Experimental design approach for the quantitative analysis of multicomponents by single marker and HPLC fingerprinting of Thunbergia laurifolia aqueous extract.

INTRODUCTION: Thunbergia laurifolia is used in traditional Thai medicine to reduce fever and treat mouth ulcers. However, the quantitative analysis of chemical markers has not yet been officially defined. OBJECTIVE: The objective of this study is to develop a high-performance liquid chromatography (HPLC) method using a design of experiment (DoE) for the quantitative analysis of multicomponents by single marker (QAMS) and fingerprinting of the T. laurifolia aqueous extract. MATERIALS AND METHODS: Critical variables were screened using a two-level fractional factorial design, followed by the optimization of the selected variables using a central composite design. The validated method was applied for quality assessment based on QAMS and fingerprinting of the extract. RESULTS: Optimum conditions of DoE for the analysis of caffeic acid, vicenin-2, and rosmarinic acid were determined. The relative correction factors for caffeic acid and vicenin-2 were calculated using rosmarinic acid as an internal reference standard, and their contents in 30 samples were determined. The differences between the external standard method (ESM) and QAMS were compared. No significant difference was observed in the quantitative determination, proving the consistency QAMS and ESM. HPLC fingerprints of T. laurifolia were established with 8 of 12 characteristic peaks that were structurally characterized using HPLC-diode array detection-electrospray ionization/tandem mass spectrometry. The similarity of the fingerprints in all samples was ≥0.74, and the pattern recognition of the characteristic peaks was satisfied. CONCLUSION: The proposed method efficiently detected multiple components of the T. laurifolia extract. Thus, the method is beneficial in providing references for enhancing the quality control of other herbal medicines.

Determination of Phytochemical Contents in Extracts from Different Growth Stages of Oroxylum indicum Fruits Using HPLC-DAD and QAMS Methods.

Flavones are major compounds found in several parts of Oroxylum indicum (O. indicum). The quantification of multiple components by one marker (QAMS) method and the high-performance liquid chromatography (HPLC) method were developed for the quantitative analysis of extracts from the young fruits, green mature fruits, dry pod coats and seeds of O. indicum. Oroxin A, oroxin B and chrysin-7-O-glucuronide were identified in the O. indicum extracts. Oroxylin A and 5-hydroxymethylfurfural were isolated and structurally identified from the pod coat and young fruit extracts, respectively. From the HPLC analysis of the seven major flavones in the extracts, baicalin was the major compound in all extracts investigated (0.4-11% w/w of the extract). All flavone contents were low in the young fruit extract (<1% w/w of the extract). The green mature fruit and dry pod coat extracts showed similar constituent compositions. They contained small amounts of baicalin and oroxylin A, which were found only in these two extracts. Oroxylin A could be used as a marker to indicate the maturity of O. indicum fruits, while 5-hydroxymethylfurfural could be used as a marker for the young fruits. Baicalin was found to be a suitable single marker to calculate the contents of all flavones in the O. indicum extracts.

Portable near-infrared and Raman spectroscopic devices as complementary tools for instantaneous quality control of turmeric powder.

INTRODUCTION: Process analytical technology (PAT) guidance is implemented in the quality assurance of phytocompounds to achieve the Industry 4.0 concept. Near-infrared (NIR) and Raman spectroscopies are feasible for rapid, reliable quantitative analysis through transparent packaging without removing the samples from their original containers. These instruments can serve PAT guidance. OBJECTIVE: This study aimed to develop online portable NIR and Raman spectroscopic methods for quantifying total curcuminoids in turmeric samples through a plastic bag. The method mimicked an in-line measurement mode in PAT compared with placing samples into a glass vessel (at-line mode). MATERIALS AND METHODS: Sixty-three curcuminoid standard-spiked samples were prepared. Then, 15 samples were randomly selected as fixed validation samples, and 40 of the 48 remaining samples were chosen as calibration set. The results obtained from the partial least square regression (PLSR) models constructed by using the spectra acquired from NIR and Raman were compared with the reference values from high-performance liquid chromatography (HPLC). RESULTS: The optimum PLSR model of at-line Raman was achieved with three latent variables and a root mean square error of prediction (RMSEP) of 0.46. Meanwhile, the PLSR model of at-line NIR with one latent variable offered an RMSEP of 0.43. For the in-line mode, PLSR models created from Raman and NIR spectra had one latent variable with RMSEP of 0.49 and 0.42, respectively. The R2 values for prediction were 0.88-0.92. CONCLUSION: The models established from the spectra from portable NIR and Raman spectroscopic devices with the appropriate spectral pretreatments allowed the determination of total curcuminoid contents through plastic bag.

