Traditional uses, pharmacological activities, and phytochemical constituents of the genus Syzygium: A review.

The genus Syzygium comprises 1200-1800 species that belong to the family of Myrtaceae. Moreover, plants that are belonged to this genus are being used in the traditional system of medicine in Asian countries, especially in China, India, and Bangladesh. The aim of this review is to describe the scientific works and to provide organized information on the available traditional uses, phytochemical constituents, and pharmacological activities of mostly available species of the genus Syzygium in Bangladesh. The information related to genus Syzygium was analytically composed from the scientific databases, including PubMed, Google Scholar, Science Direct, Web of Science, Wiley Online Library, Springer, Research Gate link, published books, and conference proceedings. Bioactive compounds such as flavanone derivatives, ellagic acid derivatives and other polyphenolics, and terpenoids are reported from several species of the genus Syzygium. However, many members of the species of the genus Syzygium need further comprehensive studies regarding phytochemical constituents and mechanism-based pharmacological activities.

Identification of Polyphenolics from Loranthus globosus as Potential Inhibitors of Cholinesterase and Oxidative Stress for Alzheimer's Disease Treatment.

Mistletoes are considered to be the potential medicinal herbs due to their rich traditional uses. Loranthus globosus is a Bangladeshi mango mistletoe that has been reported as folk medicine for various ailments and diseases. In an attempt to explore its effectiveness in Alzheimer's disease (AD), we investigated the antioxidant and acetylcholinesterase inhibitory activity of L. globosus. We report that the crude methanol extract (CME) of the plant contains a good amount of polyphenolics and possesses antioxidant and cholinesterase inhibitory activity. Fractionation of CME with solvents of varying polarity revealed the highest activity and polyphenolic content in the ethylacetate fraction (EAF). Correlation analysis revealed a significant (P < 0.05) association of polyphenolics with the antioxidant and cholinesterase inhibitory properties. Using column chromatography with diaion resin, the polyphenolics (EAF-PP) were isolated from the EAF that displayed the potent antioxidant and cholinesterase inhibitory activities. Kinetic analysis showed that EAF-PP exhibited a competitive type of inhibition. A total of thirty-six compounds including catechin and its different derivatives were identified in the EAF-PP by LC/MS analysis. Bioactivity-guided separation approach afforded the isolation of the two major active compounds catechin and catechin dimer from the EAF-PP. Hence, EAF-PP represents a potential source of antioxidants and cholinesterase inhibitors, which can be used in the management of AD.

Trichosanthes dioica seed lectin inhibits Ehrlich ascites carcinoma cells growth in vivo in mice by inducing G0 /G1 cell cycle arrest.

Trichosanthes dioica seed lectin (TDSL), having a molecular mass of 57 ± 2 kDa was purified in an alternative way. For the purification process, the galactose-sepharose-4B affinity column was used. The purified TDSL agglutinated human and mouse erythrocytes at the minimum concentration of 8  µg/ml. d-lactose and d-galactose were the most potent inhibitory sugars as observed. The purified lectin was a glycoprotein having 3.0% of a neutral sugar. The lectin exhibited maximum activity up to 60°C and pH range from 7.0 to 10.0 and stable up to 4.0 M urea as tested. The lectin demonstrated mild toxicity when administered against brine shrimp nauplii, and the LC50 value was calculated to be 84.0 µg/ml. Minimum agglutination of Ehrlich ascites carcinoma (EAC) cells caused by the lectin was found at the protein concentration of 1.56 µg/ml. TDSL inhibited 7, 50.2%, and 60.3% of the EAC cells growth in vivo in mice when administered with 0.75, 1.5, and 3.0 mg kg-1  day-1 (i.p.), respectively, for five consecutive days. After lectin treatment, red blood cell (RBC) and hemoglobin levels were increased significantly toward the normal compared with EAC cells-bearing control and normal mice. The tumor burden reduced to 29.5% and 67% after treatment with 1.5 and 3.0 mg kg-1  day-1 of the lectin. TDSL triggered the cell cycle arrest at the G0 /G1 phase, which was observed using flow cytometry. In conclusion, TDSL can be a candidate for the potent anticancer agents that exerts low toxicity toward brine shrimp nauplii. PRACTICAL APPLICATIONS: A 57 ± 2 kDa lectin (designated TDSL) was purified from Trichosanthes dioica seeds using a galactose-sepharose-4B affinity column. The lectin demonstrated mild toxicity and agglutinated Ehrlich ascites carcinoma (EAC) cells. The lectin inhibited 50.2% and 60.3% of the EAC cell growth in vivo in mice when administered with 1.5 and 3.0 mg kg-1  day-1 (i.p.), respectively, for five consecutive days. The lectin increased RBC and hemoglobin level toward the normal compared with lectin-treated EAC cells-bearing, EAC cells-bearing control and normal mice. The tumor burden reduced to 29.5% and 67% after treatment with 1.5 and 3.0 mg kg-1  day-1 lectin. TDSL triggered the cell cycle arrest at the G0 /G1 phase. The lectin can be a candidate for potent anticancer agents.

