Structurally modified Cyclovirobuxine-D Buxus alkaloids as effective analgesic agents through Cav3.2 T-Type calcium channel inhibition.

Cyclovirobuxine-D (CVB-D) is a Buxus alkaloid and a major active constituent in the Chinese medicinal herb Buxus microphylls. Traditionally, the natural alkaloid cyclovirobuxine-D has a long history of use as a traditional Chinese medicine for cardiovascular diseases as well as to treat a wide variety of medical conditions. As we found that CVB-D inhibited T-type calcium channels, we designed and synthesized a variety of fragments and analogues and evaluated them for the first time as new Cav3.2 inhibitors. Compounds 2-7 exhibited potency against Cav 3.2 channels, and two of them were more active than their parent molecules. As a result of the in vivo experiments, both compounds 3 and 4 showed significantly reduced writhes in the acetic acid-induced writhing test. Studies of molecular modeling have identified possible mechanism(s) of Cav3.2 binding. Moreover, the relationship between structure and activity was studied in a preliminary manner. Our results indicated that compounds 3 and 4 could play an important role in the discovery and development of novel analgesics.

Caged polycyclic polyprenylated acylphloroglucinols as Cav3.2 low voltage-gated Ca2+ channel inhibitors from Hypericum curvisepalum.

Five caged polycyclic polyprenylated acylphloroglucinols including an unprecedented octahydro-2,5-methanoindene skeleton (1) were discovered from Hypericum curvisepalum. Biologically, 1 and 2 are potent Cav3.2 T-type Ca2+ channel inhibitors with negligible effect on the cardiovascular antitarget, the human ether-à-go-go-related gene potassium channel. Additionally, 2 indicates strong antinociception in the mouse acetic acid writhing test.

Cyclovirobuxine D, a cardiovascular drug from traditional Chinese medicine, alleviates inflammatory and neuropathic pain mainly via inhibition of voltage-gated Cav3.2 channels.

Cyclovirobuxine D (CVB-D), the main active constituent of traditional Chinese medicine Buxus microphylla, was developed as a safe and effective cardiovascular drug in China. B. microphylla has also been used to relieve various pain symptoms for centuries. In this study, we examined and uncovered strong and persistent analgesic effects of cyclovirobuxine D against several mouse models of pain, including carrageenan- and CFA-induced inflammatory pain and paclitaxel-mediated neuropathic hypersensitivity. Cyclovirobuxine D shows comparable analgesic effects by intraplantar or intraperitoneal administration. Cyclovirobuxine D potently inhibits voltage-gated Cav2.2 and Cav3.2 channels but has negligible effects on a diverse group of nociceptive ion channels distributed in primary afferent neurons, including Nav1.7, Nav1.8, TRPV1, TPRA1, TRPM8, ASIC3, P2X2 and P2X4. Moreover, inhibition of Cav3.2, rather than Cav2.2, plays a dominant role in attenuating the excitability of isolated dorsal root ganglion neurons and pain relieving effects of cyclovirobuxine D. Our work reveals that a currently in-use cardiovascular drug has strong analgesic effects mainly via blockade of Cav3.2 and provides a compelling rationale and foundation for conducting clinical studies to repurpose cyclovirobuxine D in pain management.

Novel Meroterpenoids from Hypericum patulum: Highly Potent Late Nav1.5 Sodium Current Inhibitors.

Hypulatones A and B (1 and 2), two racemic meroterpenoids possessing an unprecedented spiro[benzofuran-2,1'-cycloundecan]-4'-ene-4,6(5H)-dione core, were characterized from Hypericum patulum. Compound 2 was found to significantly inhibit the late current of Nav1.5 (late INa, IC50 = 0.2 µM). Importantly, 2 exhibited remarkable separation (>100-fold) of late INa relative to peak INa and notable selectivity over Cav3.1, Kv1.5, and hERG. 1 showed comparable inhibition on late INa compared to that of 2 with poorer selectivity.

Congenetic Hybrids Derived from Dearomatized Isoprenylated Acylphloroglucinol with Opposite Effects on Cav3.1 Low Voltage-Gated Ca2+ Channel.

