Neolignans from North American Magnolia Species with Cyclooxygenase 2 Inhibitory Activity

Objectives:  Based upon reported ethnomedicinal use by Native Americans, extracts and pure isolates from leaves and seeds of Magnolia grandiflora, M. virginiana, M. acuminata and M. macrophylla, all native to the Southeastern United States, were investigated for their anti-inflammatory potential against cyclooxygenase 2 (COX-2). Material and methods:  The extracts and pure compounds from Magnolia species were tested for their production of prostaglandin E₂ (PGE₂) using a mouse macrophage (RAW 264.7) assay where cells were stimulated by lipopolysaccharide. Results:  Leaf extracts were moderately active (44–58% inhibition at 50 μg/ml) whereas seed extracts showed significant activity of 54–88% inhibition, respectively. In the seed extract of M. grandiflora, honokiol, magnolol and 4’-O-methylhonokiol strongly inhibited COX-2 (IC₅₀: 1.2–2.0 μg/ml), 3-O-methylmagnolol was moderately active while a new compound was inactive towards COX-2. The neolignans were not cytotoxic to macrophages (RAW 264.7) and kidney fibroblast (VERO) cells in vitro. Conclusions:  The results indicate that the reported ethnomedicinal use of the investigated Magnolia species is in agreement with anti-inflammatory activity of their respective compounds. Received 8 December 2007; accepted 16 January 2009     

ACAT抑制剂的研究进展

酰基辅酶A-胆固醇酰基转移酶(Acyl-CoA:Cholesterol Acyltransferase,ACAT)在生物体内催化胆固醇与长链脂肪酸连接形成胆固醇酯,在胆固醇的吸收、运输和贮存过程中起重要作用.抑制ACAT可降低血浆总胆固醇及低密度脂蛋白胆固醇水平,防止胆固醇酯化,减少胆固醇酯在动脉壁上积蓄,阻止动脉粥样硬化的形成.A-CAT抑制剂可能成为一类新型的降血脂与抗动脉粥样硬化药物.综述近几年来ACAT抑制剂的研究状况.     

Subacute Toxicity of a Novel Inhibitor of Acyl-CoA: Cholesterol Acyltransferase in Beagle Dogs

PD 132301–2 is a substituted urea hypolipidemic and antiatheroscleroticagent that is a potent inhibitor of acyl-CoA:cholesterol acyltransferase(ACAT). To determine its subacute toxicity, PD 132301–2was administered orally to beagle dogs at 0, 6, 12, 25, 50,200, 400, or 800 mg/kg/day for 2 weeks. Clinicopathologic evaluationswere completed on all dogs. Liver and adrenal total and esterifiedcholesterol concentrations, adrenocorticotrophic hormone (ACTH)responsiveness, and adrenal ultrastructure were determined at0, 6, 12, and 25 mg/kg. At 12 mg/kg or greater, salivation,epiphora, conjunctivitis, emesis, anorexia or decreased foodconsumption, and soft to mucoid feces and/or diarrhea were noted.Suppression of ACTH response occurred by Day 6 at all doses.Adrenocortical degeneration and/or necrosis in zona fasciculataand reticularis was seen at all doses; adrenal free and esterifiedcholesterol were normal at 6 mg/kg and decreased at 12 and 25mg/kg. Increases in serum alanine aminotransferase (2- to 15-fold),aspartate aminotransferase (2- to 12-fold), and alkaline phosphatase(2- to 7-fold) were noted at 50 mg/kg or greater. Periportalhepatocellular hypertrophy and hypereosinophilia occurred at50 mg/kg or greater; hepatic cholesterol values were not significantlyaffected by treatment. Dose-dependent ultrastructural alterationsin adrenocortical cells included decreased numbers of mitochondriaand smooth endoplasmic reticulum profiles, qualitative and quantitativechanges in lipid globules, and increased numbers of autolysosomes.PD 132301-2 or one of its metabolites has potent adrenocorticolyticproperties and limited hepatotoxic properties by mechanism(s)that are likely independent of systemic ACAT inhibition.     