Phytochemical Analysis and Cholinesterase Inhibitory and Antioxidant Activities of Enhydra fluctuans Relevant in the Management of Alzheimer's Disease.

Enhydra fluctuans, a popular vegetable in Bangladesh, is used in folk medicine to treat diseases of the nervous system. The objective of this study was to investigate the phytochemical profile and cholinesterase inhibitory and antioxidant potential of the extracts of E. fluctuans. Among the four tested extracts, the chloroform extract was found to exert the highest inhibition against both the acetylcholinesterase and butyrylcholinesterase enzymes with the IC50 (concentration required for 50% inhibition) values of 83.90 µg/mL and 48.14 µg/mL, respectively. Likewise, the chloroform extract showed the highest radical scavenging activity and reducing power. In DPPH radical scavenging assay, the IC50 value was found to be 113.27 µg/mL, and in reducing power assay, the absorbance was found to be 1.916 at a concentration of 50 µg/mL. Phytochemical analyses revealed that the chloroform extract contained 19.16 mg gallic acid equivalent (GAE)/g extract of phenolics and 41.84 mg catechin equivalent (CE)/g extract of flavonoids, which appeared to be the highest among the extracts. A significant correlation was observed between phenolic content and butyrylcholinesterase inhibition and antioxidant activity, while a moderate correlation was seen between flavonoid content and cholinesterase inhibition and antioxidant activity. These findings suggest that E. fluctuans is a natural source of cholinesterase inhibitors and antioxidants, which could be utilized as functional foods for Alzheimer's disease management.

Vanda roxburghii chloroform extract as a potential source of polyphenols with antioxidant and cholinesterase inhibitory activities: identification of a strong phenolic antioxidant.

BACKGROUND: Alzheimer's disease (AD) is a progressively developing neurodegenerative disorder of the brain in the elderly people. Vanda roxburghii Rbr. root has been used traditionally in Bangladesh as tonic to brain and in the treatment of nervous system disorders including AD. Therefore, we aimed to investigate the cholinesterase inhibitory activities and antioxidant properties of the extracts from V. roxburghii. METHODS: The crude methanol extract from the roots of plant was sequentially fractionated with petroleum ether, chloroform, ethylacetate and water to yield their corresponding extracts. The extracts were assessed for acetylcholinesterase and butyrylcholinesterase inhibitory activity by modified Ellman method and antioxidant property by several assays including ferric reducing antioxidant power, scavenging of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical and hydroxyl radical, and inhibition of lipid peroxidation. Endogenous substances in the extracts were analyzed by the standard phytochemical methods and active compound was isolated by the chromatographic methods. RESULTS: Chloroform extract was shown to demonstrate strong ferric-reducing antioxidant power and scavenging activity against DPPH and hydroxyl free radicals when compared with the other extracts and the reference standard catechin. The antioxidant effect was further verified by inhibition of lipid peroxidation in rat brain homogenates. Likewise, the chloroform extract exhibited the highest inhibition against both the acetylcholinesterase and butyrylcholinesterase enzymes with IC50 values of 221.13 and 82.51 µg/ml, respectively. Phytochemical screening revealed a large amount of phenolics and flavonoids in the chloroform extract. Bioactivity guided separation techniques led to the isolation of a strong antioxidant from the chloroform extract and its structure was determined as gigantol on the basis of spectral studies. CONCLUSION: These results suggest that the chloroform extract of V. roxburghii, possibly due to its phenolic compounds, exert potential antioxidant and cholinesterase inhibitory activities, which may be useful in the treatment of AD.

