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The first rate-limiting enzyme of the serine synthesis pathway (SSP), phosphoglycerate dehydrogenase (PHGDH), is hyperactive in multiple tumors, which leads to the activation of SSP and promotes tumorigenesis. However, only a few inhibitors of PHGDH have been discovered to date, especially the covalent inhibitors of PHGDH. Here, we identified withangulatin A (WA), a natural small molecule, as a novel covalent inhibitor of PHGDH. Affinity-based protein profiling identified that WA could directly bind to PHGDH and inactivate the enzyme activity of PHGDH. Biolayer interferometry and LC–MS/MS analysis further demonstrated the selective covalent binding of WA to the cysteine 295 residue (Cys295) of PHGDH. With the covalent modification of Cys295, WA blocked the substrate-binding domain (SBD) of PHGDH and exerted an allosteric effect to induce PHGDH inactivation. Further studies revealed that with the inhibition of PHGDH mediated by WA, the glutathione synthesis was decreased and intracellular levels of reactive oxygen species (ROS) were elevated, leading to the inhibition of tumor proliferation. This study indicates WA as a novel PHGDH covalent inhibitor, which identifies Cys295 as a novel allosteric regulatory site of PHGDH and holds great potential in developing anti-tumor agents for targeting PHGDH., Graphical abstract WA is a novel covalent-allosteric inhibitor of phosphoglycerate dehydrogenase (PHGDH), and inhibits the serine synthesis pathway and proliferation of colon cancer cells with overexpression of PHGDH.Image 1

Graphical abstract, Introduction Previously, we have reported a withanolide-type steroid, named tubocapsenolide A (TA), which shows potent anti-proliferative activity in several cancer cell lines. However, its inhibitory effect on the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway and therapeutic potential on osteosarcoma have not been reported. Objectives In the present study, we aimed to investigate the effect and molecular mechanism of TA in osteosarcoma. Methods The biological functions of TA in U2OS cells were investigated using colony formation, 5-ethynyl-20-deoxyuridine (EDU) staining, and cell cycle/apoptosis assays. The interaction between TA and Src homology 2 phosphatase 2 (SHP-2) was detected by enzyme activity and validated by target-identification methods such as drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), and biolayer interferometry (BLI). The in vivo anti-tumor efficacy of TA was analyzed in the xenograft tumor model. Western blotting analysis was performed to detect the protein expression levels. Results TA exhibited antitumor activity against osteosarcoma both in vitro and in vivo by regulating the JAK/STAT3 signaling pathway. Mechanically, TA interacted with SHP-2 directly and activated its phosphatase activity. Importantly, protein tyrosine phosphatase (PTP) inhibitor, SHP-2 inhibitor, and SHP-2 siRNA could reverse the inhibitory effect of TA on the JAK/STAT3 signaling pathway and restored the TA-induced cell death. Conclusion TA activated the phosphatase activity of SHP-2, which resulted in the inhibition of the JAK/STAT3 pathway and contributed to the antitumor efficacy of TA. Collectively, these findings suggested that TA could serve as a novel therapeutic agent for the treatment of osteosarcoma.

Six new oligomeric neolignans including two trimeric neolignans (1 and 2) and four dimeric neolignans (3-6) were isolated from the leaves of Magnolia officinalis var. biloba. Their structures were determined based on HR-ESIMS and NMR data, as well as electronic circular dichroism (ECD) calculations. Compound 1 is formed from two obovatol moieties directly linked to an aromatic ring of the remaining obovatol moiety, which is an unprecedented type of linkage between monomers. All isolates were assessed for their inhibitory effects on NO production in LPS-stimulated RAW 264.7 macrophage cells. Compounds 1 and 3 showed significantly inhibitory activities with IC50 values of 6.04 and 3.26 μmol·L-1, respectively.

Four new highly oxidized sesquiterpene lactones, Argyoxynolides A–D, along with two known analogs were isolated from the leaves of Artemisia argyi H.Lev. & Vaniot. Their structures were characterized utilizing extensive spectroscopic methods. All isolates were evaluated for the LPS-induced nitric oxide (NO) production inhibitory activity and one compound exhibited activity with IC50 = 37.95 ± 4.39 μM.

On-demand therapy following effective tumor detection would considerably reduce the side effects of traditional chemotherapy. DT-diaphorase (DTD), whose level is strongly elevated in various tumors, is a cytosolic flavoenzyme that promotes intracellular reactive oxygen species (ROS) generation via the redox cycling of hydroquinones. Incorporation of the DTD-responsive substrate to the structures of the probe and prodrug may facilitate the tumor detection and therapy. Herein, we established an multifunctional drug delivery nanosystem (HTLAC) that rapidly responds to the DTD enzyme, leads to the early-stage precise detection and termination of tumors. Firstly, the synthesis of DTD-responsive withaferin A (DT-WA) and indocyanine green (DT-Cy5) was performed. In the presence of DTD, WA, which produces ROS in cells, was released from DT-WA, and the red fluorescence of DT-Cy5 was detected for tumor imaging. Additionally, these DTD enzyme reaction processes of DT-WA and DT-Cy5 induced ROS. The self-burst of ROS generation by the two enzyme reaction processes as well as the released WA then led to the apoptosis of tumor cells. To increase the bioavailability and tumor targeting of drugs, cell-penetrating peptide and hyaluronic acid functionalized liposomes were used to encapsulate the drugs. The detailed in vitro and in vivo assays showed that HTLAC achieved enhanced tumor detection and superior antitumor efficiency. According to above outcomes, results showed that HTLAC might provide an efficacious approach for the fabrication of enzyme-triggering nanosystems to detect tumor and induce the self-burst of ROS for an efficient tumor treatment. STATEMENT OF SIGNIFICANCE: We have fabricated a HTLAC nanosystem to address the need of bursting reactive oxygen species (ROS) generation within tumor site. Our goal uniquely aims at not only augmentation of ROS-inducing anticancer efficacy, but also to meet the challenges of tumor dynamic detection in the clinical practices. In this work, the DT-diaphorase responsive withaferin A (DT-WA) and indocyanine green (DT-Cy5) are synthesized, and observed more specifically toward DTD under physiological conditions. As the cell-penetrating peptide and hyaluronic acid functionalized liposome, the HTLAC not only induces antiproliferative activity by generating self-burst of ROS, but also effectively accumulate and restore its fluorescence at the tumor site because of the HA actively targeting tumor along with the prolonged presence in blood circulation. Besides, this enzyme-triggering nanosystem exhibited an effective tumor inhibition with a low systemic toxicity.

