GC-MS and NMR spectroscopy based metabolite profiling of Panchvalkal kwath (polyherbal formulation).

Panchvalkal kwath (PK) is a bark formulation of five pharmacologically important plants, i.e., Ficus benghalensis, Ficus racemosa, Ficus religiosa, Thespesia populnea, and Ficus lacor. The Ayurvedic formulation is being used since ancient times to cure diabetes, bacterial infections and heal wounds. The present study aims to identify the metabolite profiles of PK which could explain its properties and its mode of action against specific diseases and disorders. The aqueous extract of Panchvalkal is prepared through a hot maceration process. The extract is subjected to preliminary identification of phytoconstituents and FTIR spectroscopy to recognize functional groups. GC-MS analysis reveals that the extract is enriched with 24-Norursa-3,12-diene (25.16%); Lup-20(29)-en-3-one (16.76%); 2-methyl-3-(4-propan-2-ylphenyl) propanal (7.04%); 2-(hydroxymethyl)-2-nitropropane-1,3-diol (11.21%) and 3,5-dihydroxy-6-methyl-2,3-dihydropyran-4-one (4.15%). The presence of three new phytocompounds that are 4-(hydroxymethyl)-7-methyl-1,3-dioxepane-5,6-diol; 1-(4-isopropylphenyl)-2-methylpropylacetate and 4,4,6 A,6B,8A,11,11,14B-octamethyl-1,4,4A,5,6,6A,8,8a,910,11,12,12a,12b,13,14,14a,14b-ctadecahydro-3(2H)-picenone are detected in the extract. Metabolite profiles of the extract also constitute isoeugenol, stigmasterol, ergosterol, ocimene, myrcene, squalene, sphingosine, betulin, methyl ferulate and cis-jasmone, which are unraveled by 1 D 1H and 2 D 1H-13C HSQC NMR spectroscopy. This article focuses on the presence of different phytocompounds in PK in order to demonstrate its efficacy as a therapeutic formulation for a variety of diseases.

Differences in the serum metabolic profile to identify potential biomarkers for cyanotic versus acyanotic heart disease.

BACKGROUND: Growth retardation, malnutrition, and failure to thrive are some of the consequences associated with congenital heart diseases. Several metabolic factors such as hypoxia, anoxia, and several genetic factors are believed to alter the energetics of the heart. Timely diagnosis and patient management is one of the major challenges faced by the clinicians in understanding the disease and provide better treatment options. Metabolic profiling has shown to be potential diagnostic tool to understand the disease. OBJECTIVE: The present experiment was designed as a single center observational pilot study to classify and create diagnostic metabolic signatures associated with the energetics of congenital heart disease in cyanotic and acyanotic groups. METHODS: Metabolic sera profiles were obtained from 35 patients with cyanotic congenital heart disease (TOF) and 23 patients with acyanotic congenital heart disease (ASD and VSD) using high resolution 1D 1H NMR spectra. Univariate and multivariate statistical analysis were performed to classify particular metabolic disorders associated with cyanotic and acyanotic heart disease. RESULTS: The results show dysregulations in several metabolites in cyanotic CHD patients versus acyanotic CHD patients. The discriminatory metabolites were further analyzed with area under receiver operating characteristic (AUROC) curve and identified four metabolic entities (i.e. mannose, hydroxyacetone, myoinositol, and creatinine) which could differentiate cyanotic CHDs from acyanotic CHDs with higher specificity. CONCLUSION: An untargeted metabolic approach proved to be helpful for the detection and distinction of disease-causing metabolites in cyanotic patients from acyanotic ones and can be useful for designing better and personalized treatment protocol.

Nuclear magnetic resonance spectroscopy reveals dysregulation of monounsaturated fatty acid metabolism upon SPINK1 attenuation in colorectal cancer.

Metabolic reprogramming, a key hallmark of cancer, plays a pivotal role in fulfilling the accelerated biological demands of tumor cells. Such metabolic changes trigger the production of several proinflammatory factors, thereby inciting cancer development and its progression. Serine protease inhibitor Kazal Type 1 (SPINK1), well known for its oncogenic role and its upregulation via acute-phase reactions, is highly expressed in multiple cancers including colorectal cancer (CRC). Here, we show accumulation of lipid droplets in CRC cells stained with Oil Red O upon SPINK1 silencing. Furthermore, NMR spectroscopy analysis revealed an accretion of monounsaturated fatty acids (MUFAs) and phosphatidylcholine in these CRC cells, while the levels of polyunsaturated fatty acids remained unaltered. This alteration indicates the presence of MUFAs with the triglycerides in the lipid droplets as observed in SPINK1-silenced CRC cells. Considering the role of MUFAs in the anti-inflammatory response, our data hint that suppression of SPINK1 in CRC leads to activation of an anti-inflammatory signaling milieu. Conclusively, our study uncovers a connection between lipid metabolism and SPINK1-mediated CRC progression, hence paving the way for further exploration and better prognosis of SPINK1-positive CRC patients.

