TIM-3/Galectin-9 interaction and glutamine metabolism in AML cell lines, HL-60 and THP-1.

BACKGROUND: T cell immunoglobulin and mucin-domain containing-3 (TIM-3) is a cell surface molecule that was first discovered on T cells. However, recent studies revealed that it is also highly expressed in acute myeloid leukemia (AML) cells and it is related to AML progression. As, Glutamine appears to play a prominent role in malignant tumor progression, especially in their myeloid group, therefore, in this study we aimed to evaluate the relation between TIM-3/Galectin-9 axis and glutamine metabolism in two types of AML cell lines, HL-60 and THP-1. METHODS: Cell lines were cultured in RPMI 1640 which supplemented with 10% FBS and 1% antibiotics. 24, 48, and 72 h after addition of recombinant Galectin-9 (Gal-9), RT-qPCR analysis, RP-HPLC and gas chromatography techniques were performed to evaluate the expression of glutaminase (GLS), glutamate dehydrogenase (GDH) enzymes, concentration of metabolites; Glutamate (Glu) and alpha-ketoglutarate (α-KG) in glutaminolysis pathway, respectively. Western blotting and MTT assay were used to detect expression of mammalian target of rapamycin complex (mTORC) as signaling factor, GLS protein and cell proliferation rate, respectively. RESULTS: The most mRNA expression of GLS and GDH in HL-60 cells was seen at 72 h after Gal-9 treatment (p = 0.001, p = 0.0001) and in THP-1 cell line was observed at 24 h after Gal-9 addition (p = 0.001, p = 0.0001). The most mTORC and GLS protein expression in HL-60 and THP-1 cells was observed at 72 and 24 h after Gal-9 treatment (p = 0.0001), respectively. MTT assay revealed that Gal-9 could promote cell proliferation rate in both cell lines (p = 0.001). Glu concentration in HL-60 and α-KG concentration in both HL-60 (p = 0.03) and THP-1 (p = 0.0001) cell lines had a decreasing trend. But, Glu concentration had an increasing trend in THP-1 cell line (p = 0.0001). CONCLUSION: Taken together, this study suggests TIM-3/Gal-9 interaction could promote glutamine metabolism in HL-60 and THP-1 cells and resulting in AML development.

Phytochemical study of Seriphidium khorassanicum (syn. Artemisia khorassanica) aerial parts: sesquiterpene lactones with anti-protozoal activity.

Two new eudesmane-type sesquiterpene lactones, 1ß,3α,8α-trihydroxy-11ß,13-dihydroeudesma-4(15)-en-12,6α-olide (1) and 1ß,4α,8α-trihydroxy-11ß,13-dihydroeudesma-12,6α-olide (2), and an unprecedented elemane-type sesquiterpene lactone, 1ß,2ß,8α-trihydroxy-11ß,13-dihydroelema-12,6α-olide (3) along with a known eudesmanolide artapshin (4) were isolated from Seriphidium khorassanicum. Structures were elucidated by NMR, HR-ESI-MS, and ECD spectral data analysis. The anti-protozoal activity was evaluated against Leishmania major promastigotes and amastigote-infected macrophages. They showed dose- and time-dependent activity against L. major amastigotes with IC50 values in the range of 4.9 to 25.3 µM being favourably far below their toxicity against normal murine macrophages with CC50 values ranging from 432.5 to 620.7 µM after 48 h of treatment. Compound 3 exhibited the strongest activity and the highest selectivity index (SI) with IC50 of 4.9 ± 0.6 µM and SI of 88.2 comparable with the standard drug, meglumine antimoniate (Glucantime), with IC50 and SI values of 15.5 ± 2.1 µM and 40.0, respectively.

Bioassay-guided isolation in Salvia abrotanoides Karel. stem based on its anti-fungal and anti-trichomonas activity.

