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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Rhinacanthin C Inhibits Osteoclast Differentiation and Bone Resorption: Roles of TRAF6/TAK1/MAPKs/NF-κB/NFATc1 Signaling.

Rhinacanthin C is a naphthoquinone ester with anti-inflammatory activity, found in Rhinacanthus nasutus (L) Kurz (Acanthaceae). We found that rhinacanthin C inhibited osteoclast differentiation stimulated by the receptor activator of nuclear factor-κB ligand (RANKL) in mouse bone marrow macrophage cultures, although the precise molecular mechanisms underlying this phenomenon are unclear. In this study, we investigated the inhibitory mechanisms of rhinacanthin C in osteoclastogenesis. Rhinacanthin C suppressed RANKL-induced nuclear factor of activated T cells c1 (NFATc1) expression. Phosphorylation of ERK, JNK, and NF-κB, but not p38, was inhibited by rhinacanthin C, which also inhibited RANKL-stimulated TRAF6-TAK1 complex formation. Thus, the anti-osteoclastogenic effect of rhinacanthin C is mediated by a cascade of inhibition of RANKL-induced TRAF6-TAK1 association followed by activation of MAPKs/NF-κB; this leads to suppression of c-Fos and NFATc1, which regulate transcription of genes associated with osteoclast differentiation. In vivo, rhinacanthin C also reduced RANKL-induced osteoclast formation and bone resorption in mouse calvaria. Rhinacanthin C also suppressed LPS-stimulated osteoclastogenesis and bone resorption in vitro and in vivo. Rhinacanthin C may provide a novel therapy for abnormal bone lysis that occurs during inflammatory bone resorption.

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.

Identification of the country of growth of Sophora flavescens using direct analysis in real time mass spectrometry (DART-MS).

In order to identify the country of growth of Sophora flavescens by chemical fingerprinting, extracts of plants grown in China and Japan were analyzed using direct analysis in real time mass spectrometry (DART)-MS. The peaks characteristic of each country of growth were statistically analyzed using a volcano plot to summarize the relationship between the p-values of a statistical test and the magnitude of the difference in the peak intensities of the samples in the groups. Peaks with ap value < 0.05 in the t-test and a ≥ 2 absolute difference were defined as characteristic. Peaks characteristic of Chinese S. flavescens were found at m/z 439 and 440. In contrast, peaks characteristic of Japanese S. flavescens were found at m/z 313, 423, 437 and 441. The intensity of the selected peaks was similar in Japanese samples, whereas the m/z 439 peak had a significantly higher intensity than the other peaks in Chinese samples. Therefore, differences in selected peak patterns may allow identification of the country of growth of S. flavescens.

Search of new cytotoxic crude materials against human oral squamous cell carcinoma using 1H NMR-based metabolomics.

BACKGROUND: The 5-year survival rate of the oral cancer patients has remained at approximately the 50% level during the past 30 years, possibly due to the poor tumor-selectivity of conventional anticancer drugs. This prompted us to search new plant extracts that have higher cytotoxicity against cancer cells than normal cells. MATERIALS AND METHODS: Two human oral squamous cell carcinoma cell lines (HSC-2 and HSC-4) and two normal oral cells (gingival and periodontal ligament fibroblasts; HGF and HPLF) were incubated for 48 h with various concentrations of crude plant extract and the viable cell number was determined by the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. The 50% cytotoxic concentration (CC50) was determined from the dose-response curve. Tumor-specificity (TS) was determined by the following equation: TS=mean CC50 (normal cells)/mean CC50 (cancer cell lines). Metabolic profiling techniques based on (1)H nuclear magnetic resonance (NMR) were applied to gain the chemical structural insight for cytotoxicity induction. RESULTS: Among 24 plant extracts, Camptotheca acuminate leaf, a well-known source for camptothecin, showed the highest TS value (88.3), followed by Vitis s.p.p. (>3.5), Sasa veitchii (>2.3) and Phellodendron amurense (>2.1), whereas other plant extracts showed much lower TS value (<2). These cytotoxic extracts made cluster on principal component analysis (PCA) score plot. CONCLUSION: The TS value determined by the present method seems to reflect the anti-tumor potential of each plant extract, while a part of the cytotoxic compounds present in these extracts may have common chemical structures.

