Molecular docking and enzyme kinetic studies of dihydrotanshinone on metabolism of a model CYP2D6 probe substrate in human liver microsomes

The effects of Danshen and its active components (tanshinone I, tanshinone IIA, dihydrotanshinone and cryptotanshinone) on CYP2D6 activity was investigated by measuring the metabolism of a model CYP2D6 probe substrate, dextromethorphan to dextrorphan in human pooled liver microsomes. The ethanolic extract of crude Danshen (6.25-100 μg/ml) decreased dextromethorphan O-demethylation in vitro (IC(50)=23.3 μg/ml) and the water extract of crude Danshen (0.0625-1 mg/ml) showed no inhibition. A commercially available Danshen pill (31.25-500 μg/ml) also decreased CYP2D6 activity (IC(50)=265.8 μg/ml). Among the tanshinones, only dihydrotanshinone significantly inhibited CYP2D6 activity (IC(50)=35.4 μM), compared to quinidine, a specific CYP2D6 inhibitor (IC(50)=0.9 μM). Crytotanshinone, tanshinone I and tanshinone IIA produced weak inhibition, with IC(20) of 40.8 μM, 16.5 μM and 61.4 μM, respectively. Water soluble components such as salvianolic acid B and danshensu did not affect CYP2D6-mediated metabolism. Enzyme kinetics studies showed that inhibition of CYP2D6 activity by the ethanolic extract of crude Danshen and dihydrotanshinone was concentration-dependent, with K(i) values of 4.23 μg/ml and 2.53 μM, respectively, compared to quinidine, K(i)=0.41 μM. Molecular docking study confirmed that dihydrotanshinone and tanshinone I interacted with the Phe120 amino acid residue in the active cavity of CYP2D6 through Pi-Pi interaction, but did not interact with Glu216 and Asp301, the key residues for substrate binding. The logarithm of free binding energy of dihydrotanshinone (-7.6 kcal/mol) to Phe120 was comparable to quinidine (-7.0 kcal/mol) but greater than tanshinone I (-5.4 kcal/mol), indicating dihydrotanshinone has similar affinity to quinidine in binding to the catalytic site on CYP2D6.     

Effects of major tanshinones isolated from Danshen (Salvia miltiorrhiza) on rat CYP1A2 expression and metabolism of model CYP1A2 probe substrates

This study explored the effects of Danshen on metabolism/pharmacokinetics of model CYP1A2 substrates and hepatic CYP1A2 expression in rats. The effects of Danshen and tanshinones on CYP1A2 activity was determined by metabolism of model substrates in vitro (phenacetin) and in vivo (caffeine). HPLC was used to determine model substrates/metabolites. The effect of Danshen on CYP1A2 expression was determined by Western blot. Tanshinones (1.25–50 μM) competitively inhibited phenacetin O-deethylation in vitro. Inhibition kinetics studies showed the K_i values were in the order: dihydrotanshinone (3.64 μM), cryptotanshinone (4.07 μM), tanshinone I (22.6 μM) and tanshinone IIA (23.8 μM), furafylline (35.8 μM), a CYP1A2 inhibitor. The Ki of Danshen extract (mainly tanshinones) was 72 μg/ml. Acute Danshen extract treatment (50–200 mg/kg, i.p.) decreased metabolism of caffeine to paraxanthine, with overall decrease in caffeine clearance (14–22%); increase in AUC (11–25%) and plasma T_1/2 (12–16%). Danshen treatment with (100 mg/kg/day, i.p. or 200 mg/kg/day, p.o.) for three or fourteen days showed similar pharmacokinetic changes of the CYP1A2 probe substrate without affecting CYP1A2 expression. This study demonstrated that major tanshinones competitively inhibited the metabolism of model CYP1A2 probe substrates but had no effect on rat CYP1A2 expression.     

