New cyclomyrsinol diterpenes from Euphorbia aellenii with their immunomodulatory effects.

Chloroform-acetone extract of the aerial parts of Euphorbia aellenii Rech. f. (Euphorbiaceae) was investigated for its diterpenoidal constituents. This led to the isolation of two new and one known cyclomyrsinol-type diterpenes 1-3. The structures were elucidated on the basis of 1D and 2D (1)H and (13)C NMR techniques, and in vitro immunomodulatory activity was evaluated by standard proliferation of human peripheral blood lymphocytes. Results showed that all the three compounds were found to inhibit lymphocyte proliferation significantly (p < 0.05) at 50 µg/ml concentration. Among them, compound 2 showed more activity against phytohemagglutinin-activated T-cell proliferation with an IC(50) of 40.4 ± 9.35 µg/ml.

New myrsinane-type diterpenoids from Euphorbia aellenii Rech. f. with their immunomodulatory activity.

Two new 14-desoxo-10, 18-dihydromyrinsol diterpenoids (1 and 2) were isolated and characterized from the cytotoxic chloroform fraction of Euphorbia aellenii Rech. f. (Euphorbiaceae). The structures of the new compounds were elucidated by spectroscopic methods and their immunomodulatory properties were evaluated by T-cell proliferation and phagocyte chemiluminescence assays.

RP-HPLC Method Development and Validation for Determination of Eptifibatide Acetate in Bulk Drug Substance and Pharmaceutical Dosage Forms.

A new, rapid, economical and isocratic reverse phase high performance liquid chromatography (RP-HPLC) method was developed for the determination of eptifibatide acetate, a small synthetic antiplatelet peptide, in bulk drug substance and pharmaceutical dosage forms. The developed method was validated as per of ICH guidelines. The chromatographic separation was achieved isocratically on C18 column (150 x 4.60 mm i.d., 5 µM particle size) at ambient temperature using acetonitrile (ACN), water and trifluoroacetic acid (TFA) as mobile phase at flow rate of 1 mL/min and UV detection at 275 nm. Eptifibatide acetate exhibited linearity over the concentration range of 0.15-2 mg/mL (r2=0.997) with limit of detection of 0.15 mg/mL The accuracy of the method was 96.4-103.8%. The intra-day and inter-day precision were between 0.052% and 0.598%, respectively. The present successfully validated method with excellent selectivity, linearity, sensitivity, precision and accuracy was applicable for the assay of eptifibatide acetate in bulk drug substance and pharmaceutical dosage forms.

TAMEC: a new analogue of cyclomyrsinol diterpenes decreases anxiety- and depression-like behaviors in a mouse model of multiple sclerosis.

Objectives There is a significant prevalence of affective disorders including depression and anxiety in people with multiple sclerosis (MS), resulting in reduced quality of life. Since the current treatments are not generally effective, further studies are needed to find appropriate drugs to alleviate anxiety and depression symptoms in these patients. Methods The effects of a new analog of cyclomyrsinol diterpenes (TAMEC) isolated from Euphorbia sogdiana on the anxiety (open field and elevated plus maze test) and depressive-like behaviors (sucrose preference test and forced swim test) in EAE-induced C57BL/6 mice (EAE; a mouse model of MS) were investigated. Hippocampal tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1ß and IL-10 levels were also measured by ELISA. Results The results indicated that TAMEC treatment reduced anxiety and depression-like behavior. This drug also decreased the levels of TNF-α and IL1ß and increased IL-10 level in the hippocampus. Discussion Taken together, our findings demonstrate that the drug we used here can reduce anxiety and depression-like symptoms in EAE-induced mice. However, more studies are still needed to validate, expand, and generalize these data.

Triterpene Constituents of Euphorbia Erythradenia Bioss. and their Anti-HIV Activity.

Phytochemical investigation of the aerial parts of Euphorbia erythradenia Bioss. (Euphorbiaceae), one of Iranian endemic Euphorbias, with particular attention to triterpene constituents, using methanol solvent extraction was carried out. Five known triterpenes, including four cycloartanes and oleanolic acid, were isolated for the first time and identified using NMR and Mass techniques. Anti HIV activity of the isolated triterpenes and ingenoid diterpenes was evaluated using single cycle replicable HIV-1 (SCR HIV-1) virions. Molecular features of the most active compound (IC50 = 0.008 µM, CC50 = 3.264 µM, TI = 380.64), which showed higher therapeutic index than nevirapine, was assessed using molecular docking. Docking studies demonstrated three hydrogen bonds between HIV-1 virion protease active site and this compound with a distance less than 3 A° which can be responsible for the observed anti HIV-1 activity.