Background and purpose: Salvia abrotanoides is considered a medicinal plant and has a broad distribution in Iran. In Iran's traditional medicine, it is also used to treat leishmaniasis, malaria, atherosclerosis, cardiovascular disease, and as a disinfectant. This research aimed to determine the anti-Candida component from S. abratonoides and anti-Trichomonas natural compounds from the stems of this plant. Experimental approach: The plant shoots were collected, dried, and after removing the leaves, grounded. Dried plant material was extracted in a maceration tank, concentrated by a Rotavap, degreased, and fractionated by normal column chromatography. Based on anti-fungal screening against Candida species, Fr. 4, with more anti-fungal activity, was selected for phytochemical analysis, by different chromatographic methods on the silica gel column and Sephadex LH-20. Isolated compounds were elucidated by NMR analysis, mass spectrum, and ultraviolet spectroscopy. Anti-fungal effects were investigated using the fungal suspension, incubation, and parasite-counting methods on purified compounds. Antibacterial effects were assessed using the Broth dilution test and reported according to the MIC parameter. Findings/Results: Two diterpenoid compounds named carnosol (compound 1), 11-hydroxy-12-methoxy-20-norabiata-8, 11, 13-trien (compound 2), and a flavonoid: 6,7-dimethoxy-5, 4'-dihydroxyflavone (compound 3) were isolated and identified. Compound 1 had selective anti-fungal effects against C. albicans, C. glabrata, and C. parapsilosis, but weak toxicity against Trichomonas vaginalis with IC50 of 675.8 µg/mL, less than metronidazole with an IC50 of 13.2 µg/mL. Conclusion and implications: Carnosol as the main component was assayed against Candida, Aspergillus, Rhizopus, and Trichomanas species. The results confirmed its effect on Candida compared to standard drugs.

A double-blind randomized clinical trial of Dracocephalum kotschyi Boiss. in the patients with diarrhea-predominant irritable bowel syndrome.

Background and purpose: Irritable bowel syndrome (IBS) is a disease that shows its impacts on many populations worldwide. It is known as a functional disorder of the gastrointestinal tract followed by diarrhea and fecal inconsistency. Due to the lack of treatment in the allopathic medicine system for IBS, people in the western world use different herbs as alternative medicine. In the present study, we evaluated the dried extract of Dracocephalum kotschyi against IBS. Experimental approach: In a randomized, double-blinded, placebo-controlled clinical trial, 76 diarrhea-predominant IBS patients were randomly assigned to two equal groups: the control group (given the placebo capsule containing 250 mg of dibasic calcium phosphate) and the treatment groups (given the capsule containing 75 mg of the dry extract of D. kotschyi and 175 mg of dibasic calcium phosphate as filler). The study was conducted based on Rome III criteria. We studied symptoms included in Rome III criteria and divided the study into the duration of drug administration and four weeks after drug administration. These groups were compared with those of the control group. Findings/Results: Significant improvements were found in the quality of life, temperament, and IBS symptoms throughout the treatment duration. Quality of life, temperature, and IBS symptoms were slightly decreased in the treatment group 4 weeks after stopping the treatment. While concluding the study, we found D. kotschyi effective against IBS. Conclusion and implications: Whole extract of D. kotschyi modulated symptoms of IBS patients and improved their quality of life.

Changes in polyphenolic composition, physiological characteristics, and yield-related traits of Moshgak (Ducrosia anethifolia Boiss.) populations in response to drought stress.

Ducrosia anethifolia (DC.) Boiss. is an aromatic medicinal plant that has been traditionally used as an analgesic to treat headaches, backaches, colic, and cold. This study evaluated the yield, physiological, and phytochemical traits of 24 populations for 2 consecutive years under the water stress condition. The seed yield and physiological traits demonstrated the highest values in the first and second year, respectively. Hydrogen peroxide (H2O2), proline, malondialdehyde (MDA), and antioxidant activity enzymes were elevated, while chlorophyll, carotenoids, relative water content (RWC), and yield decreased under drought stress. High-performance liquid chromatography (HPLC) was also applied to assess the changes in some polyphenolic compounds in response to water stress. The increase in some phenolic compounds, such as p-coumaric acid, was recorded due to drought stress, while there was a decrease in flavonoids, that is luteolin and quercetin. Among the populations, Abarkuh2 indicated the highest increase in p-coumaric acid (96%) in response to drought stress. In general, high diversity among the studied populations provides new insights into choosing the beneficial populations for medicinal and food purposes. HIGHLIGHTS: • Changes in polyphenolics of Moshgak populations were obtained in response to water stress. • Gallic acid, ferulic acid, p-coumaric acid and vanillic acid were the major components. • The phenolic compounds was increased due to drought stress while flavonoids were decreased High variation was obtained between Moshgak populations.