PXR-mediated transcriptional activation of CYP3A4 by cryptotanshinone and tanshinone IIA

Danshen (Radix Salvia miltiorrhiza) is a famous Traditional Chinese Medicine used widely for the treatment of coronary heart disease and cerebrovascular disease. Diterpenoid tanshinones including tanshinone I, tanshinone IIA and cryptotanshinone are the major bioactive components from Danshen herb. Previous reports have demonstrated that Danshen extracts could induce the expression of CYP3A in rodents, however, the constituents responsible for Danshen-mediated CYP3A induction and the underlying molecular mechanisms remain unknown. The discovery of a family of nuclear receptors such as pregnane X receptor (PXR), constitutive androstane receptor (CAR) and glucocorticoid receptor (GR) gives insight into the molecular explanation of CYP3A induction by xenobiotics. In the present study, interactions between Danshen constituents and human PXR were evaluated using a reporter gene assay. Our observations showed that Danshen ethanol extract could activate human PXR and induce the CYP3A4 reporter construct in HepG2 cells. Tanshinone IIA and cryptotanshinone were identified as efficacious PXR agonists, and cryptotanshinone activated the CYP3A4 promoter more strongly than tanshinone IIA. Furthermore, CAR and GR were also involved in the induction of CYP3A4 expression by tanshinones, though their roles seemed not as important as PXR. Treatment of LS174T cells with cryptotanshinone or tanshinone IIA resulted in a significant increase of CYP3A4 mRNA, which was consistent with the results from the reporter gene assay. Collectively, activation of PXR and the resultant CYP3A4 induction mediated by cryptotanshinone and tanshinone IIA provide a molecular mechanism for previously observed CYP3A induction by Danshen extracts, and our findings also suggest that caution should be taken when Danshen products are used in combination with therapeutic drugs metabolized by CYP3A4.     

Inhibition of human drug metabolizing cytochrome P450 enzymes by plant isoquinoline alkaloids

The human cytochrome P450 (CYP) enzymes play a major role in the metabolism of endobiotics and numerous xenobiotics including drugs. Therefore it is the standard procedure to test new drug candidates for interactions with CYP enzymes during the preclinical development phase. The purpose of this study was to determine in vitro CYP inhibition potencies of a set of isoquinoline alkaloids to gain insight into interactions of novel chemical structures with CYP enzymes. These alkaloids (n = 36) consist of compounds isolated from the Papaveraceae family (n = 20), synthetic analogs (n = 15), and one commercial compound. Their inhibitory activity was determined towards all principal human drug metabolizing CYP enzymes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4. All alkaloids were assayed in vitro in a 96-well plate format using pro-fluorescent probe substrates and recombinant human CYP enzymes. Many of these alkaloids inhibited the CYP3A4 form, with 30/36 alkaloids inhibiting CYP3A4 with at least moderate potency (IC₅₀ < 10 μM) and 15/36 inhibiting CYP3A4 potently (IC₅₀ < 1 μM). Among them corydine, parfumine and 8-methyl-2,3,10,11-tetraethoxyberbine were potent and selective inhibitors for CYP3A4. CYP2D6 was inhibited with at least moderate potency by 26/34 alkaloids. CYP2C19 was inhibited by 15/36 alkaloids at least moderate potently, whereas CYP1A2, CYP2B6, CYP2C8, and CYP2C9 were inhibited to a lesser degree. CYP2A6 was not significantly inhibited by any of the alkaloids. The results provide initial structure-activity information about the interaction of isoquinoline alkaloids with major human xenobiotic-metabolizing CYP enzymes, and illustrate potential novel structures as CYP form-selective inhibitors.     

In vitro inhibitory effects of ethanol extract of Danshen (Salvia miltiorrhiza) and its components on the catalytic activity of soluble epoxide hydrolase