Methoxylated Flavones from Salvia Mirzayanii Rech. f. and Esfand with Immunosuppressive Properties.

Aerial parts of Salvia Mirzayanii was extracted with methanol. Methanol extract was suspended in water and defatted with petroleum ether. The defatted part was then partitioned between ethyl acetate and water. The ethyl acetate partition was chromatographed on a silica gel column to afford several fractions. Lymphocyte proliferation inhibitory assay of the resulted fractions was compared in-vitro and the fraction with more immunosuppressive activity was subjected to more purification to yield three methoxylated flavones: 5,7-dihydroxy,6,4'- dimethoxyflavone(1), 5-hydroxy,6,7,3',4'-tetramethoxyflavone(2) and 5,3'-dihydroxy,6,7,4'- trimethoxyflavone (3). Compounds 2 and 3 potently suppressed the proliferation of human blood lymphocytes with IC50 values of 1.3 ± 0.04 µg/mL and 1.3 ± 0.21 µg/mL in comparison with prednisolone as one of the lymphocyte suppressor drugs (IC50=1.45 ± 0.6 µg/mL). In phagocyte chemiluminescence assay, compounds 1 and 3 in peripheral mononuclear cells (PMNCs) exerted suppressive moderate activity against ROS with IC50 of 55.3 ± 0.4 and 36.2 ± 0.7 µg/mL, respectively, while compound 2 showed weak activity with IC50 values more than 100 µg/mL. In conclusion, compounds 2 and 3 have a similar suppressive effect more than compound 1 on PHA-activated lymphocyte proliferation, which might be because of their C-3' oxidation pattern of ring B. It is indicated that the presence of 3'-OH or 3'-OMe in flavone ring B, caused more anti-proliferation activity than 3'-H. Oxidative burst assay showed more activity for compound 1 which is less methoxylated than others. It also showed more activity for compounds 3 than 2, which differ only in 3'-OH instead of 3'-OMe.

Total Phenolic Content and Antibacterial Activity of Five Plants of Labiatae against Four Foodborne and Some Other Bacteria.

The aim of this study was to evaluate the antibacterial effects of Thymus vulgaris, Thymus caramanicus, Zataria multiflora, Ziziphora clinopodioides and Ziziphora tenuior against four foodborne and four other bacteria including Staphylococcus aureus, Shigella dysenteriae, Salmonella typhimurium, Escherichia coli, Staphylococcus epidermidis, Bacillus subtilis, MRSA and Pseudomona aeruginosa and measuring the amount of total phenolics of the plants. The extracts were prepared by maceration method. Pre-evaluation of the antimicrobial effect was utilized by cup-plate technique and then Minimum Inhibitory Concentration was determined by agar dilution method according to NCCLS. The total phenolics as a possible cause of antibacterial effect, was measured by Folin-Ciocalteucolorimetry. The results showed that T. caramanicus and Z. multiflora were the most effective ones with MIC values between 0.78-3.125 mg/mL against all of the Bactria and Z. tenuior and Z. clinopodioides had the minimum antimicrobial activity. Total phenolic contents of these five plants were different and followed the general pattern of the antimicrobial effect. The antibacterial effects and the total phenolic content of T. caramanicus and Z. multiflora were remarkable and should be investigated more in future studies.

Cycloartane Triterpenoids from Euphorbia Macrostegia with their Cytotoxicity against MDA-MB48 and MCF-7 Cancer Cell Lines.

The dried plant was extracted with dichloromethane and after defatting with hexane, transferred repeatedly on silica columns using dichloromethane-hexane and ethyl acetate-hexane as mobile phases. Finally the fractions were purified by high performance liquid chromatography using a Pack-Sil column and hexane: Ethyl acetate as mobile phase. The structures of the isolated compounds included: cycloart-25-ene-3ß, 24-diol (1), cycloart-23(Z)-ene-3ß, 25-diol (2), cycloart-23(E)-ene-3ß, 25-diol (3), and 24-methylene-cycloart-3ß-ol (4) were elucidated by (13)C- and (1)H-NMR as well as IR and by the aid of mass fragmentation pattern and comparing with the literature. The biological effects of the compounds were done by the MTT assay on two different cancer cell lines including MDA-MB48 and MCF-7. Among these compounds, cycloart-23(E)-ene-3ß,25-diol (3) was the most active compound on MDA-MB468 cell line (LD50 = 2.05 µgmL(- 1) ) and cycloart-23(Z)-ene-3ß, 25-diol (2) was the most active compound on MCF-7 cell line (LD50 = 5.4 µgmL(- 1)).

