HPLC. The contents of anthraquinones were compared with those in the decoction of compatible herbs and single RRR. Results: The results of UV-Vis showed that total anthraquinones got the lowest amount when RRR and CR were boiled together while the highest appeared when RRR and SR were boiled together; the contents of total anthraquinones were increased when the pH value was increased. The results of HPLC showed that the five anthraquinones got the lowest dissolving-out quantity when RRR and CR were boiled together while the highest appeared when RRR and ALRP were boiled together. Under the conditions of different pH values, the highest dissolving-out quantity was got when the pH value reached 5.6. Conclusion: Both the compatible herbs and pH value could affect the dissolution of anthraquinones during the extraction. However, the effects of various compatible herbs and different pH value conditions adjusted by hydrochloric acid are different, and the pH value conditions have greater effects.