Multigram-scale HPLC enantioseparation as a rescue pathway for circumventing racemization problem during enantioselective synthesis of ethyl 3,4-dihydro-2H-1,4-benzoxazine-2-carboxylate.

Racemic ethyl 3,4-dihydro-2H-1,4-benzoxazine-2-carboxylate is a key synthon for the design of promising therapeutic drugs. It is mainly synthesized from racemic ethyl 2,3-dibromopropionate and 2-aminophenol in presence of K2 CO3 in refluxed acetone. Unfortunately, synthesis of (R)- and (S)-enantiomers using the enantioselective version of this reaction, which should normally be performed with a double SN 2 mechanism, is unsuitable due to a racemization process involving the dehydrobromination of enantiopure ethyl 2,3-dibromopropionate into ethyl 2-bromoacrylate. For the first time, the enantioselective version is studied (ee ≈ 55-66%), and the percentage of racemization process has estimated to around 34-46% after determination of the optimal experimental conditions for analytical HPLC enantioseparation of racemate. The influence of the experimental and purification conditions on the racemization rate is also studied. The results indicate that racemization occurs faster at the beginning of the reaction but the initiation of the double SN 2 process takes place more faster to limit the racemization rate. The study of the influence of experimental conditions (reaction times, temperature, solvent or type of base, etc.) on the degree of racemization of the (R)- enantiomer is performed and shows that despite changes in the experimental conditions, the synthesis of the (R)- enantiomer is always accompanied by a racemization rate which is difficult in reducing. In parallel, (R)- and (S)-enantiomers are obtained in high enantiopurity (ee > 99.5%) by preparative HPLC enantioseparation of racemate on multigram scale and characterized by optical rotation measurements, ECD and UV spectra.

In vitro anti-pancreatic cancer activity of HPLC-derived fractions from Helicteres hirsuta Lour. stem.

Pancreatic cancer (PC) is one of the leading causes of cancer death in Western societies. The absence of specific symptoms, late diagnosis and the resistance towards chemotherapy result in significant treatment difficulties. As such, it is important to find more effective therapeutic agents for the treatment of PC. Helicteres hirsuta Lour. (H. hirsuta) has been traditionally used in many countries for the treatment of various ailments, indicating that it contains potential therapeutic agents. This study aimed to derive different fractions from the saponin-enriched extract of H. hirsuta stem using RP-HPLC and examine the in vitro anti-pancreatic cancer activity of the derived fractions (F0-F5). With the exception of F0, the five fractions (F1-F5) possessed strong inhibitory activity against PC cells at IC50 values of 3.11-17.12 µg/mL. The flow cytometry assays revealed the active fractions caused cell cycle arrest at S phase and promoted apoptosis in MIAPaCa-2 PC cells. The LC/MS analysis revealed that the isolated fractions contained bioactive compounds, such as caffeic acid, rosmarinic acid, sagerinic acid, usnic acid, cucurbitacins and absinthin. It can be concluded that the fractions isolated from H. hirsuta stem exhibit potent in vitro anti-pancreatic cancer activity and thus warrant further in vivo studies to assess their activity against PC followed by isolation of individual bioactive compounds and the evaluation of their anti-pancreatic cancer activity.

Enantiopure ethyl 2,3-dibromopropionate: Enantioselective synthesis vs preparative HPLC enantioseparation of racemate on multigram scale.

Racemic ethyl 2,3-dibromopropionate, commercially available at low price, is a key intermediate used in the synthesis of several heterocycle fragments, which are present in many biologically active compounds. Surprisingly, the enantiomers are not commercially available and have never been described in the literature. In this work, we undertook two different strategies to obtain these enantiomers, which are enantioselective synthesis and preparative HPLC enantioseparation of commercially available racemate on multigram scale. The first strategy has proved inadequate because racemization occurred during the synthesis (ee ≈ 9-50%). Conversely, the second strategy produced a very good enantioseparation of commercially available racemate (ee > 99.5% for both enantiomers) on multigram scale.

Phytochemical, antioxidant, anti-proliferative and antimicrobial properties of Catharanthus roseus root extract, saponin-enriched and aqueous fractions.

