Lp-PLA2 inhibitory activities of fatty acid glycerols isolated from Saururus chinensis roots

(R)-Glycerol-monolinoleate 4 and (R)-glycerol-monostearate 5 were isolated from the ethyl acetate extracts of Saururus chinensis roots and (R)- or (S)-fatty acid glycerols 4 and 5 were synthesized for confirming their structures and evaluating their inhibitory activities against Lp-PLA(2). The (R)-4 and (S)-4 exhibited Lp-PLA(2) inhibitory activities with IC(50) values of 45.0 and 52.0 microM, respectively.     

Inhibition of NF-IL6 activity by manassantin B, a dilignan isolated from Saururus chinensis, in phorbol myristate acetate-stimulated U937 promonocytic cells

Mannasantin B, a dilignan structurally related to manssantin A, is an inhibitor of NF-kappaB transactivation. In the present study, we found that it inhibited PMA-induced expression of IL-1beta, IL-1beta mRNA, and IL-1beta promoter activity in U937 cells with IC50 values of about 50 nM. It also inhibited NF-IL6- and NF-kappaB-induced activation of IL-1beta, with IC50 values of 78 nM and 1.6 microM, respectively, revealing a potent inhibitory effect on NF-IL6. Electrophoretic mobility shift assays showed that manassantin B had an inhibitory effect on DNA binding by NF-IL6, but not by NF-kappaB. Further analysis revealed that transactivation by NF-IL6 was also inhibited. Our results indicate that manassantin B suppresses expression of IL-1beta in promonocytic cells by inhibiting not only NF-kappaB but also NF-IL6 activity. Furthermore, our observations suggest that manassantin B may be clinically useful as a potent inhibitor of NF-IL6 activity.     

Antioxidant activities of a new lignan and a neolignan from Saururus chinensis

A new diarylbutane lignan, 2'-hydroxy dihydroguaiaretic acid (4), and a known 8-O-4'-type neolignan, machilin D (5), were isolated from the ethyl acetate extracts of the underground parts of Saururus chinensis. Compounds 4 and 5 exhibited low-density lipoprotein (LDL)-antioxidant activity in the thiobarbituric acid-reactive substances (TBARS) assay (4: IC(50)=3.3 microM and 5: IC(50)=3.8 microM), the lag time of conjugated diene production, the relative electrophoretic mobility (REM) of ox-LDL, the apoB-100 fragmentation on copper-mediated LDL oxidation and the macrophage-mediated LDL oxidation, and radical DPPH scavenging activity.     

Mass-production of human ACAT-1 and ACAT-2 to screen isoform-specific inhibitor: a different substrate specificity and inhibitory regulation

Recently, acyl-CoA:cholesterol acyltransferase was found to be present as two isoforms, ACAT-1 and ACAT-2, in mammalian tissues with different metabolic functions and tissue-specific locations. In this study, the isoforms were mass-produced individually from insect cells to establish a more sensitive and reliable screening method for specific inhibitors against each isoform. The expressed hACAT-1 and hACAT-2 appeared as a 50 kDa- and a 46 kDa-band on SDS-PAGE, respectively, from Hi5 cells and they preferred to exist in oligomeric form, from dimer to tetramer, during the purification process. They also exhibited an approximate 3.4 to 3.7-fold increase in activities when compared to rat liver microsomal fractions at the same protein concentration. Known ACAT inhibitors, pyripyropene A, oleic acid anilide, and diethyl pyrocarbonate, were tested to evaluate the inhibitory specificity and sensitivity of the expressed enzymes. Interestingly, pyripyropene A inhibited only the hACAT-2 fraction with IC(50)=0.64 microM but not the hACAT-1 fraction; whereas the fatty acid anilide did not show a significant difference in inhibitory activity with either hACAT-1 or hACAT-2. Furthermore, cholesterol was more rapidly utilized by hACAT-1, but hACAT-2 esterified other cholic acid derivatives more efficiently. These results suggest that the specificity of each substrate and inhibitor was highly different, depending on each isoform from the viewpoint of the regulatory site and the substrate binding site location.     

