Sesquiterpenoids from the leaves of Chimonanthus nitens.

Two new sesquiterpenoids (1 and 3), one new natural product (2), and two known compounds (4 and 5) were isolated from the leaves of Chimonanthus nitens. Their structures were elucidated by spectroscopic analysis, and the absolute configuration of compound 3 was determined by the X-ray single-crystal diffraction analysis. The cytotoxicity of compounds 1-5 was evaluated at three concentrations on two human breast cancer cell lines (MDA-MB-468 and MDA-MB-231) by MTT assay. As a result, we found that the cytotoxicity was weak even with a concentration of these compounds up to 100 µM.

Six new cadinane-type sesquiterpenoids from the leaves of Chimonanthus nitens Oliv.

Six new cadinane-type sesquiterpenoids, named Chimnitensin A-F (1-6) were isolated from the leaves of Chimonanthus nitens Oliv. Their structures were elucidated by comprehensive spectroscopic analyses and comparison with structurally related known analogues. In vitro MTT assay showed that all six compounds had cytotoxicity against two selected human breast cancer cell lines (MDA-MB-468 and MDA-MB-231), which indicate their potential of developing into anticancer drugs.

Antibacterial mechanism of the synergistic combination between streptomycin and alcohol extracts from the Chimonanthus salicifolius S. Y. Hu. leaves.

ETHNOPHARMACOLOGICAL RELEVANCE: Chimonanthus salicifolius S. Y. Hu. Is a unique traditional medicinal plant in ancient China, and it can eliminate turbid pathogens with aromatics, clear heat, detoxify, prevent colds and influenza, Xinhua Compendium of Materia Medica records that. AIM OF THE STUDY: In previous study, we investigated the regulation of ethanol extracts (EEs) from C. salicifolius S. Y. Hu. leaves on three common antibiotics (chloramphenicol, streptomycin, imipenem) by the checkerboard method. The combination exhibited the best synergy among all combinations, which were composed of streptomycin and 50% EE (SE) from the C. salicifolius S. Y. Hu. leaves. The aim of this study was to investigate the antibacterial mechanism of the SE against Escherichia coli (E. coli, G-) and Staphylococcus aureus (S. aureus, G+). MATERIALS AND METHODS: The antibacterial mechanism of the SE was explored by the time-kill test, the phosphorus metabolism, cell membrane integrity assays, the SDS-PAGE, the SEM and TEM observation. RESULTS: The time-kill test illustrated that the SE was bacteriostatic with a time-dependent relationship, not sterilization. The phosphorus metabolism indicated that the SE lowered phosphorus consumption. The cell membrane integrity assays demonstrated that the cell membrane was damaged, with the nucleic acid flowing out. The SDS-PAGE analysis found that the SE inhibited the synthesis of the total protein. The SEM and TEM results revealed that the surface and internal ultrastructure of bacteria were damaged. The surface of the bacteria was shriveled and deformed, and the internal structure of the cells was also mutilated. CONCLUSIONS: The SE damaged the cell membrane, with the cytoplasm flowing out, disturbed the synthesis of total protein and phosphorus metabolism, and ultimately killed the bacteria.

Anticomplement and antitussive activities of major compound extracted from Chimonanthus nitens Oliv. leaf.

Chimonanthus nitens Oliv. leaf (CNOL), as a traditional Chinese medicine, has been widely used for the treatment of influenza and colds over a long history. However, the mechanism of colds related to the effects of CNOL have been little studied. In this study, the anticomplement and antitussive activities of different polarity extracts of CNOL were evaluated. Ethyl acetate extract (EAE) among different extracts not only significantly decreased cough times by 21-58% (P < 0.01), but also had anticomplement effects demonstrated by the CH50 values of 0.100 mg/ml. A total of 28 constituents (10 coumarins, 13 flavonoids and five phenolics) were identified in EAE based on the ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry technique. Eight compounds in EAE were evaluated by an ammonia-induced cough model to reveal the antitussive mechanisms and classical anticomplement pathway. The results indicated that the antitussive effects of scopoletin, kaempferol-3-O-rutinoside and kaempferol may depend on central mechanisms and that flavonoids such as compounds of kaempferol-3-O-rutinoside and kaempferol have better anticomplementary activity than coumarins like compounds of scopolin, scopoletin and isofraxidin. Taken together, kaempferol-3-O-rutinoside and kaempferol could be important chemical markers in the present study that might be used to evaluate the quality and biological activity of CNOL.

