Bioactive terpenoids and guggulusteroids from Commiphora mukul gum resin of potential anti-inflammatory interest

Guggulu, the gum resin from Commiphora mukul, is one of the components of various formulations of traditional Ayurvedic medicine to treat inflammation, obesity, and lipid disorders. In most preparations of Ayurvedic medicine in India, guggulu is boiled prior to its use. Therefore, guggulu was boiled with H2O prior to extractions in our study. Bioassay-guided isolation of compounds from the hexane-soluble portion of the MeOH extract of guggulu yielded cembrenoids, 1-6, a bicyclic diterpene, 7, guggulusterone derivatives, 8-11, myrrhanone derivatives, 12, myrrhanol derivative, 13, and a lignan, 14. The structures of these compounds were confirmed by spectroscopic methods. Compounds 5, 6, 7, 10, and 12-14 are novel. These compounds were assayed for lipid peroxidation and cyclooxygenase (COX) enzyme inhibitory activities. At 100 ppm, compounds 3, 6, and 14 inhibited the lipid peroxidation by 79, 57, and 58%, respectively, and the rest of isolated compounds showed 20-40% inhibitory activity with respect to the controls. In COX-1 and COX-2 enzyme inhibitory assays, compound 3 showed 79 and 83%, and compound 8 gave 67 and 54% of inhibition, respectively, at 100 ppm. All fourteen compounds inhibited COX-1 enzyme at 100 ppm. The lipid peroxidation and COX enzyme inhibitory activities exhibited by compounds isolated from C. mukul may substantiate its use in traditional medicine.     

铁筷子提取物对肿瘤细胞增殖及COX-2 mRNA表达的抑制作用

研究不同铁筷子提取物对肿瘤细胞增殖及 COX-2 mRNA 表达的抑制作用。以铁筷子醇总提取物(TKZ1)、正丁醇萃取部位(TKZ2)、乙酸乙酯萃取部位(TKZ3)分别作用于 DU145、PC3、HeLa、HT-29、HepG2等肿瘤细胞,应用噻唑蓝实验(MTT 法)计算其对细胞增殖的抑制作用,应用荧光定量 PCR 技术检测TKZ1、TKZ2、TKZ3处理后的各肿瘤细胞中 COX-2 mRNA 的表达情况。结果表明：TKZ1、TKZ2、TKZ3均能显著抑制多种肿瘤细胞的增殖,与阴性对照组比较,其可以在 mRNA 水平上抑制 COX-2的表达,且呈明显的量效关系。说明铁筷子提取物对体外肿瘤细胞的增殖具有显著的抑制作用,其抗瘤机制可能与抑制肿瘤细胞中 COX-2 mRNA 的表达有关。     

Constituents in Easter lily flowers with medicinal activity

Easter lily (Lilium longiflorum) flowers have been used in traditional medicine for alleviating many ailments. However, the chemical basis of its bioactivity has not been investigated. We have determined bioactive components in Easter lily flowers using lipid peroxidation and cyclooxygenase enzyme inhibitory assays and found to be kaempferol (1), kaempferol glycosides (2, 3, 4, 8, 9 and 10), quercetin glycosides (5, 6 and 7), a regaloside (11), a chalcone (12) and a fatty acid fraction (13). The structures of compounds were determined by NMR, IR, UV/VIS and mass spectroscopic studies. Compound 1 showed the highest COX-1 inhibition (94.1%) followed by 3, 8 and 12 with 38.7, 30.8 and 32.4%, respectively. Only compound 1 inhibited COX-2 enzyme by 36.9% at 80 ppm. In lipid peroxidation inhibitory assay, kaempferol showed 37 and 100 % inhibitions at 1 and 10 ppm, respectively. At 10 ppm, more than 20% inhibition was observed for compounds 4, 7, 10, 11 and 12 and 53% for compound 3. The compounds reported in here are isolated for the first time from Easter lily flowers including novel compounds 10, 11 and 12. Our results suggest that kaempferol and quercetin flavonoids contributed to the anecdotal medicinal properties of Easter lily flowers.     

