Ptehosides A-I: Nine undescribed iridoids with in vitro cytotoxicity from the whole plant of Pterocephalus hookeri (C.B. Clarke) Höeck.

Nine previously undescribed iridoids, ptehosides A-I (1-9), together with 12 known ones (10-21), were isolated from Pterocephalus hookeri (C.B. Clarke) Höeck. Their structures were elucidated using various spectroscopic methods including HR-ESI-MS, NMR, UV, IR and CD, etc. The cytotoxic activities of all isolates were evaluated using MTT method in three human cancer cell lines (Caco2, Huh-7, and SW982). As result, compound 9 exhibited substantial inhibitory activity on Caco2, Huh-7, and SW982 cells with IC50 values of 1.17 ± 0.05, 1.15 ± 0.05 and 1.14 ± 0.04 µM, respectively. A preliminary mechanism study showed that 9 arrested the cell cycle of SW982 cells in the G0/G1 phase and induced apoptosis by upregulating Bax expression and downregulating Bcl-2 expression.

Discovering a mitochondrion-localized BAHD acyltransferase involved in calystegine biosynthesis and engineering the production of 3ß-tigloyloxytropane.

Solanaceous plants produce tropane alkaloids (TAs) via esterification of 3α- and 3ß-tropanol. Although littorine synthase is revealed to be responsible for 3α-tropanol esterification that leads to hyoscyamine biosynthesis, the genes associated with 3ß-tropanol esterification are unknown. Here, we report that a BAHD acyltransferase from Atropa belladonna, 3ß-tigloyloxytropane synthase (TS), catalyzes 3ß-tropanol and tigloyl-CoA to form 3ß-tigloyloxytropane, the key intermediate in calystegine biosynthesis and a potential drug for treating neurodegenerative disease. Unlike other cytosolic-localized BAHD acyltransferases, TS is localized to mitochondria. The catalytic mechanism of TS is revealed through molecular docking and site-directed mutagenesis. Subsequently, 3ß-tigloyloxytropane is synthesized in tobacco. A bacterial CoA ligase (PcICS) is found to synthesize tigloyl-CoA, an acyl donor for 3ß-tigloyloxytropane biosynthesis. By expressing TS mutant and PcICS, engineered Escherichia coli synthesizes 3ß-tigloyloxytropane from tiglic acid and 3ß-tropanol. This study helps to characterize the enzymology and chemodiversity of TAs and provides an approach for producing 3ß-tigloyloxytropane.

Dracotangusions A and B, two new sesquiterpenes from Dracocephalum tanguticum Maxim. with anti-inflammatory activity.

Two new guaiane sesquiterpenoids were isolated from the dried aerial parts of Dracocephalum tanguticum Maxim., named as dracotangusions A (1) and B (2), together with four known sesquiterpenoids, which were identified as Curcumenone (3), (4Z,7Z,9Z)-11-Hydroxy-4,7,9-germacratriene-1,6-dione (4), Kobusone (5), and (1S,10S), (4S, 5S)-(+)-germacrone-1(10)-4-diepoxide (6). The structures of isolates were determined by UV, IR, HR-ESI-MS, and NMR analysis. What is noteworthy is that four known sesquiterpenoids were isolated for the first time from the genus of Dracocephalum L. All compounds inhibited the extremely significant difference (p < 0.01) in anti-inflammatory activity, suggesting that these compounds may be promising candidates as an anti-inflammatory agent.

Berberisides A-D: three novel prenylated benzoic acid derivatives and a clerodane glycoside from Berberis tsarica aherndt.

Three undescribed prenylated benzoic acid derivatives berberisides A-C (1-3) and a new clerodane glycoside berberiside D (4) were isolated from Berberis tsarica Aherndt. Their structures were elucidated on the basis of extensive NMR and HR-ESI-MS analysis. The in vitro cytotoxic activities of all isolates were studied against lung carcinoma A549, hepatocellular carcinoma HepG2 and breast carcinoma MDA-MB-231 cell lines. Among them, compounds 1 and 4 exhibited anti-proliferative effects against three tumor cell lines with IC50 ranging from 28.97 ± 2.18 to 35.83 ± 0.72 µM.

Pterocephin A, a novel Triterpenoid Saponin from Pterocephalus hookeri induced liver injury by activation of necroptosis.