Toxicity of Chimonanthus nitens flower extracts to the golden apple snail, Pomacea canaliculata.

We studied the molluscicidal activity of Chimonanthus nitens extracts on Pomacea canaliculata (Ampullariidae). The degree of hepatopancreatic tissue damage, and its physiological and biochemical effects, was evaluated on individuals exposed to petroleum ether extracts (PEEEs). The PEEEs, ethyl acetate extract (EAEE) and water saturated n-butyl extract (SBEE) of C. nitens also had toxic effects on P. canaliculata but PEEE had the greatest molluscicidal activity. After exposure to PEEE for 24 h, the hepatopancreas of P. canaliculata had a large necrotic area. The levels of soluble sugar, soluble protein and albumin (Alb) in the hepatopancreas of P. canaliculata decreased with increasing PEEE concentration, while the activities of glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT) and acetylcholinesterase (AchE) increased with increasing PEEE concentration. A total of 29 compounds were identified from the PEEE of C. nitens by gas chromatography-mass spectrometry analysis. The main components were esters (48.13%), alcohols (18.43%) and the compound Chimonanthine (14.70%). The results of the molluscicidal assay, histological experiments and the physiological and biochemical experiments show that the PEEE of C. nitens could potentially be used for P. canaliculata management.

Non-volatile constituents and pharmacology of Chimonanthus: A review.

Chimonanthus plants widely distributed in southern area of China, which have a long history of edibles and medicine. Phytochemical investigations have shown that Chimonanthus produced 143 non-volatile constituents, including alkaloids, flavonoids, terpenoids, coumarins and others, which exhibit significant anti-oxidant, anti-bacterial, anti-cancer, anti-inflammatory, antihyperglycemic, antihyperlipidemic and other biological activities. On the basis of systematic reviewing of literatures, this article overviews the non-volatile constituents and pharmacology of Chimonanthus from domestic and foreign over the last 30 years (until June 2018), and may provide a useful reference for the further development of Chimonanthus.

[In vivo metabolism of three coumarins from Chimonanthi Radix based on UHPLC-QTOF-MS/MS].

Scopolin (SC-1), scopoletin (SC-2) and isofraxidin (IS-1) are the main active constituents in Chimonanthi Radix. However, the in vivo metabolism of SC-1, SC-2 and IS-1 have not been comprehensively clarified. In this study, the in vivo metabolic profiles of these three coumarins in the rat plasma, urine and feces were analyzed. Ultra-high performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS/MS) method was applied to characterize the prototypes and metabolites of SC-1, SC-2 and IS-1 in rat feces, urine, and plasma after intravenous administration. A total of 11 metabolites of the three parent compounds were tentatively identified. The main metabolic pathways were analyzed by identification of metabolites, and it was found that these three coumarins underwent multiple in vivo metabolic reactions including glucuronidation, sulfonation, isomerism and reduction. In this study, the analysis of metabolites of three coumarins basically demonstrated their in vivo metabolic process, providing basis for the further pharmacokinetics and pharmacological evaluations of SC-1, SC-2 and IS-1.

Novel sesquiterpenoids isolated from Chimonanthus praecox and their antibacterial activities.

