Isolation of Pro-Osteogenic Compounds from Euptelea polyandra That Reciprocally Regulate Osteoblast and Osteoclast Differentiation.

Plants contain a large number of small-molecule compounds that are useful for targeting human health and in drug discovery. Healthy bone metabolism depends on the balance between bone-forming osteoblast activity and bone-resorbing osteoclast activity. In an ongoing study searching for 22 plant extracts effective against osteoporosis, we found that the crude extract of Euptelea polyandra Sieb. et Zucc (E. polyandra) had osteogenic bioactivity. In this study, we isolated two compounds, isoquercitrin (1) and astragalin (2), responsible for osteogenic bioactivity in osteoblastic MC3T3-E1 cells from the leaf of E. polyandra using column chromatography and the spectroscopic technique. This is the first report to isolate astragalin from E. polyandra. Compounds (1) and (2) promoted osteoblast differentiation by increasing alkaline phosphatase (ALP) activity and alizarin red S stain-positive calcium deposition, while simultaneously suppressing tartrate-resistant acid phosphatase (TRAP)-positive osteoclast differentiation in RAW264.7 cells at non-cytotoxic concentrations. Isoquercitrin (1) and astragalin (2) increased the expression of osteoblastic differentiation genes, Osterix, ALP, and Osteoprotegerin in the MC3T3-E1 cells, while suppressing osteoclast differentiation genes, TRAP, Cathepsin K, and MMP 9 in the RAW264.7 cells. These compounds may be ideal targets for the treatment of osteoporosis due to their dual function of promoting bone formation and inhibiting bone resorption.

p-Coumaroyl Malate Derivatives of the Pandanus amaryllifolius Leaf and Their Isomerization.

A novel p-coumaroyl dimethyl malate (1) was isolated from the Pandanus amaryllifolius leaf in addition to three known analogs of p-coumaroyl dimethyl malate (2-4), and their structures were elucidated by analysis of the spectroscopic data. The p-coumaroyl malate derivatives were isolated as a mixture of E and Z isomers. To determine the cause of isomerization, the p-coumaroyl malate isolated in this study was synthesized. We concluded that the Z isomer might be an artifact generated from the E isomer through purification steps.

Development of chiral building blocks having a bicyclo[3.3.0]octane framework using a diastereomeric resolution-selective deprotection.

A protocol is presented for an efficient and practical approach to the synthesis of enantiomerically pure bicyclo[3.3.0]octane derivatives from achiral Cs-symmetric bicyclo[3.3.0]octane-2,8-dione using a diastereomeric resolution-selective deprotection method. This method affords chiral building blocks having bicyclo[3.3.0]octane framework with the same site of diastereotopic carbonyl functional group.

Attempt to synthesize 2,3,5,4'-tetrahydroxystilbene derived from 2,3,5,4'-tetrahydroxystilbene-2-O-ß-glucoside (THSG).

An attempt to synthesize aglycone 1 derived from 2,3,5,4'-tetrahydroxystilbene-2-O-ß-glucoside (THSG) via the Wittig reaction and Mizoroki-Heck reaction is described. In the Wittig protocol, 2,3,5,4'-tetramethoxystilbene 2 was obtained. Additionally, a palladium-catalyzed Mizoroki-Heck reaction strategy yielded 2-aryl-2,3-dihydrobenzofuran 13 instead of derivative 12 in good yield.

Novel cytotoxic phenanthrenequinone from Odontioda Marie Noel 'Velano'.

A new phenanthrenequinone, 5-hydroxy-2,3-dimethoxy-1,4-phenanthrenequinone (1), was isolated along with a known 9,10-dihydrophenanthrenequinone, ephemeranthoquinone B (2) from an MeOH extract of Odontioda Marie Noel 'Velano' through bioassay-guided fractionation. Their structures were elucidated by spectroscopic analysis, and the compounds were tested for in vitro cytotoxic activity. The compounds showed slightly higher cytotoxicity in human oral squamous cell carcinoma and leukemic cell lines as compared with human oral normal cells. The results suggest that apoptosis may not be involved in the cytotoxicity induction.