Fortuneicyclidins A (1) and B (2), a pair of epimeric pyrrolizidine alkaloids containing an unprecedented 7-azatetracyclo[5.4.3.0.02,8]tridecane core, were isolated from the seeds of Cephalotaxus fortunei, along with two biogenetically relative known analogues, 3 and 4. The structures were determined by multiple spectral techniques and chemical derivatization methods. Compound 1 showed inhibitory activity against α-glucosidase.

Eight new germacranolides, minusolides A–H (1–8), along with two known analogues, 9 and 10, were isolated from the whole plant of Carpesium minus. Their structures were elucidated by spectroscopic ...

The thioredoxin system plays an important role in cancer cells. Inhibiting thioredoxin reductase (TrxR) has emerged as an effective strategy to selectively target cancer cells. Withangulatin A (WA), a natural product extracted from the whole herb of Physalis angulata L. (Solanaceae), exhibits potent anticancer activity and other diverse pharmacological activities. To improve activity and targeting, we designed and prepared 41 semisynthetic analogues of WA. Biological evaluation indicated that the most promising compound 13a displayed the most significant effect on HT-29 cells (human colon cancer cells) (IC50 = 0.08 μM). A structure-activity relationship study indicated that α,β-unsaturated ketones and ester are necessary groups, allowing 13a to undergo Michael addition reactions with mercaptan and selenol. Liquid chromatography-mass spectrometry (LC-MS) analysis confirmed that 13a modified selenocysteine 498 (U) residues in the redox centers of TrxR, resulting in enzyme inhibition. Therefore, compound 13a acts as a novel TrxR inhibitor and may be a promising candidate for cancer intervention.

(±)-Melipatulinones A–C (1–3), three unique enantiomeric pairs of lignan–phloroglucinol hybrids along with the biogenetically related compound melipatulignan A (4), were isolated from the leaves of Melicope patulinervia. Melipatulinones A (1) and B (2) share a novel spiro[hydrobenzofuran-2,3′-furan] 5/5/6 tricyclic ring system, while melipatulinone C (3) features an unprecedented spiro[cyclopenta[b]hydrofuran-2,3′-furan] 5/5/5 tricyclic framework. Their structures were determined by spectroscopic methods, electronic circular dichroism (ECD) calculations, and X-ray diffraction. Compounds 1–3 exhibited a pancreatic lipase inhibitory effect.

Taxodisones A and B, C30-terpenes with an unprecedented tetracyclo[12.4.0.0.2,709,14]octodecane core, were isolated from the seeds of Taxodium distichum. Their structures, including their configurations, were unambiguously determined. Their biomimetic synthesis suggests that they stem from diterpenes and monoterpenes, and not from squalene or oxidosqualene. In addition, their bioactivities were also evaluated.

Five new spirocyclic polycyclic polyprenylated acylphloroglucinols, Hyperpatulones C-G (1–5), were obtained from the leaves of Hypericum patulum. Their structures were characterized by the comprehensive analysis of their IR, NMR, CD spectra and HRESIMS data. All the new compounds were evaluated for the α-glycosidase inhibitory activities. Among them, compounds 3–5 showed α-glucosidase inhibitory activities, with IC50 values of 14.06–37.69 μM.

Development of noninvasive bioimaging fluorescent probes for detecting particular enzyme activity is greatly recommendable for preclinical diagnosis of cancer. Given that the elevated β-gal activity is positively correlated with several tumors, developing a fluorescent probe for the sensing of β-gal is therefore highly desirable for cancer diagnosis. Herein, a new enzyme-activatable near-infrared (NIR) turn-on fluorescent probe (DMC-βgal) was developed for accurately detecting β-gal activity characterized by excellent selectivity, high sensitivity (LOD = 0.298 U/L), and low toxicity. More importantly, DMC-βgal qualifies remarkable NIR excitation (725 nm) and emission wavelength (770 nm), an ideal tool for restrained photodamage and suppressed autofluorescence. The above excellent performance of DMC-β-gal allowed for the accurate monitoring of endogenous β-gal in living cells. Moreover, the probe was successfully applied to detect intracellular β-gal activity in different types of cancer cells, verifying that SKOV-3 cells had a higher level of β-gal activity than those of A549, HCT-116, MCF-7, and HepG2 cells. Furthermore, DMC-βgal could real-time visualize endogenously β-gal in tumor-bearing nude mice with low auto-fluorescence interference. All results fully demonstrated that DMC-βgal has potential value as a promising strategy for diagnosis of β-gal-related diseases.

