Structurally diverse amides from Chloranthus henryi var. hupehensis and their anti-inflammatory activities by blocking Akt phosphorylation.

Eleven new amides, four racemic pairs of (±)-chlorahupetamides A, B, D, E (1, 2, 4, 5) and chlorahupetamides C, F, G (3, 6, 7), have been isolated from Chloranthus henryi var. hupehensis. Compounds 1-3 are the first naturally occurring dimers via an unprecedented [2 + 2] cycloaddition derived from two dissimilar cinnamic acid amides, while compounds 4 and 5 represent the first examples of lignanamides in Chloranthus; together with two new hydroxycinnamic acid amide monomers (6-7), these compounds were obtained. Their structures were characterized by nuclear magnetic resonance (NMR), electronic circular dichroism (ECD), and X-ray diffraction analysis. Meanwhile, an LPS-induced BV-2 cell inflammatory model was used to determine the potential anti-inflammatory activity of all the isolated compounds. Intriguingly, compound -1 treatment showed a much greater inhibition of TNF-α expression with an EC50 value of 1.80 µM, while compound + 1 had more advantages in reducing IL-1ß expression with an EC50 value of 19.93 µM. Moreover, compounds + 1 and -1 could significantly suppress inflammation and inhibit the Akt signaling pathway by decreasing the phosphorylated protein levels of Akt.

Lindenane sesquiterpenoid dimers from Chloranthus japonicus improve LDL uptake by regulating PCSK9 and LDLR.

UPLC-TOF-MS/MDF directed phytochemical research of Chloranthus japonicus led to the isolation of 46 lindenane sesquiterpenoid dimers, which included 13 new analogs. Their structures with absolute configurations were elucidated by analysis of spectroscopic data. Fourteen compounds with ester chains significantly decreased PCSK9 protein level in medium of HepG2 cells, especially for compounds 14 and 29 (5 µM) with inhibition rates of 69.0% and 72.8%, respectively. Compound 14 in HepG2 cells was evaluated via DiI-LDL uptake assays and found to increase LDL uptake by upregulating LDLR mRNA and protein level. Meanwhile, 14 decreased the secretion of PCSK9 protein in medium and downregulated intracellular PCSK9 protein and mRNA level. The discovery of these natural small molecule compounds provides a novel structure basis for design PCSK9 regulators, making them a promising lead for development of new lipid-lowering agents.

Discovery of sesquiterpenoids from the roots of Chloranthus henryi Hemsl. var. hupehensis (Pamp.) K. F. Wu and their anti-inflammatory activity by IKBα/NF-κB p65 signaling pathway suppression.

Phytochemical analysis of Chloranthus henryi var. hupehensis roots led to the identification of a new eudesmane sesquiterpenoid dimer, 18 new sesquiterpenoids, and three known sesquiterpenoids. Among the isolates, 1 was a rare sesquiterpenoid dimer that is assembled by a unique oxygen bridge (C11-O-C8') of two highly rearranged eudesmane-type sesquiterpenes with the undescribed C16 carbon framework. (+)-2 and (-)-2 were a pair of new skeleton dinorsesquiterpenoids with a remarkable 6/6/5 tricyclic ring framework including one γ-lactone ring and the bicyclo[3.3.1]nonane core. Their structures were elucidated using spectroscopic data, single-crystal X-ray diffraction analysis, and quantum chemical computations. In the LPS-induced BV-2 microglial cell model, 17 suppressed IL-1ß and TNF-α expression with EC50 values of 6.81 and 2.76 µM, respectively, indicating its excellent efficacy in inhibiting inflammatory factors production in a dose dependent manner and without cytotoxicity. In subsequent mechanism studies, compounds 3, 16, and 17 could reduce IL-1ß and TNF-α production by inhibiting IKBα/p65 pathway activation.

Extraction and Isolation of Two Polysaccharides from Chloranthus japonicus Sieb. and Evaluation of Their Anti-Gastric Cancer Activities.