BACKGROUND: Pterocephalus hookeri (C. B. Clarke) Höeck, a Tibetan medicine widely used for treatment of rheumatoid arthritis, was recorded in Chinese Pharmacopoeia (2020 version) with slight toxicity. The liver injury was observed in mice with administration of n-butanol extract (BUE) in our previously study. However, the toxic components and the mechanism were still unrevealed. PURPOSE: The present study was aimed to isolate and structural elucidate of the toxic compound pterocephin A (PA), as well as evaluate its liver toxicity and investigate its mechanism. METHODS: PA was isolated from the BUE of P. hookeri. Its structure was determined by analysis of HRMS, NMR and ECD data. L-02 cellular viability, LDH, ALT, AST, ROS, intracellular Ca2+ and the fluidity of cell membrane were assessed by multifunctional microplate reader. The PI staining, cell membrane permeability assessment, and mitochondrial fluorescence staining analysis were determined through the fluorescence microscope. Liver samples for mice were assessed by pathological and immunohistochemistry analysis. Expression levels of indicated proteins were measured by western blotting assays. RESULTS: PA was determined as a previously undescribed oleanolane-type triterpenoid saponin. In vitro study revealed PA significantly induced hepatotoxicity by inhibition of L-02 cell growth, abnormally elevation of ALT and AST. Mechanically, PA induced the damage of cell membrane, fragmentation of mitochondria, and subsequently increase of intracellular Ca2+ and ROS levels, which trigged by necroptosis with the activation of RIP1 and NF-κB signaling pathways. In vivo study confirmed PA could induce liver injury in mice with observation of the body weight loss, increasing of serum ALT and AST, and the histopathological changes in liver tissues. CONCLUSION: Our present study indicated that PA was an undescribed toxic constituent in P. hookeri to induce liver injury in mice by activation of necroptosis and inflammation. And the findings are of great significance for the clinical use safely of this herb.

Ptehoosines A and B: Two new sesamin-type sesquilignans with antiangiogenic activity from Pterocephalus hookeri (C.B. Clarke) Höeck.

Two undescribed sesamin-type sesquilignans ptehoosines A (1) and B (2), together with 4 known lignans (3-6), were isolated from Pterocephalus hookeri (C.B. Clarke) Höeck which was widely used as traditional Tibetan medicine for treatment of rheumatoid arthritis. Their structures were determined by HR-ESI-MS, NMR analysis and CD experiment. The in vitro antiangiogenic effect of all isolated compounds against human umbilical vein endothelial cells (HUVECs) were evaluated by CCK-8 assay. Among them, compound 1 exhibited significant proliferative inhibition on HUVECs with IC50 value of 32.82 ± 0.99 µM. Further in vitro study indicated 1 could arrest cell cycle at G0/G1 phase and reduce the migration of HUVECs. In vivo experiment exhibited 1 could inhibit tail vessels plexus in zebrafish. The above finding suggested that 1 was a promising lead compound against RA by inhibiting of angiogenesis.

Dehydrodiconiferyl alcohol, a lignan from Herpetospermum pedunculosum, alleviates cholestasis by activating pathways associated with the farnesoid X receptor.

BACKGROUND: In our previous study, we demonstrated the hepatoprotective effect of Herpetospermum pedunculosum in cholestatic rats. A bioassay-guided study also led to the identification and isolation of a lignan, dihydrodiconiferyl alcohol (DA) from the seeds of H. pedunculosum. PURPOSE: To investigate whether DA could alleviate cholestasis and determine the mechanisms underlying such action. METHODS: Male Sprague-Dawley (SD) rats were administered with DA (10, 20 or 40 mg/kg) intragastrically once daily for 7 days prior to treatment with α-naphthylisothiocyanate (ANIT) (60 mg/kg). We then evaluated the levels of a range of serum indicators, determined bile flow, and carried out histopathological analyses. Western blotting was then used to investigate the levels of inflammatory mediators and the Farnesoid X Receptor (FXR), proteins involved in the downstream biosynthesis of bile acids, and a range of transport proteins. Molecular docking was used to simulate the interaction between DA and FXR. Cell viability of human hepatocytes (L-02) cells was determined by MTT. Then, we treated guggulsterone-inhibited L-02 cells, Si-FXR L-02 cells, and FXR-overexpression cells with the FXR agonist GW4064 (6 µM) or DA (25, 50 and 100 µM) for 24 h before detecting gene and protein expression by RT-PCR and western blotting, respectively. RESULTS: DA significantly attenuated ANIT-induced cholestasis in SD rats by reducing liver function indicators in the serum, increasing bile flow, improving the recovery of histopathological injuries in the liver, and by alleviating pro-inflammatory cytokines in the liver. DA also increased the expression levels of FXR and altered the levels of downstream proteins in the liver tissues, thus indicating that DA might alleviate cholestasis by regulating the FXR. Molecular docking simulations predicted that DA was as an agonist of FXR. In vitro mechanical studies further showed that DA increased the mRNA and protein expression levels of FXR, Small Heterodimer Partner 1/2, Bile Salt Export Pump, Multidrug Resistance-associated Protein 2, and Na+/taurocholate Co-transporting Polypeptide, in both guggulsterone-inhibited and Si-FXR L-02 cells. Moreover, DA enhanced the mRNA and protein expression of FXR, and its downstream genes and proteins, in L-02 cells containing an FXR-overexpression plasmid. CONCLUSION: DA may represent an effective agonist for FXR has significant therapeutic potential for the treatment of cholestatic liver injury.