Effects of eicosatrienoic acid (20:3 n-9, Mead's acid) on some promalignant-related properties of three human cancer cell lines

The essential fatty acid deficiency (EFAD) is a metabolic condition related to cancer development. We studied the effect of eicosapentaenoic acid (EPA, 20:5 n-3) and eicosatrienoic acid (ETA, 20:3 n-9), an essential fatty acid (EFA) and non-EFA respectively, on tumour cells parameters linked to tumour progression and metastases. Human tumour cell lines (T-24 from urothelium, MCF-7 from breast and HRT-18 from colon) were used. EPA showed an anti-proliferative effect on the three lines. ETA showed the following effects: in T-24, the lipid peroxidation was decreased and E-cadherin was undetectable; in MCF-7, increased E-cadherin expression enhanced the lipid peroxidation and decreased cell proliferation; on HRT-18, the E-cadherin expression and lipid peroxidation diminished, whereas cell proliferation was increased. In conclusion, EFA (20:5 n-3) exhibited beneficial effects, whereas unusual ETA showed an opposite effect on some tumour parameters. The possible riskiness of EFA-deprivation, along with the potential of EFA as natural nutrapeutic products for human tumour prevention and treatment, makes EFA worthy of further consideration.     

半枝莲抗肝癌活性部位的化学成分研究

前期研究发现,半枝莲(Scutellaria barbata)全草醇提物的乙酸乙酯萃取部位经大孔吸附树脂处理,其70%乙醇洗脱部位具有较好的抗肝癌活性。为明确其活性成分,该研究采用硅胶柱色谱、Sephadex LH-20柱色谱、制备TLC、半制备液相色谱等对活性部位进行分离和纯化,运用多种波谱分析方法鉴定了单体化合物结构,并利用CCK-8法评价了所有单体化合物对人肝癌HepG2细胞体外增殖抑制活性,同时利用分子对接技术考察了活性最好的化合物与肝癌靶标的结合情况。结果表明:(1)从该活性部位共分离得到14个化合物,包括12个新克罗烷型二萜类化合物和2个黄酮类化合物,分别鉴定为scutefolide C(1)、6-乙酰氧基-7-烟酸酰氧基半枝莲碱G(2)、scutestrigillosin D(3)、 scutehenanine D(4)、半枝莲碱A(5)、半枝莲碱B(6)、7-烟酸酰氧基半枝莲碱H(7)、半枝莲碱N(8)、半枝莲碱Y(9)、barbatin A(10)、barbatin B(11)、barbatin D(12)、5, 7, 6''-三羟基-2''-甲氧基黄酮醇(13)和5, 8-二羟基-6, 7-二甲氧基黄酮(14)。其中,化合物1-3、13、14为首次从该植物中分离得到。(2)活性测试结果显示,化合物4、7、10-12表现出较弱的HepG2细胞增殖抑制活性,化合物6的细胞增殖抑制活性和阳性对照(顺铂)活性接近,而化合物5表现出比顺铂更强的细胞增殖抑制活性。(3)分子对接结果显示,化合物5和化合物6与肝癌靶蛋白VEGF-2均具有良好的结合力。该研究结果不仅丰富了半枝莲的化学物质类群,也为进一步深入研究活性化合物抗肝癌的作用机制提供了参考。     

The effect of nitrogen and sucrose concentrations on the growth of Eucomis autumnalis (Mill.) Chitt. plantlets in vitro, and on subsequent anti-inflammatory activity in extracts prepared from the plantlets

Large amounts of anti-inflammatory activity are present in extractsprepared from Eucomis plants. Extracts prepared from in vitroplantlets grown on a modified Murashige and Skoog medium supplementedwith 1 mg &ell^–1 NAA and 1 mg &ell^–1 BA, were tested intwo cyclooxygenase assays (COX-1 and COX-2). Ethanol extracts showedhigh levels of COX-1 and COX-2 inhibitory activity, with a COX-2/COX-1inhibition ratio of 1.1. Further experimental work aimed to determine thefactors affecting the accumulation of anti-inflammatory compounds inin vitro plantlets. High concentrations of sucrose (40 g &a,p;ell^–1) inthe culture medium significantly increased the number of shoots initiated,but had no effect on the subsequent anti-inflammatory activity. Lowconcentrations of sucrose (10 g &ell^–1) led to a significantdecrease in COX-1 inhibition. Changig the amount of nitrogen in the medium(but not the ratio of nitrate to ammonium ions) had no significant effect onthe COX-1 inhibitory activity of the extracts.     