In the present study, four new sesquiterpenoids, chimonols A-D (compounds 1-4), together with four known compounds (5-8) were isolated from the EtOAc extract of Chimonanthus praecox Link. The structures of these new compounds were elucidated on the basis of spectroscopic techniques (UV, IR, MS, and 1D and 2D NMR), and their absolute configurations were established by comparing experimental and calculated electronic circular dichroism (ECD) spectra. Compounds 1-8 were evaluated for antimicrobial activities and the minimum inhibitory concentrations (MICs) were determined by the broth microdilution method in 96-well culture plates. Compounds 1, 2, and 7 exhibited weak antibacterial effects for S. aureus (ATCC 6538), E. coli (ATCC 11775), and P. aeruginosa (ATCC 10145) with MIC values being 158-249 µg·mL-1. Compounds 3-7 showed activities against C. glabrata (ATCC 2001) and S. aureus (ATCC 43300) with MIC values being 128-197 µg·mL-1. Compounds 1-4 showed activity against S. aureus (ATCC 25923) with MIC values being 162-254 µg·mL-1. The present study provided a basis for future evaluation of these compounds as antibacterial agents.

Chimonanthus nitens Oliv. leaf extract exerting anti-hyperglycemic activity by modulating GLUT4 and GLUT1 in the skeletal muscle of a diabetic mouse model.

The present study aimed to explore the potent molecular mechanisms behind the hypoglycemic effect of Chimonanthus nitens Oliv. leaf extract (COE) in combination with a high-glucose-fat diet-fed and streptozotocin-induced diabetic mouse model. COE (50 and 200 mg per kg body weight per day) was given to the diabetic-model mice by intragastric administration for 4 weeks. It was found that the fasting blood glucose level (FBG), serum insulin level (FINS), and insulin sensitivity index (ISI) were significantly improved in the COE-treated diabetic-model mice. Glucose metabolism genes expression analysis of the skeletal muscle showed that COE exerted a glucose-lowering effect through the following two ways: on the one hand, COE enhanced insulin sensitivity by upregulating the transcription level of GLUT4, and in addition, it enhanced the insulin signaling pathway to promote the translocation of GLUT4 and upregulated thermogenesis genes expression, including PGC-1α and UCP-1; while on the other hand, GLUT1 expression was also increased in both the transcription and translation levels in the presence of COE. These two ways may result in promoting glucose uptake in skeletal muscle, thus leading to the reduction of the blood glucose level. The results suggested that COE ameliorated hyperglycemia in the diabetic-model mice through regulating glucose transporters, and then was likely to increase glucose uptake, which provided more evidence for applying COE to treat anti-hyperglycemia.

[Research advances on chemical constituents from Calycanthaceae plants and their pharmacological activities].

Calycanthaceae family comprises of four genera including Chimonanthus, Sinocalycanthus, Calycanthus, and Idiospermum. The plants of Calycanthaceae are popular ornamental shrubs and used as foods and medicines, which are mainly distributed in China, North America, and Australia. The plants of Calycanthaceae are rich in volatile components, alkaloids, sesquiterpenes and coumarins. Dimeric piperidinoquinoline and dimeric pyrrolidinoindoline alkaloids, dimeric and/or trimeric coumarins are characteristic compositions in these plants. In order to provide timely reference for further investigation and development of Calycanthaceae plants, we made a systemic review on chemical constituents, i.e. alkaloids, terpenoids, flavonoids, coumarins, and steroids, from Calycanthaceae plants, focusing on their chemical structures and pharmacological activities.

Structural characterization and discrimination of Chimonanthus nitens Oliv. leaf from different geographical origins based on multiple chromatographic analysis combined with chemometric methods.

