Hydroxylation and glycosylation of Δ9-tetrahydrocannabinol by Catharanthus roseus cell suspension culture

Δ9-tetrahydrocannabinol is the active constituent in Cannabis sativa, with reported analgesic, anti-emetic, anti-oxidative, neuroprotective, and anti-inflammatory activities. Δ9-THC has been used to treat a number of disease states including pain, anxiety, asthma, glaucoma, and hypertension. Poor water solubility of Δ9-THC greatly reduces its clinical effectiveness. Consequently, there is a need to modify the compound to increase its polarity and pharmaceutical efficacy. The aim of this study was to test the capability of Catharanthus roseus suspension cultured cells to convert Δ9-THC into more polar derivatives. The transformed metabolites were analyzed and isolated by HPLC. Structures of some new derivatives were proposed on the basis of molecular ion peaks and fragmentation patterns obtained from LC-MS and UV spectra obtained by HPLC, respectively. Δ9-THC was rapidly absorbed by Catharanthus roseus cultured cells and upon biotransformation new glycosylated and hydroxylated derivatives were isolated by preparative HPLC. In addition, cannabinol was detected as degradation product, including its glycosylated derivative. Based on these results, it is concluded that Catharanthus cultured cells have great potential to transform Δ9-THC into more polar derivatives and can be used for the large scale production of new cannabinoids, which can be a source of new compounds with interesting pharmacological profiles

Prognostically Significant Fusion Oncogenes and Gene Mutations in Pakistani AML Patients

Background: The onset and progression of leukemia is associated with many genetic abnormalities including gene mutations and production of fusion oncogenes. Molecular studies on fusion oncogenes and mutations in different populations have been done. However, not much research on correlation of the fusion oncogenes with acute myeloid leukemia have been done in Pakistan.Methods: Genetic analysis of 105 AML patients was done to investigate AML1-ETOand CBFB-MYH11 fusion oncogenes and mutations in NPM1 and NRAS genes. The genomic DNA and cDNA were subjected to amplification, electrophoresis, and Sanger sequencing.Results: The frequency of AML1-ETO was 26% in AML patients and 34.2% in AML-M2 patients. CBFB-MYH11 was present in 11.4% AML patients. A total of six mutations in 4 regions of NPM1 gene and 2 regions of NRAS gene were detected. 3’UTR of NPMI gene had three variants; g.1128C>T (57.1%), g.1185-/T insertion (80.95%), and g.1163A>T (57.14%) while c.867_871subGTGGA >CAAGTTTGC (2.86%) was present in exon 12. NRAS gene had two mutations c.12C>T (51.4%) and c.33A>T (11.43%) in exon 2. c.867_871subGTGGA >CAAGTTTGC , and g.1163A>T in NPM1 gene and c.33A>T in NRAS gene were the novel findings in this ethnic population.Conclusion: This genetic analysis may help to modulate the treatment strategies and improve survival of patients.Keywords: Acute Myeloid Leukemia; Fusion Oncogenes; AML; AML1-ETO; CBFB-MYH11; AML-M2; Nucleophosmin, NPM1; NRAS

Enhanced production of butyric acid by solid-state fermentation of rice polishings by a mutant strain of Clostridium tyrobutyricum

Purpose: To enhance butyric acid production by solid-state fermentation with a hyper-producing mutant of Clostridium tyrobutyricum generated by random mutagenesis.Methods: Wild type C. tyrobutyricum was mutagenized with UV irradiation, nitrous acid, and ethidium bromide to obtain a hyper-producing strain. Various physiochemical parameters were optimized to increase the butyric acid yield.Results: The UV-induced mutant (C.TUV) produced significantly higher concentrations of butyric acid than the wild type parent, nitrous acid-induced, and ethidium bromide-induced strains. C.TUV increased butyric acid production 1.4-fold more than the parent strain. Fermentation with C.TUV with 2.5 g of rice polishings (w/w), a 2 % inoculum volume (v/v), and a 48-h incubation period at 37°C under anaerobic conditions produced 11.63 mg/100 g of butyric acid. The addition of 0.6 % corn steep liquor as a nitrogen source increased the butyric acid concentration to 26.09 mg/100 g.Conclusion: These optimized fermentation parameters on a small scale can be used on a commercial scale to mass-produce butyric acid.Keywords: Butyric acid, Mutant, Clostridium tyrobutyricum, Mutagen, Solid-state fermentatio

Bioactive constituents of Zanthoxylum rhetsa bark and its cytotoxic potential against B16-F10 melanoma cancer and normal human dermal fibroblast (HDF) cell lines