Cathepsin B inhibitory activities of phthalates isolated from a marine Pseudomonas strain

Two cathepsin B inhibitors were isolated from the culture supernatant of a marine Pseudomonas sp. PB01 (GenBank Accession No. EU126129). Their structures were elucidated by spectroscopic analyses as dibutyl phthalate and di-(2-ethylhexyl) phthalate. Both dibutyl phthalate and di-(2-ethylhexyl) phthalate showed dose-dependent cathepsin B inhibitions with IC(50) of 0.42 and 0.38 mM, respectively. It is also observed from kinetic analyses that dibutyl phthalate and di-(2-ethylhexyl) phthalate acted as noncompetitive inhibitors with K(i) values of 0.64 and 0.42 mM, respectively. Furthermore, both of them caused inactivation of the pericellular cathepsin B of murine melanoma cell with no acute cytotoxicity. The IC(50) values were found to be 0.23 mM for dibutyl phthalate and 0.14 mM for di-(2-ethylhexyl) phthalate, respectively, and were 50% compared to that of purified cathepsin B.     

三白草中ATG4B抑制剂的发现及活性测定

验证从三白草中提取的两个化合物XGN56和XGN59对自噬关键蛋白ATG4B酶活性的影响及对自噬的调节作用。分子对接的方法验证化合物与游离ATG4B及ATG4B-LC3复合体的氢键结合作用;SDS-PAGE法及荧光共振能量转移法(FRET)测定化合物(10μmol/L)抑制ATG4B的IC50值;LC3融合GFP荧光标签检测化合物(10μmol/L)对LC3荧光聚集的影响,并设置正常组、给药组和药物联用Baf(0.5μmol/L)组;过表达GFP-LC3的WT-MEF及ATG5-/--MEF细胞检测化合物诱导LC3荧光点的情况。结果显示,XGN56和XGN59能分别与游离ATG4B和ATG4B-LC3复合体形成氢键作用,且两者均能剂量依赖地抑制ATG4B的酶切活性,体外IC50分别为7.74μmol/L和8.00μmol/L,同时能够ATG5依赖地促进GFP标记的自噬体的生成(P<0.001)。结果表明,两个化合物可能是通过一定程度地抑制ATG4B的酶活性从而促进细胞自噬水平。     

Cathepsin B inhibitory activities of three new phthalate derivatives isolated from seahorse, Hippocampus Kuda Bleeler

Three new phthalate acid derivatives, 2,12-diethyl-11-methylhexadecyl 2-ethyl-11-methylhexadecyl phthalate (1), 2-ethyldecyl 2-ethylundecyl phthalate (2), and bis(2-ethyldodecyl) phthalate (3), were isolated from seahorse, Hippocampus Kuda Bleeler, together with a known natural analog bis(2-ethylheptyl) phthalate (4). The structures of these compounds were elucidated mainly by means of the comprehensive analysis of their NMR spectroscopic data. The four phthalate derivatives showed dose-dependent cathepsin B inhibitions activities with IC(50) values of 0.13 mM (1), 0.21 mM (2), 0.18 mM (3), and 0.29 mM (4), respectively.     

Evaluation of antioxidant and anticancer activities of chemical constituents of the Saururus chinensis root extracts

Evaluation of antioxidant and anticancer activities were screened by various Saururus chinensis root extracts. Four solvents (ethyl acetate, methanol, ethanol, and water) extracts were investigated for their total flavonoids, phenol contents and their antioxidant activity of DPPH (2,2-diphenyl-1-picrylhydrazyl), NO (nitric oxide), H₂O₂ (hydrogen peroxide), ABTS 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonicacid)diammonium assays, FRAP (ferric reducing ability of plasma) assays and anticancer activity. The total phenolic and flavonoid content of extracts were determined by using FC (Folin–Ciocalteu) and AlCl₃ colorimetric assay method. Total flavonoid content in these plants ranged from 24.7 to 72.1 mg g^?1 and amount of free phenolic compounds was between 11.2 and 67.1 mg g^?1 extract. The all extracts have significant levels of phenolics and flavonoids content. Anticancer activity was screened for MCF-7 breast cancer cell line. Ethanol extract shows significant of antioxidant activity and water extract shows significant of anticancer activity compared with standard (BHT) butylated hydroxy toluene. These ethanol and water extracts could be considered as a natural source for using antioxidant, and anticancer agents compared to commercial available synthetic drugs.     