Momordica charantia seed lectin: toxicity, bacterial agglutination and antitumor properties.

In last three decades, several studies were carried out on the D-galactose-specific lectin of Momordica charantia seeds (MCL). In the present study, in vitro growth inhibition (8-23 %) at different concentrations (6-24 µg/ml) of MCL was observed against Ehrlich ascites carcinoma (EAC) cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MCL also showed 28, 45, and 75 % growth inhibitions against EAC cells when administered 1.2, 2.0, and 2.8 mg/kg/day (i.p.), respectively for five consequent days in vivo in mice. After lectin treatment, the level of red blood cell and hemoglobin was increased significantly with the decrease of white blood cell and maintained the normal level when compared with EAC-bearing control and normal mice without EAC cells. Although MCL caused cell cycle arrest at G0/G1 phase of EAC cells, any irregular shape or apoptotic morphological alterations in the lectin-treated EAC cells was not observed by an optical and fluorescence microscope. Lectin showed toxicity against brine shrimp nauplii with an LC50 value of 49.7 µg/ml. Four out of seven pathogenic bacteria were agglutinated by MCL in the absence of inhibitory sugar D-lactose/D-galactose. In conclusion, MCL showed strong cytotoxic effect and therefore can be used as a potent anticancer chemotherapeutic agent.

In vitro acetylcholinesterase inhibitory activity and the antioxidant properties of Aegle marmelos leaf extract: implications for the treatment of Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder clinically characterized by loss of memory and cognition. The effective therapeutic options for AD are limited and thus there is a demand for new drugs. Aegle marmelos (Linn.) (A. marmelos) leaves have been used in traditional medicine to promote intellect and enhance memory. In this study, we evaluated A. marmelos for its acetylcholinesterase (AChE) inhibitory activity and antioxidant property in vitro in the treatment of AD. METHODS: A crude methanol extract and four fractions (petroleum ether, chloroform, ethyl acetate and aqueous) were prepared from the leaves of A. marmelos. The preparations were assessed for AChE inhibitory activity by the Ellman method, and their antioxidant properties were assessed by several assays: reducing power, scavenging of 1,1-diphenyl-2-picrylhydrazyl free radical and hydroxyl radical, and inhibition of lipid peroxidation. Qualitative and quantitative analyses of endogenous substances in A. marmelos were performed by the standard phytochemical methods. RESULTS: Among the different extracts tested, the ethyl acetate fraction exhibited the highest inhibition of AChE activity. In the same way, ethyl acetate fraction showed the highest reducing activity and radical scavenging ability towards the 1,1-diphenyl-2-picrylhydrazyl (half maximal inhibitory concentration = 3.84 µg/mL) and hydroxyl free radicals (half maximal inhibitory concentration = 5.68 µg/mL). The antiradical activity of the ethyl acetate fraction appeared to be similar to that of the reference standard butylated hydroxytoluene and catechin used in this study. In addition, the ethyl acetate fraction displayed higher inhibition of brain lipid peroxidation. Phytochemical screening of different extractives of A. marmelos showed the presence of phenols and flavonoids, alkaloid, saponin, glycoside, tannin and steroids. Quantitative analysis revealed higher contents of phenolics (58.79-mg gallic acid equivalent/g dried extract) and flavonoids (375.73-mg gallic acid equivalent/g dried extract) in the ethyl acetate fraction. CONCLUSION: The results suggest that the ethyl acetate fraction of A. marmelos is a significant source of polyphenolic compounds with potential AChE inhibitory property and antioxidant activity and, thus, may be useful in the treatment of AD.

Evaluation of antioxidant and anticancer properties of the seed extracts of Syzygium fruticosum Roxb. growing in Rajshahi, Bangladesh.

