Watoo Phrompittayarat

Neuroprotective effect of Bacopa monnieri on beta-amyloid-induced cell death in primary cortical culture.

AIM OF THE STUDY Bacopa monnieri (Brahmi) is extensively used in traditional Indian medicine as a nerve tonic and thought to improve memory. To examine the neuroprotective effects of Brahmi extract, we tested its protection against the beta-amyloid protein (25-35) and glutamate-induced neurotoxicity in primary cortical cultured neurons. MATERIALS AND METHODS Neuroprotective effects were determined by measuring neuronal cell viability following beta-amyloid and glutamate treatment with and without Brahmi extract. Mechanisms of neuroprotection were evaluated by monitoring cellular oxidative stress and acetylcholinesterase activity. RESULTS Our result demonstrated that Brahmi extract protected neurons from beta-amyloid-induced cell death, but not glutamate-induced excitotoxicity. This neuroprotection was possibly due to its ability to suppress cellular acetylcholinesterase activity but not the inhibition of glutamate-mediated toxicity. In addition, culture medium containing Brahmi extract appeared to promote cell survival compared to neuronal cells growing in regular culture medium. Further study showed that Brahmi-treated neurons expressed lower level of reactive oxygen species suggesting that Brahmi restrained intracellular oxidative stress which in turn prolonged the lifespan of the culture neurons. Brahmi extract also exhibited both reducing and lipid peroxidation inhibitory activities. CONCLUSIONS From this study, the mode of action of neuroprotective effects of Brahmi appeared to be the results of its antioxidant to suppress neuronal oxidative stress and the acetylcholinesterase inhibitory activities. Therefore, treating patients with Brahmi extract may be an alternative direction for ameliorating neurodegenerative disorders associated with the overwhelming oxidative stress as well as Alzheimers disease.

Stability studies of saponins in Bacopa monnieri dried ethanolic extracts.

Bacopa monnieri (L.) Wettst. (Brahmi) is currently used as a drug and food supplement for memory improvement. However, studies on the physical and chemical stability of the extract components, especially on the lead compound important for pre-formulation, have not yet been reported. In this study, the stabilities of the crude extract and the diluted crude extract were investigated at various temperatures using saponin glycosides, bacopaside I and bacoside A3 as markers for quantitative analysis. The stability testing of bacopaside I and bacoside A3 standard solution was performed at various temperatures and pH values. The quantity of both compounds under all conditions was analyzed using HPLC techniques. The moisture adsorption of the crude extract was determined at 5, 40, 60 and 80 degrees C at 75 % relative humidity using gravimetric methods. The results revealed that the crude extract quickly adsorbed moisture up to 54 % w/w at both 40 and 80 degrees C, while it only slowly adsorbed moisture at 5 degrees C. The amounts of intact bacopaside I and bacoside A3 in the crude extract decreased drastically at 80 degrees C, slowly at 40 and 60 degrees C, and remained unchanged at 5 degrees C during the period of investigation. Moreover, the amount of both compounds in the standard solution dropped sharply at a pH of 1.2 but slowly at pH 6.8 and 9.0, respectively. The pre-formulation data could be further used for improvement of the final product quality.

Improvement of Pseudojujubogenin Glycosides Production from Regenerated Bacopa monnieri (L.) Wettst. and Enhanced Yield by Elicitors

Abstract Bacopa monnieri (L.) Wettst. was studied for shoot induction and regeneration on Murashige and Skoog (MS) medium supplemented with different plant growth regulators. Stem explants cultured on medium containing 0.1 mg/l thidiazuron (TDZ) resulted in the highest number of shoots (117 shoots/explant). Regenerated plants from medium with 0.5 mg/l TDZ contained the highest level of pseudojujubogenin glycosides [(30.62 d 1.29) mg/g dry wt] which was 2-fold higher than that of in vitro grown plants of the same age [(16.96 d 1.49) mg/g dry wt]. Plantlets regenerated from 0.1 mg/l TDZ also showed a high level of pseudojujubogenin glycosides [(27.94 d 1.19) mg/g dry wt]. The effect of elicitor on pseudojujubogenin glycosides accumulation in B. monnieri whole plant cultures was investigated. Chitosan at 150 mg/l and yeast extract at 2 mg/ml increased the pseudojujubogenin glycosides production [(40.83 d 2.24) mg/g dry wt and (40.05 d 2.37) mg/g dry wt, respectively] after 7 days, which was 6-fold higher than in the control cultures.

Immunochromatographic assay for the detection of pseudojujubogenin glycosides.

