Cynandione A

Cynandione A causes a dynamic change in SIRT1 nuclear trafficking via PKA signaling and beige adipocyte differentiation in 3T3-L1 cells.[Pubmed:34329615]

Eur J Pharmacol. 2021 Oct 15;909:174382.

Inducible brown-like adipocytes, also known as beige adipocytes, dissipate energy through thermogenesis. Although recent reports suggest that silent information regulator 2 homolog 1 (SIRT1) promotes beige adipocyte differentiation (beiging), the activation mechanism of SIRT1 remains unknown. Here, we report that Cynandione A (CA), a major component of Cynanchum wilfordii, causes dynamic changes in SIRT1 nuclear trafficking via protein kinase cAMP-dependent (PKA) signaling and induces the beiging process in adipocyte lineage cells. SIRT1 is located in both the cytoplasm and the nucleus of 3T3-L1 cells. Using cell fractionation and RNA interference experiments, we found that the translocation of SIRT1 from the cytoplasm to the nucleus was enhanced after CA treatment and was followed by upregulation of beige adipocyte-related gene expression. Moreover, we found that CA-induced SIRT1 nuclear trafficking is dependent on the PKA signaling pathway. These results suggest a novel mechanism of CA by which PKA signaling promotes SIRT1 nuclear trafficking, which permits the docking of SIRT1 to its nuclear substrates, leading to beiging in 3T3-L1 cells.

Cynandione A and PHA-543613 inhibit inflammation and stimulate macrophageal IL-10 expression following alpha7 nAChR activation.[Pubmed:33992630]

Biochem Pharmacol. 2021 Aug;190:114600.

Cynandione A, an acetophenone isolated from Cynanchum Wilfordii Radix, attenuates inflammation. The present study aimed to study the mechanisms underlying Cynandione A-induced antiinflammation. Treatment with Cynandione A and the specific alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) agonist PHA-543613 remarkably reduced overexpression of proinflammatory cytokines, including tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and IL-1beta in lipopolysaccharide (LPS)-treated RAW264.7 cells and primary peritoneal macrophages, and endotoxemic mice. Both Cynandione A and PHA-543613 also stimulated IL-10 expression in naive and LPS-treated macrophages and endotoxemic mice. Cynandione A- and PHA-543613-inhibited proinflammatory cytokine expression was completely blocked by the alpha7 nAChR antagonist methyllycaconitine and the IL-10 antibody. The stimulatory effect of Cynandione A and PHA-543613 on IL-10 expression were suppressed by methyllycaconitine and knockdown of alpha7 nAChRs using siRNA/alpha7 nAChR. Cynandione A significantly stimulated STAT3 phosphorylation, which was attenuated by methyllycaconitine and the IL-10 neutralizing antibody. The STAT3 activation inhibitor NSC74859 also blocked Cynandione A-inhibited proinflammatory cytokine expression. Taken together, our results, for the first time, demonstrate that Cynandione A and PHA-543613 inhibit inflammation through macrophageal alpha7 nAChR activation and subsequent IL-10 expression.

Cynandione A Alleviates Neuropathic Pain Through alpha7-nAChR-Dependent IL-10/beta-Endorphin Signaling Complexes.[Pubmed:33584292]

Front Pharmacol. 2021 Jan 27;11:614450.