BACKGROUND: The use of plants and their derived substances increases day by day for the discovery of therapeutic agents owing to their versatile applications. Current research is directed towards finding naturally-occurring antioxidants having anticancer properties from plant origin since oxidants play a crucial role in developing various human diseases. The present study was designed to investigate the antioxidant and anticancer properties of Sygygium fruticosum (Roxb.) (abbreviated as SF). METHODS: The dried coarse powder of seeds of SF was exhaustively extracted with methanol and the resulting crude methanolic extract (CME) was successively fractionated with petroleum ether, chloroform and ethyl acetate to get petroleum ether (PEF), chloroform (CHF), ethyl acetate (EAF) and lastly aqueous (AQF) fraction. The antioxidant activities were determined by several assays: total antioxidant capacity assay, DPPH free radical scavenging assay, hydroxyl radical scavenging assay, ferrous reducing antioxidant capacity and lipid peroxidation inhibition assay. The in vivo anticancer activity of SF was determined on Ehrlich's Ascite cell (EAC) induced Swiss albino mice. RESULTS: All the extractives showed strong antioxidant activities related to the standard. The total antioxidant capacity (TAC) of the fractions was in the following order: EAF>AQF>CME>PEF>CHF. The TAC of EAF at 320 µg/mL was 2.60±0.005 which was significantly higher (p < 0.01) than that of standard catechin (1.37 ± 0.005). The ferrous reducing antioxidant capacity of the extracts was in the following order: EAF>AQF>CME>AA>CHF>PEF. In DPPH free radical scavenging assay, the IC50 value of EAF was 4.85 µg/mL, whereas that of BHT was 9.85 µg/mL. In hydroxyl radical scavenging assay and lipid peroxidation inhibition assay, the EAF showed the most potent inhibitory activity with IC50 of 43.3 and 68.11 µg/mL, respectively. The lipid peroxidation inhibition assay was positively correlated (p < 0 .001) with both DPPH free radical scavenging and hydroxyl radical scavenging assay. The total phenolic contents of SF were also positively correlated (p < 0 .001) with DPPH free radical scavenging, hydroxyl radical scavenging and lipid peroxidation inhibition assay. Based on antioxidant activity, EAF was selected for cytotoxic assay and it was found that EAF inhibited 67.36% (p < 0.01) cell growth at a dose of 50 mg/kg (ip) on day six of EAC cell incubation. CONCLUSIONS: Our results suggest that EAF of seeds of SF possess significant antioxidant and moderate anticancer properties. Seeds of SF may therefore be a good source for natural antioxidants and a possible pharmaceutical supplement.

Comprehensive analysis of alternative splicing and functionality in neuronal differentiation of P19 cells.

BACKGROUND: Alternative splicing, which produces multiple mRNAs from a single gene, occurs in most human genes and contributes to protein diversity. Many alternative isoforms are expressed in a spatio-temporal manner, and function in diverse processes, including in the neural system. METHODOLOGY/PRINCIPAL FINDINGS: The purpose of the present study was to comprehensively investigate neural-splicing using P19 cells. GeneChip Exon Array analysis was performed using total RNAs purified from cells during neuronal cell differentiation. To efficiently and readily extract the alternative exon candidates, 9 filtering conditions were prepared, yielding 262 candidate exons (236 genes). Semiquantitative RT-PCR results in 30 randomly selected candidates suggested that 87% of the candidates were differentially alternatively spliced in neuronal cells compared to undifferentiated cells. Gene ontology and pathway analyses suggested that many of the candidate genes were associated with neural events. Together with 66 genes whose functions in neural cells or organs were reported previously, 47 candidate genes were found to be linked to 189 events in the gene-level profile of neural differentiation. By text-mining for the alternative isoform, distinct functions of the isoforms of 9 candidate genes indicated by the result of Exon Array were confirmed. CONCLUSIONS/SIGNIFICANCE: Alternative exons were successfully extracted. Results from the informatics analyses suggested that neural events were primarily governed by genes whose expression was increased and whose transcripts were differentially alternatively spliced in the neuronal cells. In addition to known functions in neural cells or organs, the uninvestigated alternative splicing events of 11 genes among 47 candidate genes suggested that cell cycle events are also potentially important. These genes may help researchers to differentiate the roles of alternative splicing in cell differentiation and cell proliferation.

