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The diagnosis and confirmation of oral SCC (squamous cell carcinoma) is still dependent on histopathology report in spite of development of radiological investigations. It is, thus important to understand the underlying molecular mechanisms and how the alterations in metabolic pathways effect the tumor development and progression. The simultaneous and comprehensive information about the presence and absence of small molecule metabolites and their relative concentrations has been provided by 1 H HR-MAS NMR spectroscopy on tissue specimens. In this paper a unique case study was presented in order to correlate histological and NMR spectroscopic findings. The patient’s initially lesion was found to be non-malignant in nature based on histological findings but its periodic localized recurrence even after laser ablation prompted us to perform HR-MAS based analysis and its role in identifying the metabolic alterations in known pathways occurring during its progressions. Thus it was confirmed after analysis that HR-MAS NMR can also be used as an analytical tool which is reliable in order to distinguish between malignant and non-malignant tissues, in combination with histopathology.

A rapid and reliable method was developed and validated for determining betulin and betulinic acid in bark in Betula utilis by high-resolution magic angle spinning 1H nuclear magnetic resonance (HR-MAS NMR) spectroscopy. HR-MAS NMR spectroscopy clearly distinguished the resonances of betulin and betulinic acid in the bark of all accessions of B. utilis. The concentrations of betulin and betulinic acid were calculated and added to the spectra. The determination of the targeted metabolites in chloroform extract of bark of each accession of B. utilis was performed by high-performance liquid chromatography (HPLC). Quantitatively, betulin was present at higher concentrations than betulinic acid in all accessions. The HR-MAS NMR and HPLC results showed that betulin and betulinic acid varied significantly among accessions of B. utilis. Principal component analysis of the NMR and HPLC results provided classification into three metabolic groups in which the betulin concentration was high, moderate, or low....

Commiphora wightii (Arn.) Bhandari, known as guggul, produces a medicinally important gum resin which is used extensively by Ayurvedic physicians to treat various ailments. However, most of the studies on C. wightii have been limited to its gum resin. Comprehensive metabolic profiling of leaves, stem and gum resin samples was undertaken to analyse aqueous and non-aqueous metabolites from three distinct chemotypes (NBRI-101, NBRI-102 and NBRI-103) shortlisted from different agro-climatic zones. GC-MS, HPLC and NMR spectroscopy were used for comprehensive metabolomics. Multivariate analysis showed characteristic variation in quinic and citric acids, myo-inositol and glycine (aqueous metabolites) and 2,6-di-tert-butyl-phenol, trans-farnesol and guggulsterones (non-aqueous metabolites) amongst the three chemotypes. Quinic acid, citric acid and myo-ionositol were detected in substantial quantities from leaves and stem samples which provide opportunities for novel nutraceutical and pharmaceutical formulations. Quinic acid, from the leaves, was identified as a marker metabolite for early selection of high guggulsterones-yielding cultivars.

To achieve goals in metabolomics investigations, it is necessary to produce a comprehensive metabolic profiling from biological samples. Identification of metabolites is one of the important steps in metabolomics studies and the conclusion drawn from such studies depends on how exactly the metabo- lites are identified. NMR is one of the most selective analytical techniques which offers structural infor- mation of molecules. But, due to complex biological sample matrix, metabolic identification step needs application of advanced NMR techniques and analytical strategies for better accuracy. This review covers the analytical methods and strategies used for identification of metabolites in NMR-based metabolomics. The specific problems and troubleshoots associated with identification of metabolites in biological samples are discussed in details.

Periodontitis is a chronic, non-reversible inflammatory disease of the oral cavity leading to destruction of periodontal tissues. Thus, the estimation of bacterial metabolite, tissue damage and secretory metabolites of the triggered inflammatory cells likely to yield results. It may be of value for understanding the pathophysiology of the disease by metabolic profiling of saliva samples using high-resolution NMR spectroscopy. The study will evaluate the difference in salivary metabolites in healthy and periodontal condition along with fetching of possible biomarkers in case of chronic periodontitis. 1H- NMR spectroscopy has been employed in 114 saliva samples in search of distinctive differences and spectral data were further subjected to multivariate analysis. One-hundred metabolites were characterised and assigned in the 1H NMR spectra of saliva. The statistical analysis of control (Healthy subjects) and diseased (Periodontal subjects) using PLS-DA model resulted in R2 of 0.84 and Q2 of 0.79. There was an elevation in the concentration of statistically discriminant metabolites. The twenty newly identified metabolites in saliva indicates bacterial population shift along with change in homeostasis. These disturbs the biofilm, a real protector against any possible bio-damage on tooth surface. These newly identified metabolites could define better geographically diversified periodontal condition. Analysis clearly differentiates healthy subjects from the diseased ones. Few newly identified metabolites along with the pool of metabolites may serve as biomarkers for distinguishing the severity and complexity of periodontitis.

Cranial irradiation is widely used as a treatment modality or prophylactic treatment in cancer patients, but it is frequently related to neurocognitive impairment in cancer survivors. Though most of radiation-induced changes occur during early and late delayed phase of radiation sickness, recent reports have supported the evidence of impaired neurogenesis within 24-48 h of radiation exposure that may implicate changes in acute phase as well. Inspection of these acute changes could be considered important as they may have long lasting effect on cognitive development and functions. In the present study, (1)H NMR spectroscopy based metabolomic approach was used to obtain comprehensive information of hippocampus metabolic physiology during acute phase of radiation sickness in a mouse model for single dose 8 Gy cranial irradiation. The analysis demonstrated reduced metabolic activity in irradiated animals compared to controls, typically evident in citric acid cycle intermediates, glutamine/glutamate and ketone bodies metabolism thus providing strong indication that the hippocampus is metabolically responsive to radiation exposure. The data suggested reduced glucose utilization, altered intermediary and neurotransmitter metabolism in hippocampus tissue extract. To the best of our knowledge this is the first metabolomic study to document cranial irradiation induced acute metabolic changes using in vitro(1)H NMR spectroscopy.

