Xanthones from Gentianella acuta (Michx.) Hulten Ameliorate Colorectal Carcinoma via the PI3K/Akt/mTOR Signaling Pathway.

Colorectal carcinoma (CRC) is a kind of malignant tumor closely related to ulcerative colitis. Xanthone derivatives are one of the most promising therapeutic drugs which have been used in phase I/II clinical trials for cancer therapy. Our previous study indicated that the aerial parts of Gentianella acuta Michx. Hulten (GA) was rich in xanthones and showed a good therapeutic effect on ulcerative colitis in mice, suggesting that GA xanthones might have some therapeutic or ameliorative effects on CRC. However, no relevant study has been reported. This study aims to find the effective substances of GA inhibiting CRC and clarify their mechanism. Solvent extraction, column chromatographic separation, and LC-MS analysis were used to characterize the 70% EtOH extract of GA and track xanthones abundant fraction XF. MTT assay was carried out to clarify the activity of GA fractions; the result showed XF to be the main active fraction. LC-MS analysis was executed to characterize XF, 38 xanthones were identified. Network pharmacology prediction, in vitro activity screening, and molecular docking assay were combined to predict the potential mechanism; the PI3K/Akt/mTOR signaling pathway was found to be most important. Western blot assay on the main active xanthones 1,3,5-trihydroxyxanthone (16), 1,3,5,8-tetrahydroxyxanthone (17), 1,5,8-trihydroxy-3-methoxyxanthone (18), and 1,7-dihydroxy-3,8-dimethoxyxanthone (19) was used to verify the above prediction; these xanthones were found to inhibit the PI3K/Akt/mTOR signaling pathway, and 17 played a significant role among them through Western blot assay using PI3K/AKT/mTOR agonist IGF-1. In conclusion, this study demonstrated that GA xanthones were effective compounds of GA inhibiting CRC by regulating PI3K/Akt/mTOR signaling pathway transduction, at least. Importantly, 1,3,5,8-tetrahydroxyxanthone (17), the most abundant active xanthone in GA, might be a candidate drug for CRC.

Bioactive triterpenes of jujube in the prevention of colorectal cancer and their molecular mechanism research.

BACKGROUND: Jujube, a popular fruit from the Rhamnaceae family, relieves colorectal inflammation caused by spleen deficiency and has been used in many formulas in clinical for decades to treat colorectal cancer (CRC). As of yet, the therapeutic substances and mechanism of their action are unknown. PURPOSE: The purpose of this study is to define the therapeutic substances of jujube and its mechanism of action in treating CRC. METHODS: The pharmacological effects of jujube extract and its fractions were evaluated in vivo using a CRC mouse model induced by AOM/DSS. The DAI value, colon length, mortality, tumor burden, and histological tumor size of the treated animals were compared. To explore the potential therapeutic substances, LC-MS analysis was conducted to characterize the serum migration components. A network pharmacology experiment was carried out for potential molecular targets. To verify the therapeutic substances as well as the molecular mechanism of jujube intervening CRC, cellular MTT assay of the serum migration components, Western blot and IHC tests were conducted. RESULTS: The in vivo pharmacological studies showed that compared to AOM/DSS treated mice, the mortality and DAI value, tumor burden, and histological tumor size of jujube extract and its fat-soluble fraction (mainly contained triterpenes) treated mice were significantly reduced, and their colon lengths were obviously longer than AOM/DSS treated mice. The targeted-LC/MS analysis supposed triterpenes 3, 7, 9, 11, 12, 14, 17 - 21, and 25 - 28 to be the serum migration components, which might be the potential therapeutic substances. In the network pharmacology experiment, the GO annotation and enrichment analysis of the KEGG pathway indicated that PI3K-Akt pathway and inflammatory reaction were important factors for jujube inhibiting CRC. Cellular MTT assay of serum migration components indicated that the potential effective substances from fat-soluble fraction to be triterpenes 3, 7, 17, 19, 20, and 25. The Western blot and IHC assays implied that the jujube extract, its fat-soluble fraction, and triterpenes 7, 17, and 20 showed inhibition on the expression of PI3K/Akt/NF-κB signaling pathway-related proteins. Additionally, it was noted in the pharmacodynamic experiment that ZJL's effectiveness was more apparent than ZJH and SQL in tumor burden rate, colon length, and spleen weight, which indicated that the efficacy of ZJ is contributed from CD and SQ, and they may have a synergistic effect on anti-CRC. CONCLUSION: These results for the first time provide evidence that jujube triterpenes possess an anti-CRC effect, their mechanism was involving the control of the PI3K/Akt/NF-κB signaling pathway. What's more, the potential synergistic effect of the fat-soluble and water-soluble components found in this study provided a solid foundation for our deep understanding of how jujube can ameliorate CRC.