Inhibition of human tumour cell proliferation by analogues of adenosine

The effects of adenosine and several structural analogues of adenosine upon thymidine incorporation into human tumour cells and rat cervical lymphocytes were investigated. The analogue NECA, which has equal specificity for the A₁ and A₂ receptor, had the most inhibitory effect on lymphocyte proliferation while the A₁ agonists had limited effects, suggesting that these cells possess principally A₂ adenosine receptors. In the case of human tumour cells, however, the most inhibitory effect on proliferation was obtained with the A₁-specific analogues. The general order of inhibitory effects of adenosine analogues on thymidine incorporation in human tumour cells was: S-ENBA>CPA=R-PIA>S-PIA>NECA. These findings suggest that in the cells presently studied the A₁ adenosine receptor predominates. Removal of exogenous adenosine by growth in the presence of adenosine deaminase inhibited thymidine incorporation. The effect of adenosine removal lends further support to the proposal that adenosine has some, as yet unidentified, regulatory role in the control of human tumour cell proliferation. © 1997 John Wiley & Sons, Ltd.     

Synthesis of modified homo-N-nucleosides from the reactions of mesityl nitrile oxide with 9-allylpurines and their influence on lipid peroxidation and thrombin inhibition

9-(3-Mesityl-4,5-dihydroisoxazol-5-yl) homo-N-nucleosides were prepared from the 1,3-dipolar cycloaddition reactions of mesityl nitrile oxide with 9-allyl derivatives of 6-chloropurine, 6-piperidinylpurine, 6-morpholinylpurine, 6-pyrrolidinylpurine, and 6-N,N-dibenzoyladenine. The new compounds were tested in vitro for their ability: (i) to interact with 1,1-diphenyl-2-picryl-hydrazyl (DPPH) stable free radical, (ii) to inhibit lipid peroxidation, (iii) to scavenge the superoxide anion, (iv) to inhibit the activity of soybean lipoxygenase, and (v) to inhibit in vitro thrombin. Most of them found to be potent thrombin inhibitors and to inhibit in vitro lipid peroxidation. The majority of the compounds showed significant lipoxygenase inhibitory activity.     

Design,synthesis and molecular docking of benzophenone conjugated with oxadiazole sulphur bridge pyrazole pharmacophores as anti inflammatory and analgesic agents

The prostaglandins (PG) a group of physiologically active lipid compounds having diverse hormone like effects are important mediators of the body’s response to pain and inflammation, and are formed from essential fatty acids found in cell membranes. This reaction is catalyzed by cyclooxygenase, a membrane associated enzyme occurring in two isoforms, COX-1 and COX-2. Nonsteroidal anti-inflammatory drugs (NSAIDs) act by inhibiting the activity of COX. In view of this, a series of novel benzophenones conjugated with oxadiazole sulphur bridge pyrazole moiety 8a-l were designed, synthesized, characterized and subsequently evaluated for anti-inflammatory and analgesic property. The investigation of novel analogues 8a-l for potential anti-inflammatory activity showed high levels of COX-1 and COX-2 inhibitory activity. Among the series, compound 8i with electron withdrawing fluoro group at the para position of the benzoyl ring of benzophenone was characterized by highest IC₅₀ values for both COX-1 and COX-2 inhibition, which is comparable to the standard drug. Further, molecular docking studies have been performed for the potent compound.     

Effect of ginger constituents and synthetic analogues on cyclooxygenase-2 enzyme in intact cells.

Seventeen pungent oleoresin principles of ginger (Zingiber officinale, Roscoe) and synthetic analogues were evaluated for inhibition of cyclooxygenase-2 (COX-2) enzyme activity in the intact cell. These compounds exhibited a concentration and structure dependent inhibition of the enzyme, with IC(50) values in the range of 1-25 microM. Ginger constituents, [8]-paradol and [8]-shogaol, as well as two synthetic analogues, 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)decane and 5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)dodecane, showed strong inhibitory effects on COX-2 enzyme activity. The SAR analysis of these phenolic compounds revealed three important structural features that affect COX-2 inhibition: (i) lipophilicity of the alkyl side chain, (ii) substitution pattern of hydroxy and carbonyl groups on the side chain, and (iii) substitution pattern of hydroxy and methoxy groups on the aromatic moiety.     