Background: Soluble epoxide hydrolase (sEH) has been demonstrated to be a key enzyme involved in the pathologic development of several cardiovascular diseases and inflammation, and inhibition of sEH is therefore very helpful or crucial for the treatment of ischemia-reperfusion injury, cardiac hypertrophy, hypertension and inflammation. Danshen, the dried root of Salvia miltiorrhiza (Fam. Labiatae), has been used for the treatment of cardiovascular and cerebrovascular diseases in China and other countries for hundreds of years. Recent studies indicated that Danshen and its preparations also have potential for the management of inflammation. However, little information is available about the possibility of Danshen and its components on sEH inhibition.Purpose and methods: Danshen extracts and its constituents were tested for sEH inhibition using its physiological substrate, 8,9-EET, based on a LC–MS/MS assay in this study.Results: Among the tested 15 compounds, tanshinone IIA and cryptotanshinone were found to be the potent (K_i = 0.87 μM) and medium (K_i = 6.7 μM) mixed-type inhibitors of sEH, respectively. Salvianolic acid C (K_i = 8.6 μM) was proved to be a moderate noncompetitive sEH inhibitor. In consistent with the inhibition results of the pure compounds, the 75% ethanol extract of Danshen (EE, IC₅₀ = 86.5 μg/ml) which contained more tanshinone IIA and cryptotanshinone exhibited more potent inhibition on sEH than the water extract (WE, IC₅₀ > 200 μg/ml) or 1 M NaHCO₃ (BE, IC₅₀ > 200 μg/ml) extract.Conclusion: These data indicated that using the ethanol fraction of Danshen and increasing the amounts of tanshinone IIA, cryptotanshinone and salvianolic acid C, especially the contents of tanshinone IIA in Danshen extract or preparations to enhance the inhibitory effects on sEH might be efficient ways to improve its cardiovascular protective and anti-inflammatory effects, and that herbal medicines could be an untapped reservoir for sEH-inhibition agents and developing sEH inhibitors from the cardiovascular protective and anti-inflammatory herbs is a promising approach.     

Danshensu is the major marker for the antioxidant and vasorelaxation effects of Danshen (Salvia miltiorrhiza) water-extracts produced by different heat water-extractions

Some of the major components of Danshen (Salvia miltiorrhiza), a widely used Chinese herbal medicine rich in phenolic acids, are thermosensitive and may degrade to other phenolic acids during extractions with heating. The chemical profiles of Danshen water-extract may vary with different heat water extraction at different temperatures, affecting the composition and bioactivity of the extracts. In this study, six water-extracts of Danshen obtained from heat reflux water extraction and microwave-assisted extraction with water (MAE-W) at different temperatures were tested for their composition and pharmacological effects. Among these extracts, the third-round MAE-W (100 °C) extract had the highest phenolic acids and tanshinones contents, with the strongest antioxidant activity in 2,2-diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl (DPPH) assay and ferric reducing/antioxidant potential (FRAP) assay. This extract also showed the strongest inhibitory effects on 2,2′-azobis-2-amidinopropane (AAPH)-induced hemolysis in human red blood cells, hydrogen peroxide-induced apoptosis in rat heart H9c2 cells and the highest relaxation effects on rat basilar artery. The antioxidant effects of Danshen water-extracts linearly correlated to their relaxation effects (r = 0.895–0.977). Through multiple linear regression analysis, danshensu was found to be the most significant marker in the antioxidant and vasodilation effects of Danshen water-extract, while tanshinone IIA as the marker on hydrogen peroxide-induced apoptosis in rat heart H9c2 cells. Danshensu is, therefore, a useful marker for the quality control of Danshen water-extracts in antioxidant and vasodilation, while tanshinone IIA for anti-apoptotic potential of different extracts.     

Bursera fagaroides, effect of an ethanolic extract on ornithine decarboxylase (ODC) activity in vitro and on the growth of Entamoeba histolytica

The effect of an ethanolic extract from the stem bark of Bursera fagaroides on ornithine decarboxylase (ODC) activity in vitro and on the growth of Entamoeba histolytica was evaluated. For this purpose, increasing concentrations of the extract, up to 8.0 mg/mL, were added to amoeba cultures or ODC reaction mixtures, which were incubated at 37 °C. Metronidazole and G418 were added as controls. After 1.5 and 72 h, the ODC activity in vitro and growth, respectively, were determined. Results revealed a strong inhibition of growth with IC₅₀ values on the order of 0.05 mg/mL. ODC activity, on the other hand, was inhibited by 12% and 50% at concentrations of 4.0 and 8.0 mg/mL, respectively.     