HPLC quantitative analysis of protocatechuic acid contents in 11 Phellinus mushroom species collected in Thailand

Abstract Phellinus mushrooms have been traditionally used for various medicinal purposes. Protocatechuic acid, which was previously reported to be a component in some Phellinus mushrooms, has some pharmacological effects. This study aimed to validate a HPLC method for the quantitative analysis of the protocatechuic acid contents in the extracts from different Phellinus mushroom species collected in Thailand. HPLC was carried out using a C18 column and the gradient mobile phases of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. Method validation was performed to assure the linearity, accuracy, precision, limit of detection and limit of quantitation of the analytical method. The linearity range of protocatechuic acid was 1 - 10 µg/ml. The average recovery was 104.16%. The method was shown to be precise with the RSD of repeatability and intermediate precision at less than 3%. The protocatechuic contents in 11 Phellinus mushrooms were in the range of less than 0.0099 - 0.4121 %w/w of the extract. The developed HPLC method was reliable and suitable for the quantitative analysis of protocatechuic acid content in Phellinus mushrooms.

Flavone-Rich Fractions and Extracts from Oroxylum indicum and Their Antibacterial Activities against Clinically Isolated Zoonotic Bacteria and Free Radical Scavenging Effects.

Oroxylum indicum extracts from the seeds collected from Lampang and Pattani provinces in Thailand, and young fruits and flowers exhibited in vitro display antioxidant and antibacterial activities against clinically isolated zoonotic bacteria including Staphylococcus intermedius, Streptococcus suis, Pseudomonas aeruginosa, ß-hemolytic Escherichia coli and Staphylococcus aureus. The orange crystals and yellow precipitates were obtained from the preparation processes of the seed extracts. The orange-red crystals from the seeds collected from Lampang province exhibited strong in vitro 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging effects (EC50 value = 25.99 ± 3.30 µg/mL) and antibacterial effects on S. intermedius and ß-hemolytic E. coli while the yellow precipitate from the same source exhibited only antioxidant activity. Quantitative analysis of phytochemicals in O. indicum samples by spectrophotometric and HPLC techniques showed that they contained different amounts of total phenolic, total flavonoid and three major flavones; baicalin, baicalein and chrysin contents. Young fruit extract, which contained low amounts of flavone contents, still promoted antibacterial effects against the tested bacteria with IC50 values lower than 1 mg/mL and MIC values between 4 to 10 mg/mL in S. intermedius, S. aureus and S suis while higher IC50 and MIC values against P. aeruginosa and ß-hemolytic E. coli were found. From scanning electron microscopy, the extract of the young fruit of O. indicum promoted morphological changes in the bacterial cells by disrupting the bacterial cell walls, inducing leakage of the cellular content, and generating the abnormal accumulation of cells. The mechanism of action of the extract for this antibacterial effect may be the disruption of the cell membrane and abnormal cell aggregations. Regression analysis of the results suggests the correlation between total phenolic and total flavonoid contents and antioxidant and antibacterial effects. Baicalin was found to have a high correlation with an inhibitory effect against ß-hemolytic E. coli while three unidentified peaks, which could be flavones, showed high correlations with an inhibitory effect against S. intermedius, S. suis, P. aeruginosa and S. aureus.