Improving quality control of yucca extracts used as food additives by screening antimicrobial activity using NMR metabolomics.

Yucca schidigera is mainly distributed in southwestern US and the northern desert of Mexico. Its extract is widely used as a food additive for its antimicrobial activity. However, this antimicrobial activity is subject to significant variability across production lots. Yucca extracts are natural products and their composition is affected by their cultivation area and weather. Manufacturer deal with natural products such as food additives pay particularly close attention to quality control. In the present study, NMR metabolomics methods were used to screen the antimicrobial activity of yucca extracts. Yucca extracts were subjected to principal component analysis (PCA) and categorized on a score plot of their 1H NMR spectral data according to their antimicrobial activity. Furthermore, hierarchical cluster analysis (HCA) was also used to classify yucca extracts based on their antimicrobial activity. Classification using PCA and HCA was dependent upon saponin content, particularly that of schidigera-saponin A1 and D1, which was further confirmed by HPLC analysis of the yucca extracts. We demonstrated that NMR-based metabolomics is a potentially useful tool to use in combination with conventional quality control methods for yucca extracts used as food additives. We envisage this method as tool for initially screening the extracts prior to carrying out the officially recommended quality control tests.

Screening for natural medicines effective for the treatment of osteoporosis.

Bone-forming osteoblasts are differentiated from mesenchymal stem cells and dysregulation of this differentiation can lead to osteoporosis. Meanwhile, bone-resorbing osteoclasts are both differentiated and multinucleated from hematopoietic precursor cells of monocyte and/or macrophage lineage. Bone resorption inhibitors such as bisphosphonates and estrogen are used to treat osteoporosis. However, the adverse effects of the long-term use of these medicines are of concern, and so the development of new therapies to ameliorate osteoporosis is desirable. Therefore, in the present study, we screened 22 plant extracts and found that nine methanolic extracts of plants promote the differentiation of MC3T3-E1 cells to osteoblasts. These nine extracts were then evaluated for their inhibitory activity on osteoclast differentiation in RAW264.7 mouse macrophage cells. Of the nine extracts, Daucus carota, Vitis spp., Sasa veitchii, Euptelea polyandra, and Sesamum indicum exhibited pro-osteoblastic and anti-osteoclastic activity with low cytotoxicity, suggesting their potential effectiveness against osteoporosis.

Rapid identification of Aconitum plants based on loop-mediated isothermal amplification assay.

OBJECTIVE: Aconitum plants (Ranunculaceae) exhibit toxicity, and accidental ingestion of the plants has been reported in Japan. Identifying the cause of poisoning is important for emergency medical treatment, and a rapid and simple detection technique is required for the identification of poisoning cause. In the present study, we developed a rapid and simple method for detecting Aconitum plant DNA using a loop-mediated isothermal amplification (LAMP) assay. RESULTS: Specific LAMP primers for Aconitum plants were designed based on the trnL-trnF intergenic spacer region. Using the LAMP primers, the LAMP assay included an initiation reaction of 10 min followed by amplification for 20 min at the isothermal reaction temperature of 65 °C. The LAMP reaction was demonstrated to be specific and highly sensitive to Aconitum plants, given that the assay can be used for 1 pg of purified DNA. Using raw extracted DNA as template, the entire detection procedure from DNA extraction to final detection required only 30 min. Moreover, the protocol identified samples containing approximately 5 mg of Aconitum plants cooked and digested with artificial gastric juice. The currently proposed protocol exhibits good potential as a screening method of Aconitum plant poisoning for emergency medical care.

Change in Amino Acid Pools During Neuronal Differentiation of PC12 Cells.