Triterpenoids from the Flower of Campsis grandiflora K. Schum. as Human Acyl-CoA: Cholesterol Acyltransferase Inhibitors

The flower of Campsis grandiflora K. Schum. was extracted with 80% aqueous MeOH, and the concentrated extract was partitioned with EtOAc, n-BuOH and H2O. From the EtOAc fraction, seven triterpenoids were isolated through the repeated silica gel, ODS column chromatographies and preparative HPLC. From the result of physico-chemical data including NMR, MS and IR, the chemical structures of the compounds were determined as 3beta-hydroxyolean-12-en-28-oic acid (oleanolic acid, 1), 3beta-hydroxyurs-12-en-28-oic acid (ursolic acid, 2), 3beta-hydroxyurs-12-en-28-al (ursolic aldehyde, 3), 2alpha,3beta-dihydroxyolean-12-en-28-oic acid (maslinic acid, 4), 2alpha,3beta-dihydroxyurs-12-en-28-oic acid (corosolic acid, 5), 3beta,23-dihydroxyurs-12-en-28-oic acid (23-hydroxyursolic acid, 6) and 2alpha,3beta,23-trihydroxyolean-12-en-28-oic acid (arjunolic acid, 7). These teriterpenoids were isolated for the first time from this plant. Also, compounds 4, 5, 6, and 7 revealed relatively high hACAT-1 inhibitory activity with the value of 46.2+/-1.1, 46.7+/-0.9, 41.5+/-1.3 and 60.8+/-1.1% at the concentration of 100 microg/mL, respectively.     

Difference between Normal and WHHL Rabbits in Susceptibility to the Adrenal Toxicity of an Acyl-CoA:Cholesterol Acyltransferase Inhibitor, FR145237

The adrenal toxicity of an acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor, FR145237, was investigated using Japanese White (normal) and low density lipoprotein (LDL) receptor deficient Watanabe heritable hyperlipidemic (WHHL) rabbits. In the normal rabbits, severe necrosis of the cells in the zona fasciculata and reticularis was observed 24 hr after intravenous injection of 3.2 mg/kg of FR145237, whereas no morphological changes could be found in the adrenal cells of the WHHL rabbits in spite of a higher plasma concentration of the drug. Since most of the FR145237 (87%) in the plasma of the WHHL rabbits was recovered in the LDL fraction 1 hr after intravenous injection of the drug (3.2 mg/kg), it was hypothesized that the delivery of the drug to the adrenal cells may be limited by the LDL receptor deficiency. However, the concentration of FR145237 in the adrenal gland of the WHHL rabbits (13.3 μg/g) was identical to that in the normal rabbits (13.6 μg/g). These results suggest that the susceptibility of the adrenal cells of the WHHL rabbits to the toxicity of FR145237 truly differs from that of normal rabbits, and that the WHHL rabbit may be a useful animal model for the investigation of the mechanisms of the adrenal toxicity of ACAT inhibitors.     

Lignans from the flower buds of Magnolia liliflora Desr

Six new lignans, 1- 6, along with six known compounds were obtained from the flower buds of Magnolia liliflora Desr. The new lignans were elucidated as (1 S*,2 R*,5 S*,6 S*)-2-(3,5-dimethoxyphenyl)-6-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (1), (1 R*,2 R*,5 R*,6 S*)-2-(3,5-dimethoxyphenyl)-6-(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (2), (1 R*,?2 R*,5 R*,6 S*)-2,6-bis (3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (3), (1 R*,2 S*,5 R*,6 R*)-2-(3,4-methylenedioxyphenyl)-6-(3,5-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane ( 4), (7' S*,8 R*,8' R*)-3,5'-dimethoxy-3',4,9'-trihydroxy-7',9-epoxy-8,8'-lignan (5), and (7' R*,8' S*)-3,3',4,5'-tetramethoxy-7-en-7',9-epoxy-8,8'-lignan (6), by the analysis of 1D and 2D-NMR as well as HRESIMS data. The capacity of compound 1 to protect against damages to the DNA of rat lymphocyte cells induced by UV irradiation was assessed by the comet assay. It showed stronger antigenotoxicity than ascorbic acid from 6×10(-3)?mmol·L(-1) to 6×10(-6)?mmol·L(-1).     

Lignans from the bark of Machilus thunbergii and their DNA topoisomerases I and II inhibition and cytotoxicity

Activity-guided fractionation based on topoisomerase I inhibitory activity lead to the isolation of ten lignans (1-10) from the methylene chloride extract of the bark of Machilus thunbergii SIEB. et ZUCC. (Lauraceae). These were identified as machilin A (1), erythro-austrobailignan-6 (2), meso-monomethyl dihydroguaiaretic acid (3), meso-dihydroguaiaretic acid (4), galbacin (5), machilin F (6), nectandrin A (7) nectandrin B (8), (-)-acuminatin (9) and (7S,8S)-7-(4-hydroxy-3-methoxyphenyl)-1'-formyl-3'-methoxy-8-methyldihydrobenzofuran (10) by spectral evidence. In DNA topoisomerase I and II assays in vitro at a concentration of 100 microM, 4 showed the most potent inhibitory activity, 93.6 and 82.1% inhibition, respectively, and 8 showed 79.1 and 34.3% inhibition, respectively. All of these compounds exhibited weak or no cytotoxicities against either the human colon carcinoma cell line (HT-29) or the human breast carcinoma cell line (MCF-7).     