Flavones Contents in Extracts from Oroxylum indicum Seeds and Plant Tissue Cultures.

Oroxylum indicum (L.) Benth. ex Kurz or Pheka, is a plant in the Bignoniaceae family with various traditional uses. The mature fruits promote anti-helminthic and stomachic effects, while the seeds have been used as a purgative and for the relief of tonsil pain. The young fruits are popularly consumed as vegetables, while the seeds are one of the components in traditional drink formulations. To develop new plant raw material sources, a plant tissue culture technique was used to generate plant tissue cultured samples from the seeds of O. indicum. Plant tissue cultured samples were collected from three different growth stages; 4 days, then at 3 and 9 weeks, and prepared as crude extracts by maceration with ethanol, along with the seed raw material sample. A high performance liquid chromatographic (HPLC) method was used for quantitative analysis of the contents of the three major flavones; baicalin, baicalein, and chrysin in the extracts from the seeds and plant tissue cultured samples of this plant. Baicalin was found in the highest amount among these three flavones in all extracts. The seed extract contained the highest baicalin content (24.24% w/w in the extract), followed by the shoot extract from tissue-cultured plant at week 3 (14.78% w/w of the extract). The amounts of chrysin in all O. indicum showed the same trend as the contents of baicalin, but the amounts were lower, while baicalein was accumulated at the lowest amount among three flavonoids and the amounts were quite stable in all O. indicum extracts. From the results, O. indicum seed and plant tissue cultured extracts have potential as sources of flavones, which could be further developed as health products in the future.

Chemical Standardization and Anti-Proliferative Activity of Ardisia elliptica Fruit against the HCT116 Human Colon Cancer Cell Line.

The present study is intended to carry out the chemical standardization and evaluation of the anti-proliferative activity of A. elliptica fruit extract. A. elliptica fruit powder was extracted with ethanol. The obtained extract was assessed for total phenolic content using the Folin-Ciocalteu method. Moreover, a simple, accurate, and precise reversed phase high-performance liquid chromatographic method was developed and validated to determine the embelin content of A. elliptica fruit extract. Then, the extract and embelin were investigated for their anti-proliferative effect against HCT-116 cells. Finally, the mechanisms of inhibition of the extract and embelin on the mRNA expression of pro-apoptotic genes Bad, Bax, and Caspase-8 and anti-apoptotic genes c-IAP1, Mcl-1, and XIAP were determined by real-time qRT-PCR. The phenolic content and embelin content of the extract were 5.20 ± 0.01 g of gallic acid equivalent per 100 g of dried fruit (g% GAE) and 5.57 ± 0.56 mg/g of extract, respectively. The extract and embelin showed strong anti-proliferative effects on HCT-116 cells with 50% inhibition concentration (IC50) values of 19.16 ± 1.09 µg/mL and 25.93 ± 1.75 µg/mL, respectively. The A. elliptica extract exhibited a significant increase in the mRNA level of Bad, Bax, and Caspase-8 and a significant decrease in c-IAP1, Mcl-1, and XIAP. Embelin showed a significant decrease in Mcl-1 and XIAP.

Inhibitory Effects on Clinical Isolated Bacteria and Simultaneous HPLC Quantitative Analysis of Flavone Contents in Extracts from Oroxylum indicum.

