Piyanuch Rojsanga

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 GF254 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 (r2 = 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.

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.

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 α7 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 [3H] 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 α7nAChR 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 α7nAChR, only one compound (NSC34352) demonstrated partial agonistic effect at low dose (10 µM), and two compounds (NSC36369 and NSC34352) were selective antagonists on α7nAchR with moderate potency. These hits serve as novel templates/scaffolds for development of more potent and specific in the AChR systems.

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.

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 Alzheimers 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.

Thunbergia laurifolia extract ameliorates cognitive and emotional deficits in olfactorectomized mice

Abstract 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 Alzheimers 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.

High performance liquid chromatography fingerprinting and chemometric analysis of antioxidant quality of Thunbergia laurifolia leaves

Abstract High performance liquid chromatography (HPLC) method was optimized using Design of Experiment (DOE) process and validated for quantitative analysis of caffeic acid and rosmarinic acid contents in extracts from Thunbergia laurifolia leaves collected from 10 different locations in Thailand. Antioxidant qualities of all extracts were evaluated by various chemometric analysis methods from the results of HPLC analysis both fingerprints and active component contents, in vitro antioxidant activities determined by 2,2-diphenyl-1-picrylhydrazyl scavenging assay and total phenolic and total flavonoid contents determined by Folin-Ciocaltue and aluminium chloride methods, respectively. It was found that HPLC fingerprints of T. laurifolia leaf extracts analyzed by similarity analysis and chemical pattern recognition had moderate similarity (r = 0.76) with the same 11 major peaks. Total phenolic contents calculated as both caffeic acid equivalent (CAE) and rosmarinic acid equivalent (RAE) showed the highest correlation to antioxidant effects of T. laurifolia leaf extracts (R2 > 0.9). This study is the first demonstration of the analytical method for simultaneous quantitative analysis of 2 active component contents in leaf decoction extract from T. laurifolia and the uses of chemometric methods to evaluate and compare the quality, especially the chemical and biological qualities of raw materials collected from different locations. Graphical Abstract

Anticancer Effects of Some Medicinal Thai Plants

Ethanolic extracts from thirty Thai edible plants collected from Sa Keao province, Thailand, were screened for in vitro antiproliferative effect on HCT-116 human colon cancer cell line using cell titer 96 aqueous one solution cell proliferation assay. It was found that leaf extract of Crateva adansnii, fruit and leaf extracts of Ardisia elliptica, shoot extract of Colocasia esculenta, leaf extract of Cratoxylum fomosum, and leaf extract of Millettia leucantha exhibited antiproliferative activities. The fruit extract of Ardisia elliptica showed the highest antiproliferative activity. Ethanolic extract of the stems from C. fenestratum and its dichloromethane and aqueous fractions showed antiproliferative activity to human colorectal cancer cells (HCT-116) determined by cell growth assay. Berberine, one of the major alkaloid in the stems of C. fenestratum, also promoted antiproliferative effect. Extracts from the leaves of three Azadirachta species in Thailand, A. indica, A. indica var. siamensis, and A. excelsa, were reported to promote in vitro antioxidant effects determined by various methods. Ten Russula mushroom collected from northeastern part of Thailand were tested for in vitro antioxidant activities using photochemiluminescence assay for both lipid-soluble and water-soluble antioxidant capacities. R. medullata extract exhibited the highest antioxidant effects in both lipid-soluble and water-soluble models.

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-β1-mediated production of MMPs, collagen-1, and α-SMA in hepatic stellate cells.

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.