Cynandione A, an acetophenone isolated from Cynanchum Wilfordii Radix, exhibits antineuropathic pain effect. This study further explored the target molecule and signaling mechanisms underlying cynandione-A-induced antineuropathic pain. Intrathecal injection of Cynandione A significantly attenuated mechanical allodynia in neuropathic rats and substantially increased spinal expression of IL-10 and beta-endorphin but not dynorphin A. Cynandione A treatment also enhanced expression of IL-10 and beta-endorphin but not alpha7 nicotinic acetylcholine receptors (nAChRs) in cultured microglia. The IL-10 antibody attenuated cynandione-A-induced spinal or microglial gene expression of beta-endorphin and mechanical allodynia, whereas the beta-endorphin antiserum blocked cynandione-A-induced mechanical antiallodynia but not spinal or microglial IL-10 gene expression. The alpha7 nAChR antagonist methyllycaconitine significantly reduced cynandione-A-induced mechanical antiallodynia and spinal or microglial expression of IL-10 and beta-endorphin. Furthermore, Cynandione A stimulated microglial phosphorylation of PKA, p38, and CREB in an alpha7-nAChR-dependent manner, and treatment with their inhibitors attenuated cynandione-A-induced mechanical antiallodynia and spinal or microglial expression of IL-10 and beta-endorphin. In addition, Cynandione A stimulated spinal phosphorylation of the transcription factor STAT3, which was inhibited by methyllycaconitine, the PKA activation inhibitor or IL-10 antibody. The STAT3 inhibitor NSC74859 also abolished cynandione-A-induced mechanical antiallodynia and spinal expression of beta-endorphin. These findings suggest that Cynandione A suppresses neuropathic pain through alpha7-nAChR-dependent IL-10/beta-endorphin signaling pathway in spinal microglia.

Cynandione A from Cynanchum wilfordii inhibits hepatic de novo lipogenesis by activating the LKB1/AMPK pathway in HepG2 cells.[Pubmed:31463669]

J Nat Med. 2020 Jan;74(1):142-152.

Cynandione A (CA), isolated from ethyl acetate extract of Cynanchum wilfordii (CW), is a bioactive phytochemical that has been found to be beneficial for the treatment of several diseases. Hepatic de novo lipogenesis is one of the main causes of non-alcoholic fatty liver disease (NAFLD), which is thought to be a hepatic manifestation of certain metabolic syndromes. However, it has not yet been reported if CA has any therapeutic value in these diseases. Here, we investigated whether CA can inhibit hepatic lipogenesis induced by liver X receptor alpha (LXRalpha) using an in vitro model. We found that the extract and ethyl acetated layer of CW decreased the mRNA levels of sterol regulatory element-binding protein-1c (SREBP-1c), which plays a crucial role in hepatic lipogenesis. Additionally, we observed that CA could suppress the level of SREBP-1c, which was increased using two commercial LXRalpha agonists, GW3954 and T0901317. Moreover, the enzymes that act downstream of SREBP-1c were also inhibited by CA treatment. To understand the mechanism underlying this effect, the levels of phosphorylated AMP kinase (pAMPK) were measured after CA treatment. Therefore, CA might increase the pAMPK level by inducing phosphorylation of liver kinase B1 (LKB1), which can then convert AMPK to pAMPK. Taken together, we conclude that CA has an alleviative effect on hepatic lipogenesis through the stimulation of the LKB1/AMPK pathway.

Chemical constituents of Patrinia heterophylla Bunge and selective cytotoxicity against six human tumor cells.[Pubmed:30853646]

J Ethnopharmacol. 2019 May 23;236:129-135.