Two uncommon C37 heterodimeric diterpenoids, taicrypnacids A (1) and B (2), and a known labdane-type diterpenoid (3) were isolated from the leaves of Taiwania cryptomerioides. Several techniques, such as comprehensive spectroscopic analysis, chemical conversion, X-ray crystallography, and ECD data, were employed to define the structures. The two new compounds displayed cytotoxicity against human breast cancer (MCF-7), osteosarcoma (U-2 OS), and human colon carcinoma (HCT-116) cell lines, while the methyl ester 1a showed no activity. Compound 1 induced Ca2+-ROS pathway-mediated endoplasmic reticulum stress, and excessive stress led to cell death by activating apoptosis and autophagy.

Artemisianins A-D (1–4), four stereoisomers of sesquiterpenoid dimers, forming via [4+2] cycloaddition from a 1, 10-seco-guaianolide dienophile and a guaianolide diene, along with two biosynthetically related precurors 5 and 6, were isolated from the famous traditional Chinese medicine Artemisia argyi. The structures of 1–4, including their absolute configurations, were elucidated by extensive spectroscopic data and ECD/TDDFT calculation analysis. Compounds 1–4 exhibited cytotoxicity with IC50 values ranging from 7.2 to 23.3 μM. The accumulation of Ca2+ in cytoplasm and enlarged endoplasmic reticulum (ER) indicated that 1 mediated HT-29 cancer cell apoptosis through improvement of ER-stress, which was further proved by unfolded protein response (UPR) pathway on basis of the upregulation of IRE1α, p-PERK, ATF6, and CHOP.

Background. Pharbitis Semen, the seeds ofPharbitis nil, is widely used as a traditional purgative medicine in China, Korea, and Japan. This study investigated the laxative effects of a purified resin glycoside fraction obtained in our previous study from Pharbitis Semen in vivo and in vitro.Materials and Methods. After orally administering a purified resin glycoside fraction from Pharbitis Semen (RFP) to rats, the content of fecal water, AQP3, NF-κB, COX-2 expression, and the prostaglandin E2(PGE2) concentrations in the colon were examined. Moreover, human intestinal epithelial cells (HT-29) were used to investigate the mechanism of RFP decreasing the AQP3 expression.Results. Results obtained showed that treatment with RFP increased the feces excretion and fecal water content of rats in a dose-dependent manner. More interestingly, AQP3 expression was suppressed by RFP treatment both in the rat colons and in HT-29 cells, while the NF-κB pathway-mediated PGE2production was activated. Interestingly, pretreating rats with BAY-11-7082 (NF-κB inhibitor) or indomethacin (COX-2 inhibitor) and RFP neither induced diarrhea nor decreased the AQP3 expression in the colon.Conclusions. The purgative property of the purified resin glycoside fraction was attributed to NF-κB activation in the colon, which increased the COX-2-mediated secretion of PGE2. PGE2decreased AQP3 expression which inhibits water absorbed from the intestine to the blood vessel side, resulting in the laxative effect of RFP.

Five novel formyl-phloroglucinol-β-caryophyllene meroterpenoids, namely littordials A–E (1–5), were isolated from the leaves of Psidium littorale. Their structures were established by spectroscopic data and computational methods, and the structure of 1 was confirmed by X-ray crystallography. Littordials A–E feature unusual acyl phloroglucinol units, and 5 possesses a rare 6/7/9/4-fused tetracyclic ring system. Compounds 2, 3 and 5 exhibited cytotoxic activities on two cancer cell lines (MDA-MB-231 and B16) with IC50 values from 6.6 ± 1.5 to 9.2 ± 1.7 μM.

Withangulatin A (WA) has been reported to exhibit potent antitumor activity. However, its possible mechanism and direct proteomic targets remain unknown. Herein we report the subcellular localization of WA by designing and synthesizing its fluorescent analogues with coumarin moieties. Furthermore, sarco/endoplasmic reticulum calcium-ATPase (SERCA)2 was identified as the potential target of WA for its antitumor activity by chemical proteomics.

A pair of novel C23 terpenoid epimers, officinalins A (1) and B (2), together with a biogenetically related known diterpenoid (3), was isolated from the leaves of Salvia officinalis. The epimers feature an unprecedented carbon skeleton with a tetracycline-[9.6.0.03,8.012,16]-heptadecane core and a peroxide bridge. Their structures were unequivocally established by extensive spectroscopic analyses, including electronic circular dichroism (ECD) calculations and ROESY correlation analogy with model compounds for the configuration assignment. Possible biosynthetic pathways of 1 and 2 were proposed. Compound 1 exhibited potent NO inhibitory activity with an IC50 value of 2.02 ± 0.87 μM.

Five new prenylated acetophenones, melicoptelins A-E (1–5), along with one known congener (6) were isolated from the roots of Melicope ptelefolia. Among them, compounds 2a/2b, 3a/3b, and 4a/4b were obtained as inseparable interconverting mixtures of keto and enol tautomers. Their structures were elucidated on the basis of extensive spectroscopic methods, including 1D, 2D NMR and HRESIMS. Compouds 2a/2b, 4a/4b and 5 exhibit protein tyrosine phosphatase 1B (PTP1B) inhibitory activity with IC50 values of 34.4, 55.2 and 66.6 μM, respectively.

Six new Cephalotaxus alkaloids, including five cephalotaxine-type alkaloids, and one homoerythrina-type alkaloid, along with six known analogues, were isolated from the seeds of Cephalotaxus fortunei. Their structures were elucidated by combination of spectroscopic data analyses, time-dependent density functional theory (TDDFT) ECD calculation, and single-crystal X-ray diffraction. Cephalofortine B represents the first example of C-5 epi-cephalotaxine-type alkaloid. All isolated compounds were tested for cytotoxicities against HCT-116, A375, and SK-Mel-28 cell lines. Cephalofortine E showed moderate activity against HCT-116 cell line, with an IC50 value of 7.46 ± 0.77 μM.