The paucity of biomarkers for malignant obstructive jaundice results in formidable morbidity and mortality rates. Therefore, alternative diagnostic measures are required for improved clinical interpretation and better peri-operative management of patients. In the present study, 1H NMR-based metabolomic approach has been applied to investigate serum and bile based metabolic biomarkers in benign and malignant causes of obstructive jaundice (OBJ). Serum and bile specimens from benign OBJ patients (n = 28), malignant OBJ patients (n = 36) and serum of healthy controls (n = 57) were analysed by 1H NMR spectroscopy. Quantitation of eight serum metabolites (isobutyrate, lactate, alanine, acetone, glutamine, creatine, threonine and 1-methylhistidine) was carried out. A newer and rapid single step NMR based semi-quantitative ratio analysis of serum total cholesterol (tCho), cholesterol (Chol) and cholesterol ester (CE) were performed in deuterated dimethyl sulfoxide-d6. In bile, total bile acids, cholesterol, phosphatidylcholine, glycerophosphatidylcholine and urea were quantified. The effect of benign and malignant OBJ on small metabolites and lipids were statistically analysed by Mann–Whitney U test and multivariate discriminant function analysis. It was found that malignancy could be differentiated from benign cases of OBJ with a correct classification of 85.7 % when eight serum metabolites in combination with ratios of serum cholesterol were analysed. Significant alterations in serum tCho, Chol, CE and serum metabolites may have potential for early and differential non-invasive diagnosis of malignant and benign OBJ cases. It will further augment the novel insights of local and systemic effects in OBJ patients.

Meningitis, a morbidly infectious central nervous system pathology is accompanied by acute inflammation of the meninges, causing raised intracranial pressure linked with serious neurological sequelae. To observe the variation in the metabolic profile, that may occur in serum and urine along with CSF in adults using 1H NMR spectroscopy, with an attempt of appropriate and timely treatment regimen. The 1H NMR-based metabolomics has been performed in 115 adult subjects for differentiating bacterial meningitis (BM) and tubercular meningitis (TBM). The discriminant function analysis (DFA) of the three bio-fluids collectively identified 3-hydroxyisovalerate, lactate, glucose, formate, valine, alanine, ketonic bodies, malonate and choline containing compounds (choline and GPC) as significant metabolites among cases versus control group. The differentiation of bacterial meningitis and tuberculous meningitis (BM vs. TBM) can be done on the basis of identification of 3-hydroxyisovalerate, isobutyrate and formate in case of CSF (with a correct classification of 78 %), alanine in serum (correct classification 60 %), valine and acetone in case of urine (correct classification 89.1 %). The NMR spectral bins based orthogonal signal correction principal component analysis score plots of significant metabolites obtained from DFA also provided group classification among cases versus control group in CSF, serum and urine samples. The variable importance in projection scores also identified similar significant metabolites as obtained from DFA, collectively in CSF, serum and urine samples, responsible for differentiation of meningitis. The CSF contained metabolites which are formed during infection and inflammation, and these were also found in significant quantity in serum and urine samples.

Amaranthus hypochondriacus L. infected with Ageratum enation virus (AEV) was investigated for identifying alteration in the anatomical structures, sap translocation and metabolomic variations using light microscopy, magnetic resonance imaging, NMR spectroscopy and GC–MS, respectively. Combination of GC–MS and NMR spectroscopy identified 68 polar and non-polar metabolites that were present in different levels in healthy and virus-infected A. hypochondriacus. Contrast of T1 and T2 weighted MR images showed significant differences in the spatial distribution of water, lipids and macromolecules indicating alterations in the cortical region and disruption of vascular bundles in virus-infected stem tissues. MRI observations are supported by light microscopic examination. Microscopic examination of AEV infected stem revealed severe hyperplasia with a considerable reduction in size of stem cells. The NMR spectroscopy and GC–MS analysis indicated that viral infection significantly affected the plant primary and secondary metabolism resulting in decreased glucose and sucrose content and increase in the concentration of β-sitosterol and stigmasterol. Higher accumulation of TCA cycle intermediates such as citric acid and malic acid in AEV infected plants indicated enhanced rate of respiratory metabolism. The viral stress significantly increases the concentration of erythritol and myo-inositol as compared to healthy ones. Lower concentration of glucose and sucrose in viral-infected stem tissues suggests decreased translocation of photosynthates in the plants. The results demonstrated potential of MRI, NMR spectroscopy and GC–MS for studying anatomical and metabolic variations in virus-infected plants.

Gall bladder tissue specimens obtained from 112 patients were examined by high resolution magic angle spinning (HR-MAS) NMR spectroscopy. Fifty one metabolites were identified by combination of one and two-dimensional NMR spectra. To our knowledge, this is the first report on metabolic profiling of gall bladder tissues using HR-MAS NMR spectroscopy. Metabolic profiles were evaluated for differentiation between benign Chronic Cholecystitis (CC, n = 66) and xantho-granulomatous cholecystitis (XGC, n = 21) and malignant gall bladder cancer (GBC, n = 25). Increase in choline containing compounds, amino acids, taurine, nucleotides and lactate as common metabolites were observed in malignant tissues whereas lipid content was found low as compared to benign tissues. Principal component analysis obtained from the NMR data showed clear distinction between CC and GBC tissue specimens; however, 27 % of XGC tissues were classified with GBC. The partial least square discriminant analysis (PLS-DA) multivariate analysis between benign (CC, XGC) and malignant (GBC) on the training data set (CC; n = 51, XGC; n = 15, GBC; n = 19 tissues specimens) provided 100 % sensitivity and 94.12 % specificity. This PLS-DA model when executed on the spectra of unknown tissue specimens (CC; n = 15, XGC; n = 6, GBC; n = 6) classified them into the three histological categories with more than 95 % of diagnostic accuracy. Non-invasive in vivo MRS technique may be used in future to differentiate between benign (CC and XGC) and malignant (GBC) gall bladder diseases.