Polycyclic phloroglucinols as PTP1B inhibitors from Hypericum longistylum: Structures,PTP1B inhibitory activities,and interactions with PTP1B

Protein tyrosine phosphatase 1B (PTP1B) has been regarded as a target for the research and development of new drugs to treat type II diabetes and PTP1B inhibitors are potential lead compounds for this type of new drugs. A phytochemical investigation to obtain new PTP1B inhibitors resulted in the isolation of four new phloroglucinols, longistyliones A–D (1–4) from the aerial parts of Hypericum longistylum. The structures of 1–4 were elucidated on the basis of extensive 1D and 2D NMR spectroscopic data analysis, and the absolute configurations of these compounds were established by comparing their experimental electronic circular dichroism (ECD) spectra with those calculated by the time-dependent density functional theory method. Compounds 1–4 possess a rare polycyclic phloroglucinol skeleton. The following biological evaluation revealed that all of the compounds showed PTP1B inhibitory effects. The further molecular docking studies indicated the strong interactions between these bioactive compounds with the PTP1B protein, which revealed the possible mechanism of PTP1B inhibition of bioactive compounds. All of the results implied that these compounds are potentially useful for the treatment of type II diabetes.     

Triterpenoid saponins from Clematis chinensis and their inhibitory activities on NO production

Four new triterpenoid saponins, clematochinenoside H–K (1–4), and five known structures (5–9), were isolated from the roots and rhizomes of Clematis chinensis. Their structures were elucidated on the basis of spectroscopic evidence and hydrolysis products. All isolates were evaluated for inhibitory effects against nitric oxide (NO) production in LPS-induced RAW 246.7 macrophages. Monodesmosidic saponins (1–3, 5, and 6) with a free carboxylic acid function at C-28 exhibited potent inhibitory activities with IC₅₀ values in the range of 12.9–32.3 μM, where as bisdesmosidic saponin (4, and 7–9) showed modest inhibitory effects with the inhibition ratios (%) from 39.9 to 59.0 at 50 μM. In addition, the hydroxyl group at C-21 showed negative effect on the NO production inhibitory activity.     

PTP1B inhibitory activity of kaurane diterpenes isolated from Siegesbeckia glabrescens

Protein tyrosine phosphatase 1B (PTP1B) is considered as a therapeutic target for the treatment of diabetes and obesity. In our preliminary screening study, a MeOH extract of the aerial part of Siegesbeckia glabrescens was found to inhibit PTP1B activity at 30 μg/mL. Bioassay‐guided fractionation led to the isolation of two active diterpenes, ent-16βH,17-isobutyryloxy-kauran-19-oic acid (1) and ent-16βH,17-acetoxy-18-isobutyryloxy-kauran-19-oic acid (2), along with ent-16βH,17-hydroxy-kauran-19-oic acid (3). Compounds 1 and 2 inhibited the PTP1B activity with IC₅₀ values of 8.7 ± 0.9 and 30.6 ± 2.1 μM, respectively. Kinetic studies suggest that both 1 and 2 are non-competitive inhibitors of PTP1B. However, compound 3 substituted with a hydroxyl group at C-17 in kaurane-type showed no inhibitory effects towards PTP1B.     