Increasing evidence have reported that NLRP3 inflammasome has a crucial role in various kinds of immunological diseases including colitis. However, there have only a few drug candidates directly targeting inflammasomes for the therapy of colitis. Here, we first reported that Tubocapsanolide A (TA), a natural small molecule, as a novel inhibitor of NLRP3 inflammasome for the treatment of colitis. TA inhibited the activation of NLRP3 inflammasome and suppressed the secretion of IL-1β and IL-18 in macrophages. Moreover, the ASC oligomerization was inhibited by TA. The assembly of the NLRP3 inflammasome was also restrained by TA, while had little effects on potassium and chloride efflux. Biolayer interferometry analysis showed that TA could directly bind to NLRP3. Importantly, LC-MS/MS analysis further demonstrated that TA covalently bound to the cysteine 514 residue (Cys514) of NLRP3. In vivo experiments showed that TA remarkably ameliorated DSS-induced experimental colitis in mice. However, the protection of TA against DSS-induced experimental colitis was abrogated in NLRP3-deficient (Nlrp3-/-) mice. Taken together, this study indicates TA as a novel inhibitor of NLRP3, which identifies Cys514 as a novel regulatory site of NLRP3 and suggests TA as a promising candidate compound for the treatment of colitis.

Background and purpose Liver fibrosis is one of the leading causes of morbidity and mortality worldwide but lacks any acceptable therapy. The transcription factor glioma-associated oncogene homologue 1 (GLI1) is a potentially important therapeutic target in liver fibrosis. This study investigates the anti-fibrotic activities and potential mechanisms of the phytochemical, physalin B. Experimental approach Two mouse models (CCl4 challenge and bile duct ligation) were used to assess antifibrotic effects of physalin B in vivo. Mouse primary hepatic stellate cells (pHSCs) and human HSC line LX-2 also served as in vitro liver fibrosis models. Liver fibrogenic genes, GLI1 and GLI1 downstream genes were examined using Western blot and quantitative real-time PCR (qRT-PCR). GLI1 acetylation and LAP2α-HDAC1 interaction were analysed by co-immunoprecipitation. Key results In vivo, physalin B administration attenuated hepatic histopathological injury and collagen accumulation and decreased expression of fibrogenic genes. Physalin B dose-dependently suppressed fibrotic marker expression in LX-2 cells and mouse pHSCs. Mechanistic studies showed that physalin B inhibited GLI activity by non-canonical Hedgehog signalling. Physalin B blocked formation of lamina-associated polypeptide 2α (LAP2α)/histone deacetylase 1 (HDAC1) complexes, thus inhibiting HDAC1-mediated GLI1 deacetylation. Physalin B up-regulated acetylation of GLI1, down-regulated expression of GLI1 and subsequently inhibited HSC activation. Conclusion and implications Physalin B exerted potent antifibrotic effects in vitro and in vivo by disrupting LAP2α/HDAC1 complexes, increasing GLI1 acetylation and inactivating GLI1. This indicates that the phytochemical physalin B may be a potential therapeutic candidate for the treatment of liver fibrosis.

Harringtonolide (HO), a natural product isolated from Cephalotaxus harringtonia, exhibits potent antiproliferative activity. However, little information has been reported on the systematic structure−activity relationship (SAR) of HO derivatives. Modifications on tropone, lactone, and allyl positions of HO (1) were carried out to provide 17 derivatives (2–13, 11a–11f). The in vitro antiproliferative activity against four cancer cell lines (HCT-116, A375, A549, and Huh-7) and one normal cell line (L-02) was tested. Amongst these novel derivatives, compound 6 exhibited comparable cell growth inhibitory activity to HO and displayed better selectivity index (SI = 56.5) between Huh-7 and L-02 cells. The SAR results revealed that the tropone and lactone moieties are essential for the cytotoxic activities, which provided useful suggestions for further structural optimization of HO.

Two new phthalide dimers (1 and 2) were obtained from the rhizomes of Ligusticum sinense Oliv., along with three known dimeric phthalides (3–5). Their structures were determined with the aid of the spectroscopic data, and their absolute configurations were elucidated based on the comparison of calculated and experimental electronic circular dichroism (ECD) spectra. All the compounds were evaluated in vitro for their inhibitory activities against NO production in LPS-treated RAW264.7 macrophages. Among them, compounds 1 and 3 showed potent NO prohibitive activity with IC50 values at 4.86 ± 0.29 μM and 4.87 ± 0.32 μM, respectively.

Novel withangulatin A (WA) derivatives were synthesized and evaluated for antiproliferative activity against four human cancer cell lines (U2OS, MDA-MB-231, HepG2, and A549). Among these derivatives, 10 exhibited the most potent antiproliferative activity, with an IC50 value of 74.0 nM against the human breast cancer cell line MDA-MB-231 and potency that was 70-fold that of WA (IC50 = 5.22 µM). Moreover, 10 caused G2-phase cell cycle arrest in a concentration-dependent manner and induced the apoptosis of MDA-MB-231 cells by increasing intracellular reactive oxygen species (ROS). Compound 10 showed a high selectivity index (SI = 267.03) for breast cancer MDA-MB-231 cells. These results suggest that 10 is a promising anticancer agent.