The effects of multivalent metal ions (Cu(2+)/Zn(2+)/Al(3+)) on the aggregation of salmon calcitonin (sCT)--a therapeutic peptide used worldwide in the treatment of osteoporosis and Paget's disease--have been studied in vitro using NMR (both solution state and solid state), TEM, ThT-fluorescence, and FT-IR spectroscopy. Overall, the various results indicated that the metal-ions-induced conformational transitions in the peptide--mostly toward the β-sheet--facilitate the aggregation of sCT in solution. First, the solution NMR has been used to check the interaction between the peptide and the metal ions. Following this, the formation and characterization of calcitonin aggregates has been performed using TEM, solid state NMR, and FT-IR spectroscopy. The TEM and ThT-fluorescence results revealed that the sCT peptide incubated with Cu(2+) and Zn(2+) metal ions (in aqueous environment) forms globular aggregates, while that with Al(3+) ions forms fibrils. The solid state NMR and FT-IR studies revealed the presence of a substantial amount of β-sheet content in sCT aggregates (formed in the presence of these metal ions) compared to the monomeric sCT, indicating that the metal binding is concomitant with conformational changes. The present study becomes crucial while prescribing this drug peptide under physio-pathological conditions associated with an abnormal accumulation of metal ions (Cu(2+)/Zn(2+)/Al(3+)) in the body (i.e., abnormal metal ion homeostasis).

Highlights: Black-Right-Pointing-Pointer Molecular iodine (I{sub 2}) causes non-apoptotic cell death in MDA-MB231 breast tumor cells. Black-Right-Pointing-Pointer Autophagy is activated as a survival mechanism in response to I{sub 2} in MDA-MB231. Black-Right-Pointing-Pointer Autophagy inhibition sensitizes tumor cells to I{sub 2}-induced apoptotic cell death. Black-Right-Pointing-Pointer Autophagy inhibitor potentiates apoptosis and tumor regressive effects of I{sub 2} in mice. -- Abstract: Estrogen receptor negative (ER{sup -ve}) and p53 mutant breast tumors are highly aggressive and have fewer treatment options. Previously, we showed that molecular Iodine (I{sub 2}) induces apoptosis in hormone responsive MCF-7 breast cancer cells, and non-apoptotic cell death in ER{sup -ve}-p53 mutant MDA-MB231 cells (Shrivastava, 2006). Here we show that I{sub 2} (3 {mu}M) treatment enhanced the features of autophagy in MDA-MB231 cells. Since autophagy is a cell survival response to most anti-cancer therapies, we used both in vitro and in vivo systems to determine whether ER{sup -ve} mammary tumors could be sensitized to I{sub 2}-induced apoptosis by inhibiting autophagy. Autophagy inhibition with chloroquine (CQ) and inhibitors for PI3K (3MA, LY294002) and H+/ATPase (baflomycin) resulted in enhanced cell death in I{sub 2} treated MDA-MB231 cells. Further, CQ (20 {mu}M) in combination with I{sub 2}, showed apoptotic features such as increased sub-G1more » fraction ({approx}5-fold), expression of cleaved caspase-9 and -3 compared to I{sub 2} treatment alone. Flowcytometry of I{sub 2} and CQ co-treated cells revealed increase in mitochondrial membrane permeability (p < 0.01) and translocation of cathepsin D activity to cytosol relative to I{sub 2} treatment. For in vivo studies ICRC mice were transplanted subcutaneously with MMTV-induced mammary tumors. A significant reduction in tumor volumes, as measured by MRI, was found in I{sub 2} and CQ co-treated mice relative to I{sub 2} or vehicle treated mice. These data indicate that inhibition of autophagy renders ER{sup -ve} breast tumor cells more sensitive to I{sub 2} induced apoptosis. Thus, I{sub 2} together with autophagy inhibitor could have a potential tumorostatic role in ER{sup -ve} aggressive breast tumors that may be evaluated in future studies.« less

Withania somnifera (L.) Dunal (Solanaceae), commonly known as Ashwagandha, is one of the most valued Indian medicinal plants with a number of pharmaceutical and nutraceutical applications. Metabolic profiling has been performed by HR-MAS NMR spectroscopy on fresh leaf and root tissue specimens from four chemotypes of W. somnifera. The HR-MAS NMR spectroscopy of lyophilized defatted leaf tissue specimens clearly distinguishes resonances of medicinally important secondary metabolites (withaferin A and withanone) and its distinctive quantitative variability among the chemotypes. A total of 41 metabolites were identified from both the leaf and root tissues of the chemotypes. The presence of methanol in leaf and root tissues of W. somnifera was detected by HR-MAS NMR spectroscopy. Multivariate principal component analysis (PCA) on HR-MAS 1H NMR spectra of leaves revealed clear variations in primary metabolites among the chemotypes. The results of the present study demonstrated an efficient method, which can be utilized for metabolite profiling of primary and secondary metabolites in medicinally important plants. Copyright © 2011 John Wiley & Sons, Ltd.

Pus samples obtained from 109 patients with liver abscess were examined by NMR spectroscopy. To our knowledge this is the first report on metabolic profiling of liver abscesses. Fifty metabolites were identified by combination of one (1D) and two-dimensional (2D) NMR spectra. Metabolic derangements were evaluated for differentiation between amoebic (ALA) and pyogenic liver abscess (PLA). The NMR results indicate that aspartate, asparagine and galactose, integral components of lipoproteophophoglycans (LPG) of the cell wall of Entamoeba histolytica are metabolic biomarkers of ALA. On the other hand, acetate, propionate, butyrate, succinate and formate, the fermentation products the facultative anaerobes are significantly prevalent in PLA. The NMR based metabolic profile of ALA and PLA are evaluated taking polymerase chain reaction (PCR) and bacterial culture as gold standard method. However, when NMR results were compared with culture and PCR methods, a correct diagnosis of 94.11% in ALA (n = 85) and 100% in PLA (n = 10) cases were observed. NMR spectroscopy in conjunction with PCR and culture can expedite in differentiating ALA from PLA.