A hybrid of dearomatized isoprenylated acylphloroglucinol (DIAP) and monoterpenoid, hypatone A (1), together with its biosynthetic analogues 2-4 is characterized from Hypericum patulum. Structurally, 1 possesses an unprecedented spiro[bicyclo[3.2.1]octane-6,1'-cyclohexan]-2',4',6'-trione core as elucidated by extensive spectroscopic and X-ray crystallographic analyses. Biological studies reveal that compounds 1 and 2-4 produce opposite effects on Cav3.1 low voltage-gated Ca2+ channel, with 1 and 4, respectively, being the most potent Cav3.1 agonist and antagonist from natural products. Further studies suggest that compound 1 and its biogenetical precursor, 2, have the same binding site on Cav3.1 and that the rigid cagelike moiety at C-5 and C-6 is a key structural feature responsible for 1 being an agonist. Furthermore, 1 can normalize the pathological gating of a mutant Cav3.1 channel found in spinocerebellar ataxia 42 (SCA42), a hereditary neurodegenerative disorder with no available therapy. Collectively, our findings provide valuable tools for future studies on Cav3.1 physiology and pathophysiology, as well as afford possible leads for developing new drugs against SCA42, epilepsy, and pain.

Valepotriates From the Roots and Rhizomes of Valeriana jatamansi Jones as Novel N-Type Calcium Channel Antagonists.

The roots and rhizomes of Valeriana jatamansi have long been used as folk medicine in Asia and usually named as "Zhizhuxiang" in Chinese for the treatment of abdominal distention and pain. However, its active ingredients and molecular targets for treatment of abdominal pain remain unrevealed. Inhibitors of Cav2.2 N-type voltage-gated calcium channels (VGCCs) are actively sought after for their potential in treating pain, especially chronic pain. As far as we know, the method used for seeking analgesic active ingredient from plant material has rarely been reported. The analgesic potentials of the EtOH extract (0.01 mg/ml) of the roots and rhizomes of V. jatamansi and its EtOAc, n-BuOH and H2O soluble parts (0.01 mg/ml, respectively) were tested herein on Cav2.2, using whole-oocyte recordings in vitro by tow-electrode voltage clamp. The results indicated that the EtOAc-soluble part exhibited the most potent inhibition of Cav2.2 peak current (20 mv). The EtOAc-soluble part was then subjected to silica gel column chromatography (CC) and giving 9 fractions. Phytochemical studies were carried out by repeated CC and extensive spectroscopic analyses after the fraction (0.01 mg/ml) was identified to be active and got seventeen compounds (1-17). All isolates were then sent for further bioactive verification (1 and 3 at concentration of 10 µM, others at 30 µM). In addition, the selectivity of the active compounds 1 and 3 were tested on various ion channels including Cav1.2, Cav2.1 and Cav3.1 VGCCs and Kv1.2, Kv2.1, Kv3.1 and BK potassium channels. The results indicated that compound 1 and 3 (an abundant compound) inhibited Cav2.2 with an EC50 of 3.3 and 4.8 µM, respectively, and had weaker or no effect on Cav1.2, Cav2.1 and Cav3.1 VGCCs and Kv1.2, Kv2.1, Kv3.1 and BK potassium channels. Compounds 1 and 3 appear to act as allosteric modulators rather than pore blockers of Cav2.2, which may play crucial role in attenuating nociception. The results of present research indicated that the ethnopharmacological utilization of V. jatamansi for relieving the abdominal distention and pain may mediate through Cav2.2 channel. Our work is the first demonstration of inhibition of Cav2.2 by iridoids, which may provide a fresh source for finding new analgesics.

Pepluanols C-D, Two Diterpenoids with Two Skeletons from Euphorbia peplus.

Pepluanols C and D (1 and 2), featuring unprecedented 5/5/10 with out,out-[7.2.1]bicylcododecane core and 6/6/7/3 fused-ring skeletons, respectively, were isolated from Euphorbia peplus. Their chemical structures and absolute configurations were determined by a series of extensive spectroscopic methods, and X-ray diffraction analysis. In addition, pepluanols C and D showed 31.6 ± 8.3% and 30.5 ± 2.8% peak current inhibition on the Kv1.3 potassium channel at 30 µM.

New potential beneficial effects of actein, a triterpene glycoside isolated from Cimicifuga species, in breast cancer treatment.