The transcriptional repressor Snail trriggers epithelial-mesenchymal transition (EMT), the process allowing cancer cells with invasive and metastasis properties. In this study, we screened medicinal plants for the Snail inhibitory active components by high content screen (HCS) and found that the crude extract of Xylopia vielana leaves showed potential activity. Subsequently, bioassay-guided isolation of the extract of Xylopia vielana was performed to obtain twenty-four dimeric guaianes (1-24), including 16 new analogues (1-5, 8-11, 13-15, 17, 18, 21, and 22). Their structures were elucidated by the comprehensive application of multiple spectroscopic methods. Compounds 1, 11, 12, and 16 were initially identified as the active compounds. Wound healing assay, transwell migration assay and western blot experiments verified that compounds 1 and 12 inhibited the expression of Snail in a concentration-dependent manner, and compound 12 was verified as a potent tumor migration inhibitory agent. This work showed a practical strategy for the discovery of new Snail inhibitors from natural products and provided potential insights for dimeric guaianes as anticancer lead compounds specifically targeting Snail protein.

(±)-Patulignans A–C (1–3), three unique pairs of lignan enantiomers were isolated from the leaves of Melicope patulinervia. Patulignan A (1) possesses an unprecedented dimethyloxonane moiety in nature, meanwhile patulignans B (2) and C (3) are epimers carrying a novel dimethyl-1,6-dioxaspiro[4.5]decane skeleton. Their structures were established by spectroscopy methods and electronic circular dichroism (ECD) calculations. Compounds 1–3 showed significant inhibitory activity against α-glucosidase.

Background Hepatic fibrosis is considered integral to the progression of chronic liver diseases, as it leads to the development of cirrhosis and hepatocellular carcinoma. The activation of hepatic stellate cells (HSCs) is the dominant event in hepatic fibrogenesis. The transforming growth factor-β1 (TGF-β1) and Yes-associated protein (YAP) pathways play a pivotal role in HSC activation, hepatic fibrosis and cirrhosis progression. Therefore, targeting the TGF-β/Smad and YAP signaling pathways is a promising strategy for antifibrotic therapy. Purpose The present study investigated the protective effects of Physalin D (PD), a withanolide isolated from Physalis species (Solanaceae), against liver fibrosis and further elucidated the mechanisms involved in vitro and in vivo. Study design/methods We conducted a series of experiments using carbon tetrachloride (CCl4)- and bile duct ligation (BDL)-induced fibrotic mice and cultured LX-2 cells. Serum markers of liver injury, and the morphology, histology and fibrosis of liver tissue were investigated. Western blot assays and quantitative real-time PCR were used to investigate the mechanisms underlying the antifibrotic effects of PD. Result PD decreased TGF-β1-induced COL1A1 promoter activity. PD inhibited TGF-β1-induced expression of Collagen I and α-smooth muscle actin (α-SMA) in human hepatic stellate LX-2 cells. PD significantly ameliorated hepatic injury, including transaminase activities, histology, collagen deposition and α-SMA, in CCl4- or BDL-induced mice. Moreover, PD markedly decreased the expression of phosphorylated Smad2/3 in vitro and in vivo. Furthermore, PD significantly decreased YAP protein levels, and YAP knockdown did not further enhance the effects of PD, namely α-SMA inhibition, Collagen I expression and YAP target gene expression in LX-2 cells. Conclusion These results clearly show that PD ameliorated experimental liver fibrosis by inhibiting the TGF-β/Smad and YAP signaling pathways, indicating that PD has the potential to effectively treat liver fibrosis.

It is significant to precisely isolate potential active compounds from medicinal herbs containing multiple compounds. Herein, a new strategy for precise separation of lysine-specific demethylase 1 (LSD1) inhibitors from the rhizome of Corydalis yanhusuo (RCY) using counter-current chromatography (CCC) guided by molecular docking and liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis was established. First, representative alkaloids from RCY were docked with LSD1 for screening active skeleton compounds. Simultaneously, the crude extract of RCY was preliminarily separated via pH-zone refining CCC. Subsequently, guided by LC-MS/MS analysis of the fragmentation pathways, three potential active fractions were obtained, followed by further online-storage and recycling CCC separation. Finally, three high-purity target quaternary alkaloids compound 3 (dehydrocorydaline), 7 (coptisine), and 8 (columbamine) were successfully isolated as a new class of potential natural LSD1 inhibitors by only one CCC instrument with multiple modes. Compound 3, with the highest LSD1 inhibition ratio of 2.44 μM, was tested for its ability to inhibit tumor invasion and metastasis in U2OS cells. Therefore, the CCC separation guided by virtual screening is a promising method for the targeted isolation of enzyme inhibitors from medicinal herbs.

Synephrine, a chiral adrenergic agonist, exists as R- (more active) and S-enantiomers (less active) that are difficult to separate because of their alkalinity and high polarity. Herein, we show that although this enantioseparation can hardly be achieved by high-performance liquid chromatography, acceptable preparative separation can be realized using high-speed counter-current chromatography (HSCCC) and pH-zone-refining counter-current chromatography (CCC) under optimal conditions. Specifically, 23.0 mg of S-synephrine and 25.0 mg of R-synephrine were isolated from 60.0 mg of racemic synephrine by conventional HSCCC, while 70.0 mg of S-synephrine and 69.0 mg of R-synephrine were obtained from 200.0 mg of racemic synephrine by pH-zone-refining CCC. The pH-zone-refining CCC was identified as the most efficient enantioseparation technique for synephrine and can be applied for the preparative enantioseparation of other β-amino alcohols.