The effect of Allium hirtifolium bulb on serum lipid profile in adult patients with hyperlipidemia: A randomized double-blind placebo-controlled clinical trial.

BACKGROUND: One of the first goals of reducing the risk of cardiovascular disease (CVD) is to achieve effective therapies for hyperlipidemia. This study aimed to investigate the effect of Allium hirtifolium bulbs on serum lipid profile in patients with hyperlipidemia. METHODS: This clinical trial was conducted on hyperlipidemic patients in two drug (Allium; n = 25) and placebo (n = 24) groups. The first group received 500 mg of A. hirtifolium bulbs twice daily for 6 weeks, while the second group received placebo for the same frequency and duration. Cholesterol, triglycerides (TG), low-density lipoprotein (LDL), high-density lipoprotein (HDL), fasting blood sugar (FBS), creatinine, blood urea nitrogen (BUN), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured at the beginning and the end of study and were compared between the groups. RESULTS: A. hirtifolium reduced total cholesterol and LDL significantly compared to the placebo (P < 0.05). However, no significant effects on other parameters were observed. Furthermore, the use of A. hirtifolium had no effects on renal and liver function tests. CONCLUSION: The use of A. hirtifolium bulbs can decrease serum total cholesterol and LDL in hyperlipidemic adult patients.

New Cytotoxic Premyrsinane-Type Diterpenes from Euphorbia aleppica Against Breast Cancer Cells.

Euphorbia is used in traditional medicine to remove warts, possibly due to its cytotoxic or antiviral effects. This study investigated its phytochemistry and bioactive compounds. Euphorbia aleppica from the Euphorbiaceae family was collected from Kuhdasht, Lorestan, Iran. Plant material was dried and ground. Extraction was performed by maceration using a dichloromethane-acetone solvent. After removing fatty contents, fractionation was done by open column chromatography. Based on the initial H-NMR spectra, fractions containing diterpenoid compounds were identified. The Sephadex column and HPLC performed isolation. The HPLC was done with a regular YMC silica column using a hexane: Ethyl acetate (70: 30) solvent. The selected sub-fractions were identified by one and two-dimensional corelative NMR spectra. Accurate mass spectra confirmed the molecular formula of the obtained structures. Cytotoxicity was assessed using a standard MTT assay against breast cancer cells. The NMR and mass analysis identified compound 1 as a newly described and compound 2 as a pre-defined compound as 3, 7, 15ß-triacetyl-5α-tigliate-13(17)-α-epoxy-14-oxopremyrsinane and 3, 7, 14, 15, 17-pentaacetyl-5-tigliate-13(17)-epoxypremyrsinane, respectively. Compound 1 showed moderate cytotoxicity, and compound 2 exhibited a potent cytotoxic effect dose-dependently against MCF-7 and MDA-MB 231 breast cancer cells, probably because of 14-O-acetyl and 17-O-acetylated hemiacetal groups.

Phytochemical analysis of Artemisia kopetdaghensis: Sesquiterpene lactones with proapoptotic activity against prostate cancer cells.

Phytochemical investigation of the aerial parts of Artemisia kopetdaghensis resulted in the isolation and characterization of three undescribed eudesmane-type sesquiterpene lactones, persianolide A, 4-epi-persianolide A, and 3α,4-epoxypersianolide A, together with three previously described eudesmane-type sesquiterpene lactones, 11-epi-artapshin, 1ß,8α-dihydroxy-11α,13-dihydrobalchanin, and 1ß-hydroxy-11-epi-colartin. The abundantly obtained 11-epi-artapshin was oxidized to undescribed 11α,13-dihydroeudesma-12,6α-olide-1,8-dione and 8ß-hydroxy-11α,13-dihydroeudesma-12,6α-olide-1-one and acetylated to the undescribed 1,8-O-diacetyl-11α,13-dihydroeudesma-12,6α-olide. Structures were elucidated based on extensive spectral data analyses, including 1D and 2D NMR and HRESIMS. The absolute configuration was determined using calculated and experimental ECD spectral data. Compounds were subsequently subjected to the MTT assay to evaluate their cytotoxicity against prostate cancer cells (DU-145 and LNCaP). Related factors associated with the sequence of apoptosis were tested by ELISA, western blotting, and biochemical assay. Results suggested that 11-epi-artapshin hinders the growth of DU-145 cells through mitochondria-mediated apoptosis initiated by stimulation of ROS build-up, ΔΨm depletion, regulation of the Bax/Bcl-2 ratio, and activation of caspase 3, respectively.