Oroxylum indicum is a medicinal plant in Thailand, which has been used as a tonic and for the treatment of various diseases. Extracts from various parts of O. indicum were reported as promoting in vitro antioxidant and antibacterial effects. Phytochemical analysis suggested that this plant contained some flavones. O. indicum fruit and seed water and ethanol extracts and their major flavonoids including baicalein, baicalin, and chrysin were tested for in vitro antibacterial activities on four clinical isolated bacteria, namely, Staphylococcus intermedius, Streptococcus suis, Pseudomonas aeruginosa, and ß-Escherichia coli, using a broth micro-dilution assay. The amounts of these three major flavonoids were also quantitatively analyzed using the high-performance liquid chromatographic (HPLC) method. O. indicum fruit ethanol extract from Nakhon Pathom province (OFNE) promoted the strongest antimicrobial activity against four clinical pathogenic bacteria, including S. intermedius (IC50 = 1.30 mg/mL), S. suis (13.59% inhibition at 7.81 mg/mL), P. aeruginosa (IC50 = 39.20 mg/mL), and ß-E. coli (IC50 = 66.85 mg/mL). Baicalin showed high in vitro antibacterial effect to all tested bacteria. From the optimized and validated HPLC method, baicalin, baicalein, and chrysin contents in O. indicum extracts were 0.19 ± 0.00 - 9.45 ± 0.13, 0.14 ± 0.00 - 1.27 ± 0.02, and 0.02 ± 0.00 - 0.96 ± 0.02 g/100 g extract, respectively. Baicalin was found to be the major compound in O. indicum seed extract followed by baicalein, whereas chrysin was found in lower amounts than the amounts of the other two flavonoids in all O. indicum extracts.

Comparative Phytochemical Profiling and In Vitro Antioxidant Activity of Extracts from Raw Materials, Tissue-Cultured Plants, and Callus of Oroxylum indicum (L.) Vent.

Extracts from raw materials from different plant parts, tissue-cultured plants, and callus cultures of Oroxylum indicum were analyzed for in vitro antioxidant activities determined by DPPH radical scavenging assay and evaluated for phytochemical profiles by TLC and LC-MS methods. The results were analyzed by principal component analysis (PCA) to evaluate the similarity. Stalk, pedicel, flower, seed, and whole fruit and callus extracts promoted strong antioxidant activity with high total phenolic and total flavonoid contents. The main phytochemicals found in extracts were baicalin, baicalein, and chrysin. Baicalein and baicalin promoted strong antioxidant effects and existed in most extracts while chrysin, which promoted very low antioxidant activity, was a major flavonoid in the leaves and tissue-cultured plants. From PCA analysis by total phenolic and total flavonoid contents, four main clusters including callus and tissue-cultured plant groups from different growth stages, flower group, and whole fruit and leaf group could be organized. When the results were analyzed by PCA using antioxidant activity with total phenolic or total flavonoid contents, all O. indicum samples could be grouped together except the extracts from the root of tissue-cultured plants which separated from the rest due to their low phytochemical contents and weak antioxidant activities.

Hypoglycaemic activity of Mathurameha, a Thai traditional herbal formula aqueous extract, and its effect on biochemical profiles of streptozotocin-nicotinamide-induced diabetic rats.

BACKGROUND: The Thai traditional herbal formula-Mathurameha, consisting of 26 medicinal plants, has been used as an alternative and complementary medicine for diabetes treatment in Wangnamyen Hospital, Thailand. To provide scientific evidences on the efficacy and safety of this herbal formula, in vivo hypoglycaemic activity, effect on serum biochemical profiles and acute toxicity were investigated. METHODS: Experimental type 2 diabetes was induced in male Sprague-Dawley rats by intraperitoneal injection of nicotinamide 15 min prior to intravenous injection of streptozotocin. The most effective extract from the oral glucose tolerant test (OGTT) was administered daily via the oral route to diabetic rats for 2 weeks. Two-hour postprandial plasma glucose (2h-PPG) levels were measured on days 0, 7, and 14. Biochemical data were measured at the end of daily oral administration experiment. RESULTS: Aqueous extract of the herbal formula was the most potent extract for improving glucose tolerance of streptozotocin-nicotinamide-induced diabetic rats after single oral administration. After 2 weeks of daily oral administration, the aqueous extract showed a dose-dependent glucose lowering effect. At doses of 12.5, 25, and 50 mg/kg, the 2h-PPG level of diabetic rats decreased by 3.32%, 15.78%, and 17.94%, respectively. Most of the biochemical profiles of diabetic rats were improved, including the total cholesterol (TC), alkaline phosphatase (ALP), total protein, albumin, globulin, creatinine, and uric acid levels. The significantly increased triglyceride (TG) level observed in treated diabetic rats indicated a lack of a beneficial effect of the extract on lipid homeostasis. Nevertheless, there were no signs or symptoms of acute toxicity observed after oral administration of aqueous extract (5 g/kg) to both male and female rats. CONCLUSIONS: The results revealed that the herbal formula aqueous extract has hypoglycaemic activity, beneficial effects on biochemical profiles and a lack of acute toxicity. This study confirms the efficacy and safety of the Mathurameha herbal formula used for treating type 2 diabetes mellitus.