The Chimonanthus nitens Oliv. leaf (CNOL). is a widely used traditional Chinese medicine (TCM) used for treating colds and influenza. In the present study, a comprehensive strategy integrating multiple chromatographic analysis and chemometric methods was firstly proposed for structural characterization and discrimination of CNOL from different geographical origins. It consists of three steps: Firstly, the ultra-high-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS) method was applied for comprehensive profiling of characterization constituents in CNOL by high-resolution diagnostic product ions/neutral loss filtering, and a total of 40 constituents were identified. Secondly, chemical fingerprints were established by HPLC coupled with photodiode array detector (PDA), and similarity analyses were calculated based on nineteen common characteristic peaks. Subsequently, the nine major constituents, including coumarins, flavonoids, and phenolic acids were quantified, and the quantitative data further analyzed by principal component analysis (PCA) and orthogonal partial least-squares discriminant analysis (OPLS-DA). Thirdly, a hot map visualization was conducted for clarifying the distribution of major compounds among different geographical origins. Also, nine constituents detected could be used as chemical markers for discrimination of CNOL from different provinces. Collectively, these results indicated that our proposed platform was a powerful tool for chemical profiling and discrimination of herbs with multiple botanical origins, providing promising perspectives in tracking the formulation process of TCMs products.

Constituent analysis of the ethanol extracts of Chimonanthus nitens Oliv. leaves and their inhibitory effect on α-glucosidase activity.

The ethanol extracts of Chimonanthus nitens Oliv. leaves were prepared sequentially by ethanol gradient elution and tested for their α-glucosidase inhibitory. The fraction of 50% ethanol eluate (EE) exhibited the notable inhibition with IC50 of 0.376mg/mL. Also, 50% EE was chemically characterized by liquid chromatography-mass spectrometry (LC-MS) analysis. Eight compounds including rutin (1), hyperin (2), isoquercitrin (3), luteoloside (4), astragalin (6), quercetin (13), naringenin (14), kaempferol (15) were identified by compared with standard substances as well as proper luteolin-5-O-glucoside (5), kaempferol-7-O-rhamnoside (9), 5,7,8-trihydroxy-2-methoxyl-flavone-7-O-glucoside (10), kaempferol-7-O-acetyl-galactoside (11). The experiments of ultra-filtration combined with liquid chromatography-mass spectrometry (UF-LC-MS) guided quercetin and kaempferol as the key factors for 50% EE showing highly inhibitory activity on α-glucosidase. Quercetin and kaempferol inhibited yeast α-glucosidase in a mixed-type manner with IC50 of 66.8 and 109µg/mL, respectively. These results would provide theoretical underpinning for the C. nitens Oliv. leaves ethanol extracts used as nutraceutical health supplement in the management of type 2 diabetes.

Study on quality standards for Chimonanthus nitens.

Chimonanthus nitens Oliv. is a commonly used traditional Chinese medicine. Terpenoids, flavonoids, and coumarins are usually considered its main bioactive ingredients. Thus, qualitative and quantitative analyses of these compounds are crucial in quality control studies of Chimonanthus nitens. In this study, five compounds were identified by double-development thin layer chromatography (TLC) and the content of four compounds was determined by high performance liquid chromatography; the detection wavelength was set to 344 nm and the column temperature was 40°C. All calibration curves showed good linear regression (R2 > 0.9995). The average recoveries ranged from 97.06 to 104.44%. The RSD was below 4.2%. Four compounds remained stable over 24 h and the relative standard deviation (RSD) of the precision of their measurement was less than 1.5%. The developed method was reproducible, sensitive, and simple, and could be used for quality control of Chimonanthus nitens.

Preparative Separation of Phenolic Compounds from Chimonanthus praecox Flowers by High-Speed Counter-Current Chromatography Using a Stepwise Elution Mode.