Antiperoxidative Activity of Neolignans from Magnolia obovata

Honokiol and magnolol, neolignans in Magnolia obovata, have been evaluated as antioxidants. Microsomal lipid peroxidation induced by Fe(III)-ADP/NADPH and mitochondrial lipid peroxidation induced by Fe(III)-ADP/NADH were inhibited by these compounds. These neolignans protected mitochondrial respiratory chain enzyme activity against NADPH-induced peroxidative stress and protected red cells against oxidative haemolysis. The anti-oxidative activity of honokiol was more potent than that of magnolol. Neolignans in M. obovata were shown to be effective in protecting biological systems and functions against oxidative stress.     

一株南沙群岛柳珊瑚来源真菌 Aspergillus terreus 中化合物及 mPTPB 酶抑制活性研究

目的 从1株南沙群岛柳珊瑚来源真菌 Aspergillus terreus (NS02-09)中分离鉴定海洋天然产物,对所得化合物进行结核分枝杆菌酪氨酸磷酸激酶 (mPTPB) 抑制活性评价。方法 运用多种色谱手段分离纯化化合物,利用NMR、CD等现代波谱分析方法,对化合物进行结构鉴定、,通过衍生物制备获得两个乙酰化衍生物(2a和2b);并对化合物2及其衍生物2a和2b进行mPTPB酶抑制活性测试。结果 鉴定了1个土曲霉酮(1)和1个丁烯酸内酯 (2) 的结构; 2具有较强的mPTPB酶抑制活性,而其乙酰化产物(2a和2b)的mPTPB 酶抑制活性显著降低。运用Sybyl X 1.3 软件,对2与mPTPB酶的模拟对接计算发现,丁烯酸内酯环及环上的羟基是化合物2发挥酶抑制活性的重要作用基团。结论 从柳珊瑚来源真菌 A. terreus (NS02-09) 中发现了具有mPTPB 酶抑制活性的丁烯酸内酯类化合物,并对其作用机制进行了计算研究,该类化合物的相关研究对抗结核药物先导化合物发现具有借鉴作用。     

Biological Activity of Lignans from the Seeds of Centaurea scabiosa

Antibacterial, antioxidant, ecdysteroid agonist/antagonist activities, and general toxicity of two major lignans, matairesinol and matairesinoside, of the seeds of Centaurea scabiosa have been assessed.     

Inhibitory Effect of Vanadium Compounds on Glutamate Dehydrogenase Activity in Mitochondria and Hepatocytes Isolated from Rabbit Liver

Abstract: The effect of orthovanadate, vanadyl sulphate and vanadyl acetylacetonate on glutamate dehydrogenase activity was studied in liver mitochondria and isolated hepatocytes of rabbit. In pcrmeabilized mitochondria with free access of substrates and drugs to glutamate dehydrogenase, orthovanadate and vanadyl sulphate at 200 μM concentrations decreased both glutamate synthesis and glutamate deamination by 80 and 50 %, respectively, while vanadyl acetylacetonate was less potent. In view of kinetic data obtained at various ammonium concentrations, orthovanadate appeared to be a competitive inhibitor (K_i= 40 × 3 μM), while vanadyl sulphate was a non–competitive one (K_i = 147 × 10 μM). In contrast to orthovanadate, vanadyl sulphate augmented the inhibitory action of increased above 0.5 mM 2–oxoglutarate concentrations. All these effects on the enzyme activity were partially reversed in the presence of L–leucine and ADR which are allosteric activators of glutamate dehydrogenase. Moreover, all compounds studied suppressed both glutamate formation and glutamate deamination in isolated hepatocytes incubated under various metabolic conditions, as concluded from decreased rates of glutamate and urea synthesis, respectively. In view of these observations it seems likely that vanadiumcontaining compounds may be potent inhibitors of glutamate metabolism in liver.     

Biological Activity and Aryltetraline Lignans of Linum persicum.