Effective targeting of mitochondria has emerged as a beneficial strategy in cancer therapy. However, the development of mitochondria-targeting ligands is difficult because of the low permeability of the mitochondrial double membrane. We found that hypericin (HY), a natural product isolated from Hypericum perforatum L., is an effective mitochondria-targeting ligand. HY-functionalized graphene oxide (GO) loaded with doxorubicin (GO-PEG-SS-HY/DOX) increased the synergistic anticancer efficacy of phototherapy and chemotherapy in the absence of apparent adverse side effects. In vitro and in vivo assays suggested GO-PEG-SS-HY/DOX induced the expression of the key proteins of the mitochondria-mediated apoptosis pathway and caused apoptosis of breast carcinoma cells. In addition, GO vehicle exhibited low toxicity toward normal cells, indicating high safety of functionalized GO preparations in antitumor therapy. Therefore, HY-functionalized GO can be successfully used as a platform technology to target mitochondria in cancer cells and improve the therapeutic efficacy of chemotherapeutic drugs. Statement of Significance Induction of mitochondria-mediated apoptosis is a promising approach in cancer therapy. However, mitochondria are difficult to access and permeate because of their negative membrane potential and highly dense double membrane. Mitochondria-targeting ligands can be conjugated to nanoparticles or small-molecule drugs to enhance their antitumor effect. Here, we showed that the natural photosensitizer hypericin is a novel mitochondria-targeting ligand and that graphene oxide particles co-loaded with hypericin and the chemotherapeutic agent doxorubicin exhibited a synergistic antitumor effect mediated by the mitochondrial-mediated apoptosis. Treatment with such particles in combination with laser irradiation led to apoptosis of the tumor MDA-MB-231 and MCF-7 cells in vitro and in vivo. Furthermore, treatment with hypericin/doxorubicin-functionalized graphene oxide had low cellular toxicity.

γ-Glutamyltranspeptidase (GGT) plays critical roles in regulating various physiological/pathophysiological processes including the intracellular redox homeostasis. However, an effective fluorescent probe for dissecting the relationships between GGT and oxidative stress in vivo remains largely unexplored. Herein, we present a light-up fluorescent probe (DCDHF-Glu) with long wavelength emission (613 nm) for the highly sensitive and selective detection of GGT using dicyanomethylenedihydrofuran derivative as the fluorescent reporter and γ-glutamyl group as the enzyme-active trigger. DCDHF-Glu is competent to real-time image endogenous GGT in live cells and mice. In particular, DCDHF-Glu enables the direct real-time visualization of the upregulation of GGT under drug-induced oxidative stress in the HepG2 cells and the LO2 cells, as well as in vivo, vividly implying its excellent capacity in elucidation of GGT function in GGT-related biological events.

Peyronellones A and B (1 and 2), a pair of rare tetracyclic caged adducts of azaphilone with pyruvic acid, along with four new analogues (3–6), were isolated from solid cultures of the endophytic fungus Peyronellaea glomerata. Their structures were elucidated through spectroscopic analysis, and their absolute configurations were unambiguously determined by a combination of single-crystal X-ray crystallography, Rh2(OCOCF3)4-induced ECD experiments, ECD calculations, and modified Mosher methods. Compound 2 (5 μM) was found to have a significant hypoxia-protective effect that improved the survival rate of hypoxia/reoxygenation-treated human umbilical vein endothelial cells from 35% to 70%, which was equal to the potency of the positive control, verapamil. Flow cytometry analysis suggested 2 could inhibit H/R-induced late-stage apoptosis of this cell line.

The reactivity-based screening (RBS) was developed for directed discovery of cytotoxic withanolides. In this study, a thiol probe, 4-chlorobenzenethiol, was used to selectively attack cytotoxic withanolides containing potential pharmacophore, 2(3)-en-1-one in ring A (AEO) and 5β,6β-epoxy in ring B (BE), from the plant extract of Physalis angulata var. villosa. The screening was performed based on the potential mechanism of 4-chlorobenzenethiol nucleophilic addition to AEO, followed by detection of adducts using liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-Q-TOF-MS). Guided by RBS, eleven target withanolides, including five new compounds, physagulides R-V (10–14) and six known ones (2, 7–9, 15, 16) were discovered. All of them exhibited cytotoxicity against the both tested cell lines, especially, compounds 2, 7, 8 and 14 showed potent activities with IC50 values of 1.57–6.29 μM. The results suggested that RBS was efficient and accurate for rapid identification of cytotoxic withanolides and could guide isolation of target components from the complex medicinal plant extract.

Ten new highly oxidized monomeric (1–8) and dimeric guaianolides (9 and 10), along with two known guaianolide derivatives (11 and 12), were isolated from the aerial parts of Chrysanthemum indicum using a bioassay-guided fractionation procedure. The new compounds were characterized by the basic analysis of the spectroscopic data obtained, and the absolute configurations were determined by both empirical approaches and ECD calculations. Inhibitory effects of 1–12 on nitric oxide production were investigated in lipopolysaccaride (LPS)-mediated RAW 264.7 cells, and most of them (1–8 and 11) displayed IC50 values in the range 1.4–9.7 μM. Moreover, a mechanistic study revealed that the potential anti-inflammatory activity of compound 1 appears to be mediated via suppression of an LPS-induced NF-κB pathway and down-regulation of MAPK activation.

Biogenesis-inspired chemical research of the leaves of Taiwania cryptomerioides afforded four unprecedented dimeric diterpenes, featuring a tetracyclic [7. 75, 9. 4. 05, 10. 08, 9] octodecane core: taiwanoids A-D (1-4). The structures of these compounds were determined on the basis of comprehensive spectral analysis, chemical conversions and X-ray crystallography. A possible biosynthetic pathway for compounds 1-4 was proposed. Compounds 2 and 3 exerted a 5.37 and 6.26-fold potentiation effect on bortezmib (BTZ) susceptibility at a tested concentration of 20 μM, respectively.