Biophysical Investigation of the Interplay between the Conformational Species of Domain-Swapped GB1 Amyloid Mutant through Real-Time Monitoring of Amyloid Fibrillation.

Mutant polypeptide GB1HS#124F26A, which is known to aggregate into amyloid-like fibrils, has been utilized as a model in this study for gaining insights into the mechanism of domain-swapped aggregation through real-time monitoring. Size exclusion with UV monitoring at 280 nm and dynamic light scattering (DLS) profiles through different time points of fibrillation reveal that the dimer transitions into monomeric intermediates during the aggregation, which could further facilitate domain swapping to form amyloid fibrils. The 1D 1H and 2D 1H-13C HSQC nuclear magnetic resonance (NMR) spectra profiling through different time points of fibrillation reveal that there may be some other species present along with the dimer during aggregation which contribute to different trends for the intensity of protons in the spectral peaks. Diffusion NMR reveals changes in the mobility of the dimeric species during the process of aggregation, indicating that the dimer gives rise to other lower molecular weight species midway during aggregation, which further add up to form the oligomers and amyloid fibrils successively. The present work is a preliminary study which explores the possibility of utilizing biophysical methods to gain atomistic level insights into the different stages of aggregation.

Interactions Between Amyloid-ß (1-42) and Hydroxyapatite-Cholesterol Spherules Associated with Age-Related Macular Degeneration.

Drusen deposition on sub-retinal pigment epithelium is the causal factor for age-related macular degeneration for the old-aged individuals. These deposits contain hydroxyapatite-cholesterol spherules on which several proteins and lipids accumulate to cover the retina and choroid, causing blurred vision and blindness. Amyloid-ß, the known culprit in Alzheimer's disease, is one among the few major proteins known to occur in these deposits. In the present article, we report preliminary analyses of interactions between amyloid-ß and hydroxyapatite-cholesterol composites using Thioflavin-T binding kinetics, solid-state NMR and transmission electron microscopy (TEM). Thioflavin-T fluorescence kinetics shows that amyloid-ß (1-42) aggregates only under certain conditions of concentration of cholesterol in the hydroxyapatite-cholesterol composites prepared by two different methods. These results were confirmed by 1D 13C CPMAS solid-state NMR. TEM imaging revealed that there is an exposure of the cholesterol surface in the composites prepared by sonication method. These imaging experiments explain the dependence of aggregation kinetics on the exposure and availability of cholesterol surface in the composites to a certain extent.

Interactions between hydroxyapatite and cholesterol associated with calcification in age-related macular degeneration.

Hydroxyapatite deposition and calcification occurs over cholesterol-containing lipid droplets between Bruch's membrane and sub-retinal pigment epithelium (sub - RPE) in the eyes of patients affected by age-related macular degeneration (AMD) as spherules, nodules, and Bruch's membrane plaques. In the present study, an attempt has been made to prepare a composite containing hydroxyapatite and cholesterol to elucidate interactions involved in the formation of such organic-inorganic interphase. To understand the mechanism of hydroxyapatite deposition on cholesterol, we have applied various biophysical techniques such as dynamic light scattering (DLS) measurements, transmission electron microscopy (TEM) imaging, Fourier transform infrared (FTIR) spectroscopy and solid-state nuclear magnetic resonance (ssNMR) spectroscopy on the prepared composite. Our results give molecular level insight into the mechanism of biocalcification in the disease system.

NMR-Based Metabolomics in Gallbladder Cancer Research.

Gallbladder cancer (GBC) is a common form of malignancy, which has high incidence rates in the northern parts of India, South America, Japan, and in Native American populations. A few metabolomic studies have revealed changes in the composition of biofluids, tissues, and gallstones by nuclear magnetic resonance (NMR), which may prove useful in understanding GBC. In this chapter, we focus on the use of NMR in unveiling the metabolomics of GBC.

Nuclear magnetic resonance (NMR)-based metabolomics for cancer research.

Nuclear magnetic resonance (NMR) has emerged as an effective tool in various spheres of biomedical research, amongst which metabolomics is an important method for the study of various types of disease. Metabolomics has proved its stronghold in cancer research by the development of different NMR methods over time for the study of metabolites, thus identifying key players in the aetiology of cancer. A plethora of one-dimensional and two-dimensional NMR experiments (in solids, semi-solids and solution phases) are utilized to obtain metabolic profiles of biofluids, cell extracts and tissue biopsy samples, which can further be subjected to statistical analysis. Any alteration in the assigned metabolite peaks gives an indication of changes in metabolic pathways. These defined changes demonstrate the utility of NMR in the early diagnosis of cancer and provide further measures to combat malignancy and its progression. This review provides a snapshot of the trending NMR techniques and the statistical analysis involved in the metabolomics of diseases, with emphasis on advances in NMR methodology developed for cancer research.