Abstract

A total extract of Linum persicum Kotschy ex Boiss. was investigated for biological activity. The extract was not active against Gram-negative and Gram-positive bacteria. The extract exhibited significant activity against brine shrimp (Artemia salina). Cytotoxic activity of the extract was evaluated by the MTT assay on five different cell lines. The total extract showed the highest cytotoxicity against human chronic myeloid leukemia (K-562), T-cell lines (Jurkat), and lung carcinoma (A5). Three aryltetralin lignans, podophyllotoxin, 5-methoxypodophyllotoxin, and 5-methoxy podophyllotoxin acetate, were isolated from the methanol extract of the aerial parts of L. persicum, and the structures were confirmed by spectroscopic methods.     

Isolation and Inhibitory Effects of Eupatilin,a Flavone Isolated from Artemisia monosperma Del., on Rat Isolated Smooth Muscle

The isolation for the first time from Artemisia monosperma (Compositae) of the known flavone eupatilin and its effects on rat isolated smooth muscle preparations are described. In concentrations from 10 -7 M to 3×10 -4 M, eupatilin (5, 7-dihydroxy-6, 3', 4'-trimethoxyflavone) inhibited in a reversible manner the phasic contractions and the tone of rat isolated ileum, uterus, and urinary bladder. It relaxed the tonic contractions of the phenylephrine-precontracted pulmonary artery and acetylcholine-precontracted trachea. Eupatilin also shifted the concentration-effect curves of acetylcholine and calcium chloride on rat isolated ileum, oxytocin concentration-effect curve on uterine smooth muscle and phenylephrine concentration-effect curve on pulmonary artery smooth muscle. These observations indicate that eupatilin possesses a nonspecific antispasmodic effect on rat isolated smooth muscle. They also suggest that the inhibitory effect of eupatilin may be mediated by changes in Ca 2+ metabolism in these preparations.     

In-vitro Metabolism of YM17E,an Inhibitor of Acyl Coenzyme A: Cholesterol Acyltransferase,by Liver Microsomes in Man

Because YM17E (1,3-bis[[1-cycloheptyl-3-(p-dimethylaminophenyl)ureido]methyl]benzene dihydrochloride) inhibits acyl coenzyme A:cholesterol acyltransferase (ACAT) it has potential application in the treatment of hypercholesterolaemia. In man and animals YM17E is extensively metabolized, via N-demethylation, to five active metabolites (M1, M2-a, M2-b, M3 and M4). The main objectives of this study were to examine inhibition of YM17E metabolism by the products and identify the cytochrome P450 isoforms in liver microsomes which catalyse in-vitro YM17E metabolism in man. In microsomes in man, N-demethylation of YM17E to M1 occurred enzymatically; for up to 45 s the rate was linearly proportional to the microsomal protein concentration. This reaction was inhibited by metabolites M2-a, M2-b, M3 and M4. Further, N-demethylation of [¹⁴C]-YM17E was also inhibited by its product, M1. These results showed that primary metabolism of YM17E was inhibited by its products, and supported the finding that the non-linear increase in plasma concentration of the parent drug and metabolites observed in an in-vivo study was due to inhibition by these products. Metabolic activity in microsomes from ten individual human livers demonstrated that YM17E N-demethylase activity correlated closely with testosterone 6β-hydroxylase activity. When cytochrome P450 isozyme-specific substrates and chemical inhibitors were used to inhibit YM17E N-demethylase activity, CYP3A-specific substrate and inhibitors such as nifedipine, ketoconazole and triacetyloleandomycin strongly inhibited this activity, whereas CYP1A-specific substrate or inhibitor, ethoxyresorufin and α-naphthoflavone, inhibited weakly. Other CYP inhibitors, in contrast, had few or no effects. An inhibition study using anti-rat CYP1A1, CYP2B1, CYP2C11, CYP2E1 and CYP3A2 antibodies demonstrated that only anti-rat CYP3A2 antibody inhibited YM17E metabolism, to 40% of control level, with no other antibodies showing an inhibitory effect. Of seven cDNA-expressed P450 isoforms in man (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2D6, CYP2E1 and CYP3A4), CYP3A4, CYP2D6 and CYP1A2 isozyme exhibited substantial catalytic activity of N-demethylation of YM17E. These results indicate the predominant role of CYP3A4 in liver metabolism of YM17E in man.     