Seven new phloroglucinol derivatives, uraliones L-R (1-7), were isolated from the whole plant of Hypericum urialum. The structures of new compounds were unambiguously elucidated by comprehensive spectroscopic analysis, and the absolute configurations of the vic-diol groups were determined by Mo2(OAc)4-induced electronic circular dichroism experiments. Their capability to reversal the multidrug resistance in doxorubicin-induced resistant MCF-7/DOX sublines showed that compounds 1, 5, 6, 7 exerted a potential effect of doxorubicin susceptibility.

// Xu-Wei Zhou 1, * , Yuan-Zheng Xia 1, * , Ya-Long Zhang 1 , Jian-Guang Luo 1 , Chao Han 1 , Hao Zhang 1 , Chao Zhang 1 , Lei Yang 1 and Ling-Yi Kong 1 1 Jiangsu Key Laboratory of Bioactive Natural Product Research and State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China * These authors have contributed equally to this work Correspondence to: Lei Yang, email: dorothy19802003@gmail.com Ling-Yi Kong, email: cpu_lykong@126.com Keywords: tomentodione M; meroterpenoid; multidrug resistance; P-glycoprotein; p38 Received: July 02, 2017 Accepted: October 03, 2017 Published: October 19, 2017 ABSTRACT In this study, we investigated the mechanism by which tomentodione M (TTM), a novel natural syncarpic acid-conjugated monoterpene, reversed multi-drug resistance (MDR) in cancer cells. TTM increased the cytotoxicity of chemotherapeutic drugs such as docetaxel and doxorubicin in MCF-7/MDR and K562/MDR cells in a dose- and time-dependent manner. TTM reduced colony formation and enhanced apoptosis in docetaxel-treated MCF-7/MDR and K562/MDR cells, and it enhanced intracellular accumulation of doxorubicin and rhodamine 123 in MDR cancer cells by reducing drug efflux mediated by P-gp. TTM decreased expression of both P-gp mRNA and protein by inhibiting p38 MAPK signaling. Similarly, the p38 MAPK inhibitor SB203580 reversed MDR in cancer cells by decreasing P-gp expression. Conversely, p38 MAPK-overexpressing MCF-7 and K562 cells showed higher P-gp expression than controls. These observations indicate that TTM reverses MDR in cancer cells by decreasing P-gp expression via p38 MAPK inhibition.

Artemisians A-D (1-4), the first examples of [4 + 2] Diels-Alder type adducts presumably biosynthesized from a rare 1, 10-4, 5-diseco-guaianolide and a guaianolide diene, along with their possible precursor 5, were isolated from the traditional Chinese medicine Artemisia argyi. The structures of 1-4 were elucidated by extensive spectroscopic analyses and calculated electronic circular dichroism. Compound 2, with an IC50 value of 3.21 μM, exhibited significant antiproliferative activity via apoptosis induction and G2/M arrest in MDA-MB-468 cells.

Seven sucrose esters, physakengoses K-Q ( 1 – 7 ) were isolated from the aerial parts of Physalis alkekengi var. franchetii . Their structures were elucidated on the basis of extensive spectroscopic analyses and chemical methods. These new compounds were tested for their antimicrobial abilities against Staphylococcus aureus , Bacillus subtilis , Pseudomonas aeruginosa and Escherichia coli . Among the isolated sucrose esters, compounds 1 – 5 showed potent antibacterial activity with MIC values ranging from 2.16 to 12.76 μ g/mL.

Six novel calyxins, named calyxin T-W, ent-calyxin T and ent-calyxin U were isolated from the seeds of Alpinia katsumadai Hayata. Their relative configurations were elucidated by means of detailed UV, IR, NMR and MS spectroscopic data. Their absolute configurations were assigned by collaborative studies on single crystal X-ray diffraction analysis, Mosher's method, electronic circular dichroism (ECD), optical rotation and theoretical calculations. These compounds are Friedel-Cranft alkylation adducts composed of coexisted diarylheptanoids and flavanone from the seeds of Alpinia katsumadai. The antiproliferative activity of the six compounds against NCI-H460, HeLa, SMMC-7721 and HCT-116 cell lines was also reported, and most of them showed moderate to strong activities.

A pair of new tetranorlabdane diterpenoid epimers, named elettarins A (1) and B (2), together with five known labdane diterpenes (3–7), were isolated from Elettaria cardamomum Maton. The structures of elettarins A and B were established based on spectroscopic data (HRESIMS, 1D/2D NMR), and ECD experimentation. All isolates were evaluated for their inhibitory activities against nitric oxide (NO) production on BV-2 microglia cells.

Xylopiana A (1), a dimeric guaiane with an unprecedented pentacyclo[5.2.1.01,2.04,5′.05,4′]decane-3,2′-dione core, and three biosynthetically related intermediates, compounds 2–4, were isolated from the leaves of Xylopia vielana. Their structures and absolute configurations were determined by a combination of spectroscopic data, X-ray crystallography, electronic circular dichroism calculations, and chemical conversion. The structure of known vielanin A was revised to be compound 3. Compound 4 exerted a 3.7-fold potentiation effect on doxorubicin susceptibility at the tested concentration of 10 μM.