Investigation of cytochrome P450 1A2 and 3A inhibitory properties of Danshen tincture

Danshen (Salvia miltiorrhiza Bunge) as a famous Traditional Chinese medicine is widely used in the treatment of cardiovascular and cerebrovascular diseases in the world. Danshen tincture (DT), extracted from Danshen root with a mixture of water and alcohol, is a commonly used preparation method for human consumption. The aim of this study was to investigate the effects of DT on the cytochrome P450 (CYP) 1A2 and 3A activities by human and rat liver microsomes. Effects of DT were assessed with use of Danshen ethanolic extract (DEE) and selective substrates, markers of CYP activities. DEE (0.5-10 μg/ml) competitively inhibited human and rat liver microsomal CYP1A2 activity with inhibition constant (K(i)) values at 3.40 and 5.16 μg/ml, respectively. At the same time, DEE (2.5-20 μg/ml) not only noncompetitively inhibited human liver microsomal CYP3A4/5 activity with a K(i) of 11.9 μg/ml, but also competitively inhibited rat liver microsomal CYP3A1/2 activity with a K(i) of 52.1 μg/ml. The data indicate that DEE inhibited the metabolism of CYP1A2 and 3A substrates in human and rat liver in vitro with different mode of inhibition. This study may be helpful for clinical application of Danshen tincture.     

Insulin-sensitizing activities of tanshinones,diterpene compounds of the root of Salvia miltiorrhiza Bunge

In this study, the effects of the extract and four tanshinone compounds from the dried root of Salvia miltiorrhiza Bunge (Labiatae) on the tyrosine phosphorylation of the insulin receptor (IR) β-subunit and the downstream signaling were examined in Chinese-hamster ovary cells expressing human insulin receptors (CHO/IR cells) as well as in 3T3-L1 adipocytes. In addition the translocation of the glucose transporter 4 was investigated in 3T3-L1 adipocytes. Total extract of Danshen (1–10 μg/ml) and the four tanshinones (10 μM) did not show any activity, but the total extract and the tanshinone I, IIA and 15, 16-dihydrotanshinone I except cryptotanshinone enhanced the activity of insulin (1 nM) on the tyrosine phosphorylation of the IR as well as the activation of the downstream kinases Akt, ERK1/2, and GSK3β. In the adipocytes the same IR-downstream signaling and the translocation of glucose transporter 4 were demonstrated by the three tanshinones in the presence of insulin. These insulin-sensitizing activities of tanshinones may be useful for developing a new class of specific IR activators as anti-diabetic agents.     

Determination of Tanshinones in Danshen (Salvia miltiorrhiza) by High‐Performance Liquid Chromatography with Fluorescence Detection after pre‐Column Derivatisation

Introduction

Tanshinones are a major class of bioactive ingredients in the traditional herbal medicines, Danshen (Salvia miltiorrhiza). A sensitive and reliable determination method for tanshinones is useful to ensure the quality of Danshen.

Objective

To develop a sensitive and selective analytical method for tanshinones by high‐performance liquid chromatography (HPLC) with fluorescence detection after pre‐column derivatisation.

Methodology

The proposed method depends on derivatisation reaction of tanshinones with 4‐carbomethoxybenzaldehyde and ammonium acetate forming intensely fluorescent imidazole derivative.

Results

The proposed method provided excellent sensitivity with the detection limits of 3.3 nM (66 fmol/injection), 3.2 nM (64 fmol/injection) and 2.0 nM (40 fmol/injection) for cryptotanshinone, tanshinone I and tanshinone IIA, respectively, without the necessity of complicated instrumentations. The developed method is successfully applied to quantify the contents of tanshinones in Danshen.

Conclusion

The developed method is the first analytical method for tanshinones by fluorescence detection. Since the derivatisation reaction is selective for the o‐quinone structure of tanshinone, the developed method will become a suitable mean for the discovering of tanshinone type diterpenoids from herbal samples. Copyright © 2017 John Wiley & Sons, Ltd.     