辛夷不同组分抗过敏作用活性比较研究

目的通过比较辛夷不同组分抗过敏作用活性的强弱,确定辛夷抗过敏的有效组分,为指导临床用药及重大创新药物的研发提供参考及思路。方法分别给小鼠辛夷醇提组分、水提组分、挥发油组分,观察小鼠抗过敏性毛细血管通透性增强实验和皮肤被动过敏反应实验,比较辛夷不同组分抗过敏作用活性的大小。结果通过比较获知辛夷不同提取组分抗过敏作用强度为：醇提组分作用最强,水提组分和挥发油组分作用次之,两者差异不明显;醇提组分与水提组分或与挥发油组分比较,均呈现明显差异。结论辛夷不同组分抗过敏作用活性大小为：醇提组分〉水提组分〉挥发油组分,抗过敏活性导向下辛夷的有效组分为醇提取组分,但是鉴于中药多组分、多靶点、多途径的药效特点,以及辛夷多组分之间的相互作用及作用机制并不是十分明确,因此有必要进行多组分物质基础下的辛夷多组分之间药理作用及分子机制相关性研究,为更好的指导临床用药及创新药研发提供参考和思路。     

857-860Anti-inflammatory Effect of Magnolol,Isolated from Magnolia officinalis,on A23187-induced Pleurisy in Mice

In the present study, A23187-induced pleurisy in mice was used to investigate the anti-inflammatory effect of magnolol, a phenolic compound isolated from Chinese medicine Hou p'u (cortex of Magnolia officinalis). A23187-induced protein leakage was reduced by magnolol (10mgkg^?1, i.p.), indomethacin (10mgkg^?1, i.p.) and BW755C (30mgkg^?1, i.p.). A23187-induced polymorphonuclear (PMN) leucocyte infiltration in the pleural cavity was suppressed by magnolol and BW755C, while enhanced by indomethacin. Like BW755C, magnolol reduced both prostaglandin E₂ (PGE₂) and leukotriene B₄ (LTB₄) levels in the pleural fluid of A23187-induced pleurisy, while indomethacin reduced PGE₂ but increased LTB₄ formation. In the rat isolated peripheral neutrophil suspension, magnolol (3.7 μM) and BW755C (10 μM) also suppressed the A23187-induced thromboxane B₂ (TXB₂) and LTB₄ formation. These results suggest that magnolol, like BW755C, might be a dual cyclo-oxygenase and lipoxygenase inhibitor. The inhibitory effect of magnolol on the A23187-induced pleurisy is proposed to be, at least partly, dependent on the reduction of the formation of eicosanoids mediators in the inflammatory site.     

Isolation of the Lignan 6-Methoxypodophyllotoxin from Linum boissieri. and Biosynthesis of Lignans in this Species

Abstract

We were able to establish a suspension culture of Linum boissieri. that produces 6-methoxypodophyllotoxin (6MPT). As a first step to gain insight into the lignan biosynthesis in L. boissieri. cell cultures, we were able to measure phenylalanine ammonia-lyase (PAL) activity in raw protein extracts. PAL is a key enzyme in the early part of the general phenylpropanoid pathway, leading (beside others) to the precursors for lignan biosynthesis.     

Anti-Inflammatory and Anti-Superbacterial Activity of Polyphenols Isolated from Black Raspberry

The fruit of the black raspberry (Rubus coreanus Miquel) has been employed in traditional medicine, and recent studies have demonstrated its measureable biological activities. However, the root of the black raspberry has not been studied. Therefore, in this study, we evaluated the anti-inflammatory and antibacterial properties of the root and unripe fruit polyphenols of the black raspberry. Both polyphenols proved to have anti-inflammatory activity as evidenced by the decreased nitric oxide (NO), cytokines (IL-1β , IL-6, and IL-10) and prostaglandin E2 (PGE₂) levels in lipopolysaccharide (LPS)-stimulated RAW 264.7 murine macrophages. However, root polyphenols showed stronger anti-inflammatory activity than fruit polyphenols. LPS-induced mRNA and protein expressions of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 levels were also decreased, confirming the anti-inflammatory activity. Root polyphenols showed lethal activity against methicillin-resistant Staphylococcus aureus (MRSA), carbapenem-resistant Acinetobacter baumannii (CRAB), and Bacillus anthracis. In contrast, the black raspberry fruit did not demonstrate these properties. These data provide the first demonstration that black raspberry root has potential anti-inflammatory and anti-superbacterial properties that can be exploited as alternatives for use in the food and cosmetic industries and/or as pharmaceuticals.     

Guineensine, an Acyl-CoA: cholesterol acyltransferase inhibitor, from the fruits of Piper longum

Bioactivity-guided fractionations for ACAT inhibitor led to the isolation of guineensine from the CHCl (3) extract of Piper longum. Its structure was identified by spectroscopic means (IR, UV, MS and NMR). Guineensine inhibited ACAT activity in a dose-dependent manner with an IC (50) value of 3.12 micro M.