Synthesis and antioxidative activity of metalloporphyrins bearing 2,6-di-tert-butylphenol pendants

The novel metalloporphyrins (M = HH, Fe, Mn, Co, Cu, Zn) bearing 2,6-di-tert-butylphenol pendants as antioxidant substituents, and a long chain hydrocarbon palmitoyl group have been synthesized. The oxidation of compounds by PbO₂ leads to the formation of the corresponding 2,6-di-tert-butylphenoxyl radicals studied by EPR. The activity of porphyrins in lipid peroxidation has been examined using (1) in vitro lipid peroxidation induced by tert-butylhydroperoxide in respiring rat liver mitochondria, (2) in vitro lipid peroxidation in liver homogenates of Wistar strain rats, and (3) a model process of peroxidation of (Z)-octadec-9-enic (oleic) acid as a structural fragment of lipids. The activity of these compounds depends dramatically on the nature of metal and might be changed from antioxidative (M = HH, Mn, Cu, Zn) to indifferent (M = Co), and to pro-oxidative one (M = Fe). The anti- or pro-oxidative action of these compounds may be derived from the concurrence between the involvement of 2,6-di-tert-butylphenol pendants acting as radical scavengers and redox active metal center promoting oxidation processes. The results of this study suggest that the polytopic compounds combining in one molecule 2,6-di-tert-butylphenol pendants, metalloporphyrin moiety, and a palmitoyl group, are membrane active compounds and might be studied in an effort to find novel pharmaceutical agents.     

The growth inhibitory effect of conjugated linoleic acid on a human hepatoma cell line, HepG2, is induced by a change in fatty acid metabolism, but not the facilitation of lipid peroxidation in the cells

We investigated the growth inhibitory effect of conjugated linoleic acid (CLA) on HepG2 (human hepatoma cell line), exploring whether the inhibitory action occurs via lipid peroxidation in the cells. When the cells were incubated up to 72 h with 5-40 microM of CLA (a mixture of 9c,11t-18:2 and 10t,12c-18:2), cell proliferation was clearly inhibited in a dose and time dependent manner but such an inhibition was not confirmed with linoleic acid (LA). In order to evaluate the possible contribution of lipid peroxidation exerted by CLA to cell growth inhibition, alpha-tocopherol (5-20 microM) and BHT (1-10 microM) as potent antioxidants were added to the medium with CLA (20 microM), which did not restore cell growth at all. Furthermore, after 72 h incubation, the membranous phospholipid hydroperoxide formation in the CLA-supplemented cells was suppressed respectively to 25% and 50% of that in LA-supplemented cells and control cells. No difference was observed by a conventional lipid peroxide assay, the TBA test, between CLA-supplemented cells and LA-supplemented cells. Although the cellular lipid peroxidation was not stimulated, lipid contents (triacylglycerol, total cholesterol and free cholesterol) and fatty acid contents (palmitic acid, palmitoleic acid and stearic acid) markedly increased in CLA-supplemented cells compared with LA-supplemented and control cells. Moreover, supplementation with 20 microM LA and 20 microM arachidonic acid profoundly interfered with the inhibitory effect of CLA in HepG2. These results suggest that the growth inhibitory effect of CLA on HepG2 is due to changes in fatty acid metabolism but not to lipid peroxidation.     

Response of Barley Seedlings to UV-B Radiation as Affected by Proline and NaCl

Barley (Hordeum vulgare L. cv. Alfa) seedlings were treated for 4 d before UV-B irradiation with 0.05 mM proline or 150 mM NaCl. UV-B exposure induced synthesis of yellow coloured compounds with maximum absorbance at 438 nm. The content of these compounds was increased in proline-treated and decreased in NaCl-treated plants. UV-B radiation reduced chlorophyll/carotenoids ratio, oxygen evolution rate and photochemical efficiency of PS 2 as estimated by chlorophyll fluorescence and increased proline accumulation, H₂O₂ generation and lipid peroxidation. Exogenous proline had no effect on the parameters studied and did not change the response of plants to UV-B radiation. NaCl inhibited photochemical efficiency of PS 2, reduced oxygen evolution and increased H₂O₂ concentration and lipid peroxidation. The combination of NaCl and proline treatment led to lowering the inhibitory effect of NaCl in non UV-B irradiated seedlings. There was not relationship between the level of UV-B-induced compounds and UV-B tolerance of barley seedlings.     