New mirsinane-type diterpenes from Euphorbia microsciadia Boiss. with inhibitory effect on VEGF-induced angiogenesis.

Euphorbia microsciadia (Euphorbiaceae) is a perennial plant growing in Iran. Two new cyclomyrsinol esters, 3-O-propionyl-5, 10, 14-O-triacetyl-8-O-(2'-methyl-butanoyl)-cyclomyrsinol (1) and 3, 5, 10, 14, 15-O-pentaacetyl-8-O-isobutanoyl-cyclomyrsinol (2) were isolated from the methanolic extract of its dried aerial parts. The structures were elucidated based on (13)C- and (1)H-NMR as well as 2D-NMR, IR and different MS spectra. Anti-angiogenic activity was also evaluated on vascular endothelium growth factor (VEGF)-induced angiogenesis in cultured human umbilical vein endothelial cells in vitro by assessing capillary-like tube network formation.

New immunosuppressive cyclomyrsinol diterpenes from Euphorbia kopetdaghi Prokh.

Aceton: chloroform (1:2) extracts of the aerial parts of Euphorbia kopetdaghi Prokh. (Euphorbiaceae) were investigated for its diterpenoids and afforded three new five-membered ring, pentacyclic myrisinane polyester comprised of 3,5,10-O-triacetyl-8-O-isobutanoyl-14-O-benzoylcyclomyrsinol (1), 3,5,10,14-O-tetraacetyl-8-O-(2'-methyl butanoyl)-cyclomyrsinol (2) and 3,5,10,14-O-tetracetyl-8-O-isobutanoylcyclomyrsinol (3). The structures were elucidated based on (13)C- and (1)H-NMR as well as 2D-NMR, IR and different MS spectra and the immunomodulation activity for compound 1 was evaluated through lymphocyte proliferation assay, IL-2 assay, oxidative burst of phagocytic leukocytes and through their cytotoxicity on two cell lines. Compound 1 showed significant suppressive activity against phytohemagglutinin-activated T-cell proliferation with an IC(50) of 1.83 µg mL(-1), IL-2 suppressive activity with an IC(50) of 19.0 µg mL(-1) and oxidative burst suppressive activity with an IC(50) of 1.6 µg mL(-1) and ignorable cytotoxic effect on the CC-1 rat hepatocyte and 3T3-L1 mouse fibroblast cell-lines.

Unusual ingenoids from Euphorbia erythradenia Bioss. with pro-apoptotic effects.

Dichloromethane-acetone extract of Euphorbia erythradenia Bioss., a spurge endemic to the Iran, afforded four novel tetrahydroingenol diterpenes, one new myrsinane type diterpene, and two known triterpenes. Tetrahydroingenoids are novel compounds not only for the double bond reduction but also for their unique hydroxylation pattern at C-11 and C-13. The structures of these compounds were elucidated by spectroscopic methods, and especially 2D NMR measurements. The biological effects of the compounds were done by the MTT and Annexin V-FITC & PI staining assays on different cancer cell lines. The results obtained on EJ-138, CAOV-4, and OVCAR-3 cell lines suggested that tetrahydroingenanes inhibit cell proliferation through apoptosis in cancer cell lines. In conclusion, the new pattern of hydrogenation and hydroxylation of these compounds compared to other ingenoids, along with their apoptosis inducing properties on cancer cells, makes them of great interest for more investigation.

Phytochemical Analysis and Metal-chelation Activity of Achillea tenuifolia Lam.

Achillea tenuifolia Lam. (Asteraceae) afforded a dichloromethane fraction from which three known compounds ß-sitosterol (compound1), 5-hydroxy, 4',6,7- trimethoxy flavone (salvigenin compound 2), and methyl-gallate (compound 3) were isolated for the first time. The structure of isolated compounds was elucidated by different spectroscopic methods. Applying the molar-ratio method, the complexation of salvigenin with Fe (III), Cu(II) and Zn(II), the most abundant type of metal ions in the body, were then evaluated. It was determined that stoichiometric ratio of salvigenin with these cations were as Fe(Salvigenin)2 (H2O)2 and Cu(Salvigenin)2(H2O)2 in methanolic solution without pH control, while zinc ions didn`t form significant complexes. The results were confirmed more, by computational molecular modeling of the structure of proposed ligand-complexes by semi-imperical PM3 calculations, which determined negative heat of formation for the complexes Fe(III) and Cu(II) ions as -689.7 and -573.5, respectively and proposed chelating affinity of salvigenin in the following order: Fe(III) > Cu(II) >> Zn(II).