Tyrosinase activity of Greyia flanaganii (Bolus) constituents

Hyper-pigmentation of the skin is a common problem that is prevalent in middle aged and elderly people. It is caused by over production of melanin. Tyrosinase is known to be the key enzyme in melanin production. Ethanolic extract of Greyia flanaganii leaves showed significant (P < 0.05) antityrosinase activity exhibiting the IC₅₀ of 32.62 μg/ml. The total extract was further investigated for its toxicity and effect on melanin production by melanocytes cells, and showed significant inhibition (P < 0.05) (20%) of melanin production at 6.25 μg/ml and low levels of cytotoxicity (IC₅₀ < 400 μg/ml). The amount of antioxidants necessary to decrease the initial DPPH absorbance by 50% (EC₅₀) by the total ethanolic extract was found to be 22.01 μg/ml. The effect of G. flanaganii against acne causing bacteria, Propionibacterium acnes, was investigated using microdilution assay. The MIC of the extract of G. flanaganii was found to be 250 μg/ml. Bioassay-guided fractionation led to the isolation of (3S)-4-hydroxyphenethyl 3-hydroxy-5-phenylpentanoate (1), 2′,4′,6′-trihydroxydihydrochalcone (2), 2′,6′,4-trihydroxy-4′-methoxydihydrochalcone (3), 2′,6′-dihydroxy-4′-methoxydihydrochalcone (4), 5,7-dihydroxyflavanone [(2S)-pinocembrin] (5), 2′,6′-dihydroxy-4′,4-dimethoxy dihydrochalcone (6) and (2R,3R)-3,5,7-trihydroxy-3-O-acetylflavanone (7). The isolated compounds were tested for their antioxidant, cytotoxicity, tyrosinase inhibition and antibacterial activities. Compound 2 exhibited significant (P < 0.05) antityrosinase activity exhibiting the IC₅₀ of 69.15 μM. The isolated compounds showed low toxicity of the cells with reduction of melanin content of the cells. All compounds tested showed good radical scavenging activity. These data indicates that G. flanaganii extract and its isolated phenolic constituents could be possible skin lightening agents.     

Plasmodium falciparum: In vitro interaction of quassin and neo-quassin with artesunate, a hemisuccinate derivative of artemisinin

Quassia amara L. (Family Simaroubaceae) is known to have several medicinal properties including the activity against malaria. An HPLC method was employed for purification of the biologically active quassinoids; quassin (Q) and neo-quassin (NQ), further characterized by MALDI-TOF analyses. Purified Q, NQ and the crude bark extract (S1) along with artesunate (AS) were studied for their in vitro anti-plasmodial activity. The in vivo toxicity studies at intraperitoneal doses with higher concentrations of the crude bark extract (S1) in Balb/C mice ruled out the apprehension of toxicity. Interaction studies between the test compounds among themselves (Q + NQ) and individually with artesunate (AS + Q, AS + NQ), were carried out in vitro at four ratios (1:5, 1:2, 2:1 and 5:1) on chloroquine sensitive (MRC-pf-20) and resistant (MRC-pf-303) strains of Plasmodium falciparum. The crude bark extracts of Q. amara exhibited higher P. falciparum inhibitory activity (IC₅₀ = 0.0025 μg/ml) as compared to that of the isolated compounds, quassin (IC₅₀ = 0.06 μg/ml, 0.15 μM), neo-quassin (IC₅₀ = 0.04 μg/ml, 0.1 μM) and also to the positive control, artesunate (IC₅₀ = 0.02 μg/ml, 0.05 μM). The in vitro drug interaction study revealed the compounds, quassin and neo-quassin to be additive to each other. At lower ratios, artesunate was found to be a potential combination partner with both the compounds. It was interesting to note that none of the combinations exhibited antagonistic interactions. This phenomenon offers the opportunity for further exploration of novel therapeutic concentrations and combinations.     

An unusual dimeric guaianolide with antiprotozoal activity and further sesquiterpene lactones from Eupatoriumperfoliatum

The CH₂Cl₂ extract of aerial parts of Eupatorium perfoliatum L. exhibits antiprotozoal activity under in vitro conditions, especially against Plasmodium falciparum (IC₅₀ = 2.7 μg/ml). The search for active compounds yielded seven sesquiterpene lactones: Four structurally similar guaianolides, one dimeric guaianolide, and two germacranolides. The guaianolides differ in the degree of oxidation at C-14, ranging from a hydroxyl group up to a free carboxylic acid. The dimeric guaianolide, structurally closely related to the monomers, displays an unusual type of interguaianolide linkage between C-14 and C-4. Except for the germacranolide euperfolitin, all STLs described here were hitherto unknown. Furthermore, the flavonoid aglycones eupafolin, hispidulin, patuletin, and kaempferol were identified in the extract, which, except for kaempferol, have not been described as constituents of E. perfoliatum before. The dimeric guaianolide was shown to be the most active constituent against Plasmodium falciparum (IC₅₀ = 2.0 μM) and was less cytotoxic against rat skeletal myoblasts (IC₅₀ = 16.2 μM, selectivity index of about 8).     