BACKGROUND/AIM: Although rat PC12 cells are a well-established model to investigate neuronal differentiation, survival and function, the reports of differentiation-associated changes in the intracellular amino acid pools of neurotransmitters have been limited. In this study, possible changes in the intracellular amino acid pools were investigated during nerve growth factor (NGF)-induced differentiation of PC12 cells. MATERIALS AND METHODS: Rat PC12 cells were induced to differentiate into neuronal cells by 50 ng/ml NGF in serum-free Dulbecco's Modified Eagle's medium, followed by the addition of fresh NGF-containing medium at day 3, without medium change. Cell viability was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Intracellular amino acids were extracted by 5%trichloroacetic acid and quantified by amino acid analyzer. RESULTS: Differentiated PC12 cells showed high concentrations of excitatory neurotransmitters (glutamic acid and aspartic acid) and glutamine (energy supply). In contrast, urea and taurine levels declined with the progression of neuronal differentiation. Exogenous addition of taurine, urea, and L- and D- aspartic acid showed little or no effect on supporting viability of PC12 cells cultured in serum-free medium. CONCLUSION: The present study demonstrated dramatic changes in the composition of intracellular amino acids during neuronal differentiation.

Studies on the antimicrobial potential of the cardiovascular drug lacidipine.

The cardiovascular drug lacidipine was screened in vitro for possible antibacterial activity with respect to 389 Gram-positive and Gram-negative bacterial strains. It was noticed that most bacteria (233) failed to grow at 50-200 microg/mL concentrations of the drug. Some strains were inhibited at even lower concentrations. The bacteria could be arranged according to their decreasing order of sensitivity as follows: Staphylococcus aureus, Vibrio cholerae, Salmonella spp., Shigellae, Escherichia coli, Bacillus spp., Klebsiellae and Pseudomonas spp. Lacidipine was found to be bacteriostatic in nature against S. aureus and V cholerae. When administered to Swiss strain of white mice at doses of 30 and 60 microg/mouse, lacidipine significantly protected the animals challenged with 50 MLD of S. typhimurium NCTC 74. According to the chi-square test, the in vivo data were highly significant (p<0.001).

Combined long-term caffeine intake and exercise inhibits the development of diabetic nephropathy in OLETF rats.

This study was performed to examine the effects of long-term caffeine-intake, with and without exercise, on the progression of diabetic nephropathy (DN) in an obese diabetic rat model. Thirty-two male Otsuka Long-Evans Tokushima fatty (OLETF) rats were assigned to sedentary (OLETF-Sed), exercise (OLETF-Ex), caffeine-intake (OLETF-Caf), and combined (OLETF-Caf + Ex) groups. Caffeine-intake groups were fed rat chow containing caffeine (90.7 ± 4.7 mg/kg/day). The OLETF-Ex and OLETF-Caf + Ex groups were able to run voluntarily at any time using a rotatory wheel. Body weight (BW) and blood pressure (BP) were measured weekly from 24 to 29 wk of age. Pre- and posttreatment serum glucose, insulin, and creatinine concentrations were measured, and a 24 h urine sample was collected for measurement of creatinine clearance (Ccr) and albumin excretion (UEAlb). After treatment, the kidneys were removed for morphological analysis. The OLETF-Caf and OLETF-Caf + Ex groups exhibited no BP increase during the study. Both the caffeine-intake groups exhibited a significant increase in urine volume (UV), electrolyte excretion, and Ccr, and decreased UEAlb, following treatment. Furthermore, no structural damage was observed in the kidneys of rats from either caffeine-intake group, whereas the OLETF-Sed and OLETF-Ex groups exhibited DN progression. This study demonstrates that caffeine-intake alone and/or combined with exercise significantly decreases BW and improves glucose intolerance, without the progression of DN. Further research should be performed to examine whether the quantities of caffeine contained in a normal human daily intake also have a protective effect against kidney damage.NEW & NOTEWORTHY The present study showed that caffeine administration alone and/or combined with exercise results in an improvement of diabetic nephropathy (DN), including an increase in creatinine clearance and urinary Na excretion, a decrease in urinary protein excretion, and in renal morphological findings. To our knowledge, there are no other studies showing that caffeine administration inhibits DN progression.