Thunbergia laurifolia Exhibits Antifibrotic Effects in Human Hepatic Stellate Cells.

Leaves of Thunbergia laurifolia (TL) have been reported to have antioxidation, anti-inflammatory, detoxifying, and hepatoprotective effects. However, studies relating to antifibrotic activity have not been reported. Currently, there is no standard treatment for hepatic fibrosis. This study aimed to investigate the antifibrotic activity of TL in human hepatic stellate LX-2 cells. Results from cell viability and cell death assays showed that the extract at high concentrations was toxic to LX-2 cells. TL extract reversed the transformation of LX-2 cells to myofibroblast-like characteristics in response to stimulation by transforming growth factor-beta 1. This action may be associated with the effect of TL in suppressing α-SMA and collagen-I production observed by immunofluorescence study and western blot analysis. Additionally, TL extract significantly decreased MMP-9 activity which is consistent with the reduction of MMP-9, MMP-2, and TIMP-1 gene expression. The effect of TL in suppressing fibrosis may be associated with its ability to inhibit the activation of p38 MAPK and Erk1/2 kinases as examined by western blot analysis. Our study provides convincing evidence that TL possesses antifibrotic activity which may be through the suppression of TGF-ß1-mediated production of MMPs, collagen-1, and α-SMA in hepatic stellate cells.

Screening for Antibacterial and Antioxidant Activities and Phytochemical Analysis of Oroxylum indicum Fruit Extracts.

Oroxylum indicum, which is called Pheka in Thai, is a traditional Thai plant in the Bignoniaceae family with various ethnomedical uses such as as an astringent, an anti-inflammatory agent, an anti-bronchitic agent, an anti-helminthic agent and an anti-microbial agent. The young fruits of this plant have also been consumed as vegetables. However, there has been no report concerning its antibacterial activities, especially activities related to clinically isolated pathogenic bacteria and the in vitro antioxidant effects of this plant. Therefore, the extracts from O. indicum fruits and seeds collected from different provinces in Thailand were prepared by decoction and maceration with ethanol and determined for their in vitro antibacterial effects on two clinically isolated bacteria, Streptococcus suis and Staphylococcus intermedius, using disc diffusion assay. Ethanol extracts from O. indicum fruits collected from Nakorn Pathom province at the concentration of 1000 mg/mL exhibited intermediate antibacterial activity against S. intermedius with an inhibition zone of 15.11 mm. Moreover, it promoted moderate inhibitory effects on S. suis with an inhibition zone of 14.39 mm. The extracts prepared by maceration with ethanol promoted higher antibacterial activities than those prepared with water. The ethanol extract from the seeds of this plant, purchased in Bangkok, showed stronger in vitro antioxidant activities than the other extracts, with an EC50 value of 26.33 µg/mL. Phytochemical analysis suggested that the seed ethanol extract contained the highest total phenolic and flavonoid contents (10.66 g% gallic acid equivalent and 7.16 g% quercetin equivalent, respectively) by a significant amount. Thin layer chromatographic analysis of the extracts showed the chromatographic band that could correspond to a flavonoid baicalein. From the results, extracts from O. indicum fruits have an in vitro antioxidant effect, with antibacterial potential, on clinically pathologic bacteria and they contain an antioxidant flavonoid which could be developed for medicinal and pharmaceutical purposes in the future.