High-speed counter-current chromatography (HSCCC) has been successfully used for the separation of eight compounds from Chimonanthus praecox flowers. Firstly, the crude extract of Chimonanthus praecox flowers was dissolved in a two-phase solvent system composed of petroleum ether-ethyl acetate-methanol-H2O (5:5:3:7, v/v) and divided into two parts: the upper phase (part I) and the lower phase (part II). Then, HSCCC was applied to separate the phenolic acids from part I and part II, respectively. Considering the broad polarity range of target compounds in part I, a stepwise elution mode was established. Two optimal solvent systems of petroleum ether-ethyl acetate-methanol-H2O-formic acid (FA) (5:5:3:7:0.02, 5:5:4.3:5.7:0.02, v/v) were employed in this separation. Five phenylpropanoids and two flavonoids were successfully separated from 280 mg of part I, including 8.7 mg of 3,4-dihydroxy benzoic acid (a, 95.3% purity), 10.9 mg of protocatechualdehyde (b, 96.8% purity), 11.3 mg of p-coumaric acid (c, 98.9% purity), 12.2 mg of p-hydroxybenzaldehyde (d, 95.9% purity), 24.7 mg of quercetin (e, 97.3% purity), 33.8 mg of kaempferol (f, 96.8% purity), and 24.6 mg of 4-hydroxylcinnamic aldehyde (g, 98.0% purity). From 300 mg of part II, 65.7 mg of rutin (h, 98.2% purity), 7.5 mg of 3,4-dihydroxy benzoic acid (a, 77.4% purity), and 4.7 mg of protocatechualdehyde (b, 81.6% purity) were obtained using the solvent system EtOAc-n-butanol (n-BuOH)-FA-H2O (4:1:0.5:5, v/v). The structures of the eight pure compounds were confirmed by electrospray ionization-mass spectrometry (ESI-MS), ¹H-NMR and (13)C-NMR. To the best of our knowledge, compounds a-d and f were the first separated and reported from the Chimonanthus praecox flower extract.

A Fast and Reliable UPLC-PAD Fingerprint Analysis of Chimonanthus salicifolius Combined with Chemometrics Methods.

A novel fingerprinting approach was developed by means of ultra-high-performance liquid chromatography with photodiode array detector (UPLC-PAD) for the quality control of Chimonanthus salicifolius (C. salicifolius). All UPLC analyses were carried out on a Waters Acquity BEH Phenyl column (2.1 × 50 mm, 1.7 µm particle size) at 48°C, with a gradient mobile phase composed of 0.1% aqueous phosphoric acid and acetonitrile at a flow rate of 0.2 mL/min. The method validation results demonstrated the developed method possessing desirable precision [<0.88% relative standard deviation (RSD)], reproducibility (<1.87% RSD), stability (<1.42% RSD) and allowing fingerprint analysis in one chromatographic run within 21 min. The quality assessment was achieved by using chemometrics methods including similarity analysis, hierarchical clustering analysis and principal component analysis. The developed method can be used for further quality control of C. salicifolius.

Combination with Chemometrics and Quantification for Quality Evaluation and Variety Identification of Flos Chimonanthi Praecocis by HPLC.

Flos Chimonanthi Praecocis (FCP) is one of the popular traditional Chinese medicines, which have been widely used in China. Inconspicuous appearance differences are disadvantageous for identification, and it is difficult to evaluate the quality of FCP using the current methods. In this article, a simple method that combined chemometrics and quantitative analysis was established. The samples were obtained from three typical varieties for fingerprint analysis by high-performance liquid chromatography. Contents of rutin and quercetin were determined, and then a common pattern with 16 characteristic peaks was applied for principal component analysis, similarity analysis, and the hierarchical cluster analysis heatmap (HCA heatmap) to characterize the similarity and differences among samples for identification. Furthermore, seven characteristics peaks with higher loading values were selected for chemometrics analysis after dimensionality reduction to reduce analytical difficulty, and the new common pattern showed the similar identification effects. Overall, combination with chemometrics and quantitative analysis would provide a useful and simple method for quality control of FCP in the future.

Quantitative Determination of Principal Alkaloid and Flavonoid Constituents in Wintersweet, the Flower Buds of Chimonanthuspraecox.