ETHNOPHARMACOLOGICAL RELEVANCE: Patrinia heterophylla Bunge, known as "Mu-Tou-Hui" in China, is distributed in most provinces and regions of China. As a traditional medicinal plant, which was first found in . In many traditional herbal books, there are records of "Mu-Tou-Hui" of treatment for uterine bleeding, cancer, swelling pain, leukemia, etc. However, there are few studies on the chemical constituents of Patrinia heterophylla Bunge. AIM OF THE STUDY: To investigate the chemical constituents of P. heterophylla and the basis of their antitumor activity. MATERIALS AND METHODS: 15 compounds were isolated from roots and rhizomes of P. heterophylla by repeating various column chromatography techniques, whose structures were determined by organic spectrum analysis methods and compared with published data. The cytotoxicities were evaluated by MTT assay on six cancer cell lines: human melanoma cell (A375), human hepatocellular carcinoma cell (SMMC-7721), human gastric cancer cell (SGC-7901), human cervical cancer cell (HeLa), human colon cancer cell (HCT-116), and human breast cancer cell (MDA-MB-231). The apoptosis-inducing activities of compounds 1, 5, 12 and 15 in A375 tumor cell determined by flow cytometry. RESULT: Five phenylpropanoids, ethyl caffeate (1), coniferaldehyde (5), trans-p-coumaryl aldehyde (6), caffeic acid methyl ester (12), and 3,4-dihydroxycinnamic acid (15), four acetophenones, 1-(2,4-dihydroxyphenyl) ethanone (2), 2',5'-Dihydroxyacetophenone (3), cynanchone A (8), and Cynandione A (10), two phenols, vanillin (4) and catechol (9), two iridoids, sarracenin (7) and patriscabrol (11), one alkane, tetracosane (14), and one coumarin, scopoletin (13), were isolated from the EtOH extracts. Of them, compounds 1-10, 12 and 14-15 were isolated for the first time from the roots of P. heterophylla. Compounds 1 and 15 were reported for the first time with in vitro inhibitory activity against tumor cells. MTT assay showed that compounds 1, 5-9, 12-13 and 15 had selective cytotoxic activities (IC50 27.20-163.03muM) against tumor cells. Apoptosis detected by flow cytometry revealed that compounds 1, 5, 12 and 15 can induce apoptosis for A375 at low concentrations when the concentrations of compounds 1, 5, 12 and 15 are the value of 14, 40, 34, 108muM, the percentages of apoptotic cells were about 50%. CONCLUSIONS: Compounds 1-10, 12 and 14-15 were isolated for the first time from the P. heterophylla. This result enriches the previous studies on the chemical constituents of P. heterophylla. Compounds 1 and 15 were reported for the first time to have cytotoxic activities against tumor cells. Compounds 1, 5, 6, 7, 12, 15 showed cytotoxic activities against tumor cells. This result reveals that the active ingredient of P. heterophylla are composed of phenylpropanoids, iridoids and coumarins. This study provides some theoretical basis for the anti-tumor research of P. heterophylla.

Preliminary Quality Evaluation and Characterization of Phenolic Constituents in Cynanchi Wilfordii Radix.[Pubmed:29538318]

Molecules. 2018 Mar 14;23(3). pii: molecules23030656.

A new phenolic compound, 2-O-beta-laminaribiosyl-4-hydroxyacetophenone (1), was isolated from Cynanchi Wilfordii Radix (CWR, the root of Cynanchum wilfordii Hemsley), along with 10 known aromatic compounds, including Cynandione A (2), bungeisides-C (7) and -D (8), p-hydroxyacetophenone (9), 2',5'-dihydroxyacetophenone (10), and 2',4'-dihydroxyacetophenone (11). The structure of the new compound (1) was elucidated using spectroscopic methods and chemical methods. The structure of Cynandione A (2), including a linkage mode of the biphenyl parts that remained uncertain, was unambiguously confirmed using the 2D (13)C-(13)C incredible natural abundance double quantum transfer experiment (INADEQUATE) spectrum. Additionally, health issues related to the use of Cynanchi Auriculati Radix (CAR, the root of Cynanchum auriculatum Royle ex Wight) instead of CWR have emerged. Therefore, constituents present in methanolic extracts of commercially available CWRs and CARs were examined using UV-sensitive high-performance liquid chromatography (HPLC), resulting in common detection of three major peaks ascribed to Cynandione A (2), p-hydroxyacetophenone (9), and 2',4'-dihydroxyacetophenone (11). Thus, to distinguish between these ingredients, a thin-layer chromatography (TLC) method, combined with only UV irradiation detection, focusing on wilfosides C1N (12) and K1N (13) as marker compounds characteristic of CAR, was performed. Furthermore, we propose this method as a simple and convenient strategy for the preliminary distinction of CWR and CAR to ensure the quality and safety of their crude drugs.

Potential hypoglycemic effect of acetophenones from the root bark of Cynanchum wilfordii.[Pubmed:29480065]

Nat Prod Res. 2019 Aug;33(16):2314-2321.

Three new acetophenones, named cynwilforones A-C (1-3), together with Cynandione A (4) were isolated from the root bark of Cynanchum wilfordii (Maxim.) Hemsl. Their structures were deduced based on spectroscopic analysis and chemical methods. Compounds 1 and 4 exhibited potential hypoglycemic effects through inhibition of hepatic gluconeogenesis by down-regulating the expression of phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. This is the first report that acetophenones from the root bark of C. wilfordii possesses potential hypoglycemic activity in vitro.