Two unreported heteropolysaccharides, denoted as YCJP-1 and YCJP-2, were isolated from the herbs of Chloranthus japonicus. YCJP-1 was a heteropolysaccharide composed of glucose, galactose, arabinose, mannose, rhamnose, and a minor proportion of uronic acids, with the molecular weight mainly distributed in the 74,475-228,443 Da range. YCJP-2 was mainly composed of glucose, mannose, and galactose, with the molecular weights ranging from 848 to 5810 Da. To further evaluate the anti-gastric cancer effects of C. japonicus, the inhibitory effects of the crude polysaccharide (YCJP) and the purified polysaccharides (YCJP-1 and YCJP-2) were determined using a CCK-8 assay and colon-forming assay on MGC-803 and AGS gastric cancer cell lines. Our results showed that YCJP, YCJP-1, and YCJP-2 possess prominent inhibitory effects on the proliferation of MGC-803 and AGS cells, and the AGS cell was more sensitive to YCJP, YCJP-1, and YCJP-2. Moreover, YCJP-2 demonstrated superior anti-gastric cancer effects compared to YCJP-1. This could potentially be attributed to YCJP-2's higher glucose content and narrower molecular weight distribution.

Anti-Inflammatory Peroxidized Chlorahololide-Type Dimers Are Artifacts of Shizukaol-Type Dimers: From Phenomena Discovery and Confirmation to Potential Underlying Mechanism.

In our research on naturally occurring sesquiterpenes, eight shizukaol-type dimers, one chlorahololide-type dimer, and one sarcanolide-type dimer were isolated from the roots of Chloranthus fortunei. As the project was implemented, we accidentally discovered that shizukaol-type dimers can be converted into peroxidized chlorahololide-type dimers. This potential change was discovered after simulations of the changes in corresponding shizukaols showed that three peroxide products were generated (1-3), indicating that peroxidation reactions occurred. HPLC-HR-MS analysis results obtained for the shizukaol derivatives further demonstrate that the reaction occurred, and the type of substituent of small organic ester moieties at positions C-15' and C-13' of unit B were not decisively related to the reaction. Quantum chemical calculations of the mode dimer further demonstrated this phenomenon. The highest occupied molecular orbital (HOMO)-lowest unoccupied molecular orbital (LUMO) energy of the precursor and production revealed the advantageous yield of 4ß-hydroperoxyl production. Additionally, the potential reaction mechanism was speculated and validated using the free energy in the reaction which successfully explained the feasibility of the reaction. Finally, the anti-inflammatory activity of the precursors and products was evaluated, and the products of peroxidation showed better anti-inflammatory activity.

Constituents from Chloranthus multistachys and their cytotoxic activities against various human cancer cell lines.

Two new furanoeremophilane sesquiterpenoids, namely, 6,9-dioxo-1α,4α-dihydroxy-furanoeremophilane (1) and 4α,5α-epoxy-6,9-dioxo-1α-hydroxyl-furanoeremophilane (2), and 10 known compounds were isolated from the whole plant of Chloranthus multistachys, and compound 3 was converted to derivative 3a. Their structures were determined based on extensive spectroscopic analysis. All compounds were evaluated by using five cancer cell lines: PC3, LNcap, A549, K562, and HEL. The derivative 3a exhibited excellent cytotoxic activities, with the IC50 against HEL cells being the lowest at 1.322 ± 0.08 µM, which was comparable to that of the positive control (doxorubicin). Mechanism studies showed that the anticancer activity of 3a may be associated with cell cycle regulation.

Chlorahololide D, a Lindenane-Type Sesquiterpenoid Dimer from Chloranthus holostegius Suppressing Breast Cancer Progression.

Aimed at discovering small molecules as anticancer drugs or lead compounds from plants, a lindenane-type sesquiterpene dimer, chlorahololide D, was isolated from Chloranthus holostegius. The literature review showed that there were few reports on the antitumor effects and mechanisms of chlorahololide D. Our biological assay suggested that chlorahololide D blocked the growth and triggered apoptosis of MCF-7 cells by stimulating the reactive oxygen species (ROS) levels and arresting the cell cycle at the G2 stage. Further mechanism exploration suggested that chlorahololide D regulated apoptosis-related proteins Bcl-2 and Bax. Moreover, chlorahololide D inhibited cell migration by regulating the FAK signaling pathway. In the zebrafish xenograft model, chlorahololide D was observed to suppress tumor proliferation and migration significantly. Considering the crucial function of angiogenesis in tumor development, the anti-angiogenesis of chlorahololide D was also investigated. All of the research preliminarily revealed that chlorahololide D could become an anti-breast cancer drug.

