NMR-based stability evaluation of (E)-1-(3',4'-dimethoxyphenyl)butadiene (DMPBD) from Zingiber cassumunar Roxb. rhizome.

INTRODUCTION: The active compound (E)-1-(3',4'-dimethoxyphenyl)butadiene (DMPBD) isolated from the rhizomes of Zingiber cassumunar Roxb. has potent anti-inflammatory and anticancer activities. Although DMPBD is one of the promising drug candidates for phytomedicine, its limited stability impedes its widespread use. For the development of new drugs, the assessment of their chemical stability is essential, ensuring they maintain their properties within specified limits throughout the period from production until use. OBJECTIVE: In the present study, we aimed to evaluate the stability of DMPBD under various conditions, including different solvents, temperatures, and lighting conditions, to identify the factors affecting stability and optimize the storage and handling conditions. METHODOLOGY: DMPBD samples subjected to the different conditions tested were monitored by quantitative 1H NMR (qHNMR), using an internal standard for the determination of the absolute quantity of DMPBD as a function of time and the changes thereof within 1 month. RESULTS: Significant decomposition of DMPBD was observed in chloroform-d1, whereas its content remained constant in methanol-d4. The content of DMPBD was maintained upon storage at temperatures below 4°C, both as methanolic solution and in the crude extract. Exposure to light had a slight negative impact on its contents. Some degradation products could be identified as resulting from O2-induced cleavage of the diene moiety. CONCLUSIONS: For pharmacological/therapeutic applications, DMPBD should be stored in the form of the crude extract or as a purified material in methanolic solution. Ideally, the storage temperature should be below 4°C and O2 should be excluded.

Computational Analysis and Biological Activities of Oxyresveratrol Analogues, the Putative Cyclooxygenase-2 Inhibitors.

Polyphenols are a large family of naturally occurring phytochemicals. Herein, oxyresveratrol was isolated from ethanolic crude extracts of Artocarpus lacucha Buch.-Ham., and chemically modified to derive its lipophilic analogues. Biological screening assays showed their inhibitory potency against cyclooxygenase-2 (COX-2) with very low cytotoxicity to the MRC-5 normal cell lines. At the catalytic site of COX-2, docking protocols with ChemPLP, GoldScore and AutoDock scoring functions were carried out to reveal hydrogen bonding interactions with key polar contacts and hydrophobic pi-interactions. For more accurate binding energetics, COX-2/ligand complexes at the binding region were computed in vacuo and implicit aqueous solvation using M06-2X density functional with 6-31G+(d,p) basis set. Our computational results confirmed that dihydrooxyresveratrol (4) is the putative inhibitor of human COX-2 with the highest inhibitory activity (IC50 of 11.50 ± 1.54 µM) among studied non-fluorinated analogues for further lead optimization. Selective substitution of fluorine provides a stronger binding affinity; however, lowering the cytotoxicity of a fluorinated analogue to a normal cell is challenging. The consensus among biological activities, ChemPLP docking score and the binding energies computed at the quantum mechanical level is obviously helpful for identification of oxyresveratrol analogues as a putative anti-inflammatory agent.

Yanangdaengin, a dihydrochalcone glucoside galloyl ester as active antioxidative agent from leaves of Lysiphyllum strychnifolium (syn. Bauhinia strychnifolia).

Objective: To isolate and identify the major bioactive components from the leaves of Lysiphyllum strychnifolium, an indigenous herb used in traditional Thai medicine for detoxification, longevity, and some other health related issues. Methods: Comparative HPLC analyses of the crude extracts from three provenances were carried out for an overview of characteristic compound profiles. Isolation of the major compounds was undertaken with chromatographic methods. Chemical structures were elucidated by NMR spectroscopic techniques and mass spectrometry. DPPH scavenging assay was carried out to determine the free radical scavenging activity of isolated compounds. Results: Yanangdaengin (3), a dihydrochalcone glucoside galloyl ester, has been isolated together with its corresponding dihydrochalcone glucoside trilobatin (2) as major compounds from the leaves of L. strychnifolium. Additionally, gallic acid (1) was co-chromatographically identified. Free radical scavenging activity of isolated compounds were determined. Compound 3 exhibited higher free radical scavenging activities in comparison to Trolox and quercetin. Conclusion: The isolated compounds could be used as chemical markers for quality assessment. The present work could promote the quality control and herbal medicinal product development of this plant.

Quantitative 1 HNMR spectroscopy for the determination of oxyresveratrol in Artocarpus lacucha heartwood.

INTRODUCTION: Quantitative nuclear magnetic resonance (qNMR) spectroscopy is an analytical method based on the principles of NMR spectroscopy. The main advantages of this method are its simplicity, time efficiency, high accuracy and reproducibility, and it is a non-destructive technique. OBJECTIVE: To evaluate and standardise the quality of Artocarpus lacucha heartwood. A method for quantifying its oxyresveratrol content using qNMR was developed. METHODOLOGY: Proton (1 H)NMR (400 MHz) spectroscopy was used to analyse the methanol-d4 solution of a given amount of crude extract of A. lacucha heartwood using ethyl p-methoxycinnamate (EPMC) as an internal standard. The qNMR methodology was validated in terms of its linearity and range, limit of quantification (LOQ), stability, precision, and accuracy for the determination of the oxyresveratrol content. RESULTS: The qNMR method was validated in terms of its linearity, range, LOQ, accuracy, precision, and stability. The quantitative determination of the oxyresveratrol content in the methanolic crude extract of A. lacucha was found to be 17% based on 1 HNMR analysis, which proved to be a reliable method as the results were comparable to those obtained by high-performance liquid chromatography (HPLC) analysis. CONCLUSIONS: This study validated qNMR spectroscopy as a reliable analytical procedure to determine oxyresveratrol in A. lacucha heartwood. Therefore, this qNMR method can serve as an alternative to the classical HPLC methods for evaluating and standardising the quality of A. lacucha heartwood.