PTP1B inhibitory activity of kaurane diterpenes isolated from Siegesbeckia glabrescens

Protein tyrosine phosphatase 1B (PTP1B) is considered as a therapeutic target for the treatment of diabetes and obesity. In our preliminary screening study, a MeOH extract of the aerial part of Siegesbeckia glabrescens was found to inhibit PTP1B activity at 30 microg/mL. Bioassay-guided fractionation led to the isolation of two active diterpenes, ent-16betaH, 17-isobutyryloxy-kauran-19-oic acid (1) and ent-16betaH, 17-acetoxy-18-isobutyryloxy-kauran-19-oic acid (2), along with ent- 16betaH, 17-hydroxykauran-19-oic acid (3). Compounds 1 and 2 inhibited the PTP1B activity with IC50 values of 8.7 +/- 0.9 and 30.6 +/- 2.1 microM, respectively. Kinetic studies suggest that both 1 and 2 are non-competitive inhibitors of PTP1B. However, compound 3 substituted with a hydroxyl group at C-17 in kaurane-type showed no inhibitory effects towards PTP1B.     

Prenylflavonoids from Glycyrrhiza uralensis and their protein tyrosine phosphatase-1B inhibitory activities

Two new 2-arylbenzofurans, glycybenzofuran (1) and cyclolicocoumarone (2), together with 10 known flavonoids including licocoumarone (3), glycyrrhisoflavone (4), glisoflavone (5), cycloglycyrrhisoflavone (6), isoliquiritigenin (7), licoflavone A (8), apigenin (9), isokaempferide (10), glycycoumarin (11), and isoglycycoumarin (12), were isolated from the roots of Glycyrrhiza uralensis and their structures were determined by extensive spectroscopic analyses. Compounds 1 and 5 showed significant protein tyrosine phosphatase-1B (PTP1B) inhibitory activity in vitro with the IC₅₀ values of 25.5 and 27.9 μM, respectively. The structure–activity relationship indicated that the presence of prenyl group and ortho-hydroxy group is important for exhibiting the activity. Kinetic analysis indicated that compound 1 inhibits PTP1B by a competitive mode, whereas compound 5 by a mixed mode.     

Human acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) gene organization and evidence that the 4.3-kilobase ACAT-1 mRNA is produced from two different chromosomes

Acyl-CoA:cholesterol acyltransferase (ACAT) plays important roles in cellular cholesterol homeostasis. Four human ACAT-1 mRNAs (7.0, 4.3, 3.6, and 2.8 kilobases (kb)) share the same short 5'-untranslated region (exon 1) and coding sequence (exons 2-15). The 4.3-kb mRNA contains an additional 5'-untranslated region (1289 nucleotides in length; exons Xa and Xb) immediately upstream from the exon 1 sequence. One ACAT-1 genomic DNA insert covers exons 1-16 and a promoter (the P1 promoter). A separate insert covers exon Xa (1277 base pairs) and a different promoter (the P7 promoter). Gene mapping shows that exons 1-16 and the P1 promoter sequences are located in chromosome 1, while exon Xa and the P7 promoter sequence are located in chromosome 7. RNase protection assays demonstrate three different protected fragments, corresponding to the 4.3-kb mRNA and the two other mRNAs transcribed from the two promoters. These results are consistent with the interpretation that the 4.3-kb mRNA is produced from two different chromosomes, by a novel RNA recombination mechanism involving trans-splicing of two discontinuous precursor RNAs.     

Four new triterpenoid saponins isolated from Schefflera kwangsiensis and their inhibitory activities against FBPase1

Four new triterpenoid saponins, schekwangsiensides H–K (1–4) were isolated from the aerial parts of Schefflera kwangsiensis, together with fifteen esters (5–19) of known saponins. The structures of these compounds were elucidated on the basis of spectroscopic data analysis and chemical evidence. Furthermore, in in vitro assays, compounds 3, 8, 17, and 18 exhibited weak inhibitory activities against fructose-1,6-bisphosphatase (FBPase1).     