Two new dinor-eudesmane sesquiterpenoids from the roots of Chloranthus multistachys.

Two new dinor-eudesmane sesquiterpenoids, named multistalin A (1), and multistalin B (2), together with three sesquiterpene glycosides (3-5), and a norlabdane-type diterpene (6) were isolated from the root extract of Chloranthus multistachys Pei. Their structures were elucidated on the basis of spectroscopic analysis including 1D, 2D NMR techniques and HR-ESI-MS. In addition, the cytotoxicity activities of the isolated compounds against selected cancer cells (Hela and A-549) were evaluated by MTT assay.

Diterpenoid Compounds Isolated from Chloranthus oldhamii Solms Exert Anti-Inflammatory Effects by Inhibiting the IKK/NF-κB Pathway.

Chloranthus oldhamii Solms (CO) is a folk medicine for treating infection and arthritis pain but its pharmacological activity and bioactive compounds remain mostly uncharacterized. In this study, the anti-inflammatory compounds of C. oldhamii were identified using an LPS-stimulated, NF-κB-responsive RAW 264.7 macrophage reporter line. Three diterpenoid compounds, 3α-hydroxy-ent-abieta-8,11,13-triene (CO-9), 3α, 7ß-dihydroxy-ent-abieta-8,11,13-triene (CO-10), and decandrin B (CO-15) were found to inhibit NF-κB activity at nontoxic concentrations. Moreover, CO-9 and CO-10 suppressed the expression of IL-6 and TNF-α in LPS-stimulated RAW 264.7 cells. The inhibitory effect of CO-9 on TNF-α and IL-6 expression was further demonstrated using LPS-treated bone marrow-derived macrophages. Furthermore, CO-9, CO-10, and CO-15 suppressed LPS-triggered COX-2 expression and downstream PGE2 production in RAW 264.7 cells. CO-9 and CO-10 also reduced LPS-triggered iNOS expression and nitrogen oxide production in RAW 264.7 cells. The anti-inflammatory mechanism of the most effective compound, CO-9, was further investigated. CO-9 attenuated LPS-induced NF-κB activation by reducing the phosphorylation of IKKα/ß (Ser176/180), IκBα (Ser32), and p65 (Ser534). Conversely, CO-9 did not affect the LPS-induced activation of MAPK signaling pathways. In summary, this study revealed new anti-inflammatory diterpenoid compounds from C. oldhamii and demonstrated that the IKK-mediated NK-κB pathway is the major target of these compounds.

[Research progress on chemical constituents from Chloranthus plants and their biological activities].

The genus Chloranthus has 13 species and 5 varieties in China, which can be found in the southwest and northeast regions. Phytochemical studies on Chloranthus plants have reported a large amount of terpenoids, such as diterpenoids, sesquiterpenoids, and sesquiterpenoid dimers. Their anti-inflammation, anti-tumor, antifungal, antivirus, and neuroprotection activities have been confirmed by previous pharmacological research. Herein, research on the chemical constituents from Chloranthus plants and their biological activities over the five years was summarized to provide scientific basis for the further development and utilization of Chloranthus plants.

[A new sesquiterpene from Chloranthus henryi].

Eight sesquiterpenes were isolated and purified from the ethanol extract of Chloranthus henryi by column chromatographies over silica gel, ODS and Sephadex LH-20,and preparative HPLC. Their chemical structures were established by spectral data and physiochemical properties as(1S,6S,8S,10R)-8-ethoxy-10-methoxychlomultin C(1),tianmushanol(2),multistalide A(3),myrrhterpenoid N(4),1α,9α-dihydroxy-8,12-expoxy-eudesma-4,7,11-trien-6-one(5),4ß,10α-aromadendranediol(6),oplopanone(7),10α-hydroxycadinan-4-en-3-one(8). Among them, compound(1) was a new compound, and compounds 2-8 were isolated from Chloranthus henryi for the first time.