Re-evaluation of Culture Condition of PC12 and SH-SY5Y Cells Based on Growth Rate and Amino Acid Consumption.

BACKGROUND/AIM: Most of the previous investigators have used various types of media for the culture of nerve cells. In order to optimize the culture conditions, we compared the growth rate and amino acid consumption by two popular neuron models, rat PC12 and human SH-SY5Y, grown in DMEM or DMEM: Ham's F-12 (1:1): non-essential amino acids, supplemented with 10% fetal bovine serum (referred to DMEM and Mix, respectively). MATERIALS AND METHODS: Cell growth was monitored by the MTT method. Amino acids in the culture medium were quantitated by amino acid analysis after deproteinization. RESULTS: Efficient cell attachment could be achieved even if PC12 cells were inoculated at extreme lower cell density in a non-coated plain dish, without addition of its condition medium. Both PC12 and SH-SY5Y cells proliferated up to slightly higher cell density in DMEM than in Mix. Approximately 2-fold higher utilization rate of glutamine and essential amino acids was observed in DMEM. Amyloid peptides such as Aß1-42 and Aß25-35 suppressed their growth nearly by 50%. CONCLUSION: The present study suggests the usefulness of DMEM for the study of searching neuroprotective substances, based on its favorable effects on cell attachment, cell growth and amino acid utilization as well as amyloid peptide sensitivity.

Evaluation of Biological Activity of Mastic Extracts Based on Chemotherapeutic Indices.

BACKGROUND: Most previous mastic investigators have not considered its potent cytotoxicity that may significantly affect the interpretation of obtained data. In the present study, we re-evaluated several biological activities of mastic extracts, based on chemotherapeutic indexes. MATERIALS AND METHODS: Pulverized mastic gum was extracted with n-hexane and then with ethyl acetate or independently with methanol or n-butanol. Tumor specificity (TS) of the extracts was determined by their cytotoxicity against human malignant and non-malignant cells. Antibacterial activity was determined by their cytotoxicity against bacteria and normal oral cells. Antiviral activity was determined by their protection of viral infection and cytotoxic activity. Cytochrome P-450 (CYP) 3A4 activity was measured by ß-hydroxylation of testosterone. RESULTS: Ethyl acetate extract showed slightly higher tumor specificity (TS=2.6) and one order higher antibacterial activity (selectivity index (SI)=0.813) than other extracts (TS=1.4-2.5; SI=0.030-0.063). All extracts showed no anti-human immunodeficiency virus (HIV) activity, but some anti-herpes simplex virus (HSV) activity, which was masked by potent cytotoxicity. They showed strong inhibitory activity against CYP3A4. CONCLUSION: Ethyl acetate extraction following the removal of cytotoxic and CYP3A4 inhibitory substances by n-hexane can enhance antitumor and antibacterial activity of mastic.

Resveratrol oligomers are potent MRP1 transport inhibitors.

BACKGROUND: Knowledge of the structure-activity relationships of multidrug resistance protein 1 (MRP1, ABCC1) inhibitors may aid in developing potent inhibitors that can be used to circumvent MRP1-mediated multidrug resistance. MATERIALS AND METHODS: Six stilbenes were examined for their ability to inhibit MRP1-mediated transport of 2',7'-bis-(carboxypropyl)-5(6)-carboxyfluorescein (BCPCF) from human erythrocytes and into inside-out erythrocyte membrane vesicles (IOVs). The concentrations of stilbenes decreasing BCPCF transport by 50% during 60 min of incubation at 37 degrees C (IC50) were determined from dose-response curves. RESULTS: Stilbenes inhibited BCPCF transport in cells in the rank order (+)-alpha-viniferin (IC50 = 0.8 microM) > sophorastilbene A (IC50 = 3.1 microM) > (-)-epsilon-viniferin (IC50 = 8.9 microM) > piceatannol (IC50 = 57 microM). Resveratrol and rhaponticin were ineffective. (+)-alpha-Viniferin (IC50 = 0.8 microM), sophorastilbene A (IC50 = 3.7 microM) and (-)-epsilon-viniferin (IC50 = 3.5 microM) were also efficient BCPCF transport inhibitors in IOVs. CONCLUSION: Stilbenes may efficiently inhibit MRP1-mediated organic anion transport. This inhibitory potency of stilbenes increases with oligomerisation. The membrane is not a strong barrier for the inhibitory activity of the trimeric stilbenes.