Structural characterization of phenolic constituents from the rhizome of Imperata cylindrica var. major and their anti-inflammatory activity.

As one of raw materials, the rhizome of Imperata cylindrica var. major (Nees) C.E. Hubb. is used in kinds of preparations curing inflammation related diseases, while its effective substances are not yet clear. In this paper, its chemical constituents and their anti-inflammatory activities were investigated. As results, ten compounds, named as imperphenoside A (1), imperphenols B (2) and C (3), imperphenosides D-F (4-6), and imperlignanosides A-D (7-10), along with previously reported thirty-seven known ones (11-47) were obtained from it. Their structures were ascertained basing on the extensive spectroscopic methods and electronic circular dichroism data analysis. Meanwhile, compounds 4, 11, 12, 24, 27, 31, 32, 37, 43, 45, and 47 exhibited nitric oxide inhibitory effects in concentration dependent at 3, 10, and 30 µM on lipopolysaccharides induced RAW 264.7 cells. Moreover, the western blot analysis indicated that compounds 4, 11, 43, and 47 could restrain the phosphorylation of nuclear factor kappa-B kinase to down-regulate the protein expression of inflammatory cytokines such as inducible nitric oxide synthase, interleukin-6 and tumor necrosis factor-α. In conclusion, they might play the anti-inflammatory effects through regulating NF-κB signaling pathway.

The phenolic acids from Oplopanax elatus Nakai stems and their potential photo-damage prevention activity.

25 phenolic acids, including four new isolates, eurylophenosides A-D (1-4) and 21 known ones (5-25) were isolated and identified from the stems of Oplopanax elatus Nakai. Among the known compounds 5-9, 11-13, 16, 18-25 were isolated from the genus for the first time; 17 was first obtained from the plant; and the NMR data of 22 was reported here first. Meanwhile, the UVB-induced photodamage model of HaCaT cells was used to study the prevent-photodamage abilities of compounds 1-2, 4-8, 11-13 and 15-25 with a nontoxic concentration at 50 µM. Moreover, a dose-dependent experiment was conducted for active compounds at the concentration of 10, 25, and 50 µM, respectively. Consequently, pretreatment with compounds 1, 16, 17, 19, 20, 22, 24 and 25 could suppress the cell viability decreasing induced by UVB irradiation in a concentration-dependent manner. These results indicated that phenolic acids were one kind of material basis with prevent-photodamage activity of O. elatus.

Constituents of Morus alba var. multicaulis leaf improve lipid metabolism by activating the AMPK signaling pathway in HepG2 cells.