Cytotoxic and antioxidant activities of diterpenes and sterols from the Vietnamese soft coral Lobophytum compactum

Two new diterpenes, lobocompactols A (1) and B (2), and five known compounds (3-7) were isolated from the methanol extract of the soft coral Lobophytum compactum using combined chromatographic methods and identified based on NMR and MS data. Each compound was evaluated for cytotoxic activity against A549 (lung) and HL-60 (acute promyelocytic leukemia) human cancer cell lines. Among them, compound 5 exhibited strong cytotoxic activity against the A549 cell line with an IC₅₀ of 4.97 ± 0.06 μM. Compounds 3, 4, and 7 showed moderate activity with IC₅₀ values of 23.03 ± 0.76, 31.13 ± 0.08, and 36.45 ± 0.01 μM, respectively. The cytotoxicity of 5 on the A549 cells was comparable to that of the positive control, mitoxantrone (MX). All compounds exhibited moderate cytotoxicity against the HL-60 cell line, with IC₅₀ values ranging from 17.80 ± 1.43 to 59.06 ± 2.31 μM. Their antioxidant activity was also measured using oxygen radical absorbance capacity method, compounds 1 and 2 exhibiting moderate peroxyl radical scavenging activity of 1.4 and 1.3 μM Trolox equivalents, respectively, at a concentration of 5 μM.     

Antimalarial compounds from Schefflera umbellifera

The organic extract of the leaves of Schefflera umbellifera exhibited good antimalarial activity when tested against the chloroquine-susceptible strain (D10). Bioassay-guided fractionation of the dichloromethane fraction of the dichloromethane/methanol extract yielded an active compound, betulin, which exhibited good antiplasmodial activity with an IC₅₀ value of 3.2 µg/ml. The reference compound, chloroquine gave an IC₅₀ value of 27.2 ng/ml. Two other compounds were also isolated from the dichloromethane extract namely, 7-hydroxy-6-methoxycoumarin and ent-kaur-16-en-19-oic acid. These two compounds did not exhibit any significant antiplasmodial activity.     

Estrogenic activities of Psoralea corylifolia L. seed extracts and main constituents

Estrogenic activities of ethanol extract and its active components from Psoralea corylifolia L. were studied using various in vitro assays. The main components from ethanol extract were analyzed to be bakuchiol, psoralen, isobavachalcone, isobavachromene, and bavachinin. In a fractionation procedure, hexane and chloroform fractions showed estrogenic activity in yeast transactivation assay and E-screen assay. In yeast transactivation assay, ethanol extract, hexane, and chloroform fractions showed significantly higher activities at a concentration of 1.0 ng/ml, and bakuchiol at the concentration of 10⁻⁶ M was showed the highest activity, especially, which was higher than genistein at the same concentration. In E-screen assay, cell proliferation of bakuchiol (10⁻⁶ M) showed similar estrogenic activity with genistein (10⁻⁶ M). In ER binding assay, bakuchiol displayed the strongest ER-binding affinity (IC₅₀ for ERα = 1.01 × 10⁻⁶ M, IC₅₀ for ERβ = 1.20 × 10⁻⁶ M) and bakuchiol showed five times higher affinity for ERα than for ERβ.     

Evaluation of in vitro cytotoxicity and hepatotoxicity of platinum(II) and palladium(II) oxalato complexes with adenine derivatives as carrier ligands

In vitro antitumour activity of the [Pt(ox)(L_n)₂] (1-7) and [Pd(ox)(L_n)₂] (8-14) oxalato (ox) complexes involving N6-benzyl-9-isopropyladenine-based N-donor carrier ligands (L_n) against ovarian carcinoma (A2780), cisplatin resistant ovarian carcinoma (A2780cis), malignant melanoma (G-361), lung carcinoma (A549), cervix epitheloid carcinoma (HeLa), breast adenocarcinoma (MCF7) and osteosarcoma (HOS) human cancer cell lines was studied. Some of the tested complexes were even several times more cytotoxic as compared with cisplatin employed as a positive control. The improved cytotoxic effect was demonstrated for the platinum(II) complexes 3 (IC₅₀ = 3.2 ± 1.0 μM and 3.2 ± 0.6 μM) and 5 (IC₅₀ = 4.0 ± 1.0 μM and 4.1 ± 1.4 μM) against A2780 and A2780cis, as compared with 11.5 ± 1.6 μM, and 30.3 ± 6.1 μM determined for cisplatin, respectively. The significant in vitro cytotoxicity against MCF7 (IC₅₀ = 8.2 ± 3.8 μM for 12) and A2780 (IC₅₀ = 5.4 ± 1.2 μM for 14) was evaluated for the palladium(II) oxalato complexes, which again exceeded cisplatin, whose IC₅₀ equalled 19.6 ± 4.3 μM against the MCF7 cells. Selected complexes were also screened for their in vitro cytotoxic effect in primary cultures of human hepatocytes and they were found to be non-hepatotoxic.     