Introduction Withania somnifera (Ashwagandha) is a high-value Ayurvedic medicinal plant and an important constituent of several dietary supplements. In order to substantiate the health claims, the herb has drawn considerable scientific attention. Objective The objective of the study was to investigate the alterations in primary and secondary metabolites of W. somnifera fruits during its maturity using NMR spectroscopy. Methodology Fruits at different stages of development from one week after fertilisation until maturity, classified in seven developmental stages, were analysed by a combined use of one- and two-dimensional NMR experiments. Results Seventeen metabolites were characterised and quantified from non-polar and polar extracts of different fruit development stages of W. somnifera. The principal component analysis of polar metabolites at different stages could be grossly classified into three metabolic phases, viz. initial phase, developmental phase and maturation phase. Conclusion Qualitative and quantitative analysis of metabolites in W. somnifera fruits indicated specific stages when fruits can be harvested for obtaining substantial bioactive ingredients for desirable pharmacological activity. This study potentially provides a complementary tool for quality control of herbal medicinal products when W. somnifera fruits are used. Copyright © 2011 John Wiley & Sons, Ltd.

Oral squamous cell carcinoma (SCC) represents more than 90% of all head and neck cancers as reported by Hermans (Cancer Imaging, 5(Spec No A), S52–S57, 2005), which draws attention of investigative research for novel predictive metabolic biomarkers to understand the malignancy induced biochemical perturbations occurring at molecular level. In the present work, proton HR-MAS NMR spectroscopic studies have been performed on resected human oral SCC tumor tissues, its neighboring margins and bed tissues (n = 159), obtained from 36 patients (n = 27 training set; n = 9 unknown test set), for the identification of metabolic fingerprints. The proton NMR spectra were then subjected to chemometric unsupervised PCA and supervised OSC-filtered PCA and PLS-DA multivariate analysis. Application of PLS-DA on orthogonally signal corrected training data-set (n = 120 tissue specimens; 27 patients) allowed >95% correct classification of malignant tissues from benign samples with >98% specificity and sensitivity. The OSC-PLS-DA model thus constructed was used to predict the class membership of unknown tissue specimens (n = 39) obtained from nine patients. These tissue samples were correctly predicted in its respective histological categories with 97.4% diagnostic accuracy. The regression coefficients obtained from OSC-filtered PLS-DA model indicated that malignant tissues had higher levels of glutamate, choline, phosphocholine, lactate, acetate, taurine, glycine, leucine, lysine, isoleucine and alanine, and lower levels of creatine and PUFA, representing altered metabolic processes (lipidogenesis, protein synthesis, and volume regulation) during tumor progression. Thus proton HR-MAS MR spectroscopy could efficiently identify the metabolic perturbations of malignant tumor from non-malignant bed and margins tissue specimens, which may be helpful in understanding the extent of tumor penetration in neighboring tissues.

Aim of the study was to undertake comprehensive metabolic profiling of plant parts of Commiphora wightii during two contrasting seasons i.e. summer and winter; compared seasonal metabolic variations; and assess antioxidant activity of fractions for commercial applications. Leaves, young stems and gum-resin extracts from summer and winter seasons were analyzed using GC-MS, HPLC and NMR spectroscopy. The antioxidant activity on each set was determined by DPPH free radical scavenging assay. Complete metabolic profiling from two contrasting seasons identified one hundred and four major known and unknown metabolites. Also, two alkylated phenols, 2,4-di- tert-butyl phenol and 3-(3,5-di- tert-butyl-4-hydroxyphenyl) propanoic acid not reported earlier from this taxon were isolated from the vegetative part. Comparative analysis of seasonal metabolic profiles of leaves, young stems and gum-resin revealed significant variations in concentrations of several metabolites. Multivariate principal component analysis (PCA) showed significant qualitative and quantitative variations in the polar (glycine, quinic acid and myo-inositol) and non-polar metabolites (alkylated phenols, guggulsterones and α-tocopherol) between the two seasons. Variation amongst metabolites such as myo-inositol, quinic acid α- tocopherol and alkylated phenols that are important for nutraceutical industry in the two contrasting seasons is a useful finding. These metabolites are of medicinal and nutraceutical importance and are commonly used in nutraceuticals and dietary supplement industry. DPPH radical scavenging activity (IC50 values) of polar and non-polar extracts varied significantly between summer and winter seasons. The antioxidant activity can be attributed to major polar metabolite, quinic acid biosynthesized in excess during winter, and to non-polar metabolites like alkylated phenols and α-tocopherol present during the summer season. The study shall be useful for medicinal, nutraceutical and dietary supplement industry for selection of polar or non-polar extracts from a particular season for obtaining targeted products with optimized functionality.

The bio-transformation of 3-β-hydroxy-5, 16-dien-pregnane-20-one (CDRI 80/574, 1) a novel antagonist of farnesoid X receptor (FXR) on hepatic membranes, with Aspergillus niger produced the oxidation products at C3 and / or C11 (compound 2 and 3) while the Aspergillus ochraeus promoted the oxidation at C11 and / or C15 (compound 4 and 5). The products were characterized using 2D-NMR spectroscopy.

Though tropical sprue (TS) is common in tropics, studies on small intestinal permeability (SIP) in TS are scant.SIP was evaluated using (1)H nuclear magnetic resonance (NMR) spectroscopy of urinary lactulose and mannitol in 24 patients with TS (22 before and 15 after treatment with tetracycline and folate) and in 31 healthy subjects (HS). Effect of treatment of TS on SIP and its relationship with outcome were studied.Subjects were comparable in terms of age and gender. Before treatment, urinary lactulose (0.24 mmol, 0-1.09 mmol versus 0.09 mmol, 0-0.68 mmol, P=0.02) and lactulose-to-mannitol (L/M) ratio (0.11, 0-0.41 versus 0.042, 0-0.26, P=0.001) were higher in TS than in HS, though mannitol was comparable (2.7 mmol, 0.61-10.5 mmol versus 3.8 mmol, 1.3-16.4 mmol, P=0.08). Patients improved after treatment [stool frequency (9, 4-20/day versus 1, 1-2/day, P0.0001), weight (44.4, 32-69 kg, versus 56, 39-84 kg, P0.0001), fecal fat (10.1, 6-26 g/24 h versus 4.4, 3.0-6.7 g/24 h, P0.0001), D-xylose (0.57, 0.28-1.2 g/5 g/5 h versus 1.1, 0.2-2.1 g/5 g/5 h, P0.0001), and small intestinal bacterial overgrowth (SIBO) resolved in 10/24 (41.7%) versus 1/15 (6.6%), P=0.02]. Though urinary lactulose (0.17, 0-4.3 mmol versus 0.09, 0-0.68 mmol, P=0.11) and mannitol (2.17, 0.8-36.7 mmol versus 3.84, 1.3-16.4 mmol, P=0.06) were comparable, L/M ratio was higher in TS than in HS (0.09, 0-0.22 versus 0.042, 0-0.26, P=0.002). L/M ratio was more often abnormal (cutoff 0.078) in TS than in HS [14/22 (63.6%) versus 3/31 (9.7%); P=0.0001], which persisted even after treatment [9/15 (60%) as compared with HS; P=0.0006]. Persistently abnormal SIP was associated with less weight gain and frequent stools following treatment.SIP is often abnormal in TS and remains unchanged even after successful treatment that was associated with less weight gain and more frequent stool.