Development and Validation of LC-MS/MS Method for Quantitative Determination of Adenosine, Guanosine, Xanthine and Uric acid in Widely Consumed Vegetables in Thailand.

In this study, a triple quadrupole LC-MS/MS electrospray ionization method was developed and validated for quantitative determination of adenosine, guanosine, xanthine and uric acid in fifteen widely consumed Thai vegetables. The method was successively developed by using caffeine as internal standard. The lower limit of quantitation (LLOQ) was 0.2 µg/g for adenosine and guanosine, and 1.0 µg/g for uric acid and xanthine. The method was fully validated according to USFDA guidelines and all performance characteristics were found acceptable. Subsequently, the developed and validated LC-MS/MS method was applied to determine the four interest substances in fifteen widely consumed vegetables in Thailand. The results showed that all vegetables included in the study could be classified as low adenosine, guanosine, xanthine and uric acid containing foods since the concentrations of these substances were less than 50 mg per 100 g. This finding was enormously valuable information for hyperuricemia and gouty patients.

Antioxidant Activity and Antibacterial Effects on Clinical Isolated Streptococcus suis and Staphylococcus intermedius of Extracts from Several Parts of Cladogynos orientalis and Their Phytochemical Screenings.

The in vitro antioxidant and antibacterial assays against clinically isolated Streptococcus suis and Staphylococcus intermedius of the extracts prepared by decoction and ethanolic reflux of different parts of Chettaphangki (Cladogynos orientalis Zipp. ex Span), including the leaves, roots, and stems, using 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging assay and disc diffusion method were conducted. Quantitative analysis of total phenolic and total flavonoid contents in the extracts using spectrophotometric methods was also performed. Finally, phytochemical screening by thin layer chromatography (TLC) and high performance liquid chromatography (HPLC) was conducted. Leaf ethanolic reflux extract (100 g) contained the highest total phenolic and total flavonoid contents of 7.21 ± 0.28 µg gallic acid equivalent (GAE) and 11.51 ± 2.02 µg rutin equivalent (RE), respectively. Chettaphangki extracts promoted low antioxidant activity with EC50 values in the range of 0.27-0.48 mg/mL. Extracts and fractions from the roots and stems of this plant promoted low to intermediate antibacterial activity against S. intermedius with the inhibition zones between 7 and 14 mm. The chromatographic data suggested that the leaf extracts of C. orientalis contained rutin while the root and stem extracts contained scopoletin and chettaphanin I. Rutin promoted strong antioxidant activity while chettaphanin I showed low antibacterial activity against Staphylococcus intermedius.

Thunbergia laurifolia extract ameliorates cognitive and emotional deficits in olfactorectomized mice.

CONTEXT: Thunbergia laurifolia Lindl. (Acanthaceae) is a Thai medicinal plant used for the detoxification of poison which is likely to be beneficial for the treatment of cognitive deficits including Alzheimer's disease. OBJECTIVE: To elucidate the effects of Thunbergia laurifolia leaf extract (TLL) on cognitive dysfunction and depression-like behavior in olfactory bulbectomized mice (OBX). MATERIALS AND METHODS: OBX mice were treated daily with TLL at the dose of 250 and 500 mg/kg, tacrine, and imipramine, on the day after 10 d of OBX operation. The effects of TLL on cognitive and depression-like behavior of the animals were analyzed. After completing behavioral experiments, the expression levels of cholinergic marker genes encoding ChAT and muscarinic M1 receptor were quantitatively analyzed. RESULTS: TLL and tacrine reduced OBX-induced cognitive deficits in the object recognition test (ORT) with the time spent for the novel object two times longer than that of the familiar object. Moreover, TLL at the dose of 500 mg/kg and imipramine ameliorated depression-like behavior in the tail suspension test (TST) by reducing the duration of immobility from 25.18% to 3.16% and from 25.18% to 6.48%, respectively. TLL at the dose of 250 and 500 mg/kg reversed the OBX-induced down-regulation of ChAT mRNA expression in the hippocampus from 0.12 to 0.17 and 0.24, respectively, while the down-regulation of mRNA expression of muscarinic M1 receptor was also reversed by TLL from 0.23 to 0.38 and 0.48, respectively. CONCLUSIONS: TLL ameliorates non-spatial short-term memory deficits in OBX mice, and has the potential to exhibit an antidepressant-like action.