One new compound, 3Z-1-O-ß-D-glucopyranosyl-3-hexene-1,5-diol (1), together with 26 known isolates (2-27) were obtained from the leaf of Morus alba var. multicaulis. Among the known compounds, 7, 11, 12, 14, 15, 18, 19, 23, and 24 were firstly obtained from the Morus genus; 2-5, 8, 10, 13, and 20 were firstly isolated from M. alba. var. multlcaulis. Meanwhile, the NMR data of 20 and 23 have been reported here for the first time. Moreover, compounds 1-11, 13, 21, and 23-27 showed inhibitory effects on triglyceride (TG) accumulation in HepG2 cells. In mechanism, compound 1 could activate the phosphorylation of AMP-activated protein kinase α (AMPKα) to accelerate the ß-oxidation of fatty acids via promoting the phosphorylation of acetyl-CoA carboxylase 1 and up-regulating carnitine palmitoyl-transferase 1A. Besides, compound 1 exerted lipolysis effect by activating hormone-sensitive lipase. In brief, compound 1 might play a role by up-regulating phosphorylation of AMPKα, enhancing the fatty acid ß-oxidation and lipolysis. 27 compounds were obtained from the leaf of Morus alba var. multicaulis. Among them, 18 showed inhibitory effects on TG accumulation in HepG2 cells. Moreover, the new compound, 3Z-1-O-ß-D-glucopyranosyl-3-hexene-1,5-diol (1), was found to play a role by up-regulating phosphorylation of AMPKα, enhancing the fatty acids ß-oxidation and lipolysis.

Sesquiterpenes From Oplopanax elatus Stems and Their Anti-Photoaging Effects by Down-Regulating Matrix Metalloproteinase-1 Expression via Anti-Inflammation.

In the process of continuing to investigate ultraviolet b (UVB) irradiation protective constituents from Oplopanax elatus stems, nine new sesquiterpenes, named as eurylosesquiterpenosides A-D (1-4), eurylosesquiterpenols E-I (5-9), and ten known ones (10-19) were gained. Their structures were established by analysis of their NMR spectroscopic data, and electronic circular dichroism calculations were applied to define their absolute configurations. In addition, UVB induced HaCaT cells were used to study their anti-photoaging activities and mechanism. The results consolidated that compounds 7, 11, and 14 could improve the survival rate of HaCaT cells in concentration dependent manner at 10, 25, and 50 µM. Furthermore, western blot assay suggested that all of them could inhibit the expression of matrix metalloproteinase-1 (MMP-1), and increase the level of type I collagen markedly. Compounds 11 and 14 could reduce the phosphorylation of extracellular signal-regulated kinase and p38, respectively. Besides, compounds 7, 11, and 14 could significantly down-regulate the expression of inflammation related protein, such as tumor necrosis factor-α and cyclooxygenase-2, which indicated that they played anti-photoaging activities by reducing MMP-1 expression via down-regulating the production of inflammatory mediators and cytokines in UVB-induced HaCaT cells.

Bioactive flavonoids and stilbenes from the leaf of Morus alba var. multicaulis.

Twenty-two flavonoids and stilbenes (1-22) were obtained from the leaf of Morus alba var. multicaulis. Among them, morusalbanosides A (1), B1 (2), and B2 (3) were new compounds. Moreover, compounds 1, 3, 4-11, 15-18, and 22 displayed inhibitory effects on triglyceride (TG) accumulation in HepG2 cells in a concentration dependent manner. Furthermore, compounds 1, 3, 11, and 22 could activate the phosphorylation of AMP-activated protein kinase α (AMPKα), reduce the synthesis of TG by inhibiting the expression of fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD1). While, only compounds 1 and 11 could promote the phosphorylation of acetyl-CoA carboxylase 1 (ACC1) and accelerate the oxidation of fatty acids by up-regulating carnitine palmitoyltransferase 1A (CPT1A). In brief, this study found that most of the researched flavonoids and stilbenes could regulate TG metabolism in vitro. They might play the role by up-regulating phosphorylation of AMPKα, inhibiting TG biosynthesis, and promoting the oxidation of fatty acids.

Structurally diverse triterpenes obtained from the fruits of Ziziphus jujuba Mill. as inflammation inhibitors by NF-κB signaling pathway.