Diclofenac in the management of E. coli urinary tract infections.

E. coli is the main agent of uncomplicated urinary tract infections (UTIs) and accounts for more than 85% of recurrent cystitis and at least 35% of recurrent pyelonephritis. Despite the widespread availability of antibiotics, UTIs remain the most common bacterial infection in the human population. It is currently advised that the clinical administration of antibiotics against the pathogenic bacteria should be prohibitted due to the emergence of multidrug resistant (MDR) bacterial strains. Therefore, newer and more effective antimicrobials are in demand to treat such cases. One hundred and thirty six urine samples were collected from UTI patients. E. coli was isolated from 85 samples, out of which 33% were resistant to common antibiotics. The isolates were decreasingly resistant to ampicillin, tobramycin, augmentin, nalidixic acid, cefuroxime, nitrofurantoin, kanamycin, pipemidic acid, chloramphenicol, cefotaxime, cefamendol, ofloxacin, ceftizoxime, norfloxacin and amikacin. The anti-inflammatory drug diclofenac exhibited significant antibacterial activity against common bacterial strains both in vitro and in vivo. The present work was conducted to evaluate the in vitro inhibitory effect of this drug on the clinically isolated strains of E. coli in hospitals. All the isolates were sensitive to diclofenac, with MIC values ranging from 5-50 microg/mL. The MIC90 value of the drug was 25 microg/mL. Therefore, it may be suggested that diclofenac has the capacity to treat UTI caused by E. coli.

Qualitative analysis of MDR-reversing Anastasia Black (Russian black sweet pepper, Capsicum annuum, Solanaceae) extracts and fractions by HPLC and LC-MS-MS methods.

In earlier experiments, the MDR (multidrug resistance)-reversal activities of Anastasia Black (Russian black sweet pepper) extracts had been analysed. Recently, the most effective MDR reversing extracts and fractions have been separated by HPLC (high-performance liquid chromatography, for carotenoids) and LC-MS-MS (HPLC combined with mass spectrometry, for phenolic compounds) methods. As a result of the analytical studies, the following flavonoids had been identified: feruloyl glucopyranoside, quercetin rhamnopyranoside glucopyranoside, luteolin glucopyranoside arabinopyranoside, apigenin glucopyranoside arabinopyranoside, quercetin rhamnopyranoside, luteolin arabinopyranoside diglucopy-ranoside, hesperidine and luteolin glucuronide. According to the literature, the aglycones of these phenolic compounds exhibit MDR-reversal activity in vitro, and the connection between the phenolic content of Anastasia Black and MDR-reversal action was therefore studied by different analytical methods. The results of this study revealed that the identified flavonoids of Anastasia Black may be only partially responsible for the modulation of the MDR of mouse lymphoma cells. Other lipophilic compounds, most probably carotenoids, present in Russian black sweet pepper may act as inhibitors of MDR reversal.

Multidrug resistance reversal by 3-formylchromones in human colon cancer and human mdr1 gene-transfected mouse lymphoma cells.