[Chemical constituents of sesquiterpenes from Chloranthus multistachys].

With repeated silica gel, octadecyl silica(ODS), and Sephadex LH-20 column chromatography, normal-phase and reverse-phase high performance liquid chromatography(HPLC), etc., a pair of new enantiomers and 5 known compounds were separated from the 95% ethanol extract of Chloranthus multistachys. These compounds were identified by the nuclear magnetic resonance spectroscopy(including 1 D-NMR and 2 D-NMR), single-crystal X-ray diffraction, circular dichroism(CD) spectroscopy, mass spectrometry(MS), and some other methods as(1R,4R,5R,8S,10R)-chloraeudolide H(1 a),(1S,4S,5S,8R,10S)-chloraeudolide H(1 b), hydroxyisogermafurenolide(2), 4α-hydroxy-5α,8ß(H)-eudesm-7(11)-en-8,12-olide(3), chloraniolide A(4), chlorantene D(5), 4α,8ß-dihydroxy-5α(H)-eudesm-7(11)-en-8,12-olide(6). Compounds 1 a and 1 b are a pair of new eudesmane-type sesquiterpene enantiomers, and compounds 2-4 were isolated from C. multistachys for the first time.

Diversity and Antimicrobial Activity of Endophytic Fungi Isolated from Chloranthus japonicus Sieb in Qinling Mountains, China.

The plant Chloranthus japonicus Sieb is known for its anticancer properties and mainly distributed in China, Japan, and Korea. In this study, we firstly investigated the diversity and antimicrobial activity of the culturable endophytic fungi from C. japonicus. A total of 332 fungal colonies were successfully isolated from 555 tissue segments of the medicinal plant C. japonicus collected from Qinling Mountains, China. One hundred and thirty representative morphotype strains were identified according to ITS rDNA sequence analyses and were grouped into three phyla (Ascomycota, Basidiomycota, Mucoromycota), five classes (Dothideomycetes, Sordariomycetes, Eurotiomycetes, Agaricomycetes, Mucoromycetes), and at least 30 genera. Colletotrichum (RA, 60.54%) was the most abundant genus, followed by Aspergillus (RA, 11.75%) and Diaporthe (RA, 9.34%). The Species Richness Index (S, 56) and the Shannon-Wiener Index (H', 2.7076) indicated that C. japonicus harbored abundant fungal resources. Thirteen out of 130 endophytic fungal ethyl acetate extracts exhibited inhibitory activities against at least one pathogenic bacterium or fungus. Among of these, F8158, which was identified as Trichoderma cf. harzianum, exhibited good antagonistic capacities (the percent inhibition of mycelial growth ranged from 47.72~88.18) for different pathogens and has a potential application in biological control. In addition, it is noteworthy that the strain F8157 (Thanatephorus cucumeris, an opportunistic pathogen) showed antibacterial and antifungal activity, which is reported firstly in this study, and should be investigated further. Taken together, these results indicated that the endophytic fungi from C. japonicus may be of potential interest in screening bio-control agents and discovering of new bioactive compounds.

Reanalysis of Lindenatriene, a Building Block for the Synthesis of Lindenane Oligomers.

Lindenane oligomers isolated from Chloranthus pose significant challenges for chemical synthesis. The structure of a proposed biosynthetic monomer, lindenatriene, was recently called into question. Its attempted synthesis produced a compound whose 1H NMR spectrum differed significantly from the spectrum of a monomer produced by oligomer pyrolysis. Here we propose that the original structural assignment after pyrolysis was correct and instead the spectra of synthetic materials were misinterpreted. Reanalysis of 2D NMR data suggest that lindenatriene isomerizes (formal [1,7]-hydrogen shift) upon treatment with base.

Water-separated part of Chloranthus serratus alleviates lipopolysaccharide- induced RAW264.7 cell injury mainly by regulating the MAPK and Nrf2/HO-1 inflammatory pathways.