Alterations in the anatomical structures, sap translocation and metabolic profiles in Jatropha curcas L. (Euphorbiaceae), infected with Jatropha mosaic virus (JMV) have been investigated using MRI and HR-MAS NMR spectroscopy. The contrast of MRI images distinguishes abnormalities in anatomical structures of infected and healthy stem. The HR-MAS NMR spectroscopic analysis indicated that viral infection significantly affected the plant metabolism. Higher accumulation of TCA cycle intermediates, such as citrate and malate, in JMV-infected plants suggested a higher rate of respiration. The respiration rate was more than twofold as compared to healthy ones. The viral stress also significantly increases the concentrations of alanine, arginine, glutamine, valine, GABA and choline as compared to healthy ones. Microscopic examination revealed severe hyperplasia caused by JMV with a considerable reduction in the size of stem cells. Lower concentration of glucose and sucrose in viral-infected stem tissues indicates decreased translocation of photosynthates from leaves to stem due to hyperplasia caused by JMV. The MR images distinguished stele, cortical and pith regions of JMV-infected and healthy stems. Contrast of T(1)- and T(2)-weighted images showed significant differences in the spatial distribution of water, lipids and macromolecules in virus-infected and healthy stem tissues. The results demonstrated the value of MRI and HR-MAS NMR spectroscopy in studying viral infection and metabolic shift in plants. The present methodology may help in better understanding the metabolic alterations during biotic stress in other plant species of agricultural and commercial importance.

Four xanthones were isolated from the roots of Andrographis paniculata using a combination of column and thin-layer chromatographic methods. They were characterized as (i) 1,8-di-hydroxy-3,7-dimethoxy-xanthone, (ii) 4,8-dihydroxy-2,7-dimethoxy-xanthone, (iii) 1,2-dihydroxy-6,8-dimethoxy-xanthone and (iv) 3,7,8-trimethoxy-1-hydroxy xanthone by IR, MS and NMR spectroscopic methods. In vitro study revealed that compound 1,2-dihydroxy-6,8-dimethoxy-xanthone possessed substantial anti-plasmodial activity against Plasmodium falciparum with its IC(50) value of 4 microg ml(-1). Xanthones bearing hydroxyl group at 2 position demonstrated most potent activity while xanthones with hydroxyl group at 1,4 or 8 position possessed very low activity. In vivo anti-malarial sensitivity test of this compound on Swiss Albino mice with Plasmodium berghei infection using Peters' 4-day test gave substantial reduction (62%) in parasitaemia after treating the mice with 30 mg kg(-1) dose. In vitro cytotoxicity against mammalian cells revealed that 1,2-dihydroxy-6,8-dimethoxy-xanthone is non-cytotoxic with its IC(50) > 32 microg ml(-1).

Interception of pregnancy in its initial stage is an attractive and viable approach to contraception. A chemical agent, taken within the first few days of missed menses, intercepts the conception, which is expelled with menstrual flow. The main targets of such agents are the uterus, blastocyst and the growing trophoblasts, whose nutritional requirement is inhibited. Our previous work has identified several nonsteroidal chemical entities as pregnancy interceptives in rodents and infrahuman primates. However, none reached clinical stage due to their ineffectiveness by oral route. Nevertheless, parallel to these rationally designed synthetic compounds, a program was ongoing to identify natural product(s) that can be used as interceptives. We are reporting for the first time the detailed profile of emetine ditartrate, a compound whose pregnancy interceptive efficacy has been studied in mouse, rat, hamster, guinea pig and rabbit by oral and intravaginal routes of administration. By the oral route, the compound caused 100% resorption of the fetuses in rat, hamster and guinea pig at 6.0, 5.0 and 3.0 mg/kg, respectively, on administration during peri- and early postimplantation periods of pregnancy (depending upon the day of implantation in each species). By intravaginal route, the compound was administered once in the form of a vaginal pessary on the day of implantation in respective species; interception of pregnancy was not achieved completely in rat and hamster at doses four to five times the oral dose in multi-day schedule. However, in guinea pig and rabbit it was fully effective at 7.0 and 70.0 mg/animal, respectively. The compound was devoid of estrogenic, antiestrogenic and progestational activity but possessed mild antiprogestational activity at the high dose in vivo. In in vitro assay, however, it did not show any significant binding to estrogen and progesterone receptors. The mode of action of the compound was found to be mainly on the uterus and early embryos around implantation, possibly on the trophoblasts and endometrial cells at the attachment site. The absence of 100% efficacy in rat and hamster by intravaginal route, but not by oral route, is possibly due to poor absorption of the compound through the vagina in these species. The guinea pig and rabbit, therefore, seem the better species for evaluating the efficacy of the compound administered by the vaginal route.

The first example of a pair of interconvertible diastereomeric epimers 2α/2β-hydroxy-2- p -hydroxybenzyl-3(2 H )-benzofuranone-7-C-β- d -glucopyranoside isolated from the heartwood of Pterocarpus marsupium is reported. The predominance of 1a over 1b was supported by dynamic exchange rates and activation parameters obtained from NMR studies. The mechanism of this unique phenomenon is thought to be operative by the formation of diketone as suggested by deuterium exchange.