Alhagi maurorum extract modulates quorum sensing genes and biofilm formation in Proteus mirabilis.

Proteus mirabilis (P. mirabilis) is a frequent cause of catheter-associated urinary tract infections. This study aims to investigate the anti-infective effect of Alhagi maurorum extract (AME), the traditional medicinal plant in the middle east, on the biofilm-forming P. mirabilis isolates. Hydroalcoholic extract and oil of A. maurorum were characterized by HPLC and GC-MS. The antiproliferative, anti-biofilm, and bactericidal activity of AME at various concentrations were assessed by turbidity, crystal violet binding, and agar well diffusion assays, respectively. The AME's effect on adhesion and quorum sensing (QS) were investigated by in vitro adhesion assay on cell culture and agar overlay assay using Janthinobacterium lividum (ATCC 12472) as a biosensor strain. In addition, the expression level of selected genes involved in QS and biofilm regulation were determined by quantitative Real-Time PCR. Furthermore, the bladder phantom model was created to evaluate the assays and investigate the catheter's calcium deposition. The most effective chemical compounds found in AME were tamarixetin, quercetin, and trans-anethole. Although AME did not inhibit swarming motility, it reduced biofilm production and exerted a concentration-dependent anti-adhesive and anti-QS activity against P. mirabilis. AME also downregulated the expression level of selected genes involved in biofilm formation and QS. This study showed that AME as a natural compound reduced biofilm formation of P. mirabilis by targeting virulence factor genes, quorum sensing, and other strategies that include preventing the adhesion of P. mirabilis to the cells. The results suggest that A. maurorum extract might have the potential to be considered for preventing UTIs caused by P. mirabilis.

Myrsinane-type diterpenes from Euphorbia gedrosiaca with cell growth inhibitory activity and apoptotic effects on melanoma cancer cells.

Phytochemical analysis of Euphorbia gedrosiaca Rech.f., Aellen & Esfand., an Iranian endemic spurge, afforded the isolation of four myrsinane types diterpene polyesters. Two new compounds (1-2) were based on a myrsinane skeleton while the others (3-4) were known diterpenes based on a cyclomyrsinane backbone. Their chemical structures were elucidated by spectroscopic methods, including 1D and 2D NMR and HRESIMS. The isolated compounds were tested to evaluate their cell growth inhibitory activity and apoptotic effects on melanoma cell lines, B16F10 and A375. The IC50 values for compounds 1-4 were 58.45, 55.43, 86.52 and 82.27 µM, respectively, on B16F10, and 20.66, 21.88, 36.21 and 39.87 µM, respectively, on A375 cells. Non-treated cells were used as negative control (100% cell growth) and 5 nM Taxol were considered as a positive control.

In Vitro and In Vivo Anti-Toxoplasma Activities of Dracocephalum kotschyi Extract in Experimental Models of Acute Toxoplasmosis.

BACKGROUND: The problem of resistance to antiparasitic drugs, associated with their side effects, suggest exploring other alternatives, including medicinal plants. Dracocephalum kotschyi (D. kotschyi), for example, from Lamiaceae family, is a plant widely used in Iran and in many countries, and to which interesting pharmacological properties have been attributed. This study aimed to investigate in vitro and in vivo anti-Toxoplasma activities of D. kotschyi extract in experimental models of acute toxoplasmosis. METHODS: Anti-Toxoplasma activity of the extracts in vitro was performed on Vero Cell, using the MTT test. Vero cell were infected with (3 × 105 tachyzoites/well) following treatment with Dichloromethane (F1), dichloromethane: methanol (F2), methanol (F3), methanol: water (F4), and deionized water (F5) extracts of D. kotschyi, and pyrimethamine and sulfadiazine (positive control). MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used to measure cell viability. Effects of extracts on tachyzoites viability were evaluated by trypan blue exclusion method, followed by light microscopy. For in vivo test, 18 groups of 8-10-week-old Inbred Balb/c mice weighing 18-20 g, were intraperitoneally infected with 2 × 103 tachyzoites and then treated with sterile PBS (negative control), pyrimethamine (25 mg/kg) and sulfadiazine (500 mg/kg) as positive controls, and F1 to F5 extracts (at doses 50, 100 and 200 mg/kg). RESULTS: The 50% Inhibitory Concentration of F1 extract, F2 extract, Sulfadiazine (Positive control) and Pyrimethamine (Positive control) were 8.77 µg, 7.1 µg 391.18 µg, and 84.20 µg, respectively, while the selectivity indices were 15.667, 30.197, 1.552 and 4.064, respectively. In vivo anti-Toxoplasma activity test showed that Methanol: water (F-4) 50 extract was more active than the positive control. CONCLUSIONS: Indeed, the extract allowed a survival rate of 10% of the mice, compared to 0% for all the other groups. D. kotschyi has the potential to be valorized in the management of toxoplasmosis.