Protein tyrosine phosphatase 1B inhibitory activity of triterpenes isolated from Astilbe koreana

Bioassay-guided fractionation of a MeOH extract of the rhizomes of Astilbe koreana (Saxifragaceae), using an in vitro protein tyrosine phosphatase 1B (PTP1B) inhibitory assay, resulted in the isolation of a new triterpene, 3alpha,24-dihydroxyolean-12-en-27-oic acid (4), along with four triterpenes, 3-oxoolean-12-en-27-oic acid (1), 3beta-hydroxyolean-12-en-27-oic acid (beta-peltoboykinolic acid; 2), 3beta-hydroxyurs-12-en-27-oic acid (3), and 3beta,6beta-dihydroxyolean-12-en-27-oic acid (astilbic acid; 5). Compounds 1-5 inhibited PTP1B with IC50 values of 6.8+/-0.5, 5.2+/-0.5, 4.9+/-0.4, 11.7+/-0.9, and 12.8+/-1.1 microM, respectively. Our results indicate that 3-hydroxyl group and a carboxyl group in this type of triterpenes may be required for the activity, while addition of one more hydroxyl group at C-6 or C-24 may be responsible for a loss of activity. Thus, compounds 2 and 3 which possess only one hydroxyl group at C-3 and a carboxyl group at C-27 could be potential PTP1B inhibitors.     

Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine

By using specific anti-ACAT-1 antibodies in immunodepletion studies, we previously found that ACAT-1, a 50-kDa protein, plays a major catalytic role in the adult human liver, adrenal glands, macrophages, and kidneys but not in the intestine. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in the intestine may be largely derived from a different ACAT protein. To test this hypothesis, we produced specific polyclonal anti-ACAT-2 antibodies that quantitatively immunodepleted human ACAT-2, a 46-kDa protein expressed in Chinese hamster ovary cells. In hepatocyte-like HepG2 cells, ACAT-1 comprises 85-90% of the total ACAT activity, with the remainder attributed to ACAT-2. In adult intestines, most of the ACAT activity can be immunodepleted by anti-ACAT-2. ACAT-1 and ACAT-2 do not form hetero-oligomeric complexes. In differentiating intestinal enterocyte-like Caco-2 cells, ACAT-2 protein content increases by 5-10-fold in 6 days, whereas ACAT-1 protein content remains relatively constant. In the small intestine, ACAT-2 is concentrated at the apices of the villi, whereas ACAT-1 is uniformly distributed along the villus-crypt axis. In the human liver, ACAT-1 is present in both fetal and adult hepatocytes. In contrast, ACAT-2 is evident in fetal but not adult hepatocytes. Our results collectively suggest that in humans, ACAT-2 performs significant catalytic roles in the fetal liver and in intestinal enterocytes.     

Constituents of the stems of Macrococculus pomiferus and their inhibitory activities against cyclooxygenases-1 and -2

As part of our program directed towards the discovery of new cancer chemopreventive agents from plants, the EtOAc-soluble extract of the stems of M. pomiferus was found to inhibit the enzyme cyclooxygenase-2 (COX-2). Bioassay-directed fractionation of this extract led to the isolation of two dibenzylbutyrolactone lignans, (8R,8'R)-3'-O-demethyl-5-hydroxymatairesinol (1) and (8R,8'R)-3'-O-demethyl-5-methoxymatairesinol (2), as well as seven known compounds, (-)-5'-methoxyyatein (3), blumenol A, (-)-deoxypodophyllotoxin (anthricin), (-)-deoxypodorhizone, 2,6-dimethoxyhydroquinone, 4-hydroxybenzaldehyde, and beta-sitosterol glucoside. The structures of compounds 1 and 2 were determined using spectroscopic data (1D and 2D NMR, and HREIMS), and the 8R and 8'R absolute stereochemistry was established for both 1 and 2 on the basis of their CD spectra. All isolates obtained in the present study were evaluated for their inhibitory effects with both COX-1 and -2. Of these, only 5'-methoxyyatein (3) showed weak activity against COX-2, while all other compounds isolated were inactive. The COX-2 inhibitory activity of the EtOAc extract was also traced to the presence of several common fatty acids by LC-MS.