Actein is a triterpene glycoside isolated from the rhizomes of Cimicifuga foetida (Chinese herb "shengma") which could inhibit the growth of breast cancer cells. Nevertheless, the effect of actein on angiogenesis, which is an essential step for tumor growth and metastasis, has never been reported. Hence, this study aimed to investigate the in vitro and in vivo effects of actein on angiogenesis using human microvascular endothelial cells (HMEC-1), matrigel plug and tumor-bearing mouse models. Our results showed that actein significantly inhibited the proliferation, reduced the migration and motility of endothelial cells, and it could suppress the protein expressions of VEGFR1, pJNK and pERK, suggesting that JNK/ERK pathways were involved. In vivo results showed that oral administration of actein at 10 mg/kg for 7 days inhibited blood vessel formation in the growth factor-containing matrigel plugs. Oral actein treatments (10-15 mg/kg) for 28 days resulted in decreasing mouse 4T1 breast tumor sizes and metastasis to lungs and livers. The apparent reduced angiogenic proteins (CD34 and Factor VIII) expressions and down-regulated metastasis-related VEGFR1 and CXCR4 gene expressions were observed in breast tumors. Our novel findings provide insights into the use of actein for development of anti-angiogenic agents for breast cancer.

KHF16 is a Leading Structure from Cimicifuga foetida that Suppresses Breast Cancer Partially by Inhibiting the NF-κB Signaling Pathway.

Triterpenoids extracted from Cimicifuga foetida have been reported to inhibit cancer by inducing cell cycle arrest and apoptosis. In this study, KHF16 (24-acetylisodahurinol-3-O-ß-D-xylopyranoside), a cycloartane triterpenoid isolated from the rhizomes of C. foetida, showed potent anti-cancer activity in multiple ERα/PR/HER2 triple-negative breast cancer (TNBC) cell lines. KHF16 significantly induces cell cycle G2/M phase arrest and apoptosis in both MDA-MB-468 and SW527 TNBC cell lines. KHF16 reduces the expression levels of XIAP, Mcl-1, Survivin and Cyclin B1/D1 proteins. Importantly, KHF16 inhibits TNFα-induced IKKα/ß phosphorylation, IKBα phosphorylation, p65 nuclear translocation and NF-κB downstream target gene expression, including XIAP, Mcl-1 and Survivin, in TNBC cells. These results suggest that KHF16 may inhibit TNBC by blocking the NF-κB signaling pathway in part.

Six new physalins from Physalis alkekengi var. franchetii and their cytotoxicity and antibacterial activity.

Six new physalin steroids, 7ß-methoxylisophysalin B (1), 7ß-methoxylphysalin C (2), physalin V (3), physalin VI (4), physalin VII (5), isophysalin I (6), together with 20 known physalins (7-26) were isolated from calyces of Physalis alkekengi var. franchetii. Structures of the new compounds were revealed through 1D and 2D NMR and mass spectroscopic methods. Compounds 1-26 were evaluated for cytotoxicity against human HL-60, SMMC-7721, A-549, MCF-7 and SW-480, and the results indicated that compounds 8, 11, and 14 displayed potent cytotoxicities (IC50<5µM) in vitro. Further antibacterial assay indicated that compounds 8, 14, and 19 showed high antibacterial activities against Bacillus subtilis and Escherichia coli.

Three Minor Diterpenoids with Three Carbon Skeletons from Euphorbia peplus.

Euphorbia peplus has been used in traditional medicine to treat asthma and psoriasis. Three highly modified diterpenoids, namely, pepluacetal (1) and pepluanol A-B (2-3), have been isolated and identified from this plant. Compounds 1-3 exhibit unprecedented 5/4/7/3, 5/6/7/3, and 5/5/8/3 ring systems, respectively. Their structures with absolute configurations were determined by spectroscopic analyses, X-ray crystallography, and electronic circular dichroism calculations. Since Kv1.3 is a validated target for the treatment of autoimmune diseases, such as multiple sclerosis, type-1 diabetes, asthma, and psoriasis, Kv1.3 was studied in terms of its response to the new compounds. All three compounds inhibit Kv1.3, with compound 3 being the most effective with an IC50 value of 9.50 µM.