Cynandione A inhibits lipopolysaccharide-induced cell adhesion via suppression of the protein expression of VCAM1 in human endothelial cells.[Pubmed:29328438]

Int J Mol Med. 2018 Mar;41(3):1756-1764.

Cynandione A (CA) is one of the most active compounds in the roots of Cynanchum wilfordii, the extracts of which have been used extensively in East Asia to treat various diseases including antiischemic stroke. In the present study, the antiadherent activity of CA in lipopolysaccharide (LPS)stimulated human umbilical vascular endothelial cells (HUVECs) was investigated. CA markedly reduced the expression of vascular adhesion molecule1 (VCAM1) by LPS in HUVECs. The results also demonstrated that CA significantly reduced the expression of proinflammatory and chemoattractant cytokines, including interleukin (IL)1beta, IL6, IL8, monocyte chemoattractant protein1 and tumor necrosis factoralpha, in LPSactivated human endothelial cells. CA inhibited the phosphorylation of mitogenactivated protein kinases, including the extracellular signalregulated kinase 1/2 and p38 kinases. It was found that CA decreased the IKK/IkappaBalpha phosphorylation of inhibitor of nuclear factor (NF)kappaB kinase/inhibitor of NFkappaBalpha, suppressed translocation of the NFkappaB p65 subunit into the nucleus and inhibited the transcriptional activity of NFkappaB. CA also decreased human monocyte cell adhesion to endothelial cells in LPSstimulated conditions. These results demonstrated that CA inhibited the protein expression of VCAM1 and proinflammatory cytokines by suppressing the transcriptional activity of NFkappaB. The results also suggested that CA may be important in the development of antiinflammatory drugs by inhibiting the expression of cell adhesion molecules.

Cynandione A attenuates neuropathic pain through p38beta MAPK-mediated spinal microglial expression of beta-endorphin.[Pubmed:28189715]

Brain Behav Immun. 2017 May;62:64-77.

Cynanchi Wilfordii Radix (baishouwu), a medicinal herb, has been widely used in Asia to treat a variety of diseases or illnesses. Cynandione A isolated from C. Wilfordii is the principle acetophenone and exhibits neuroprotective and anti-inflammatory activities. This study aims to evaluate the antihypersensitivity activities of Cynandione A in neuropathy and explored its mechanisms of action. Intrathecal injection of Cynandione A dose-dependently attenuated spinal nerve ligation-induced mechanical allodynia and thermal hyperalgesia, with maximal possible effects of 57% and 59%, ED50s of 14.9mug and 6.5mug, respectively. Intrathecal injection of Cynandione A significantly increased beta-endorphin levels in spinal cords of neuropathic rats and its treatment concentration-dependently induced beta-endorphin expression in cultured primary microglia (but not in neurons or astrocytes), with EC50s of 38.8 and 20.0muM, respectively. Cynandione A also non-selectively upregulated phosphorylation of mitogen-activated protein kinases (MAPKs), including p38, extracellular signal regulated kinase (ERK1/2), and extracellular signal regulated kinase (JNK) in primary microglial culture; however, Cynandione A-stimulated beta-endorphin expression was completely inhibited by the specific p38 activation inhibitor SB203580, but not by the ERK1/2 or JNK activation inhibitors. Knockdown of spinal p38beta but not p38alpha using siRNA also completely blocked Cynandione A-induced beta-endorphin expression in cultured microglial cells. Furthermore, Cynandione A-induced antiallodynia in neuropathy was totally inhibited by the microglial inhibitor minocycline, SB203580, anti-beta-endorphin antibody, and mu-opioid receptor antagonist CTAP (but not the kappa- or delta-opioid receptor antagonist). These results suggest that Cynandione A attenuates neuropathic pain through upregulation of spinal microglial expression beta-endorphin via p38beta MAPK activation.