Background Abscess formation is a common bacterial infection and requires an immediate antimicrobial approach for apposite treatment. Delay in patient treatment is usually a common feature, as the bacterial identification of clinical samples is based on the culture, which is a time-consuming exercise. The current study was aimed at developing an alternative technique with the potential for rapid bacterial group identification. Materials and methods In the present study we performed ex vivo proton magnetic resonance spectroscopy of 40 pus samples collected from abscesses in different locations and the results have been retrospectively compared with the microorganism identified in the pus culture. In addition, the microbes obtained from the culture have been further subcultured and studied with magnetic resonance spectroscopy to identify the bacterial fingerprint in the pus sample seen on spectroscopy. Results On reviewing the spectra obtained from the various abscesses, they were found to be qualitatively similar for a particular bacterium. The similar spectral pattern of the pus with obligate aerobes/anaerobes and pure cultures of the same bacteria suggests its strict metabolism under in vivo and in vitro conditions, respectively. Conclusions The characteristic metabolite pattern of obligate anaerobes may be used as a prototype for its rapid identification. This information may be of value for more appropriate clinical management of such cases.

The structural characterization of three compounds of a mixture of taxoids from the Taxus wallichiana was achieved by employing the combinations of two-dimensional 1 H– 1 H COSY, 1 H– 13 C HMQC, 1 H– 13 C HMBC and LC–MS techniques. In the mixture of taxoids, 13-acetyl-13-decinnamoyltaxchinin ( 3 ) showed some specific ROE correlations, which were found to be different from earlier reports for a similar molecule. Besides complementing the NMR results, LC–MS data also indicated the presence of three compounds.

The metabolite patterns obtained by ex vivo proton MR spectroscopy of fluid from different locations of hydatid cysts of sheep and humans (n = 16) and cysticercus cysts of swine and humans (n = 25) were compared with an objective of differentiating the two parasites on the basis of their metabolite pattern. The spectra from hydatid fluid differed from cysticercus cyst by the absence of creatine in the former. When the hydatid cyst was fertile, malate and/or fumarate was also observed, which was absent in cysticercus cyst. The most likely explanation for the presence of creatine only in the cysticercus fluid is its active diffusion from the surrounding host tissue along with a contribution from the musculature present in the bladder wall of the cyst.

Guggul gum resin from Commiphora wightii (syn. Commiphoramukul) has been used for centuries in Ayurveda to treat a variety of ailments. The NMR and GC-MS based non-targeted metabolite profiling identified 118 chemically diverse metabolites including amino acids, fatty acids, organic acids, phenolic acids, pregnane-derivatives, steroids, sterols, sugars, sugar alcohol, terpenoids, and tocopherol from aqueous and non-aqueous extracts of leaves, stem, roots, latex and fruits of C. wightii. Out of 118, 51 structurally diverse aqueous metabolites were characterized by NMR spectroscopy. For the first time quinic acid and myo-inositol were identified as the major metabolites in C. wightii. Very high concentration of quinic acid was found in fruits (553.5 ± 39.38 mg g(-1) dry wt.) and leaves (212.9 ± 10.37 mg g(-1) dry wt.). Similarly, high concentration of myo-inositol (168.8 ± 13.84 mg g(-1) dry wt.) was observed from fruits. The other metabolites of cosmeceutical, medicinal, nutraceutical and industrial significance such as α-tocopherol, n-methylpyrrolidone (NMP), trans-farnesol, prostaglandin F2, protocatechuic, gallic and cinnamic acids were identified from non-aqueous extracts using GC-MS. These important metabolites have thus far not been reported from this plant. Isolation of a fungal endophyte, (Nigrospora sps.) from this plant is the first report. The fungal endophyte produced a substantial quantity of bostrycin and deoxybostrycin known for their antitumor properties. Very high concentrations of quinic acid and myo-inositol in leaves and fruits; a substantial quantity of α-tocopherol and NMP in leaves, trans-farnesol in fruits, bostrycin and deoxybostrycin from its endophyte makes the taxa distinct, since these metabolites with medicinal properties find immense applications as dietary supplements and nutraceuticals.

1H Magnetic resonance imaging and 31P magnetic resonance spectroscopy (MRS) have been carried out in experimental rodent filariasis, i.e., Acanthocheilonema viteae infection in the rodent host, Mastomys coucha. The T2-weighted image of the infected host shows fine hyperintense thread like structures of adult filariid nests in the cervical region. 31P MRS of normal and infected hosts, localized over the same region of interest, show seven major peaks corresponding to phosphomonoesters (including glucose-6-phosphate, fructose-6-phosphate, fructose-1-6-diphosphate, phosphorylcholine, and adenine monophosphate or AMP), inorganic phosphate, glycerophosphorylcholine, phosphoenolpyruvate, phosphocreatine and nucleoside di- and tri-phosphates. Concentrations of phosphomonoesters (PMEs) are higher in the normal rodent compared with the infected ones. In vivo 31P MRS provides a non-invasive assessment of tissue bioenergetics and phospholipid metabolism.

In vivo, ex vivo, and in vitro proton magnetic resonance spectroscopy was performed in 12 patients with intracranial tuberculomas with an aim of detecting the biochemical constituents of Mycobacterium tuberculosis in a granuloma. One dimensional (1D) single pulse and spin-echo sequences and 2D correlative spectroscopy were used for the ex vivo study to confirm the resonances seen on in vivo study. Spectroscopic studies of the perchloric acid and lipid extract of granuloma and M. tuberculosis were performed to look for similarity of resonance. In vivo study showed the presence of lipids at 0.9, 1.3, 2.0, 2.8 ppm, and phosphoserine at 3.7 ppm. All these resonances were confirmed on ex vivo study. In addition, distinct resonances of serine and phenolic lipids were seen on ex vivo and in vitro study of tuberculous granuloma, which have not been observed in other intracranial tumors. Lipid extract of granuloma and M. tuberculosis showed phenolic lipids at 7.1 and 7.4 ppm, a constituent of the cell wall of the bacteria in a tuberculoma. It appears that it may be possible to finger print the biochemicals of the cell wall of M. tuberculosis in a tuberculous granuloma and thus may help in detection and diagnosis of such lesions.