BACKGROUND: Chloranthus serratus (Chloranthaceae) has been used to treat bruises, rheumatoid and bone pain. However, the anti-inflammatory mechanisms of C. serratus in vitro have not been fully elucidated. The present study aimed to explore the anti-inflammatory activity and potential mechanisms of C. serratus's separated part of water (CSSPW) in lipopolysaccharide (LPS)-induced RAW264.7 cells. METHODS: The concentrations of CSSPW were optimized by CCK-8 method. Nitric oxide (NO) content was detected by one-step method. The levels of inflammatory cytokines were determined by enzyme-linked immunosorbent assay (ELISA). Gene expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was detected by real-time quantitative PCR (qPCR). Immunofluorescence and DCFH-DA fluorescent probes were used to detect p65 nuclear translocation and reactive oxygen species (ROS) content, respectively. Western blotting was used to assay the protein expression of mitogen-activated protein kinases (MAPK), nuclear factor-kappa B (NF-κB) and nuclear transcription factor E2 related factor 2/haem oxygenase-1 (Nrf2/HO-1) pathways. RESULTS: The final concentrations of 15 ng/mL, 1.5 µg/mL and 150 µg/mL were selected as low, medium and high doses of CSSPW, respectively. CSSPW treatment significantly reduced the generation of NO, tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), prostaglandinE2 (PGE2), iNOS mRNA and COX-2 mRNA in response to LPS stimulation. Furthermore, the protein expression of the MAPK and NF-κB pathways was suppressed by CSSPW treatment, as well as p65 nuclear translocation and ROS production. In contrast, the protein expression of the Nrf2/HO-1 pathway was markedly upregulated. CONCLUSIONS: CSSPW exerts its anti-inflammatory effect via downregulating the production of pro-inflammatory mediators, inhibiting the activation of NF-κB and MAPK pathways, as well as activating Nrf2/HO-1 pathway in LPS-induced RAW264.7 cells.

Chlojaponilactone B Attenuates Lipopolysaccharide-Induced Inflammatory Responses by Suppressing TLR4-Mediated ROS Generation and NF-κB Signaling Pathway.

The lindenane-type sesquiterpenoid chlojaponilactone B (1), isolated from Chloranthus japonicus, has been reported to possess anti-inflammatory properties. The present study aimed to further explore the molecular mechanisms underlying the anti-inflammatory activity of 1. RNA-seq analyses revealed the significant changes in the expression levels of genes related to multiple inflammatory pathways upon treatment of lipopolysaccharide (LPS)-induced RAW 264.7 murine macrophages with 1. Real time PCR (RT-PCR) and Western blotting were used to confirm the modulations in the expression of essential molecules related to inflammatory responses. Compound 1 inhibited toll like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) activation upon LPS stimulation, influencing the expression of NF-κB and pro-inflammatory mediators. Molecular docking studies showed that 1 bound to TLR4 in a manner similar to that of TAK-242, a TLR4 inhibitor. Moreover, our results showed that 1 suppressed inflammatory responses by inhibiting TLR4 and subsequently decreasing reactive oxygen species (ROS) generation, downregulating the NF-κB, thus reducing the expression of the pro-inflammatory cytokines iNOS, NO, COX-2, IL-6 and TNF-α; these effects were similar to those of TAK-242. We proposed that 1 should be considered as a potential anti-inflammatory compound in future research.

Mechanisms of Toxicity and Cardiotoxicity of Alcohol Extract from Root, Stem and Leaf of Chloranthus Serratus.