Analysis of Cynandione A's Anti-Ischemic Stroke Effects from Pathways and Protein-Protein Interactome.[Pubmed:25955557]

PLoS One. 2015 May 8;10(5):e0124632.

Ischemic stroke is the third leading cause of death in the world. Our previous study found that Cynandione A (CYNA), the main component from the root of Cynanchum bungei, exhibits anti-ischemic stroke activity. In this work, we investigated the therapeutic mechanisms of CYNA to ischemic stroke at protein network level. First, PC12 cells and cerebellar granule neurons were prepared to validate the effects of CYNA against glutamate injury. Our experiments suggested that CYNA could dose-dependently mitigate glutamate-induced neurons neurotoxicity and inhibit glutamate-induced upregulation of KHSRP and HMGB1, further confirming the neuroprotective effects of CYNA in vivo. Then, on the pathway sub-networks, which present biological processes that can be impacted directly or in periphery nodes by drugs via their targets, we found that CYNA regulates 11 pathways associated with the biological process of thrombotic or embolic occlusion of a cerebral artery. Meanwhile, by defining a network-based anti-ischemic stroke effect score, we showed that CYNA has a significantly higher effect score than random counterparts, which suggests a synergistic effect of CYNA to ischemic stroke. This study may shed new lights on the study of network based pharmacology.

An ethanol root extract of Cynanchum wilfordii containing acetophenones suppresses the expression of VCAM-1 and ICAM-1 in TNF-alpha-stimulated human aortic smooth muscle cells through the NF-kappaB pathway.[Pubmed:25716870]

Int J Mol Med. 2015 Apr;35(4):915-24.

The root of Cynanchum wilfordii (C. wilfordii) contains several biologically active compounds which have been used as traditional medicines in Asia. In the present study, we evaluated the anti-inflammatory effects of an ethanol root extract of C. wilfordii (CWE) on tumor necrosis factor (TNF)-alpha-stimulated human aortic smooth muscle cells (HASMCs). The inhibitory effects of CWE on vascular cell adhesion molecule (VCAM)-1 expression under an optimum extraction condition were examined. CWE suppressed the expression of VCAM-1 and ICAM-1 and the adhesion of THP-1 monocytes to the TNF-alpha-stimulated HASMCs. Consistent with the in vitro observations, CWE inhibited the aortic expression of ICAM-1 and VCAM-1 in atherogenic diet-fed mice. CWE also downregulated the expression of nuclear factor-kappaB (NF-kappaB p65) and its uclear translocation in the stimulated HASMCs. In order to identify the active components in CWE, we re-extracted CWE using several solvents, and found that the ethyl acetate fraction was the most effective in suppressing the expression of VCAM-1 and ICAM-1. Four major acetophenones were purified from the ethyl acetate fraction, and two components, p-hydroxyacetophenone and Cynandione A, potently inhibited the expression of ICAM-1 and VCAM-1 in the stimulated HASMCs. We assessed and determined the amounts of these two active components from CWE, and our results suggested that the root of C. wilfordii and its two bioactive acetophenones may be used for the prevention and treatment of atherosclerosis and vascular inflammatory diseases.

Cynandione A attenuates lipopolysaccharide-induced production of inflammatory mediators via MAPK inhibition and NF-kappaB inactivation in RAW264.7 macrophages and protects mice against endotoxin shock.[Pubmed:25361770]

Exp Biol Med (Maywood). 2015 Jul;240(7):946-54.