Scrokoelziside A, isolated from the aerial parts of Scrophularia koelzii was shown to be 3-O-([alpha-L-rhamnopyranosyl-(1-->3), beta-D-glucopyranosyl-(1-->2)]-beta-D-fucopyranosyl-(1-->4)-beta-D- glucopyranosyl]-13 beta,28-epoxyolean-11-en-23-ol, on the basis one- and two-dimensional NMR homo- and hetero-nuclear spectroscopic evidence.

Withania somnifera (L.) Dunal (Solanaceae), commonly known as Ashwagandha, is one of the most valued Indian medicinal plant with several pharmaceutical and nutraceutical applications. Metabolic profiling was performed by GC-MS and NMR spectroscopy on the fruits obtained from four chemotypes of W. somnifera. A combination of (1)H NMR spectroscopy and GC-MS identified 82 chemically diverse metabolites consisting of organic acids, fatty acids, aliphatic and aromatic amino acids, polyols, sugars, sterols, tocopherols, phenolic acids and withanamides in the fruits of W. somnifera. The range of metabolites identified by GC-MS and NMR of W. somnifera fruits showed various known and unknown metabolites. The primary and secondary metabolites observed in this study represent MVA, DOXP, shikimic acid and phenylpropanoid biosynthetic metabolic pathways. Squalene and tocopherol have been rated as the most potent naturally occurring compounds with antioxidant properties. These compounds have been identified by us for the first time in the fruits of W. somnifera. Multivariate principal component analysis (PCA) on GC-MS and NMR data revealed clear distinctions in the primary and secondary metabolites among the chemotypes. The variation in the metabolite concentration among different chemotypes of the fruits of W. somnifera suggest that specific chemovars can be used to obtain substantial amounts of bioactive ingredients for use as potential pharmacological and nutraceuticals agents.

A new alkaloid, 15-β-stemmadenine, was isolated from the fruits of Tabernaemontana heyneana Wall. from the ethanolic extract. The complete 1H and 13C NMR assignments of the compound were carried out using COSY, HMQC and HMBC. Stereochemistry at C-15 and C-16 was established using ROESY and temperature-dependent 1H NMR studies. Copyright © 2002 John Wiley & Sons, Ltd.

Rv3097c of Mycobacterium tuberculosis encoding lipase (LipY) was overexpressed in Mycobacterium bovis BCG. Efficacy of recombinant BCG to protect against infection of M. tuberculosis was evaluated in mice. Whereas the parent BCG vaccine protected the mice against infection, recombinant BCG overexpressing LipY offered no protection as judged by viable counts of tubercule bacilli in lungs, weight of infected mice, pathology of lungs and survival of challenged mice. Downregulation of overexpression of LipY by antisense approach considerably restored protection of infected mice as observed with parent BCG vaccine. Overexpression of lipase in BCG caused extensive hydrolysis of triacylglycerol (TG) as identified by TLC, HPLC and NMR spectroscopy. A good correlation could be inferred between hydrolysis of TG and decrease in Th1 secreted IFNγ and IL-2, proinflammatory cytokines and survival of infected mice. Mice immunized with purified LipY antigen were protected and both proinflammatory and Th1 specific cytokines were augmented. TG was found to be a poor vaccine providing no protection, which appears to be due to attenuation of Th1 and proinflammatory immune responses. In conclusion this is the first experimental report to show that immunogenicity of BCG vaccine was impaired by LipY-induced hydrolysis of specific lipids leading to suppression of host immune responses.

The structures of two new minor diterpenes, 1,9-dideoxy coleonol-B and 1-acetoxy coleosol, isolated from the roots of Coleus forskohlii have been shown to be 7-hydroxy-6-acetoxy, 13-epoxy labd-14-en-11-one and 1-acetoxy-6,9-dihydroxy, 13-epoxy labd-14-en-11-one, respectively, mainly through interpretation of 2D NMR data and X-ray analysis.

The serendipitous formation of the novel, 20 membered, cyclo[bis-oxalyl cystine] - on treatment of cystine-di-OMe with oxalyl chloride - has provided an exceptionally short route to the first synthesis of the glucocorticoid receptor zinc template via PDT mediated thiol-disulfide exchange and metal complexation.

A new diterpene acid isolated from Pogostemon auricularis has been characterized as (4R, 5S, 8R, 9S, 10S, 14R)-cleistanth-12, 15-dien-19-oic acid by spectral and X-ray crystallographic analyses. Further, the stereochemistry at C-4 of auricularic acid (7) and its 7-hydroxy (6), 7-acetoxy (5), 7-senecioxy (4) and 7-(3-methylbutyroxy) (3) derivatives has been revised.

Profiling of metabolites is a rapidly expanding area of research for resolving metabolic pathways. Metabolic fingerprinting in medicinally important plants is critical to establishing the quality of herbal medicines. In the present study, metabolic profiling of crude extracts of leaf and root of Withania somnifera (Ashwagandha), an important medicinal plant of Indian system of medicine (ISM) was carried out using NMR and chromatographic (HPLC and GC-MS) techniques. A total of 62 major and minor primary and secondary metabolites from leaves and 48 from roots were unambiguously identified. Twenty-nine of these were common to the two tissues. These included fatty acids, organic acids, amino acids, sugars and sterol based compounds. Eleven bioactive sterol-lactone molecules were also identified. Twenty-seven of the identified metabolites were quantified. Highly significant qualitative and quantitative differences were noticed between the leaf and root tissues, particularly with respect to the secondary metabolites.

A new steroidal saponin, chloragin (1), was isolated and characterised from the aerial part of Chlorophytum nimonii. The structure of chloragin (1) was established as tigogenin-3-O-alpha-L-rhamnopyranosyl-(1 --4)-beta-D-glucopyranosyl-(1 --3)-beta-D-xylopyranosyl-(1 --4)-beta-D-glucopyranosyl-(1 --4)-beta-D-xyloopyranoside on the basis of detailed chemical and spectral evidence. The saponin showed potent antihyperglycaemic and antidyslipidaemic activities in albino rats.