Ameliorative Effects of Acanthopanax trifoliatus on Cognitive and Emotional Deficits in Olfactory Bulbectomized Mice: An Animal Model of Depression and Cognitive Deficits.

Acanthopanax trifoliatus is a plant that has been traditionally used in Thailand as a vegetable and a tonic. This study investigated effects of the aqueous extract of its leaves (ATL) on cognitive and emotional deficits using an olfactory bulbectomized mouse (OBX) model. OBX mice were treated daily with ATL (250 and 500 mg/kg, p.o.) 3 days after OBX. Antidementia drug tacrine (2.5 mg/kg/day) and antidepressant drug imipramine (10 mg/kg/day) were given i.p. as reference drugs. OBX significantly impaired cognitive behavior in a novel object recognition test and a modified Y-maze test and induced depression-like behavior in a tail suspension test. ATL and tacrine treatment attenuated OBX-induced cognitive deficits, whereas ATL and imipramine improved OBX-induced depression-like behavior. Neurochemical studies conducted after completing behavioral experiments demonstrated that OBX downregulated the expression levels of cholinergic marker genes encoding choline acetyltransferase and muscarinic M1 receptor in a manner reversed by ATL and tacrine. Moreover, ATL and tacrine administration inhibited the ex vivo activity of acetylcholinesterase in the brain. These findings suggest that ATL is beneficial for the treatment of cognitive and emotional deficits related to dementia with depressive symptoms and that the antidementia effect of ATL is mediated by normalizing the function of central cholinergic systems.

The effect of crebanine on memory and cognition impairment via the alpha-7 nicotinic acetylcholine receptor.

AIMS: The aims of the present study were to investigate the effect of crebanine on memory and cognition impairment in mice and to elucidate the underlying molecular mechanisms. MAIN METHODS: The memory-enhancing effects of crebanine were assessed with a water maze test using scopolamine-induced amnesic mice. The molecular mechanism was explored in silico by docking crebanine against acetylcholine binding proteins (AChBPs) and in vitro with a radioligand competition assay using (±)-[(3)H]-epibatidine. The pharmacological behavior was assessed by observing changes to the functional activity of α7-nAChRs expressed in Xenopus oocytes and by fluorescent assays on recombinant ligand gated ion channel (LGIC) receptors expressed in mammalian cells. KEY FINDINGS: The administration of crebanine significantly improved the cognitive deficits induced by scopolamine, as measured by the water maze test. The docking results demonstrated that crebanine bound to the active binding site of the AChBP template with a good docking energy. Crebanine significantly inhibited the binding of (±)-[(3)H]-epibatidine to AChBPs with K(i) values of 179 nM and 538 nM for Ls and Ac, respectively. Further functional assays performed using two separate protocols indicated that crebanine is an antagonist of the α7-nAChR with an IC(50) of 19.1µM. SIGNIFICANCE: The observed actions of crebanine against amnesia and its effect on α7-nAChRs will be beneficial for target-based drug design; crebanine or its scaffold can be used as the starting point to develop a drug for Alzheimer's disease. The cognition-enhancing effects of crebanine and the underlying mechanism based on α7-nAChRs are consistent with its traditional use. These findings demonstrate the potential utility of crebanine in the development of neurodegenerative therapy.

Virtual screening against acetylcholine binding protein.