INTRODUCTION Bacopa monnieri contains pseudojujubogenin glycosides as pharmacologically active compounds. In order to screen large numbers of plant samples for the presence of pseudojujubogenin glycosides, a rapid and simple assay system is required for application to small quantities of test materials. Immunoassays using monoclonal antibodies could be useful for the determination of small quantities of pseudojujubogenin glycosides in plant extracts. OBJECTIVE The objective of this work was to develop a simple method for the detection of pseudojujubogenin glycosides by the immunochromatographic strip test using anti-bacopaside I monoclonal antibody. METHODOLOGY The qualitative assay was based on a competitive immunoassay in which the detector reagent consisted of a colloidal gold particle coated with the respective anti-bacopaside I MAb. The capture reagent was a bacopaside I-human serum albumin conjugate immobilised onto a test strip membrane. RESULTS The sample containing pseudojujubogenin glycosides and the detection reagent were incubated with the immobilised capture reagent. The glycosides in the sample competed in binding to the limited amount of antibodies in the detection reagent with the immobilised bacopaside I-HSA conjugates and, hence, positive samples showed no colour in the capture spot zone. The detection limit for the strip test was 125 ng/mL. CONCLUSION The assay system was found to be useful as a rapid and simple screening method for the detection of pseudojujubogenin glycosides in plants.

Thermal, mechanical and drug release characteristics of an acrylic film using active pharmaceutical ingredient as non-traditional plasticizer

Abstract The objective of this study was to investigate thermal and mechanical properties as well as in vitro drug release of Eudragit® RL (ERL) film using chlorpheniramine maleate (CPM) as either active pharmaceutical ingredient or non-traditional plasticizer. Differential scanning calorimeter was used to measure the glass transition temperature (Tg) of 0–100% w/w CPM in ERL physical mixture. Instron testing machine was used to investigate Young’s modulus, tensile stress and tensile strain (%) of ERL film containing 20–60% w/w CPM. Finally, a Franz diffusion cell was used to study drug release from ERL films obtained from four formulations, i.e. CRHP0/0, CRHP0/5, CRHP2/0 and CRHP2/5. The Tg of ERL was decreased when the weight percentage of CPM increased. The reduction of the Tg could be described by Kwei equation, indicating the interaction between CPM and ERL. Modulus and tensile stress decreased whereas tensile strain (%) increased when weight percentage of CPM increased. The change of mechanical properties was associated with the reduction of the Tg when weight percentage of CPM increased. ERL films obtained from four formulations could release the drug in no less than 10 h. Cumulative amount of drug release per unit area of ERL film containing only CPM (CRHP0/0) was lower than those obtained from the formulations containing traditional plasticizer (CRHP0/5), surfactant (CRHP2/0) or both of them (CRHP2/5). The increase of drug release was a result of the increase of drug permeability through ERL film and drug solubility based on traditional plasticizer and surfactant, respectively.

Immunochromatographic determination of bacopaside I in biological samples

We describe a novel immunochromatographic method for qualitative and quantitative analyses of bacopaside I, a bioactive constituent in Bacopa monnieri (L.) Wettst in biological samples. The assay was performed on polyethersulfone membrane using a polyclonal antibody raised against bacopaside I. The finalised method could quantitatively determine bacopaside I in the range of 31.3-1000.0ng and the detection and quantification limits were 1.0 and 31.3ng, respectively. The percentage recoveries of bacopaside I in blood and urine were nearly 100% indicating the accuracy of the extraction. The method was then applied for the determination of this compound in rat serum, urine and feces after an oral dose of 15mg/kg body weight. About 4% of the ingested dose of bacopaside I was detected in rat feces but none was detected in serum and urine which accorded with results from liquid chromatography tandem mass spectrometry. The accuracy, selectivity, sensitivity of the method are appropriate for in vivo pharmacokinetic studies.

Brahmi, a Medicinal Plant for Memory Improvement

Faculty of Pharmaceutical Sciences and Center of Excellence for Innovation in Chemistry, Naresuan University, Phitsanulok 65000, Thailand; Department of Physiology, Faculty of Medical Sciences, Naresuan University, Phitsanulok 65000, Thailand; Department of Physiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand; Department of Preclinical Science, Faculty of Medicine, Thammasat University, Pathumthani. 12120, Thailand; Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand; Department of Pharmacognosy and Pharmaceutical Chemistry, Faculty of Pharmacy, Srinakharinwiroj University, 26120 Nakhonayok, Thailand; Department of Medicinal Plant Breeding, Graduate School of Pharmaceutical Sciences, Kyushu University.Fukuoka 812-8582, Japan; E-mail: k_ingkaninan@yahoo.com

Survey of Acetylcholinesterase Inhibitory Activity in Essential Oils From Aromatic Plants

Essential oils have the potency to inhibit acetylcholinesterase (AChE). In our study, 29 essential oils from aromatic plants were screened for cholinesterase inhibitory activity using Ellman’s colorimetric method. Results revealed that the essential oils from Eucalyptus, Cajuput, Sweet magoram, Camphor tree and Rosemarry showed such activity. The compositions of these essential oils were analyzed by GC-MS. We also demonstrated that the AChE inhibitory activity of the essential oil could be attributed to 1,8-cineole.