Cynanchum wilfordii has been traditionally used in eastern Asia for the treatment of various diseases such as gastrointestinal diseases and arteriosclerosis. Cynandione A (CA), an acetophenone, is one of major constituents from roots of C. wilfordii. In the present study, the anti-inflammatory activities of CA were investigated in lipopolysaccharide (LPS)-treated RAW264.7 macrophages and LPS-administered C57BL/6 N mice. CA significantly decreased LPS-induced production of nitric oxide and prostaglandin E2 in a dose-dependent manner, while CA up to 200 muM did not exhibit cytotoxic activity. Our data also showed that CA significantly attenuated expression of iNOS and COX-2 in LPS-stimulated macrophages. CA inhibited phosphorylation of IkappaB-alpha and MAP kinases such as ERK and p38. Furthermore, we demonstrated that CA inhibited translocation of NF-kappaB to the nucleus, transcription of the NF-kappaB minimal promoter and NF-kappaB DNA binding activity. Administration of CA significantly decreased the plasma levels of pro-inflammatory cytokines such as TNF-alpha, IL-6, and IL-1beta in LPS-injected mice and improved survival of septic mice with lethal endotoxemia. These results demonstrate that CA has effective inhibitory effects on production of inflammatory mediators via suppressing activation of NF-kappaB and MAPK signaling pathways, suggesting that CA may be used as a potential anti-inflammatory agent for the prevention and treatment of inflammatory diseases.

Cynandione A from Cynanchum wilfordii attenuates the production of inflammatory mediators in LPS-induced BV-2 microglial cells via NF-kappaB inactivation.[Pubmed:25087960]

Biol Pharm Bull. 2014;37(8):1390-6.

Cynanchum wilfordii is one of most widely used medicinal plants in Oriental medicine for the treatment of various conditions. In the present study, we isolated Cynandione A (CA) from an extract of Cynanchum wilfordii roots (CWE) and investigated the effects of CA on the expression of inducible nitric oxide synthase (iNOS) and pro-inflammatory cytokines in lipopolysaccharide (LPS)-induced BV-2 microglial cells. CWE and CA significantly decreased LPS-induced nitric oxide production and the expression of iNOS in a concentration-dependent manner, while they (CWE up to 500 microg/mL and CA up to 80 microM) did not exhibit cytotoxic activity. Results from reverse transcription-polymerase chain reaction (RT-PCR) analysis and enzyme-linked immunosorbent assay (ELISA) showed that CA significantly attenuated the expression of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and IL-1beta in LPS-stimulated BV-2 cells. Furthermore, CA inhibited the phosphorylation of inhibitor kappa B-alpha (IkappaB-alpha) and translocation of nuclear factor-kappa B (NF-kappaB) to the BV-2 cell nucleus, indicating that CWE and CA may have effective anti-inflammatory activities via NF-kappaB inactivation in stimulated microglial cells.

Cynandione A mitigates ischemic injuries in rats with cerebral ischemia.[Pubmed:22309483]

J Neurochem. 2012 May;121(3):451-64.

Cynandione A, an acetophenone from the roots of Cynanchum auriculatum and other species in the genus attenuates neurotoxicity of a variety of neurotoxic agents such as l-glutamate in vitro. In this study, we sought to further characterize the neuroprotective effects of Cynandione A and other acetophenones from the roots of C. auriculatum in pheochromocytoma tumor cell line PC12 and investigate whether Cynandione A protected against ischemic injuries in rats with experimentally induced cerebral ischemia. Viability assays using the 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophen-yl)-2H-tetrazoli um monosodium salt method and lactate dehydrogenase (LDH) release assays showed that Cynandione A dose-dependently attenuated glutamate-induced cytotoxicity. Comparative proteomic analysis by two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight MS/MS of PC12 cells treated with Cynandione A showed 10 muM Cynandione A caused broad changes in protein expression in PC12 cells including down-regulation of high mobility group box 1 (HMGB1) and dihydropyrimidinase-like 2 (DPYSL2). Immunoblotting studies showed that 10 muM Cynandione A aborted glutamate-induced increase in DPYSL2 and HMGB1 levels in PC12 cells and 30 mg/kg Cynandione A also attenuated the rise in HMGB1 levels and mitigated DPYSL2 cleavage in brain tissues of rats with cerebral ischemia. Furthermore, rats with cerebral ischemia treated with 30 mg/kg Cynandione A exhibited markedly improved neurological deficit scores at 24 and 72 h compared with control and a 7.2% reduction in cerebral infarction size at 72 h (p < 0.05 vs. control). Our findings demonstrated that Cynandione A mitigated ischemic injuries and should be further explored as a neuroprotective agent for ischemic stroke.