Application of NH2 silica gel column chromatography using CH2Cl2-MeOH was found to show a satisfactory resolution for separation of the crude resin glycosides of Pharbitis Semen (the seeds of Pharbitis nil), led to the isolation of seven new acylated glycosidic acid methyl esters, Pharesinosides A-G (1–7), along with four known ones (8–11). These compounds (1–11) were considered to be generated via methyl esterification of the carboxyl group in acylated glycosidic acids. Their structures including stereochemistry were elucidated on the basis of a combination of the spectroscopic and chemical methods. All isolates were evaluated for anti-tumor migration activities with human colon cancer cell line HCT-116, and compound 7 exhibited a potent migration inhibitory activity.

Seven azaphilones, montagnuphilones A–G (1–7), together with previously known azaphilones 8–11, were encountered in Montagnulaceae sp. DM0194, an endophytic fungus isolated from submerged roots of Persicaria amphibia. The structures of 1–7 were elucidated on the basis of their MS and NMR spectroscopic analysis. Compounds 1–8 were evaluated for their cytotoxicity and ability to inhibit nitric oxide (NO) production in lipopolysaccharide-activated RAW264.7 macrophage cells. Among these, none were found to be cytotoxic to RAW264.7 cells up to 100.0 μM, but 8, 5, and 2 showed NO inhibitory activity with IC50 values of 9.2 ± 0.9, 25.5 ± 1.1, and 39.6 ± 1.8 μM, respectively.

Tooniliatone A (1), a novel limonoid with an unprecedented 6/5/6/5 tetracarbocyclic skeleton was isolated from Toona ciliata Roem. var. yunnanensis. Its rearranged ring-seco backbone was proposed to biosynthetically originate from co-isolated known toonacilin (3) via a rare electron-deficient alkene electrophilic cyclization and 1,2-alkyl shift sequence. The acid-catalyzed biomimetic conversion of 3 not only successfully afforded 1 but also another novel 6/5/6/5 tetracarbocyclic skeleton, tooniliatone B (2). The priority of 2 in this biomimetic conversion, the existence of 1 and the absence of 2 in the crude extract suggested the biosynthetic selectivity of 1 from 3. The divergent biomimetic sequence, based on a carbocation from an epoxide moiety, provided an inspiration for constructing complex polycyclic carbon skeletons.

A new peroxide of xanthone ( 1 ), together with five analogues ( 2 – 6 ), was obtained from horseradish peroxidase (HRP) catalysed biotransformation of α -mangostin. In order to increase the yield of compound 1 , the environmental pH was succinctly adjusted, and as a result, compound 1 was formed with considerable selectivity. The anticancer potential of compound 1 , a pentacyclic xanthone with a 1,2-dioxolane ring, was investigated due to its potent cytotoxic activity. The results showed that apoptosis induced by compound 1 in human hepatocellular carcinoma (HepG2) cell was associated with activation of caspase-dependent apoptotic pathway, the generation of reactive oxygen species (ROS) and the activation of c-Jun N-terminal kinases (JNK). In summary, compound 1 with a peroxide group was biosynthesized in considerable yield through pH-switched biotransformation catalysed by HRP, which could be further developed as a promising candidate in the treatment of liver cancer.

An off-on fluorescent probe (CuCIN), coumarin-naphthol linked by an imine group, for detecting Cu2+ in DMSO-HEPES buffer (1:99, v/v) solution was successfully developed. Mechanism studies suggested that CuCIN firstly formed a complex with Cu2+ in a 1:2 metal-to-ligand ratio, and then 60-fold fluorescence enhancement was observed when Cu2+ promoted the imine hydrolysis to release the fluorescence compound 7-diethylaminocoumarin-3-aldehyde. This probe displayed desired properties such as high selectivity toward other metal ions, low detection limit (12.7 nM) and low cytotoxicity. Bio-imaging study revealed that the new probe CuCIN could be applied for imaging living cells with good cell permeability. We hope to apply this probe in the biomedical research field for the imaging of disease-relevant Cu2+.

Six new obovatol trimeric neolignans, houpulignans A–F (1–6) were isolated from the leaves of Magnolia officinalis var. biloba. Their structures were determined on the basis of the interpretation of HRESIMS, NMR data, and electronic circular dichroism (ECD) calculations. Compounds 1 and 2 are the first examples of neolignans derived from three units of obovatol bearing a rare 1,4-benzodioxepane moiety. Compound 3 possesses a benzodihydropyran ring, meanwhile three units of obovatol in 4–6 are connected by an alkyl chain. Compounds 1–3 inhibited NO production in LPS-stimulated RAW264.7 cells with IC50 values of 8.01, 20.21, and 4.05 µM, respectively.

In terms of ligand fishing, the amount and the relative activity recovery of enzymes immobilized on magnetic particles and nanoparticles are not preeminent. Therefore, the metal-organic framework (MOF) UiO-66-NH2 was synthesized to immobilize the porcine pancreatic lipase (PPL) via precipitation-cross-linking, and the resulting novel biological matrices named PPL@MOF manifested high PPL loading capacity (98.31 mg/g) and relative activity recovery (104.4%). Moreover, the novel enzyme-MOF composite was applied to screen lipase inhibitors from Prunella vulgaris L. to enrich and improve the techniques of ligand fishing. As a result, 13 lipase ligands were obtained, and 12 compounds were determined by HPLC-Q-TOF-MS/MS. All of these ligands were further confirmed to be potential inhibitors through the verification of the activity assay and molecular docking. The proposed approach based on PPL@MOF was superior in terms of abundant protein loading capacity, high enzyme catalytic activity and easy preparation process. Taken together, our newly developed method provided a new platform for efficient discovering bioactive molecules from natural herbs.