Pharmacokinetics and tissue distribution study of herpetotriol in rats by ultrahigh-performance liquid chromatography with tandem mass spectrometry.

Herpetotriol, a typical lignan in Herpetospermum pedunculosum Wall's seeds that has long been used to treat icterhepatitis and indigestion and other related diseases in Tibet, is of potential hepatoprotection. This study aims to study the pharmacokinetics features of herpetotriol, including the blood drug concentration - time curve and tissue distribution. The ultrahigh-performance liquid chromatography with tandem mass spectrometry method was established to detect herpetotriol concentration in plasma and tissues, and the method showed good linearity from 10 to 2000 ng/mL (r ≥ 0.9972) and sensitivity (≥10 ng/mL). Our blood drug concentration - time curve indicated that herpetotriol was distributed quickly in rats with a Tmax value at about 0.083 h and eliminated rapidly with a clearance rate at 98.13 ± 8.05 and 137.04 ± 9.48 L·h-1·kg-1 with doses of 5 and 2.5 mg/kg, respectively. Although herpetotriol was detectable in all tested tissues, it has a higher concentration in liver than in heart, lung, spleen, and kidney, which is in line with its hepatoprotection. The pharmacokinetics features uncovered by the present study could provide more information for future pharmacological and toxicological study of herpetotriol.

Cytotoxic lignans from the stems of Herpetospermum pedunculosum.

A bioassay-guided chemical investigation on the ethyl acetate extract of the stems of Herpetospermum pedunculosum led to the isolation and identification of 22 lignans including 6 previously undescribed ones, herpetosiols A-F. Their structures including stereochemistries were elucidated by analysis of NMR, HRMS and ECD data. The in vitro cytotoxic activities of all isolates were studied against human gastric carcinoma SGC7901, lung carcinoma A549, breast carcinoma MDA-MB-231 and hepatocellular carcinoma HepG2 cell lines. Among them, eight lignans exhibited anti-proliferative effects against four tumor cell lines with IC50 ranging from 1.7 ±â€¯0.1 to 32.6 ±â€¯1.1 µM. Hedyotol-B displayed potent inhibitory effect with IC50 values of 1.7 ±â€¯0.1 µM against SGC7901 and 6.1 ±â€¯0.5 µM against A549, respectively.

Evaluation of the Liver Toxicity of Pterocephalus hookeri Extract via Triggering Necrosis.

Pterocephalus hookeri (C. B. Clarke) Höeck, recorded in the Chinese Pharmacopoeia (2015 version) as a Tibetan medicine for the treatment of various diseases, especially rheumatoid arthritis, was believed to possess a slight toxicity. However, hardly any research has been carried out about it. The present study aimed to evaluate the toxicity in vivo and in vitro. Toxicity was observed by the evaluation of mice weight loss and histopathological changes in the liver. Then, the comparison research between ethyl acetate extract (EAE) and n-butanol extract (BUE) suggested that liver toxicity was mainly induced by BUE. The mechanical study suggested that BUE-induced liver toxicity was closely associated with necrosis detected by MTT and propidium iodide (PI) staining, via releasing lactate dehydrogenase (LDH), reducing the fluidity, and increasing the permeability of the cell membrane. Western blot analysis confirmed that the necrosis occurred molecularly by the up-regulation of receptor-interacting protein kinase 1 (RIP1) and receptor-interacting protein kinase 3 (RIP3), as well as the activation of the nuclear factor-kappa-gene binding (NF-κB) signaling pathway in vivo and in vitro. This finding indicated that the liver toxicity induced by BUE from P. hookeri was mainly caused by necrosis, which provides an important theoretical support for further evaluation of the safety of this folk medicine.