Simultaneous HPLC analysis of crebanine, dicentrine, stephanine and tetrahydropalmatine in Stephania venosa

ABSTRACT Stephania venosa (Blume) Spreng., Menispermaceae, has been traditionally used as tonic drug and treatment of various diseases in South East Asian countries. In order to evaluate the quality and standardization of S. venosa roots, the HPLC method for quantification of the content of major components in S. venosa was developed and validated. The chromatographic separation was performed on a Hypersil BDS C18 column using gradient system of 100 mM ammonium acetate in water and methanol with flow rate 1 ml/min. Detection wavelength was set at 210 nm for tetrahydropalmatine, 280 nm for dicentrine and crebanine, and 270 nm for stephanine. The validated method showed good sensitivity, linearity, precision, and accuracy. The suitable solvent that yielded highest alkaloids contents from the matrix was optimized. S. venosa samples collected from various locations were analyzed. The present study provided comprehensive overview of major components in S. venosa. A remarkable variation in the accumulation of alkaloids in each population and the between individual in the same population could be observed. Our results showed the heterogeneity of S. venosa in Thailand which would need a further study for species delimitations.

Screening, Molecular Cloning, and Biochemical Characterization of an Alcohol Dehydrogenase from Pichia pastoris Useful for the Kinetic Resolution of a Racemic ß-Hydroxy-ß-trifluoromethyl Ketone.

The stereoselective synthesis of chiral 1,3-diols with the aid of biocatalysts is an attractive tool in organic chemistry. Besides the reduction of diketones, an alternative approach consists of the stereoselective reduction of ß-hydroxy ketones (aldols). Thus, we screened for an alcohol dehydrogenase (ADH) that would selectively reduce a ß-hydroxy-ß-trifluoromethyl ketone. One potential starting material for this process is readily available by aldol addition of acetone to 2,2,2-trifluoroacetophenone. Over 200 strains were screened, and only a few yeast strains showed stereoselective reduction activities. The enzyme responsible for the reduction of the ß-hydroxy-ß-trifluoromethyl ketone was identified after purification and subsequent MALDI-TOF mass spectrometric analysis. As a result, a new NADP(+) -dependent ADH from Pichia pastoris (PPADH) was identified and confirmed to be capable of stereospecific and diastereoselective reduction of the ß-hydroxy-ß-trifluoromethyl ketone to its corresponding 1,3-diol. The gene encoding PPADH was cloned and heterologously expressed in Escherichia coli BL21(DE3). To determine the influence of an N- or C-terminal His-tag fusion, three different recombinant plasmids were constructed. Interestingly, the variant with the N-terminal His-tag showed the highest activity; consequently, this variant was purified and characterized. Kinetic parameters and the dependency of activity on pH and temperature were determined. PPADH shows a substrate preference for the reduction of linear and branched aliphatic aldehydes. Surprisingly, the enzyme shows no comparable activity towards ketones other than the ß-hydroxy-ß-trifluoromethyl ketone.

Acetylcholinesterase Inhibitory Activity of Alkaloids Isolated from Stephania venosa.

Stephania venosa (Blume) Spreng or "Sa-Bu-Leud" is a Thai medicinal plant used for treatment of cancer and diabetes, and as a blood-tonic and aphrodisiac. This plant .ontains alkaloids as its major components and has been of interest for its acetylcholinesterase (AChE) inhibitory activity. Phytochemical screening - of S. venosa was made using HPLC analysis and showed the chemical variation between the same species from different provenances. Fractionation of .S..venosa extract yielded three alkaloids, namely, dicentrine, crebanine, and tetrahydropalmatine. AChE inhibitory potential of the isolated alkaloids was evaluated using Ellman's AChE inhibition assay. Dicentrine, crebanine, and tetrahydropalmatine inhibited-AChE activity with IC(50) values of 93.5, 86.6, and 168.6 µg/mL, respectively. The AChE inhibitory activity of the tertiary protoberberine alkaloid, tetrahydropalriiatine, was lower than that of the aporphine alkaloids, dicentrine and crebanine, whereas the quaternary protoberberine alkaloid, berberine, showed a higher AChE inhibitory effect than the others.

Highly enantioselective organocatalytic trifluoromethyl carbinol synthesis--a caveat on reaction times and product isolation.

Aldol reactions with trifluoroacetophenones as acceptors yield chiral α-aryl, α-trifluoromethyl tertiary alcohols, valuable intermediates in organic synthesis. Of the various organocatalysts examined, Singh's catalyst [(2S)-N-[(1S)-1-hydroxydiphenylmethyl-3-methylbutyl]-2-pyrrolidinecarboxamide] was found to efficiently promote this organocatalytic transformation in a highly enantioselective manner. Detailed reaction monitoring ((19)F-NMR, HPLC) showed that, up to full conversion, the catalytic transformation proceeds under kinetic control and affords up to 95% ee in a time-independent manner. At longer reaction times, the catalyst effects racemization. For the product aldols, even weak acids (such as ammonium chloride) or protic solvents, can induce racemization, too. Thus, acid-free workup, at carefully chosen reaction time, is crucial for the isolation of the aldols in high (and stable) enantiomeric purity. As evidenced by (19)F-NMR, X-ray structural analysis, and independent synthesis of a stable intramolecular variant, Singh's catalyst reversibly forms a catalytically inactive ("parasitic") intermediate, namely a N,O-hemiacetal with trifluoroacetophenones. X-ray crystallography also allowed the determination of the product aldols' absolute configuration (S).