15-Hydroxy-8(17),13(E)-labdadiene-19-carboxylic acid (HLCA) inhibits proliferation and induces cell cycle arrest and apoptosis in ovarian cancer cells.

AIM: 15-Hydroxy-8(17),13(E)-labdadiene-19-carboxylic acid (HLCA) isolated from Juniperus foetidissima, has been recently identified as an antiproliferative agent; however, the molecular basis of antiproliferative effects of HLCA remains unknown. To investigate it, the current study has emphasized the hypothesis that HLCA induced cell death is a consequence of intracellular reactive oxygen species (ROS) production followed by cell cycle arrest and apoptosis. MAIN METHODS: Human ovarian OVCAR-3 and Caov-4 cells were treated with various concentrations of HLCA (48 h) and the measurement of intracellular ROS was considered. Then, the potential of HLCA in promoting apoptosis was investigated via flow cytometry, western blot, and caspase activity assay. Also, the inhibitory effect of HLCA on the cell cycle was evaluated using flow cytometry and western blot analysis. KEY FINDINGS: We found intracellular (ROS) accumulation in HLCA-treated cells. Subsequent observation of the increment in pro-apoptotic Bax as well as the decrement in antiapoptotic Bcl2 revealed that the HLCA-induced cytotoxicity may be triggered by the intrinsic pathway of apoptosis. Our subsequent experiments suggested that caspase-9 and -3 were activated and led the cells to apoptosis during the process. Cell cycle disruption at the G1 phase via down-regulation of cyclin D1 and Cyclin-dependent kinase 4 (CDK4) was another proved mechanism by which HLCA exerts its antiproliferative effects on the ovarian cell lines, OVCAR-3 and Caov-4, especially at relatively lower concentrations. SIGNIFICANCE: This is the first study that reveals the apoptotic effects of HLCA, suggesting its therapeutic potential as an effective anti-tumor agent. However, further in vivo studies are required to confirm these effects.

Inhibitors of α-Synuclein Fibrillation and Oligomer Toxicity in Rosa damascena: The All-Pervading Powers of Flavonoids and Phenolic Glycosides.

There is an intense search for natural compounds that can inhibit the oligomerization and fibrillation of α-synuclein (α-Syn), whose aggregation is key to the development of Parkinson's disease (PD). Rosa damascena is a medicinal herb widely used in Middle Eastern food, ceremonies, and perfumes. The herb is known to contain many different polyphenols. Here we investigated the existence of α-Syn fibrillation inhibitors in R. damascena extract. Different HPLC fractions of the extract were assessed in α-Syn fibrillation and toxicity assays. The most active fractions led to the formation of more α-Syn oligomers but with less toxicity to SH-SY5Y cells, according to MTT and LDH assays. LC-MS analysis identified gallic acid, kaempferol 3-glucoside, kaempferol-3-O-ß-rutinoside, and quercetin which were subsequently shown to be strong α-Syn fibrillation inhibitors. Our results highlight the benefits of R. damascena extract to combat PD at the population level.

Effects of Cyperus rotundus Extract on Spatial Memory Impairment and Neuronal Differentiation in Rat Model of Alzheimer's Disease.