New anti-angiogenic leading structure discovered in the fruit of Cimicifuga yunnanensis.

Cimyunnins A-C (1-3), characterized with an unusual fused cyclopentenone ring G, together with cimyunnin D (4), possessing a highly rearranged γ-lactone ring F, were characterized from the fruit of Cimicifuga yunnanensis. Their structures were elucidated by spectroscopic analysis, X-ray diffraction, and density functional theory calculations. In addition, cimyunnin A exhibited comparable anti-angiogenic activities to those of sunitinib, a clinically-used first-line angiogenesis inhibitor, in the in vitro and ex vivo studies.

Cytotoxic 9,19-cycloartane triterpenes from the aerial parts of Cimicifuga yunnanensis.

Six new 9,19-cycloartane triterpenes (1-6) were isolated from the aerial parts of Cimicifuga yunnanensis. The new chemical structures were determined by extensive analyses of 1D and 2D NMR spectroscopy. Compounds 1 and 2 are the first 9,19-cycloartane triterpenes characterized by CH3-18 shifting from C-13 to C-12 in the Cimicifuga spp. The evaluation of inhibition activity against human HL-60, SMMC-7721, A-549, MCF-7, and SW480 cell lines indicated that compounds 1-6 showed different levels of cytotoxic activities with IC50 values ranging from 1.2 to 27.8 µm.

New 9, 19-cycloartane triterpenoid from the root of Cimicifuga foetida.

AIM: To study the 9, 19-cycloartane triterpenes from the roots of Cimicifuga foetida. METHOD: Chromatographic separations by silica gel, C18 reversed phase silica gel, and high-performance liquid chromatography (HPLC) were used. All of the structures were elucidated on the basis of spectroscopic analysis and chemical methods. RESULTS: Five 9, 19-cycloartane triterpenes, (3ß, 12ß, 15α, 24R)-12, 2'-diacetoxy-24, 25-epoxy-15-hydroxy-16, 23-dione-3-O-α-L-arabinopyranoside (1), actein (2), 23-epi-26-deoxyactein (3), asiaticoside B (4), and 12ß-hydroxycimigenol (5) were isolated from the roots of Cimicifuga foetida. CONCLUSION: Compound 1 is a new triterpene with two acetoxy groups at C-2' and C-12.

Triterpenes from the aerial parts of Cimicifuga yunnanensis and their antiproliferative effects on p53(N236S) mouse embryonic fibroblasts.

Nine new triterpene derivatives, yunnanterpenes A-F (1-6), 15,16-seco-cimiterpenes A and B (7, 8), and cimilactone C (9), and 15 known analogues (10-24) were isolated from the aerial parts of Cimicifuga yunnanensis. The new structures were established using a combination of MS, NMR, and single-crystal X-ray diffraction techniques. WT MEFs (wild-type mouse embryonic fibroblasts) and tumorigenic cell lines p53(-/-)+H-RasV12 and p53(-/-)+p53(N236S)+H-RasV12 were used for evaluating active structures, targeting p53(N236S) (corresponding to p53(N239S) in humans) mutation. Compound 5 showed nonselective activities against these cell lines, with IC50 values of 5.8, 8.6, and 6.0 µM, respectively. Compound 4 exhibited greater selectivity against the p53(-/-)+p53(N236S)+H-RasV12 cells (IC50 5.5 µM) than against the WT MEFs cells (IC50 14.3 µM).

Cytotoxic cycloartane triterpenes of the traditional Chinese medicine "shengma" (Cimicifuga dahurica).

Twelve new 9,19-cycloartane triterpenes (1-12), together with fourteen known compounds (13-26), were isolated from the roots of Cimicifuga dahurica. Their structures were determined by application of spectroscopic analyses and chemical methods. Biological evaluation of the compounds against human HL-60, SMMC-7721, A549, MCF-7, and SW-480 cell lines indicated that cimigenol-type glycosides (1-3, 19, and 20) showed broad-spectrum and moderate cytotoxicities, with IC50 values ranging from 4.2 to 14.5 µM. Meanwhile, cimigenol-type aglycones (6-8, 15, 16, and 18) exhibited broad-spectrum and week cytotoxicities, having IC50 values around 20 µM. In addition, the key points of the structure-activity relationships of aglycones with a cimigenol skeleton were discussed.