Twenty-nine triterpenes were obtained from the fruits of Ziziphus jujuba Mill. through various chromatography methods, and their stereo-structures were confirmed by spectroscopic methods. Among them, 2α,3ß,20-trihydroxylupane-28-oic acid (1) was identified as a new compound, and the 1H and 13C NMR data of 7, 8 and 23, as well as the 13C NMR data of 17 are reported here for the first time. Meanwhile, the nitric oxide (NO) inhibitory activities of all compounds were examined in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. As results, compounds 2, 7, 10-13, 15, 16, 18-21, 26-29 were found to play important roles in suppressing NO production at 5 µM. The structure-activity relationships (SARs) on NO inhibition indicated that the ursolic and oleanolic acid skeletons, p-coumaroyl group substitution, six-membered A ring, and deoxygenation (loss of C[double bond, length as m-dash]O) in the C ring showed a more positive effect on the NO inhibitory activity of triterpenes, while the reduction of the A ring C[double bond, length as m-dash]O to OH was a negative factor. Moreover, it was found that compounds 15 and 19 could suppress the phosphorylation of IκBα and NF-κB/p65 to prevent it from shifting into the nucleus and downregulate the expression of inflammatory factors, such as iNOS, IL-6 and TNF-α. Our investigations revealed that the NO inhibitory effects of the active triterpenes obtained from Z. jujuba were mediated, at least in part, through the NF-κB signaling pathway.

Phytochemistry and Comprehensive Chemical Profiling Study of Flavonoids and Phenolic Acids in the Aerial Parts of Allium Mongolicum Regel and Their Intestinal Motility Evaluation.

To clarify whether flavonoids and phenols in Allium mongolicum Regel have the effect of improving gastrointestinal function and analyze its quality, this study was designed to isolate and identify them from the aerial parts of A. mongolicum by using various chromatographic and spectrophotometric methods, a bioassay on motility of mouse isolated intestine tissue, as well as qualitative analysis using liquid chromatography/mass spectrometry (LC-MS) analysis. As a result, 31 flavonoids and phenolic acids were obtained and identified, including six new flavonoid glycosides, mongoflavonosides A1 (1), A2 (2), A3 (3), A4 (4), B1 (5), B2 (6), and four new phenolic acid glycosides, mongophenosides A1 (7), A2 (8), A3 (9), B (10). Among them, eleven flavonoids and three phenolic acids showed significant increase in the height of mouse small intestinal muscle. It was a first systematic bioactive constituents' study for A. mongolicum on gastrointestinal tract. Furthermore, according to the retention time (tR) and the exact mass-to-charge ratio (m/z), thirty-one compounds were unambiguously identified by comparing to the standard references by using LC-MS. Then, on the basis of generalized rules of MS/MS fragmentation pattern, chromatographic behaviors, as well as biosynthetic laws of the 31 isolates, five flavonoid glycosides and one phenolic acid glycoside were tentatively speculated. On the basis of the study, a fast analysis method for flavonoids and phenolic acids in A. mongolicum was established.

New Dammarane-Type Triterpenoid Saponins from Panax notoginseng Leaves and Their Nitric Oxide Inhibitory Activities.

Inflammation is a very common and important pathological process that can cause many diseases. The discovery of anti-inflammatory drugs and the treatment of inflammation are particularly essential. Dammarane-type triterpenoid saponins (PNS) were demonstrated to show anti-inflammatory effects in the leaves of Panax notoginseng. Chromatographies and spectral analysis methods were combined to isolate and identify PNS. Moreover, the nitric oxide (NO) inhibitory activities of all compounds were examined in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. As a result, eleven new dammarane-type triterpenoid saponins, notoginsenosides NL-A1-NL-A4 (1-4), NL-B1-NL-B3 (5-7), NL-C1-NL-C3 (8-10), and NL-D (11) were isolated, and their structures were identified by using various spectrometric techniques and chemical reactions. Among them, compounds 4 and 11 were characterized by the malonyl substitution at 3-position. The 3-malonyl substituted dammarane-type terpennoids were first obtained from natural products. In addition, compounds 1, 2, 5, 6, and 8-10 were found to play an important role in suppressing NO levels at 50 µM, without cytotoxicity. All inhibitory activities were found to be dose-dependent.

Synergistic Promotion on Tyrosinase Inhibition by Antioxidants.