BACKGROUND: Alzheimer's disease (AD) is one of the most common neurodegenerative diseases in the older population and characterized by progressive memory and cognitive impairment. Cyperus rotundus, a traditional medicinal herb, has analgesic, sedative, and anti-inflammatory effects and also used to increase memory in Islamic traditional medicine. This study was designed to consider the effects of C. rotundus extract on memory impairment and neurogenesis in the Beta-Amyloid rats' model. MATERIALS AND METHODS: Forty-two male Wistar rats were randomly divided into six groups (n = 7) for the evaluation of baseline training performance in the Morris water maze test. Then, amyloid-beta (Aß1-42) was injected in animal hippocampal CA1 bilaterally in four groups. The first probe trial was performed 21 days after Aß injection. Then, 250, 500, and 750 mg/kg of C. rotundus extract were administered to three Aß-injected groups for 1 month; after that, the second probe trial was performed, and rats were sacrificed after 28 days of the second probe trial. The neurogenesis was detected in the hippocampus, by immunohistochemical staining. RESULTS: This study showed that spatial memory increased in the behavioral test in AD treated group with C. rotundus extract, compared with the AD group (P = 0.02). Immunohistochemical staining revealed that neuronal differentiation has been occurred in the hippocampus in the AD-treated group with C. rotundus extract compared with the AD group (P = 0.01). CONCLUSIONS: This study showed that C. rotundus extract, repaired spatial memory impairment in the Aß rats, through increased neurogenesis in the hippocampus, which could be related to the flavonoid components in the extract.

Cycloarta-23-ene-3beta,25-diol a pentacyclic steroid from Euphorbia spinidens, as COX inhibitor with molecular docking, and in vivo study of its analgesic and anti-inflammatory activities in male swiss mice and wistar rats.

BACKGROUND AND AIMS: Euphorbia is a large genus of flowering plants. In Iran, some plants of this family have been used in the treatment of inflammatory disorders and also to relieve back pain. Euphorbia spinidens is a rich source of Cycloarta-23-ene-3beta,25-diol. Cycloartane structures are the starting material for the synthesis of plant steroids, and the aim of this study is to demonstrate COX inhibitory activity, molecular docking and in vivo approach of anti-inflammatory activity of cycloartane compound isolated from Euphorbia spinidens. MATERIAL AND METHODS: Plant material was extracted with acetone-chloroform and submitted to column chromatography for fractionation. Based on preliminary 1H-NMR spectra, cycloartane fraction was selected and purified by repeated recycle HPLC system. The structure and purity of compound were determined by 1H and 13C-NMR, HPTLC, and mass spectra. Inhibitory activities of the tested compounds on COX-1 and COX-2 were evaluated by a colorimetric COX (ovine) inhibitor screening method. Vero cells were used to assess the toxicity against the normal cells, and calculate the selectivity index. COX inhibitory activity results were evaluated and confirmed by molecular docking experiments. In the in vivo approach, analgesic activity was assessed by acetic acid-induced abdominal writhing and formalin tests. Croton oil-induced ear edema in mice and carrageenan-induced rat paw edema in rats were used to evaluate anti-inflammatory activity. Pain tests were carried out on male Swiss mice (25-35 g). Male Wistar rats (160-200 g) were used for the carrageenan test. RESULTS: Cycloart-23-ene-3ß,25-diol showedin vitro cyclooxygenase 1 and 2 inhibitory activities with more selectivity for COX-2. Molecular docking by predicting binding energies in COX protein receptors confirmed in vitro COX inhibitory results, and determined the best position for ligand in COX receptors along with its residue interactions in receptor pockets, which must be considered for designing of their inhibitors. In the in vivo studies, cycloartane inhibited significantly acetic acid-induced abdominal contractions and formalin-induced licking behavior at a dose of 200 mg/kg. The same dose reduced croton oil ear edema in mice and carrageenan-induced paw edema in rats. CONCLUSION: Therefore, according to these findings, cycloart-23-ene-3beta,25-diol showed promising analgesic and anti-inflammatory effects with low toxicity against normal cells and can be suggested as a template lead for designing anti-inflammatory compounds with good selectivity index, and potency for COX-2 inhibitory activity.

Kopetdaghinanes, pro-apoptotic hemiacetialic cyclomyrsinanes from Euphorbia kopetdaghi.