Several new 3-formylchromone derivatives proved to be modifiers of multidrug resistance in mouse lymphoma cells and in human Colo320 colon cancer cells. There is apparently a structure-activity relationship between the antiproliferative multidrug resistance-reversing effect and the chemical structure of the 3-formylchromones. The total polar surface areas and the ground state dipole moments of the molecules are presumed to play a key role in the multidrug resistance-reversing effect. The log P values can provide an adequate explanation for the selective cytotoxicity against cancer cells.

Electron Ionization Mass Spectrometry-based Metabolomics Studies of Sophoraflavescens can Identify the Geographical Origin of Root Samples.

An electron ionization mass spectrometry (EI-MS)-based metabolomic approach was applied to Sophora flavescens to identify the geographical origin of each sample. The score plot from principal component analysis using the EI-MS data showed that Japanese S. flavescens samples tended to cluster away from Chinese S. flavescens samples. Statistical techniques showed that ions arising from kurarinol and kushenol H, which we previously identified as marker molecules for Japanese S. flavescens, were characteristic of Japanese S. flavescens. Therefore, metabolomics based on EI-MS data is a valuable tool for confirming the geographical origins of S. flavescens samples. The results suggest that EI-MS-based metabolomics is suitable for the quality control of traditional medicines containing many components.

Cytotoxic Components Against Human Oral Squamous Cell Carcinoma Isolated from Andrographis paniculata.

BACKGROUND: The 5-year survival rate of patients with oral cancer has remained approximately 50% during the past 30 years, possibly due to the poor tumor selectivity of conventional anticancer drugs. This prompted us to search for new candidates for anticancer drugs that have higher cytotoxicity and tumor selectivity. MATERIALS AND METHODS: Dried leaves of Andrographis paniculata were supplied from a market in Shanghai. The methanolic fraction of A. paniculata was further fractionated to identify cytotoxic principles by spectroscopic analysis and comparison with literature values. Viable cell number was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide method, and tumor specificity was calculated by relative cytotoxicity against oral squamous cell carcinoma cell lines compared to that against normal oral cells. Apoptosis induction was detected by cleaved poly (ADP-ribose) polymerase and caspase-3 on western blot analysis. RESULTS: Major cytotoxicity in the methanol extract of a leaf of A. paniculata was recovered by partitioning with EtOAc, followed by silica gel chromatography. Further purification with reversed-phase high-performance liquid chromatography led to isolation of four known cytotoxic compounds, 14-deoxyandrographolide, andrographolide, neoandrographolide and deoxyandrographiside. Among them, andrographolide had the greatest cytotoxicity and tumor specificity, also inducing caspase-3 activation of HSC-2 oral squamous cell carcinoma cells. CONCLUSION: The present study identified andrographolide as a major antitumor principle in the methanolic extract of leaves of A. paniculata.

Modulation of MRP1 protein transport by plant, and synthetically modified flavonoids.

The influence of novel synthetic and plant origin flavonoids on activity of multidrug resistance-associated protein (MRP1) was investigated in human erythrocytes used as a cell model expressing MRP1 in plasma membrane. The fluorescent probe, BCPCF (2', 7'-bis-(3-carboxy-propyl)-5-(and-6)-carboxyfluorescein), was applied as a substrate for MRP1 multidrug resistance transporter. The effect of compounds belonging to different classes of natural flavonoids: flavone, flavonol, isoflavones and flavanolignan was compared with action of new synthetic derivatives of genistein. Most of the flavonoids showed strong or moderate ability to inhibit transport carried out by MRP1. Inhibitory properties of flavonoids were compared to the effects of indomethacin, probenecid and MK-571 known as MRP1 inhibitors. Studying the influence of new synthetic genistein derivatives on BCPCF transport we have found that the presence of hydrophobic groups substituting hydrogen of hydroxyl group at the position 4' in ring B of isoflavone is more important for inhibitory properties than hydrophobic substitution at the position 7 in ring A. In case of naturally occurring isoflavones the replacement of hydrogen at position 4' by hydrophobic ring structure seems also to be favourable for inhibition potency.