Flavonol Glycosides from Euphorbia microsciadia Bioss. with their Immunomodulatory Activities.

FOUR KNOWN FLAVONOIDS: quercetin 3-O-ß-D-rutinoside (Q3Rut), myricetin 3-O-ß-D-galactopyranoside (M3Gal), quercetin 3-O-ß-D-galactopyranoside (Q3Gal) and quercetin 3-O-ß-D-glucopyranoside (Q3Glc), for the first time were isolated from aerial parts of Euphorbia microsciadia. The chemical structure of them was elucidated on the basis of 1 and 2 D-NMR spectra and different spectroscopic techniques. The immunomodulatory activities of isolated compounds were compared using standard T-cell proliferation assay. These data showed that lymphocyte suppression activity of flavonoids (1-4) were comparatively lower than prednisolon as a standard drug. Immunosuppressive activity of flavonoids with hydroxyl groups at both 3'-and 4'-positions in their B-ring (Q3Gal) were more than those with 3'-,4'-and 5'-hydroxyl substitution (M3Gal). In these compounds, Q3Gal showed the most inhibitory activity, whereas M3Gal showed the least lymphocyte antiprolifeartive activity.

Pentacyclic Triterpenes in Euphorbia microsciadia with Their T-cell Proliferation Activity.

The ethyl acetate partition of dried methanolic extract of aerial parts of Euphorbia microsciadia (Euphorbiaceae) afforded three pentacyclic triterpenes, betulinic acid (1) from lupane type, oleanolic acid (2) from oleane type and ursolic acid (3) from ursane type triterpenes that are reported for the first time in this plant. These three compounds were structurally compared through their mass fragmentation pattern, nuclear magnetic resonance (NMR) data and their biologic immunomodulatory effects. The structures of the isolated compounds were elucidated by (13)C- and (1)H-NMR as well as 2D-NMR, IR and by the aid of mass fragmentation pattern and comparing with the literature. After running T-Cell proliferation assay, oleanolic acid stimulated proliferation of T-Cells at lower concentration 0.5 µg/mL, while betulinic acid and ursolic acid showed inhibitory activity against T-Cell proliferation with IC50- value > 50 µg/mL and 3.01 ± 0.47 µg/mL, respectively.

Cycloartanes from Euphorbia aellenii Rech. f. and their Antiproliferative Activity.

The cytotoxic chloroform fraction of Euphorbia aellenii afforded two cycloartane type triterpenes-cycloart-25-en-3ß,24-diol (1) and 24-methylene-cycloartan-3ß-ol (2)-for the first time from this plant. Preparation of cycloartane derivatives, 3ß, 24-O-diacetyl-cycloart-25-en as compound 3 and 3ß-O-acetyl-24-methylene-cycloartan (4) were conducted by acetylating of 1 and 2, respectively. The structures of the isolated compounds were elucidated by spectroscopic methods and their activities evaluated by proliferation assay on human peripheral blood lymphocytes (PBLs). Comparing the results suggested that anti-proliferation effect of these compounds on PBLs might be due to the presence of free 3-OH group while masking the free OH groups by acetylation, could induce proliferation activity.

Two new lathyrane type diterpenoids from Euphorbia aellenii.

Euphorbia aellenii Rech. f. (Euphorbiaceae) afforded a cytotoxic chloroform fraction from which two new esters of 6(17)-epoxylathyrol were identified (1 and 2). The structure of these compounds was elucidated by spectroscopic methods.

Biological screening of euphorbia aellenii.

Fractions obtained from partitioning of aerial methanolic extract of Euphorbia Aelleniiwith chloroform (E2), ethyl acetate (E3), n-butanol (E4) and water (E5) were investigated in vitro for their phytotoxicity, insecticidal, anti-leishmanial, cytotoxicity, antibacterial and antifungal activities. Sample E2 appeared to have significant phytotoxic activity. In the insecticidal screening study only one of the insects, Rhyzopertha dominica, showed acceptable mortality after treatment with E2, but weak response to E3 and E4 fractions. Leishmanicidal activity of the chloroform fraction was present, but not very significant. E2 showed favorable cytotoxic activity, while E3 had weak activity, and the other samples had no significant activity. In the antifungal screening tests; E2, E3 and E4 fractions exhibited about 25% inhibition of fungal growth against Fusariumsolani, while they were not active against other tested fungi. The results of antibacterial screening were completely negative, which may be due to resistance towards these types of constituents.