Euphorbia kopetdaghi grows wild in the Northeast parts of Iran. Phytochemical study of its aerial parts led to the isolation of two undescribed cyclomyrsinol macrocyclic diterpenes with a new tetrahydrofuran oxidation pattern containing a hemiacetal group named: kopetdaghinane A and B. The structure of the isolated compounds was elucidated by extensive spectroscopic methods. Cytotoxic activity of kopetdaghinane A was evaluated using standard MTT assay against MCF-7 breast cancer and OVCAR-3 ovary cells. HUVEC cells were used as a normal cell line for calculation of the selectivity index. The MTT showed cyclomyrsinol diterpene has a significant cytotoxic effect with good selectivity indexes against both cell lines but with more selectivity against MCF-7 cells. Apoptosis induction by cyclomyrsinol treatment was confirmed by annexin V-FITC/PI staining, and caspase-6 activation. Western blot analysis showed that the expression of Bcl-2 was noticeably decreased in response to kopetdaghinane A treatment, while the expression of Bax protein was increased. Moreover, the apoptotic effect of cyclomyrsinol was shown to be related to ROS production, and loss of mitochondrial membrane potential (ΔΨm). Taken together, these results showed that kopetdaghinane A inhibits the growth of MCF-7 breast cancer cells through the activation of the mitochondrial apoptotic pathway and may be considered as an investigational compound in breast cancer preclinical study.

Therapeutic effects of Cyperus rotundus rhizome extract on memory impairment, neurogenesis and mitochondria in beta-amyloid rat model of Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disturbance leading to memory deficit, cognitive decline, and behavioral disturbance. Deposition of Amyloid beta plaques, neurofibrillary tangle and mitochondrial impairment are common neuropathological signs in AD. In this study, the effect of standardized Cyperus rotundus(C. rotundus) extract in three different doses of 250, 500, and 750 mg/kg on memory, neurogenesis and mitochondrial mass in the beta amyloid rat model was assessed. For this purpose, 42 male Wistar rats were randomly divided into six groups (n = 7) to evaluate baseline training performance in Morris water maze test. Amyloid beta (Aß) was injected in animal hippocampal CA1 bilaterally in four groups. After 21 days, a decrease was observed in spending time in target quadrant in the first probe trial in Aß injected groups. Following that, 250, 500, and 750 mg/kg of C. rotundus extracts were administered to three out of four groups for a period of one month. BrdU (Bromodeoxyuridine) was intraperitoneally injected in all groups on the last 7 days of treatment. Then, 28 days after the last BrdU injection, the second probe trial was run, and rats were sacrificed. The neurogenesis and mitochondrial distribution were detected in hippocampus, by immunohistochemical staining. At last, it was observed that C. rotundus, almost recovered memory impairment, in addition to increasing in mitochondrial mass in CA1 and neurogenesis in dentate gyruse in the beta-amyloid rat model of Alzheimer's disease.

Centaurea cyanus extracted 13-O-acetylsolstitialin A decrease Bax/Bcl-2 ratio and expression of cyclin D1/Cdk-4 to induce apoptosis and cell cycle arrest in MCF-7 and MDA-MB-231 breast cancer cell lines.

Natural products are considered recently as one of the source for production of efficient therapeutical agents for breast cancer treatment. In this study, a sesquiterpene lactone, 13-O-acetylsolstitialin A (13ASA), isolated from Centaurea cyanus, showed cytotoxic activities against MCF-7 and MDA-MB-231 breast cancer cell lines using standard 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. To find the mechanism of action of cytotoxicity, annexin V/propidium iodide (PI) staining was performed for evaluation of apoptosis. This process was further confirmed by immunoblotting of anti- and proapoptotic, Bcl-2 and Bax, proteins. Cell cycle arrest was evaluated by measurement of fluorescence intensity of PI dye and further confirmed by immunoblotting of Cdk-4 and cyclin D1. Mitochondrial transmembrane potential (ΔΨm) and generation of reactive oxygen species (ROS) were measured using the JC-1 and DCFDA fluorescence probes, respectively. These experiments showed that 13ASA is a potent cytotoxic agent, which activates apoptosis-mediated cell death. In response to this compound, Bax/Bcl-2 ratio was noticeably increased in MCF-7 and MDA-MB-231 cells. Moreover, 13ASA induced cell cycle arrest at subG1 and G1 phases by decreasing protein levels of cyclin D1 and Cdk-4. It was done possibly through the decrease of ΔΨm and increase of ROS levels which induce apoptosis. In conclusion, this study mentioned that 13ASA inhibit the growth of MCF-7 and MDA-MB-231 breast cancer cell lines through the induction of cell cycle arrest, which triggers apoptotic pathways. 13ASA can be considered as a susceptible compound for further investigation in breast cancer study.