Identification of a xanthine oxidase-inhibitory component from Sophora flavescens using NMR-based metabolomics.

We demonstrate that NMR-based metabolomics studies can be used to identify xanthine oxidase-inhibitory compounds in the diethyl ether soluble fraction prepared from a methanolic extract of Sophora flavescens. Loading plot analysis, accompanied by direct comparison of 1H NMR spectraexhibiting characteristic signals, identified compounds exhibiting inhibitory activity. NMR analysis indicated that these characteristic signals were attributed to flavanones such as sophoraflavanone G and kurarinone. Sophoraflavanone G showed inhibitory activity towards xanthine oxidase in an in vitro assay.

New biological activities of Rhinacanthins from the root of Rhinacanthus nasutus.

BACKGROUND: We recently reported that the ethyl acetate (EtOAc)-soluble fraction of the methanol extract of the root of Rhinacanthus nasutus showed tumor-specific non-apoptotic cytotoxicity and antiosteoclastogenic activity. In the present study, we investigated whether five rhinacanthins, mostly isolated from the EtOAc-soluble fraction of this plant, are responsible for these activities. MATERIALS AND METHODS: The cytotoxic activity was determined by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method. The 50% cytotoxic concentration (CC(50)) was determined by the dose-response curve. Tumor specificity (TS) was determined by the ratio of the mean CC(50) for normal cells to that of tumor cell lines. DNA fragmentation was assayed by agarose gel electrophoresis. Caspase-3 activation was monitored by substrate cleavage assay. Osteoclastogenesis was monitored by tartrate-resistant acid phosphatase (TRAP) activity in receptor activator of NF-κB ligand (RANKL)-stimulated bone marrow-derived macrophages. RESULTS: Among five rhinacanthins (rhinacanthin C, G, N and Q, and rhinacanthone), rhinacanthin C exhibited the highest tumor specificity (TS=15.2). Rhinacanthin C did not induce internucleosomal DNA fragmentation nor caspase-3 activation, suggesting non-apoptotic cell death. Rhinacanthin C most potently inhibited the RANKL-stimulated osteoclastogenesis. CONCLUSION: The present study suggests that rhinacanthin C may be responsible for the biological activity of the EtOAc-soluble fraction prepared from the methanolic extract of R. nasutus we previously reported on.

In search of new biological activities of isolates from Odontoglossum Harvengtense 'Tutu'.

BACKGROUND: In the current study, we isolated four known compounds, two phenanthrenes, 2,5-dihydroxy-4,9-dimethoxy phenanthrene [1] and 4-methoxyphenanthrene-2,7-diol (flavanthrinin) [2], one phenanthrenequinone, 5-hydroxy-2,3-dimethoxy-1,4-phenanthrenequinone [3], and one flavone, 3,5,7-trihydroxyflavone (galangin) [4], from the ethyl acetate (EtOAc) extract of Odontoglossum Harvengtense 'Tutu' through bioassay-guided fractionation, and investigated their biological activities. MATERIALS AND METHODS: The isolated compounds were identified with spectroscopic analysis and through comparison to literature values. Cytotoxic activity towards human tumor and normal cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. Nitric oxide (NO) was determined by the Griess method. Radical scavenging activity was determined by electron spin resonance (ESR) spectroscopy. Osteoclastogenesis was monitored by tartrate-resistant acid phosphatase (TRAP) activity. RESULTS: The compounds had slightly higher cytotoxicity towards human oral squamous cell carcinoma and leukemia cell lines as compared with human normal oral cells, yielding a tumor specificity value of 1.1-2.7. Among these four compounds, 1 most potently inhibited the lipopolysaccharide (LPS)-stimulated NO production and the receptor activator of nuclear factor-κB ligand (RANKL)-stimulated osteoclastogenesis by mouse macrophage-like RAW264.7 cells. Micromolar concentrations of 1 scavenged the NO radical produced from 1-hydroxy-2-oxo-3-(N-3-methyl-3-aminopropyl)-3-methyl-1-triazene. CONCLUSION: The present study demonstrated, for the first time, that 1 inhibited both macrophage activation and osteoclast differentiation, suggesting its possible anti-inflammatory action.

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.