Zanthoxylum rhetsa is an aromatic tree, known vernacularly as "Indian Prickly Ash". It has been predominantly used by Indian tribes for the treatment of many infirmities like diabetes, inflammation, rheumatism, toothache and diarrhea. In this study, we identified major volatile constituents present in different solvent fractions of Z. rhetsa bark using GC-MS analysis and isolated two tetrahydrofuran lignans (yangambin and kobusin), a berberine alkaloid (columbamine) and a triterpenoid (lupeol) from the bioactive chloroform fraction. The solvent fractions and purified compounds were tested for their cytotoxic potential against human dermal fibroblasts (HDF) and mouse melanoma (B16-F10) cells, using the MTT assay. All the solvent fractions and purified compounds were found to be non-cytotoxic to HDF cells. However, the chloroform fraction and kobusin exhibited cytotoxic effect against B16-F10 melanoma cells. The presence of bioactive lignans and alkaloids were suggested to be responsible for the cytotoxic property of Z. rhetsa bark against B16-F10 cells

Anti-diabetic activity and metabolic changes induced by Andrographis paniculata plant extract in obese diabetic rats

Andrographis paniculata is an annual herb and widely cultivated in Southeast Asian countries for its medicinal use. In recent investigations, A. paniculata was found to be effective against Type 1 diabetes mellitus (Type 1 DM). Here, we used a non-genetic out-bred Sprague-Dawley rat model to test the antidiabetic activity of A. paniculata against Type 2 diabetes mellitus (Type 2 DM). Proton Nuclear Magnetic Resonance (1H-NMR) spectroscopy in combination with multivariate data analyses was used to evaluate the A. paniculata and metformin induced metabolic effects on the obese and obese–diabetic (obdb) rat models. Compared to the normal rats, high levels of creatinine, lactate, and allantoin were found in the urine of obese rats, whereas, obese-diabetic rats were marked by high glucose, choline and taurine levels, and low lactate, formate, creatinine, citrate, 2-oxoglutarate, succinate, dimethylamine, acetoacetate, acetate, allantoin and hippurate levels. Treatment of A. paniculata leaf water extract was found to be quite effective in restoring the disturbed metabolic profile of obdb rats back towards normal conditions. Thisstudy shows the anti-diabetic potential of A. paniculata plant extract and strengthens the idea of using this plant against the diabetes. Further classical genetic methods and state of the art molecular techniques could provide insights into the molecular mechanisms involved in the pathogenesis of diabetes mellitus and anti-diabetic effects of A. paniculata water extract

Zebrafish as a model for systems medicine R&D: rethinking the metabolic effects of carrier solvents and culture buffers determined by 1H NMR metabolomics

Zebrafish is a frequently employed model organism in systems medicine and biomarker discovery. A crosscutting fundamental question, and one that has been overlooked in the field, is the “system-wide” (omics) effects induced in zebrafish by metabolic solvents and culture buffers. Indeed, any bioactivity or toxicity test requires that the target compounds are dissolved in an appropriate nonpolar solvent or aqueous media. It is important to know whether the solvent or the buffer itself has an effect on the zebrafish model organism. We evaluated the effects of two organic carrier solvents used in research with zebrafish, as well as in drug screening: dimethyl sulfoxide (DMSO) and ethanol, and two commonly used aqueous buffers (egg water and Hank's balanced salt solution). The effects of three concentrations (0.01, 0.1, and 1%) of DMSO and ethanol were tested in the 5-day-old zebrafish embryo using proton nuclear magnetic resonance (1H NMR) based metabolomics. DMSO (1% and 0.1%, but not 0.01%) exposure significantly decreased the levels of adenosine triphosphate (ATP), betaine, alanine, histidine, lactate, acetate, and creatine (p < 0.05). By contrast, ethanol exposure did not alter the embryos' metabolome at any concentration tested. The two different aqueous media noted above impacted the zebrafish embryo metabolome as evidenced by changes in valine, alanine, lactate, acetate, betaine, glycine, glutamate, adenosine triphosphate, and histidine. These results show that DMSO has greater effects on the embryo metabolome than ethanol, and thus is used with caution as a carrier solvent in zebrafish biomarker research and oral medicine. Moreover, the DMSO concentration should not be higher than 0.01%. Careful attention is also warranted for the use of the buffers egg water and Hank's balanced salt solution in zebrafish. In conclusion, as zebrafish is widely used as a model organism in life sciences, metabolome changes induced by solvents and culture buffers warrant further attention for robust systems science, and precision biomarkers that will stand the test of time

Variation in the metabolites and αglucosidase inhibitory activity of Cosmos caudatus at different growth stages