Relationship correlation of antioxidant and antiproliferative capacity of Cyperus rotundus products towards K562 erythroleukemia cells

A Total Oligomers Flavonoids (TOFs) and ethyl acetate extracts of Cyperus rotundus were analyzed, in vitro, for their antioxidant activity using several biochemical assays: the xanthine (X)/xanthine oxidase (XO), the lipid peroxidation induced by H₂O₂ in K562 human chronic myelogenous leukemia cells and the DNA damage in pKS plasmid DNA assay induced by H₂O₂/UV-photolysis and for their apoptotic effect. TOF and ethyl acetate extracts were found to be efficient in inhibiting xanthine oxidase with IC₅₀ values of 240 and 185 μg/ml and superoxide anion with IC₅₀ values of 150 and 215 μg/ml, respectively. Also, all the extracts tested were effective in reducing the production of thiobarbituric acid reactive substances (TBARS) and were able to protect against H₂O₂/UV-photolysis induced DNA damage. The highest activity, measured as equivalents of MDA concentration, was observed in the ethyl acetate extract (MDA = 2.04 nM). In addition, the data suggest that only TOF enriched extract exerts growth inhibition on K562 cells through apoptosis induction. Therefore, these extracts were subjected to further separation by chromatographic methods. Thus, three major compounds (catechin, afzelechin and galloyl quinic acid) were isolated from the TOF enriched extract and five major compounds (luteolin, ferulic acid, quercetin, 3-hydroxy, 4-methoxy-benzoic acid and 6,7-dimethoxycoumarin) from ethyl acetate extract. Their structures were determined by spectroscopic data analysis and comparison with the literature. In addition, we evaluate the biological activities of the catechin, ferulic acid and luteolin. This investigation has revealed that the luteolin was the most active in reducing the production of TBARS (MDA = 1.5 nM), inhibiting significantly the proliferation of K562 cells (IC₅₀ = 25 μg/ml) and protecting against H₂O₂/UV-photolysis induced DNA damage. In conclusion, the study reveals that the ability of C. rotundus to inhibit the enzyme xanthine oxidase (XO), the lipid peroxidation and to exert apoptotic effect, may explain possible mechanisms by which C. rotundus exhibits its health benefits.     

α-Glucosidase inhibition and antihyperglycemic activity of prenylated xanthones from Garcinia mangostana

An ethanol extract of the fruit case of Garcinia mangostan, whose most abundant chemical species are xanthones, showed potent α-glucosidase inhibitory activity (IC₅₀ = 3.2 μg/ml). A series of isolated xanthones (1-16) demonstrated modest to high inhibition of α-glucosidase with IC₅₀ values of 1.5-63.5 μM. In particular, one hitherto unknown xanthone 16 has a very rare 2-oxoethyl group on C-8. Kinetic enzymatic assays with a p-nitrophenyl glucopyranoside indicated that one of them, compound (9) exhibited the highest activity (K_i = 1.4 μM) and mixed inhibition. Using, a physiologically relevant substrate, maltose, as substrate, many compounds (6, 9, 14, and 15) also showed potent inhibition which ranged between 17.5 and 53.5 μM and thus compared favorably with deoxynojirimycin (IC₅₀ = 68.8 μM). Finally, the actual pharmacological potential of the ethanol extract was demonstrated by showing that it could elicit reduction of postprandial blood glucose levels. Furthermore, the most active α-glucosidase inhibitors (6, 9, and 14) were proven to be present in high quantities in the native seedcase by a HPLC chromatogram.