The nicotinic acetylcholine receptors (nAChRs) are a member of the ligand-gated ion channel family and play a key role in the transfer of information across neurological networks. The X-ray crystal structure of agonist-bound α(7) acetylcholine binding protein (AChBP) has been recognized as the most appropriate template to model the ligand-binding domain of nAChR for studying the molecular mechanism of the receptor-ligand interactions. Virtual screening of the National Cancer Institute diversity set, a library of 1990 compounds with nonredundant pharmacophore profiles, using AutoDock against AChBPs revealed 51 potential candidates. In vitro radioligand competition assays using [(3)H] epibatidine against the AChBPs from the freshwater snails, Lymnaea stagnalis, and from the marine species, Aplysia californica and the mutant (AcY55W), revealed seven compounds from the list of candidates that had micromolar to nanomolar affinities for the AChBPs. Further investigation on α(7)nAChR expressing in Xenopus oocytes and on the recombinant receptors with fluorescence resonance energy transfer (FRET)-based calcium sensor expressing in HEK cells showed that seven compounds were antagonists of α(7)nAChR, only one compound (NSC34352) demonstrated partial agonistic effect at low dose (10 µM), and two compounds (NSC36369 and NSC34352) were selective antagonists on α(7)nAchR with moderate potency. These hits serve as novel templates/scaffolds for development of more potent and specific in the AChR systems.

In vitro anti-proliferative activity of alcoholic stem extract of Coscinium fenestratum in human colorectal cancer cells.

Coscinium fenestratum (Gaertn.) Colebr. is traditionally used for the treatment of cancer, arthritis and diabetes mellitus. The purpose of this study was to determine the molecular mechanisms by which this plant shows beneficial effects. An 80% ethanolic extract of C. fenestratum (80ET) was separated by its polarity into dichloromethane (DCM) and aqueous fractions (WF), and the anti-proliferative effects of 80ET, DCM and WF were investigated. Berberine, one of the major components of C. fenestratum, was used as a control. The 80ET, DCM, WF and berberine showed anti-proliferative activity as assessed by cell growth assay. Subsequently, the pro-apoptotic proteins NAG-1 and ATF3 were increased and the cell cycle protein cyclin D1 was decreased by the extract and its fractions. Interestingly, only the DCM fraction exhibited the induction of peroxisome proliferator-activated receptor γ (PPARγ) binding activity, which represents a pro-apoptotic activity in colorectal cancer cells. The overall results of this study indicate that the extract from this plant has anti-proliferative activity through the activation of pro-apoptotic proteins and PPARγ, and may have potential as a preventive regimen in the treatment of cancer.

Determination of berberine content in the stem extracts of Coscinium fenestratum by TLC densitometry.

OBJECTIVE: To develop the optimal extraction procedure (i.e. maceration, percolation or Soxhlet extraction) and thin- layer chromatographic (TLC)-densitometric method for the determination of berberine content of Coscinium fenestratum. MATERIALS AND METHODS: Maceration, percolation and Soxhlet extraction techniques were used to extract alkaloids from dried stems of C. fenestratum. The solvents used were 50 and 80% ethanol. Crude extracts and berberine content recovered from the TLC fingerprint were evaluated for chemical components of each extraction method. Precoated silica gel GF(254) plates were used as stationary phase while butanol:glacial acetic acid:water (14:3:4) was used as a mobile phase. Detection and quantitation of berberine were performed by densitometry at the wavelength of 415 nm over the linearity range of 240-840 ng (r(2) = 0.9982). The relative standard deviations from intraday and interday precisions were less than 4.13%. RESULTS: The recovery of standard berberine was 97.58-98.71% (%RSD = 3.85), and the limit of detection and quantitation were 25 and 50 ng/spot, respectively. Eighty percent ethanol gave a higher content of berberine than 50% ethanol. Berberine contents from maceration, percolation and Soxhlet extraction with 80% ethanol were 3.37+/- 0.30, 3.08+/- 0.38 and 2.67+/- 0.27% w/w, respectively. CONCLUSION: The TLC-densitometric method was simple, accurate and precise for quantitating berberine in the stem extract of C. fenestratum. Maceration with 80% ethanol gave the highest content of berberine in the extract. TLC of the extracts from different methods showed a similar pattern.