Ganglioside-dependent factor, inhibiting lipid peroxidation in rat brain synaptosomes.

Preincubation of rat brain synaptosomes with GM1, GD1a or GT1b (10(-10)-10(-6) M), as well as with phorbol 12-myristate, 13-acetate (10(-10)-10(-6) M) was found to have dose dependent inhibitory effect on Fe(2+)-ascorbate induced lipid peroxidation, while penetrating analogue of c-AMP markedly decreased the inhibitory effect of these compounds. In liposomes made of lipids isolated from synaptosomal membranes the degree of inhibition of induced LPO by gangliosides was practically absent. The inhibitory effect of GM1 on lipid peroxidation could not be revealed after thermal denaturation of synaptosomes or after treatment with polymyxin B (inhibitor of lipid-dependent protein kinases). These results and some other data provide evidence for the existence of ganglioside-dependent factor inhibiting lipid peroxidation in brain tissue. It may be suggested to be a protein kinase modulated by gangliosides.     

Artepillin C isoprenomics: design and synthesis of artepillin C isoprene analogues as lipid peroxidation inhibitor having low mitochondrial toxicity

We designed and synthesized isoprene analogues of artepillin C, a major component of Brazilian propolis, and investigated the inhibitory activity on lipid peroxidation of rat liver mitochondria (RLM) and RLM toxicity based on isoprenomics. We succeeded in the synthesis of artepillin C isoprene analogues using regioselective prenylation within the range from 22% to 53% total yield. Reactivity of artepillin C and its isoprene analogues with ABTS (2,2'-Azinobis(3-ethylbenzothiazoline-6-sulfonate)) radical cations showed only a slight difference among the molecules. The isoprene side-chain elongation analogues of artepillin C showed almost the same inhibitory activity against RLM lipid peroxidation as artepillin C. Artepillin C and its isoprene analogues had very weak RLM uncoupling activity. Moreover, artepillin C and its isoprene analogues exhibited a lower inhibitory activity against adenosine 5'-triphosphate (ATP) synthesis by about two orders of magnitude than the effective inhibitory activity against RLM lipid peroxidation. From these results we conclude that artepillin C isoprene analogues could be potent lipid peroxidation inhibitors having low mitochondrial toxicity. We also conclude that elongation of the isoprene side chain of artepillin C to increase lipophilicity had little influence on the inhibitory activity toward RLM lipid peroxidation.     

Oxidative variables and antioxidant enzymes activities in the mdx mouse brain

Dystrophin is a protein found at the plasmatic membrane in muscle and postsynaptic membrane of some neurons, where it plays an important role on synaptic transmission and plasticity. Its absence is associated with Duchenne's muscular dystrophy (DMD), in which cognitive impairment is found. Oxidative stress appears to be involved in the physiopathology of DMD and its cognitive dysfunction. In this regard, the present study investigated oxidative parameters (lipid and protein peroxidation) and antioxidant enzymes activities (superoxide dismutase and catalase) in prefrontal cortex, cerebellum, hippocampus, striatum and cortex tissues from male dystrophic mdx and normal C57BL10 mice. We observed (1) reduced lipid peroxidation in striatum and protein peroxidation in cerebellum and prefrontal cortex; (2) increased superoxide dismutase activity in cerebellum, prefrontal cortex, hippocampus and striatum; and (3) reduced catalase activity in striatum. It seems by our results, that the superoxide dismutase antioxidant mechanism is playing a protective role against lipid and protein peroxidation in mdx mouse brain.     

Efficient scavenging of hydroxyl radicals and inhibition of lipid peroxidation by novel analogues of 1,3,7-trimethyluric acid.