A cytotoxic 4α-methyl steroid from the aerial parts of Cimicifuga foetida L.

A new 4α-methyl sterol, cimisterol A (1), together with five known compounds (2-6), were isolated from the aerial parts of Cimicifuga foetida L. The new compound's structure was determined with the help of extensive 1D and 2D NMR spectroscopy. Compound 1 exhibited broad-spectrum and potent cytotoxic activities against human HL-60, Jurkat, K562, U937, HepG-2, and SGC-7091 cell lines, with IC(50) values of 7.23, 2.89, 6.88, 3.38, 4.21, and 4.89 µM, respectively. Compound 3 showed moderate to weak activities to all cell lines, except for SGC-7091, having IC(50) values ranging from 13.37 to 17.72 µM. This is the first time a cytotoxic 4α-methyl sterol constituent was discovered from Cimicifuga spp.

Cycloartane triterpenoids from Cimicifuga yunnanensis induce apoptosis of breast cancer cells (MCF7) via p53-dependent mitochondrial signaling pathway.

The present study was carried out to investigate the antitumor activity of five cycloartane triterpenoids isolated from Cimicifuga yunnanensis on the breast cancer cell line MCF7 and its corresponding drug resistant subline R-MCF7, including cimigenol-3-O-ß-D-xylopyranoside (compound 1), 25-O-acetylcimigenol-3-O-ß-D-xylopyranoside (compound 2), 25-chlorodeoxycimigenol-3-O-ß-D-xylopyranoside (compound 3), 25-O-acetylcimigenol-3-O-α-L-arabinopyranoside (compound 4) and 23-O-acetylcimigenol-3-O-ß-D-xylopyranoside (compound 5). The results showed that compounds 2-5 have relatively high antitumor activity on both MCF7 and R-MCF7 cells. The involvement of apoptosis as a major cause of cycloartane triterpenoids-induced cell death was further confirmed. The results of RT-PCR showed that compounds 2-5 increased the expression of p53 and bax, which led to the loss of mitochondrial potential and then resulted in the activation of caspase-7. These findings collectively demonstrated that compounds 2-5 induced apoptosis of MCF7 via p53-dependent mitochondrial pathway.

Cytotoxic chemical constituents from the roots of Cimicifuga foetida. [corrected].

Seven new 9,19-cycloartane triterpene glycosides, 25-O-acetylcimigenol-3-O-[2'-O-(E)-2-butenoyl]-beta-d-xylopyranoside (1), 25-O-acetylcimigenol-3-O-[4'-O-(E)-2-butenoyl]-beta-d-xylopyranoside (2), 25-O-acetylcimigenol-3-O-[3'-O-acetyl]-beta-d-xylopyranoside (3), 25-O-acetylcimigenol-3-O-[4'-O-acetyl]-beta-d-xylopyranoside (4), 25-O-acetyl-12beta-acetoxycimigenol-3-O-beta-d-xylopyranoside (5), 3'-O-acetylactein (6), and 3'-O-acetyl-23-epi-26-deoxyactein (7), together with eight known compounds (8-15), were isolated from the roots of Cimicifuga fetida. Their structures were established by spectroscopic and chemical methods. Most of these compounds showed more selective and higher cytotoxicity against the human HepG2 cell line than against the MCF7, HT29, and MKN28 cell lines. Compounds 2, 3, and 7 exhibited significant cytotoxicity against HepG2 cells, with IC(50) values of 1.29, 0.71, and 1.41 microM, respectively.

Trinor-cycloartane glycosides from the rhizomes of Cimicifuga foetida.

Three new trinor-cycloartane glycosides, 15alpha-hydroxy-16-dehydroxy-16(24)-en-foetidinol-3-O-beta-D-xylopyranoside (1), 28-hydroxy-foetidinol-3-O-beta-D-xylopyranoside (2) and foetidinol-3-O-beta-D-xylopyranosyl-(1"-->3')-beta-D-xylopyranoside (3) together with the known compound foetidinol-3-O-beta-D-xylopyranoside (4) were isolated from the n-BuOH fraction of the roots of Cimicifuga foetida. Their structures were elucidated on the basis of spectroscopic and chemical reaction data.