Catharanthus roseus (L.) G. Don (C. roseus) is a well-known medicinal plant for its source of alkaloids solely found in the leaves. Other parts including the root are usually discarded after the alkaloid extraction. This study sought to investigate phytochemical profiles, antioxidant, antimicrobial and cytotoxic properties of the C. roseus root extract (RE) and its two sub-fractions including saponin-enriched (SE) and aqueous (AQ) fractions. The results showed that the RE was a rich source of saponins (1744.44 mg ESE/g) and phenolics (51.27 mg GAE/g), which comprised of gallic acid (25.74 mg/g), apigenin (1.45 mg/g) and kaempferol (1.58 mg/g). The SE fraction was enriched with 31% of saponins and 63% of phenolics higher than those of the RE; whereas the concentrations of saponins and phenolics of the AQ fraction were lower than those of the RE by 40% and 74%, respectively. The content of gallic acid in the SE fraction was 1.4-fold and 1.5-fold higher than those of the RE or AQ fraction, respectively. The SE fraction demonstrated potent antioxidant capacity, which was significantly higher than the RE or AQ fraction, and also exhibited strong anti-proliferative activity against 11 cancer cell lines including A2780 (ovarian), H460 (lung), A431 (skin), MIA PaCa-2 (pancreas), Du145 (prostate), HT29 (colon), MCF-7 (breast), BE2-C (neuroblastoma), SJ-G2, U87 and SMA (glioblastoma) with low GI50 values (≤ 2.00 µg/mL). The SE fraction was also shown to effectively inhibit the growth of both bacteria (Escherichia coli, Enterobacter aerogenes and Staphylococccus lugdunensis) and fungi (Candida albicans and Aspergillus niger). These findings warrant further investigation to isolate major compounds from the SE fraction and further test their antioxidant, anticancer and antimicrobial activities.

In vitro antibacterial and anticancer properties of Helicteres hirsuta Lour. leaf and stem extracts and their fractions.

Helicteres hirsuta Lour. (H. hirsuta) has been considered as a herbal medicine for the treatment of malaria and diabetes but limited studies have been conducted on its anticancer and antibacterial properties. In this study, the in vitro antibacterial and anticancer properties of the leaf and stem extracts and their two sub-fractions (aqueous and saponin-enriched butanol fractions) prepared from H. hirsuta were elucidated. MTT and CCK-8 assays were employed to assess their in vitro anticancer properties against various cancer cell lines. The antibacterial activity was assessed using the disc diffusion method and minimum inhibitory concentration (MIC) values were determined. The results revealed that the saponin-enriched fractions from H. hirsuta leaves and stems showed the highest antibacterial activity against E. coli (MIC values of 2.50 and 5.00 mg/mL, respectively) and S. lugdunensis (MIC values of 0.35 and 0.50 mg/mL, respectively). Importantly, these saponin-enriched fractions possessed strong anticancer activity in vitro towards a range of cancer cell lines including MIA PaCa-2 (pancreas); A2780 (ovarian); H460 (lung); A431 (skin); Du145 (prostate); HT29 (colon); MCF-7 (breast); SJ-G2, U87, SMA (glioblastoma) and BE2-C (neuroblastoma) at low doses (GI50 values of 0.36-11.17 µg/mL). They especially revealed potent anti-pancreatic cancer activity in vitro against MIA PaCa-2, BxPC-3 and CFPAC-1 cells with IC50 values of 1.80-6.43 µg/mL. This finding provides scientific evidence of the cytotoxic activity of the extracts prepared from H. hirsuta leaves and stems, and suggests further studies to isolate active compounds for development of new anticancer agents from these plant extracts.

Leptospermum extract (QV0) suppresses pleural mesothelioma tumor growth in vitro and in vivo by mitochondrial dysfunction associated apoptosis.

Pleural mesothelioma (PM) is a highly aggressive, fast-growing asbestos-induced cancer with limited effective treatments. There has been interest in using naturally occurring anticancer agents derived from plant materials for the treatment of PM. However, it is unclear if an aqueous extract from Leptospermum polygalifolium (QV0) has activity against PM. Here we investigated the anti-cancer properties of QV0 and Defender® (QV0 dietary formula) in vitro and in vivo, respectively. QV0 suppressed the growth of eight PM cell lines in a dose-dependent manner, effective at concentrations as low as 0.02% w/v (equivalent to 0.2 mg/ml). This response was found to be associated with inhibited cell migration, proliferation, and colony formation but without evident cell cycle alteration. We observed mitochondrial dysfunction post-QV0 treatment, as evidenced by significantly decreased basal and maximal oxygen consumption rates. Ten SCID mice were treated with 0.25 mg/g Defender® daily and exhibited reduced tumor size over 30 days, which was associated with an average extension of seven days of mouse life. There was no evidence of liver toxicity or increased blood glucose post-treatment in animals treated with Defender®. Significantly enhanced tumor apoptosis was observed in the Defender®-treated animals, correlating to mitochondrial dysfunction. Lastly, the high levels of polyphenols and antioxidant properties of QV0 and Defender® were detected in HPLC analysis. To the best of our knowledge, this study constitutes the first demonstration of an improved host survival (without adverse effects) response in a QV0-treated PM mouse model, associated with evident inhibition of PM cell growth and mitochondrial dysfunction-related enhancement of tumor apoptosis.