New water-soluble analogues of 1,3,7-trimethyluric acid with N-1 methyl replaced by various groups were prepared and evaluated for their ability to scavenge hydroxyl radicals as well as their protective potential against lipid peroxidation in erythrocyte membranes. The deoxyribose degradation method indicates that all the analogues tested effectively scavenge hydroxyl radicals and some of them show better activity than uric acid and methyluric acids. These effects are shown to be concentration dependent and are more potent at low concentrations (10-50 microM). Among the analogues tested, 1-butenyl-, 1-propargyl- and 1-benzyl-3,7-dimethyluric acids show high hydroxyl radical scavenging property with a reaction rate constant (Ks) of 3.2-6.7 x 10(10) M(-1) S(-1), 2.3-3.7 x 10(10) M(-1) S(-1) and 2.4-3.7 x 10(10) M(-1) S(-1), respectively. The effectiveness of these analogues as hydroxyl radical scavengers appears to be better than mannitol (Ks, 1.9-2.5 x 10(9) M(-1) S(-1)). With the exception of 1-pentyl- and 1-(2'-oxopropyl)-3,7-dimethyluric acids, all other analogues tested are effective inhibitors of tert-butylhydroperoxide-induced lipid peroxidation in human erythrocyte membranes. All the analogues tested are susceptible to peroxidation in the presence of hemoprotein and hydrogen peroxide. The present study has pointed out that it is possible to significantly enhance the antioxidant property of 1,3,7-trimethyluric acid by structural modification at N-1 position. Such compounds may be useful as antioxidants in vivo.     

Lipid Peroxidation Induced by Phenolics in Conjunction with Aluminum Ions

Using the whole plant and model systems, we demonstrate that the aluminum ions (Al³⁺) stimulate phenolic-dependent lipid peroxidation. Lipid peroxidation in barley (Hordeum vulgare L. cv. Donor) roots was 30 % higher under AlCl₃ treatment than without Al. Major decomposition product of lipid peroxidation was 4-hydroxynonenal (4-HNE) but not thiobarbituric acid reactive substances (TBARS), a widely used markers for lipid peroxidation. Similarly, AlCl₃ stimulated lipid peroxidation of soybean liposomes in the presence of chlorogenic acid (CGA) and H₂O₂/horseradish peroxidase system which can oxidize phenolics. Al³⁺ was found to enhance lipid peroxidation induced by oxidized CGA. Intermediates of lignin biosynthesis in plants, including p-coumaric acid, ferulic acid, sinapic acid and coniferyl alcohol, also showed similar effects. These results suggest that Al³⁺ has a potential to induce oxidative stress in plants by stimulating the prooxidant nature of endogenous phenolic compounds.     

Inhibitory effects of echinoisoflavanone and sophoraisoflavanone D in Sophora chrysophylla SEEM on lipid peroxidation of mice brain homogenate by interaction of ferrous ion and hydrogen peroxide, in vitro

We present the results of an in vitro investigation of the inhibitory effects of echinoisoflavanone and sophoraisoflavanone D isolated from Sophora chrysophylla SEEM on lipid peroxidation of mice brain homogenate by interaction of ferrous ion and hydrogen peroxide, in vitro. They inhibited lipid peroxidation. The order of inhibitory effects of these isoflavanones and mannitol as a hydroxy radical scavenger was echinoisoflavanone > mannitol > sophoraisoflavanone D. The results suggest that some isoflavanones may be of use in cases where oxidative stress is present.     

Lignans from the Fruits of Forsythia suspensa (Thunb.) Vahl Protect High-Density Lipoprotein during Oxidative Stress

The objective of the present study was to investigate the beneficial properties lignan compounds obtained from the fruits of Forsythia suspensa (Thunb.) Vahl (Oleaceae) for protecting human high-density lipoprotein (HDL) against lipid peroxidation. The isolated compounds (1–8) inhibited the generation of thiobarbituric acid-reactive substances (TBARS) in a dose-dependent manner with IC₅₀ values from 8.5 to 18.7 μM, since HDL oxidation mediated by catalytic Cu²⁺. They also exerted an inhibitory effect against thermo-labile radical initiator (AAPH)-induced lipid peroxidation of HDL with IC₅₀ values from 12.1 to 51.1 μM. Compounds 1 and 5 exerted inhibitory effects against the Cu²⁺-induced lipid peroxidation of HDL, as shown by an extended lag time prolongation at the concentration of 3.0 μM. These results suggest that the antioxidative effects of F. suspensa are due to its lignans and that these constituents may be useful for preventing the oxidation of HDL.