The effect of frankincense (Boswellia serrata, oleoresin) and ginger (Zingiber officinale, rhizoma) on heavy menstrual bleeding: A randomized, placebo-controlled, clinical trial.

OBJECTIVES: To evaluate the effect of frankincense (Boswellia serrata, oleoresin) and ginger (Zingiber officinale, rhizoma) as complementary treatments for heavy menstrual bleeding (HMB) among women of reproductive age. DESIGN: Randomized, placebo-controlled, clinical trial. SETTING: Gynecology outpatient clinics. INTERVENTIONS: Patients with HMB (n = 102) were randomly assigned to three groups. All patients received ibuprofen (200 mg) and either frankincense (300 mg), ginger (300 mg), or a placebo, which contains 200 mg anhydrous lactose as the filling agent and was similar in appearance to the two other drugs. Patients received the medications three times a day for seven days of the menstrual cycle, starting from the first bleeding day and this was repeated for two consecutive menstrual cycles. MAIN OUTCOME MEASURES: Amount and duration of menstrual bleeding and quality of life (QOL). RESULTS: Duration of menstrual bleeding was decreased in the frankincense (-1.77 ± 2.47 days, P = 0.003) and ginger (-1.8 ± 1.79 days, P = 0.001) groups, but not in the placebo group (-0.52 ± 1.86 days, P = 0.42). Amount of menstrual bleeding was decreased in all (P < 0.05), with no difference among the study groups (P > 0.05). More improvement in QOL was observed in the frankincense (-25.7 ± 3.1; P < 0.001) and ginger (-29.2 ± 3.7: P < 0.001) groups compared to the placebo group (-15.07 ± 3.52; P < 0.001) and between the groups, differences were statistically significant (P = 0.02). CONCLUSIONS: Ginger and frankincense seem to be effective complementary treatments for HMB. Further studies with a larger sample size and longer follow-up are warranted in this regard.

Design, Formulation, and Physicochemical Evaluation of Vaginal Cream Containing Eucalyptus camaldulensis, Viola odorata, and Mentha piperita extracts for Prevention and Treatment of Trichomoniasis.

BACKGROUND: Trichomoniasis is a common sexually transmitted disease that is caused by infection with protozoan parasite called Trichomonas vaginalis. Metronidazole is the drug of choice for the treatment of this infection. In this study, design, formulation, and physicochemical evaluation of vaginal cream containing Eucalyptus camaldulensis, Viola odorata, and Mentha piperita extracts for the prevention and treatment of trichomoniasis has been investigated. METHODS: Ethyl acetate extract of E. camaldulensis leaves, water fraction of V. odorata root, and hydroalcoholic extract of M. piperita leaves was prepared and used for anti-trichomonas experiments. Then, based on results, different formulations of vaginal cream containing mixed extracts were prepared and physicochemical evaluation was conducted. In the next step, anti-trichomonas effect of selective formulation was tested in vitro. RESULTS: The mixed concentrates containing 2.5 mg/ml E. camaldulensis, 0.06 mg/ml V. odorata, and 1 mg/ml M. piperita showed 100% growth inhibition (GI) during 24 h. Furthermore, the mixture containing 1.25 mg/ml E. camaldulensis, 0.03 mg/ml V. odorata, and 0.5 mg/ml M. piperita showed 92% GI in the first 24 h. The selective formulation passed all of physicochemical test and also showed 100% GI for in vitro anti-trichomonas experiments in the first 24 h. CONCLUSIONS: The mixed concentrates containing 2.5 mg/ml E. camaldulensis, 0.06 mg/ml of V. odorata, and 1 mg/ml of M. piperita are the mixture which showed the highest percentage of GI (100%) after 24 h. The selective formulation of vaginal cream containing this mixture of extracts was detected 100% GI in the first 24 h.