Upregulation of immunoproteasome PSMB8 is associated with Parkinson's disease.

BACKGROUND: Immunoproteasome, a part of ubiquitin-proteasome system, is involved in immune response as well as protein degradation. However, the relationship between immunoproteasome and Parkinson's disease (PD) was not evaluated clearly. We hypothesized that the shift of immunoproteasome attributes to PD pathogenesis due to its role in inflammation and protein homeostasis. OBJECTIVE: To determine whether immunoproteasome in peripheral blood mononuclear cells (PBMC) and brain is expressed differently between patients with PD and healthy controls (HC). METHODS: Blood samples were collected from 19 HC to 40 patients with PD of comparable ages. Peripheral blood mononuclear cells were isolated and followed by RT-qPCR to measure the mRNA levels of three catalytic subunits of immunoproteasome, namely, PSMB8, PSMB9, and PSMB10. Then, the protein levels of each subunit were measured by western blot. Finally, we confirmed the altered immunoproteasome subunit in the post-mortem human brain of PD. RESULTS: In PBMCs, PSMB8 mRNA expression of PD group significantly increased compared to HC (p = 0.004), whereas PSMB9 and PSMB10 mRNA were not different between the PD and HC. The ratio of PSMB10 and PSMB8 mRNA (PSMB10/8 ratio) also reflected the significant difference between the PD and HC (p = 0.002). The PSMB10/8 ratio was well correlated with the UPDRS total and Part III score in the early stage of PD (Hoehn and Yahr ≤2.5) or drug-naïve PD subgroups. In terms of the protein level of immunoproteasome subunits in PBMCs, the increase of PSMB8 protein was observed in PD compared to HC (p = 0.0009), while PSMB9 and PSMB10 were not different between groups. Finally, we confirmed that immunoproteasome PSMB8 was expressed abundantly in the postmortem PD brain compared with normal control. CONCLUSION: Our novel findings implicate that immunoproteasome PSMB8 is engaged in PD pathomechanism.

UHPLC-Q-TOF-MS/MS Dereplication to Identify Chemical Constituents of Hedera helix Leaves in Vietnam.

Hedera helix has been reported to contain a wide range of metabolites and produce many pharmacological effects. This research demonstrates the determination and evaluation of the phytochemical profiling of H. helix grown in central Vietnam. Methanolic extract of ivy had been analyzed by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). MS, and MS/MS experiments were manipulated using both negative and positive ionization modes to provide molecular mass information and production spectra for the structural elucidation of compounds. A total of 46 compounds including 24 triterpene saponins and other compounds were successfully identified of which four established saponin structures have been reported for the first time. This study has provided a base for building a quality control of the raw materials according to the profile of triterpene saponins and assessment of pharmaceutical ingredients of H. helix planted in Vietnam.

Comparative cytotoxic activity between kaempferol and gallic acid against various cancer cell lines.

This data article indicates the in vitro cytotoxicity of kaempferol and gallic acid across different cancer cell lines including A2780 (ovarian), H460 (lung), A431 (skin), MIA PaCa-2 (pancreas), Du145 (prostate), HT29 (colon), MCF-7 (breast), BE2-C (neuroblastoma), SJ-G2, U87 and SMA (glioblastoma). The dataset showed that the inhibitory activity of kaempferol was comparatively stronger than gallic acid. Thereby, kaempferol is offered as a potent anticancer agent for further investigation and beneficial as a dietary supplement. The data within this article relates to the research article entitled "Screening phytochemical content, antioxidant, antimicrobial and cytotoxic activities of Catharanthus roseus (L.) G. Don stem extract and its fractions" (Pham et al., 2018).