Mycobacterium fortuitum is a non-tubercular fast growing pathogenic mycobacteria whose virulence factors have not been studied. Infection of M. fortuitum ATCC 6841 in a murine infection model leads to spinning of the head in 8-12 days after infection, 20-25% mortality and a constant bacillary load in the kidney of mice, suggesting persistence. From a TnphoA insertion library, a mutant MT13 was isolated which was attenuated in virulence with lesser bacterial burden, milder and delayed spinning of the head and no mortality of mice. The significant feature of the mutant was its failure to persist in kidney and thus the persistent bacillary load characteristic exhibited by the wild type strain was not observed. The insertion of transposon in MT13 was mapped in a region of the genome, which showed homology to Rv3291c of M. tuberculosis, annotated as a transcriptional regulatory factor and reported to be up regulated in nutrient starvation and anaerobic persistent states. Complementation of MT13 with rv3291c resulted in restoration of wild type characteristics including persistence in kidney suggesting the role of a Rv3291c homolog in the virulence and persistence of M. fortuitum.

Seed development in Jatropha curcas L was studied with respect to phenology, oil content, lipid profile and concentration of sterols. Seeds were collected at various stages of development starting from one week after fertilization and in an interval of five days thereafter till maturity. These were classified as stage I to stage VII. Moisture content of the seeds ranged from 8.8 to 90.3%; the lowest in mature seeds in stage VII and highest in stage 1. The seed area increased as the seed grew from stage I to stage VI (0.2-10.2 mm(2) per seed), however, the seed area shrunk at stage VII. Increase in seed area corresponded to increase in fresh weight of the seeds. H-1 NMR spectroscopy of hexane extracts made at different stages of seed development revealed the presence of free fatty acids (FFA), methyl esters of fatty acids (FAME) and triglycerol esters (TAG), along with small quantity of sterols. The young seeds synthesized predominantly polar lipids. Lipid synthesis was noticed nearly three weeks after fertilization. From the fourth week the seeds actively synthesized TAG. Stage Ill is a turning point in seed development since at this stage, the concentration of sterols decreased to negligible, there was very little FAME formation, accumulation of TAG increased substantially, and there was a Sudden decrease in FFA concentration. The findings can be helpful in understanding the biosynthesis and in efforts to improve biosynthesis of TAG and reduce FFA content in the mature seeds. (C) 2008 Elsevier Ltd. All rights reserved.

The needles of Taxus wallichiana gave a new brevifoliol derivative whose structure was established by spectral data.

S,S'-[disulfanediylbis(dialkylaminopropane-2,1-diyl)]bis- (dialkylaminothiocarbamate) (14-31) were prepared and evaluated for the spermicidal activity and antifungal activity. Dialkyldithiocarbamates (1-5) were reacted with epichlorohydrin to give 1-dialkylaminocarbothioic acid S-[(2,3-epithio)propyl]ester (7-11), these on further reaction with a secondary amine gave S,S'-[disulfanediylbis(dialkylaminopropane-2,1-diyl)]bis- (dialkylaminothiocarbamate) (14-31). Some of these compounds (16, 19-21, 23, 30, 31) were found to be very potent spermicidal agents with marginal antifungal activity. Two compounds (20, 21) were 25 times more active than nonoxynol-9 (N-9), the spermicide currently in the market.

The (1)H NMR spectroscopic method is suggested and its utility is demonstrated for the diagnosis of Klebsiella pneumoniae (K. pneumoniae) in urinary tract infection (UTI). K. pneumoniae have the specific property of metabolizing glycerol to 1,3-propanediol (1,3-PD), acetate, ethanol and succinate. The quantity of 1,3-PD produced correlates well with the viable bacterial count. Other common bacteria causing UTI (except for Citrobacter frundii), such as Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Enterobacter aerogenes, Acinetobacter baumanii, Proteus mirabilis, Enterococcus faecalis, Streptococcus gp B and Staphylococcus aureus do not metabolize glycerol under similar conditions. Citrobacter frundii (C. frundii) also gives the same NMR results but is easily differentiated as being motile on direct microscopic examination of urine and it is not common nosocomial infectious agent in urinary tract infection. The method provides a single-step documentation of K. pneumoniae (and C. frundii) qualitatively as well as quantitatively. Out of the total 614 subjects considered, clinical diagnosis of UTI was obtained in 516 cases (84%). The NMR-based screening had a sensitivity of 90%, a specificity of 100% and a false negativity of 10% relative to the conventional quantitative culture method. In the present authors' experience, the results of NMR spectroscopy based screening show a very good correlation with the diagnosis of urinary tract infected patients.

Two new limonoids, xyloccensin X (1) and xyloccensin Y (2), have been identified in mixture using NMR spectroscopy. Both limonoids were isolated in mixture from the fruit of the plant Xylocarpus molluccensis. The structures of these were proposed after extensive 2D NMR analyses in mixture. Copyright © 2006 John Wiley & Sons, Ltd.

Re-examination of the leaves of Nyctanthes arbor-tristis led to the isolation and identification of new minor iridoid glucoside, arborside D [1], as its acetyl derivative. The structure of the new compound was determined using spectral methods.

Background. Hydatid cysts, the larvae of the parasite Echinococcus granulosus , may lodge in any organ of intermediate hosts, namely, man and sheep. Complete cyst removal is the treatment of choice; however, spillage of fertile cysts during surgery leads to disease recurrence that may be prevented by preoperative detection of the fertility status of the cyst. With this perspective, ex vivo proton ( 1 H) MR spectroscopy of hydatid fluid of human and sheep origin was performed to differentiate fertile from sterile cysts on the basis of their metabolite pattern. Patients and materials. Cysts of sheep and human origin were used as source of hydatid fluid. A fraction of this fluid was tested for cyst fertility and the rest was used for ex vivo 1 H spectroscopy. Histopathology of the cyst wall was done as a gold standard for this study. Results. Of 10 sheep samples, 7 were fertile and 3 were sterile, while among 6 human samples, 5 were fertile and 1 was sterile. Spectroscopic and histopathological results corroborated each other. The fluid from microbiologically proven fertile cysts contained malate and fumarate along with other resonances and the histopathology of the fertile cyst wall demonstrated germinal lining and protoscoleces. Conclusions. The ex vivo spectroscopic differentiation of fertile and sterile cysts may be a stepping-stone for their in vivo separation in future and thus help in framing strategies for percutaneous/surgical management.