Retinoic acid treatment and cell aggregation independently regulate alternative splicing in P19 cells during neural differentiation.

To induce neural differentiation of P19 cells, two different treatments, RA (retinoic acid) and cell aggregation, are required. However, there has been no report that RA treatment alone or cell aggregation alone could control alternative splicing regulation in P19 cells. Therefore, we focused on alternative splicing effects by neural induction (RA treatment and/or cell aggregation) in P19 cells. We analysed the splicing patterns of several genes, including 5-HT3R-A (5-hydroxytryptamine receptor), Actn1 (actinin alpha1), CUGBP2 (CUG-binding protein) and PTB (polypyrimidine track-binding protein), which showed different responses during the early neural induction of P19 cells. We show here that RA treatment alone changes the alternative splice mechanism of 5-HT3R-A. Cell aggregation alone controls alternative splicing regulation of Actn1. Both treatments (RA and cell aggregation) compensate and regulate the alternative splicing mechanism of CUGBP2. However, PTB is independent of RA and cell aggregation. Taken together, our results suggest that RA treatment and cell aggregation independently regulate the alternative splicing mechanism in the early stage of P19 cells during neural differentiation.

Alternative splicing of Mef2c promoted by Fox-1 during neural differentiation in P19 cells.

Mef2c protein is one of the MADS-box type transcription factors involved in muscular differentiation and synaptic formation. Previously, it has been reported that the Mef2c gene is responsible for three alternative splicing regulations. Here, we investigated the alternative splicing variants of Mef2c during neural differentiation of P19 cells and during cardio muscular differentiation of P19 clone 6 (P19CL6). We detected that two Mef2c mRNA isoforms, using exon α1 with and without the γ region at exon 10, are mainly produced in immature P19 cells. Remarkably, Mef2c isoforms containing exon ß specifically appeared in the neural cell stage. Because most transcripts contain exon ß in the neural cell stage and in the brain, this suggests that the alternative splicing of exon ß is highly regulated. Among known regulators, Fox-1 was specifically expressed in the neural cell stage in correlation with Mef2c exon ß. Fox-1 promoted exon ß inclusion in transfection experiments using Mef2cß minigene. Moreover, we found that the promotion required RNA-binding activity of Fox-1 and GCAUG sequence located in adjacent intron of exon ß. Taken together, our results suggest that Fox-1, expressed specifically in the neural cell stage, promoted Mef2c exon ß inclusion via the GCAUG.

Expression analysis of Fgf8a &Fgf8b in early stage of P19 cells during neural differentiation.

Fgf8 is a member of the fibroblast growth factor (FGF) family that plays an important role in early neural development. Cellular aggregation and retinoic acid (RA) are needed for mouse embryonic carcinoma (EC) P19 cell neural differentiation. We have examined the Fgf8 gene in P19 cells during neural differentiation and identified 2 alternatively spliced Fgf8 isoforms, Fgf8a and Fgf8b, among the 8 known splicing isoforms in mammals. The expression of Fgf8a and Fgf8b mRNAs transiently and rapidly increased in the early stage of P19 cells during RA-induced neural differentiation, followed by a decline in expression. The relative amount of Fgf8b was clearly higher than that of Fgf8a at different time-points measured within 24h after RA treatment. Increased Fgf8b mRNA expression was cellular-aggregation dependent. The results demonstrated that cellular-aggregation-induced Fgf8b, but not Fgf8a, may play a pivotal role in early neural differentiation of P19 cells.

Antibacterial and cytotoxic compounds from the bark of Cananga odorata.

O-Methylmoschatoline, liriodenine and 3,4-dihydroxybenzoic acid isolated from the barks of Cananga odorata showed antibacterial activities against a number of Gram (+) and Gram (-) bacteria. The compounds also showed antifungal and cytotoxic activities.