Sweroside Alleviated LPS-Induced Inflammation via SIRT1 Mediating NF-κB and FOXO1 Signaling Pathways in RAW264.7 Cells.

Pterocephalus hookeri was used as a traditional Chinese medicine for the treatment of rheumatoid arthritis. Sweroside was a main iridoid isolated from P. hookeri. The present study aimed to investigate the anti-inflammatory effect mechanism of sweroside. In RAW264.7 cells induced by lipopolysaccharide (LPS), the abnormal proliferation, the NO content increase, and the downregulated Sirtuin1 (SIRT1) expression were observed. Sweroside could alleviate the inflammation by inhibiting cell proliferation through arresting the cell cycle at the G0/G1 phase, by suppressing pro-inflammatory cytokines and by promoting anti-inflammatory cytokines in LPS-induced RAW264.7 cells. Further mechanism research indicated that sweroside could activate the SIRT1, then suppress the nuclear factor-kappa B (NF-κB) and promote the Forkhead transcription factor O1 (FOXO1) signaling pathways. The present study indicated that sweroside may be the main anti-inflammatory constituent of P. hookeri and a promising candidate for anti-inflammation therapy.

[A new napthalenone from roots of Rumex nepalensis].

A new napthalenone, rumexone A (1), was isolated from the roots of Rumex nepalensis. The structure of 1 was elucidated by extensive spectroscopic analyses, including 1D and 2D NMR spectra and MS data. Its cytotoxic effect was evaluated using four clinically relevant human cancer cell lines, gastric carcinoma SGC7901, breast carcinoma MDA-MB-231, lung carcinoma A549, and hepatocellular carcinoma HepG2.

Protective effects of petroleum ether extracts of Herpetospermum caudigerum against α-naphthylisothiocyanate-induced acute cholestasis of rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The ripe seeds of Herpetospermum caudigerum have been used in Tibetan folk medicine for treatment of bile or liver diseases including jaundice, hepatitis, intumescences or inflammation. Previously reports suggested that the seed oil and some lignans from H. caudigerum exhibited protective effects against carbon tetrachloride (CCl4)-induced hepatic damage in rats, which may be related to their free radical scavenging effect. However, the protective effect of H. caudigerum against cholestasis is still not revealed. The aim of the present study was to investigate the pharmacological effect and the chemical constituents of the petroleum ether extract (PEE) derived from H. caudigerum against α-naphthylisothiocyanate (ANIT)-induced acute cholestasis in rats. MATERIALS AND METHODS: Male cholestatic Sprague-Dawley (SD) rats induced by ANIT (60mg/kg) were orally administered with PEE (350, 700 and 1400mg/kg). Levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), γ-Glutamyl transpeptidase (γ-GTP), total bilirubin (TBIL), direct bilirubin (DBIL) and total bile acid (TBA), as well as bile flow, and histopathological assay were evaluated. Hepatic malondialdehyde (MDA), myeloperoxidase (MPO), superoxide dismutase (SOD), glutathione S-transferase (GST), and nitric monoxide (NO) in liver were measured to explore the possible protective mechanisms. Phytochemical analysis of PEE was performed by gas chromatography-mass spectrometer (GC-MS). RESULTS: PEE have exhibited significant and dose-dependent protective effect on ANIT-induced liver injury by reduce the increases in serum levels of ALT, AST, ALP, γ-GTP, TBIL, DBIL and TBA, restore the bile flow in cholestatic rats, and reduce the severity of the pathological tissue damage induced by ANIT. Hepatic MDA, MPO and NO contents in liver tissue were reduced, while SOD and GST activities were elevated in liver tissue. 49 compounds were detected and 39 of them were identified by GC-MS analysis, in which long-chain fatty acids were the main constituents. CONCLUSIONS: PEE exhibited a dose-dependently protective effect on ANIT-induced liver injury in cholestatic rats with the potential mechanism of attenuated oxidative stress in the liver tissue, and the possible active compounds were long-chain fatty acids.