Background: Cosmos caudatus is an annual plant known for its medicinal value in treating several health conditions, such as high blood pressure, arthritis, and diabetes mellitus. The α-glucosidase inhibitory activity and total phenolic content of the leaf aqueous ethanolic extracts of the plant at different growth stages (6, 8. 10, 12 and 14weeks) were determined in an effort to ascertain the best time to harvest the plant for maximum medicinal quality with respect to its glucose-lowering effects. Methods: The aqueous ethanolic leaf extracts of C. caudatus were characterized by NMR and LC-MS/MS. The total phenolic content and α-glucosidase inhibitory activity were evaluated by the Folin-Ciocalteu method and α-glucosidase inhibitory assay, respectively. The statistical significance of the results was evaluated using one-way ANOVA with Duncan’s post hoc test, and correlation among the different activities was performed by Pearson’s correlation test. NMR spectroscopy along with multivariate data analysis was used to identify the metabolites correlated with total phenolic content and α-glucosidase inhibitory activity of the C. caudatus leaf extracts. Results: It was found that the α-glucosidase inhibitory activity and total phenolic content of the optimized ethanol: water (80:20) leaf extract of the plant increased significantly as the plant matured, reaching a maximum at the 10th week. The IC50 value for α-glucosidase inhibitory activity (39.18μgmL− 1) at the 10th week showed greater potency than the positive standard, quercetin (110.50μgmL− 1). Through an 1H NMR-based metabolomics approach, the 10-week-old samples were shown to be correlated with a high total phenolic content and α-glucosidase inhibitory activity. From the partial least squares biplot, rutin and flavonoid glycosides, consisting of quercetin 3-Oarabinofuranoside, quercetin 3-O-rhamnoside, quercetin 3-O-glucoside, and quercetin 3-O-xyloside, were identified as the major bioactive metabolites. The metabolites were identified by NMR spectroscopy (J-resolve, HSQC and HMBC experiments) and further supported by dereplication via LC-MS/MS. Conclusion: For high phytomedicinal quality, the 10th week is recommended as the best time to harvest C. caudatus leaves with respect to its glucose lowering potential

Genomics of Salinity Tolerance in Plants

Plants are frequently exposed to wide range of harsh environmental factors, such as drought, salinity, cold, heat, and insect attack. Being sessile in nature, plants have developed different strategies to adapt and grow under rapidly changing environments. These strategies involve rearrangements at the molecular level starting from transcription, regulation of mRNA processing, translation, and protein modification or its turnover. Plants show stress-specific regulation of transcription that affects their transcriptome under stress conditions. The transcriptionally regulated genes have different roles under stress response. Generally, seedling and reproductive stages are more susceptible to stress. Thus, stress response studies during these growth stages reveal novel differentially regulated genes or proteins with important functions in plant stress adaptation. Exploiting the functional genomics and bioinformatics studies paved the way in understanding the relationship between genotype and phenotype of an organism suffering from environmental stress. Future research programs can be focused on the development of transgenic plants with enhanced stress tolerance in field conditions based upon the outcome of genomic approaches and knowing the mystery of nucleotides sequences hidden in cells

Utilization of the ethyl acetate fraction of Zanthoxylum rhetsa bark extract as an active ingredient in natural sunscreen formulations

Sunburn, premature skin aging, skin cancers and suppression of the immune system are linked to exposure of the skin to UV light. In recent years, plant extracts are becoming a popular active ingredient in natural sunscreen formulations. In the present study, the ethyl acetate fraction of Zanthoxylum rhetsa bark (commonly called as Indian prickly Ash) was used as an active ingredient in two sunscreen cream formulations (F1 and F2). Primarily, the constituents present in the active fraction were identified using LC–MS/MS analysis. Coumaric acid, benzoic acid, p-hydroxybenzoic acid and its isomers, hesperitin, trihydroxyoctadecenoic acid and columbamine were identified in the ethyl acetate fraction of Z. rhetsa bark extract. The UV protection properties of the formulated creams were evaluated by assessment of parameters such as their SPF values (F1: 3.60 ± 0.28, F2: 6.90 ± 0.57), UVA effectiveness (moderate for both test formulations) and critical wavelengths (F1: 365.4, F2: 360.3). Moreover, the physicochemical and microbial count of the formulated creams was also assessed based on various parameters such as colour, pH, centrifugation, viscosity and microbial load over a storage period of 28 days. Both formulations showed pseudo plastic behaviour and were stable at all conditions except for samples kept at 40 °C. Altogether, these results suggested that the ethyl acetate fraction of Z. rhetsa bark has great potential to reduce exposure to harmful UVA/UVB radiations and may be utilized as an active ingredient in natural sunscreen formulation