A quantitative analytical method has been-developed for four alkaloids (1-4), identified as constituents responsible for the melanogenesis inhibitory activity of the.extracts of wintersweet, the flower buds of Chimonanthus praecox (L.) Link (Calycanthaceae). Concurrently, a quantitative analytical protocol has been developed for five flavonoids (5-9), which also exhibited inhibitory activity. To approve the validity of the developed protocols, five extracts of the flower buds collected in Chinese market were evaluated. The optimum conditions of separation and detection of these alkaloids (1-4) and flavonoids (5-9) were achieved on a common ODS column using a MeOH-H20 mobile phase with different additives [Et2NH for alkaloids (1-4); acetic acid for flavonoids (5-9)]. The results. indicated that these assays were reproducible and precise, and could be readily utilized for evaluation of the melanogenesis inhibitory activity of -wintersweet on the basis of the content of the functional species. The principal flavonoid constituents (5-9) also exhibited lipid accumulation inhibitory activity.

[Cloning and expression analysis of glucose-6-phosphate dehydrogenase 1 (G6PDH1) gene from Chimonanthus praecox].

Glucose-6-phosphate dehydrogenase is main regulatory enzyme for pentose phosphate pathway. To amplify the core sequence of G6PDH gene from Chimonanthus praecox, the primers were synthesized, based on the conserved nucleotide sequence of other reported plant G6PDH genes. The specific primers were designed according to the major fragment. The full length cDNA of the G6PDH1 gene was isolated by the 3' and 5' rapid amplification of cDNA ends approach. Transcript levels of G6PDH1 isoform was measured by real-time quantitative RT-PCR in different tissues and in responds to cold treatment. The G6PDH1 subcellular localization, transmembrane domain, three-dimensional structure, and phylogenetic analysis were predicted by different software to analysis the bioinformatics of G6PDH1 protein. The G6PDH1 cDNA sequence was 2 011 bp in length and consisted of 1 551 bp Open Reading Frame (ORF) , encoding a protein of 516 amino acids. Expression analysis results in different tissues showed that G6PDH1 was primarily observed in flowers and roots, as opposed to the leaves and stems. Cold treatment experiments indicated that cold treatment caused a rapid increase in G6PDH1 expression in flowers within 12 h. The full-length cDNA of G6PDH1 and its expression analysis will play an important role for further study on cold stress responses in Ch. praecox.

Dimeric pyrrolidinoindoline-type alkaloids with melanogenesis inhibitory activity in flower buds of Chimonanthus praecox.

A methanol extract of the flower buds of Chimonanthus praecox (L.) Link (Calycanthaceae) demonstrated inhibitory effects on melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells. From the extract, five dimeric pyrrolidinoindoline alkaloids and four sesquiterpenes were isolated, together with 16 known compounds. Among them, (-)-chimonanthine (1, IC50 = 0.93 µM), (-)-folicanthine (2, 1.4 µM), and (-)-calycanthidine (3, 1.8 µM) showed potent inhibitory effects without notable cytotoxicity at the effective concentrations. The most potent alkaloid (1) inhibited both tyrosinase and tyrosine-related protein-1 mRNA expressions, to which the melanogenesis inhibitory activity would be ascribable.

[Chemical constituents of chloroform fraction from leaves of Chimonanthus salicifolius].

To explore anti-tumor active components of Chimonanthus salicifolius, the phytochemistry of the chloroform fraction from leaves extract was investigated by repeated silica gel column chromatography. Twelve compounds were isolated and their structures were identified by physicochemical properties and spectroscopic data analysis as 9-epi-blumenol C(1), blumenol C(2), (+)-dehydrovomifoliol (3), (+)-vomifoliol (4), robinlin (5), (-)-loliolide (6), isofraxidin (7), scopoletin (8), 6,7-dimethoxycoumarin (9), 6, 7, 8-trimethoxycoumarin (10), beta-sitostenone (11), and beta-stigmasterol(12). Compounds 1-6 belonging to nor-sesquiterpenoids were isolated from the family Calycanthaceae for the first time. Compound 1 was a new natural product. Compounds 7, 11 and 12 were obtained from this plant for the first time.