When exposed to ultraviolet radiation, the human skin produces profuse reactive oxygen species (ROS), which in turn activate a variety of biological responses. Mounting ROS levels activate tyrosinase by mobilizing α-melanocyte-stimulating hormone in the epidermis and finally stimulates the melanocytes to produce melanin. Meanwhile, the Keap1-Nrf2/ARE pathway, which removes ROS, is activated at increased ROS levels, and antioxidant compounds facilitates the dissociation of Nrf2. In this study, we explored the possible suppressing effects of antioxidant compounds and tyrosine inhibitors on melanin formation and the promotory effects of these compounds on ROS scavenging. The antioxidant activity of glabridin (GLA), resveratrol (RES), oxyresveratrol (OXYR), and phenylethylresorcinol (PR) were investigated via the stable free radical 2,2-diphenyl-1-picrylhydrazyl method. The inhibitory effects of the four compounds and their mixtures on tyrosinase were evaluated. l-Tyrosine or 3-(3,4-dihydroxyphenyl)-l-alanine (l-DOPA) was used as a substrate. The results showed that all mixtures did not exhibit synergistic effects with the l-tyrosine as a substrate, suggesting that l-tyrosine is not suitable as a substrate. However, the mixtures of "GLA:RES," "GLA:OXYR," "OXYR:RES," and "PR:RES" demonstrated synergistic effects (CI < 0.9, p < 0.05), whereas "GLA:RES" and "PR:OXYR" indicated an additive effect (0.9 ditive1, p < 0.05). Furthermore, we used a molecular docking strategy to study the interactions of the four compounds with tyrosinase and l-DOPA. The molecular docking result is consistent with that of the experiment. Finally, we selected RES + OXYR and used PIG1 cells to verify whether OXYR synergistically promotes RES activity on tyrosinase. The two agents had a synergistic inhibitory effect on tyrosinase activity. These results provided a novel synergistic strategy for antioxidants and tyrosinase inhibitors, and this strategy is useful in skin injury treatment.

Mutant Nbs1 enhances cisplatin-induced DNA damage and cytotoxicity in head and neck cancer.

OBJECTIVE: Enhanced DNA double-strand break (DSB) repair could be a primary cause for development of resistance in tumor cells to cisplatin, which induces crosslinks and DNA DSBs. A protein complex consisting of hMre11, hRad50, and Nbs1 (MRN) has been identified as a critical component in repair of DNA DSBs. The present study investigates whether the expression of a truncated form of Nbs1 interrupts the function of the MRN complex and therefore enhances cisplatin-induced DNA damage and cytotoxicity in human head and neck squamous cell carcinoma (HNSCC). METHODS AND MEASURES: Two human HNSCC cell lines, JHU006 and JHU029, were used. A dominant negative recombinant adenovirus expressing domains of Nbs1 was constructed. Adenovirus-mediated mutant Nbs1 (Ad-Nbs1) gene transfer was performed with replication-defective virus (DL312) and no treatment as controls. Transgene expression and cell viability were evaluated in transfected cells. Neutral comet assay was performed and the "tail moment," the product of the amount of DNA in the tail and the distance of tail migration, was analyzed for evaluating DNA DSB damage at 24, 48, and 72 hours. RESULTS: Transgene expression of mutant Nbs1 was confirmed by Western blotting. Ad-Nbs1 gene transfer significantly increased cisplatin-induced cytotoxicity as shown by stunting of 6-day growth curves. Neutral comet analysis revealed that the mean tail moment, indicative of DNA damage, was significantly elevated in cells treated with combined cisplatin and Ad-Nbs1 compared to cisplatin alone in both cell lines. CONCLUSIONS: Expression of mutant Nbs1 significantly increases cisplatin-induced DNA DSBs and cytotoxicity. The increase in double-strand DNA damage corresponds to the level of cytotoxicity in the different treatment groups and suggests that tumor chemosensitization occurs through augmentation of DNA DSBs. CLINICAL SIGNIFICANCE: Alteration of DNA repair may provide a novel approach to enhancing sensitivity of HNSCC to chemotherapy. Our study supports the potential application of Ad-Nbs1 in combination with cisplatin for treatment of advanced and metastatic HNSCC.