ABSTRACT: Objective To compare the differences of cardiotoxicity of alcohol extract from root, stem and leaf of Chloranthus serratus in the rats, and discuss preliminarily its mechanism of toxicity. Methods Rats were randomly divided into four groups: blank, root alcohol, stem alcohol and leaf alcohol, with 8 in each group. After 14 days of continuous intragastric administration, the body mass change curves were drawn. The cardiac coefficient was calculated. The contents of creatine kinase （CK）, creatine kinase isoenzyme （CK-MB）, lactate dehydrogenase （LDH） and α-hydroxybutyrate dehydrogenase （α-HBDH） as well as the content changes of oxidative stress indexes - total superoxide dismutase （T-SOD） and malondialdehyde （MDA） in the serum of rats were detected. The cardiac pathomorphology changes in the rats were observed. The expression of intercellular adhesion molecule （ICAM-1） and heme oxygenase （HO-1） in myocardial tissue was detected. Results Body mass growth rate： stem alcohol group was the smallest, followed by leaf alcohol group. The difference of cardiac coefficient of every group had no statistical significance （P>0.05）. The myocardial tissues of stem alcohol group suffered the most serious damage, followed by the leaf alcohol group. The contents of CK, CK-MB, LDH and α-HBDH in stem alcohol group increased （P<0.05）. The increase of MDA content and decrease of T-SOD content in stem alcohol group had statistical significance compared with the blank group and root alcohol group, while the leaf alcohol group only had statistical significance in the decrease of T-SOD content compared with the blank group （P<0.05）. The positive expression of ICAM-1 enhanced and the expression of HO-1 protein decreased in every group after the intervention of different extracts. The change trend was stem alcohol > leaf alcohol > root alcohol group. Conclusion The alcohol extract from the stem has the highest cardiotoxicity, followed by the leaf extract, and its mechanism of toxicity may be related to oxidative stress.

Chemical Constituents from Chloranthus anhuiensis and Their Cytotoxic Activities.

Three hiherto unknown phenylpropanoid compounds, namely (7S,8R)-1-(1-ethoxy-2-hydroxypropyl)-2-methoxy-3,4-(methylenedioxy)benzene (1), (7S,8S)-1-(1-ethoxy-2-hydroxypropyl)-2-methoxy-3,4-(methylenedioxy)benzene (2), and (7S,8R)-1-(1-methoxy-2-hydroxypropyl)-2-methoxy-3,4-(methylenedioxy)benzene (3), along with 12 known compounds (4 - 15) were obtained from the extract of whole plant of Chloranthus anhuiensis. Among them, 7 and 13 were obtained from nature for the first time. The structures of these natural compounds were characterized by extensive spectroscopic analysis and calculated electronic circular dichroism (ECD) data. Furthermore, their cytotoxic and neuroprotective activities were evaluated using MDA-MB-231, 4T1, HepG2, and PC12 cell lines. Compounds 8 and 13 exhibited moderate cytotoxic activities against MDA-MB-231 cell line with the IC50 values of 39.7 and 25.8 µm, respectively. And all the isolated compounds have no neuroprotective activities.

[Sesquiterpenes with anti-metastasis breast cancer activity from Chloranthus henryi].

To study sesquiterpenes with anti-metastasis breast cancer activity from Chloranthus henryi, ten sesquiterpenes ,zedoarofuran (1), chlorajapolide D (2), 4ß, 8ß-dihydroxy-5α(H)-eudesm-7(11)-en-8, 12-olide (3), curcolonol (4), lasianthuslactone A (5), chlomultin C (6), (1E,4Z)-8-hydroxy-6-oxogermacra-1(10), 4, 7(11) -trieno-12, 8-lactone (7), shizukanolide E (8) , shizukanolide F (9) , 9α-hydroxycurcolonol (10), and five bis-sesquiterpenes, shizukaol B (11), shizukaol C (12) , cycloshizukaol A (13) , sarcandrolide B (14) , henriol A(15), were isolated by using different kinds of column chromatography methods from the ethyl acetate part of Ch.henryi and their structures were identified based on spectroscopic methods. Compounds 2, 8, 9, and 10 were obtained from the genus Chloranthus for the first time. Compounds 2, 5, 8-10, 12,and 14 were obtained from this plant for the first time. Some isolated compounds were subjected to evaluate the anti-metastasis breast cancer activity by using pharmacological methods, and only compounds 4, 11, and 12 were potent active.

Natural nitric oxide (NO) inhibitors from Chloranthus japonicus.

Eight new lindenane sesquiterpenoid dimers, chlojapolides A-H (1-8), along with 11 known analogues were isolated from the whole plant of Chloranthus japonicus. Their structures including absolute configurations were elucidated by spectral and chemical methods. All the compounds were examined for their inhibitory effects on the nitric oxide (NO) production induced by lipopolysaccharide (LPS) in RAW 264.7 macrophages, and compounds 1, 11, 13, and 17 exhibited pronounced inhibition with IC50 values in the range of 6.91-15.75µM, being more active than the positive control, quercetin (IC50=15.90µM).