Application of HR-MAS NMR spectroscopy for studying chemotype variations of Withania somnifera (L.) Dunal.

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 (1) H 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.

Metabolic alterations of Withania somnifera (L.) dunal fruits at different developmental stages by NMR spectroscopy.

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.

Metabolic and histopathological alterations of Jatropha mosaic begomovirus-infected Jatropha curcas L. by HR-MAS NMR spectroscopy and magnetic resonance imaging.

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.

Lipid profiling of developing Jatropha curcas L. seeds using (1)H NMR spectroscopy.

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 I. The seed area increased as the seed grew from stage I to stage VI (0.2-10.2mm(2) per seed), however, the seed area shrunk at stage VII. Increase in seed area corresponded to increase in fresh weight of the seeds. (1)H 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 III 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.

Grilling enhances antidiarrheal activity of Terminalia bellerica Roxb. fruits.

ETHNOPHARMACOLOGICAL RELEVANCE: Terminalia bellerica Roxb. fruits are rich in a variety of biologically active ingredients. Tharu and Buksa tribes of Udham Singh Nagar, Uttarakhand, India use grilled fruits of Terminalia bellerica as an effective cure for diarrhea AIM OF THE STUDY: We validated the ethnobotanical claim by comparing the antidiarrheal effect of grilled fruits (GF) with dried fruits (DF). MATERIALS AND METHODS: The 50% ethanolic extracts of GF and DF were successively fractionated; the antioxidant and bacterial inhibition activity were studied using DPPH free radical scavenging, anti-lipid peroxidation and broth dilution method respectively. Difference in metabolites of ethyl acetate fractions of GF and DF was analyzed using GC-MS, gallic acid content was determined through HPTLC. Further the in-vivo antidiarrheal effect of ethyl acetate fractions of DF and GF was studied on castor oil induced diarrhea model. RESULTS: The ethyl acetate fractions showed potential DPPH free radical scavenging (IC50 11.13µg/ml in DF and 8.56µg/ml in GF), anti-lipid peroxidation and antibacterial activity. The non-targeted metabolic profiling showed higher content of tartaric acid, valeric acid, gallic acid, succinic acid, oxalic acid, malonic acid, malic acid, 1,2,3 trisbenzene, uridine and 11-eicosenoic acid in GF. The HPTLC results indicated that gallic acid content was 2.8 (±0.14) and 4.92 (±0.28) mg/g while ellagic acid content was 4.7 (±0.32) and 4.45 (±0.45) mg/g dry powder in DF and GF respectively. According to in vivo antidiarrheal activity DF and GF (100mg/kg oral) inhibited diarrhea by 41.87% and 71.72% respectively. CONCLUSION: Grilling significantly altered the levels of metabolites in T. bellerica fruits which could be responsible for its increased therapeutic potential.

Variability in Neem (Azadirachta indica) with respect to azadirachtin content.

There is a controversy over variations in azadirachtin content in neem (Azadirachta indica) seeds among various provenances and countries. Also, variations in azadirachtins are usually attributed to climatic conditions such as temperature and humidity. The present study was undertaken to evaluate qualitative and quantitative variability in azadirachtins A and B among various neem provenances or individual neem trees. Forty-three provenances of India were examined for intraprovenance variability in azadirachtin A and B content and oil percentage. Twenty-eight individual neem trees from five provenances of different agroclimatic regions were also examined for interprovenance variability. The azadirachtins were quantified using reversed phase analytical HPLC. There were wide variations in oil and azadirachtin contents among different provenances. Azadirachtin A ranged from 556.9 to 3030.8 mg kg(-)(1) of kernels, whereas azadirachtin B was in the range 43.1-590.6 mg kg(-)(1) of kernel among the provenances investigated. Analysis of variance among various neem provenances showed significant differences in oil content, azadirachtin A, total azadirachtin (A + B), and A:B ratio. There were individuals with high and low azadirachtins within a single provenance, and this trend was observed in all of the provenances selected from five agroclimatic regions of the country. Variations among individual trees of a particular provenance indicated that climatic factors such as rainfall, humidity, or temperature did not influence azadirachtin content in the neem trees. The present study shows that there are individual genetic differences among neem trees. A systematic study for tree improvement with a population of mother trees with desired traits should be undertaken by performing half-sib progeny trials and further selections by clonal propagations. The role of genetic makeup needs further research.

Changes in the metabolome and histopathology of Amaranthus hypochondriacus L. in response to Ageratum enation virus infection.

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 ß-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.

Occurrence of aflatoxins in mahua (Madhuca indica Gmel.) seeds: synergistic effect of plant extracts on inhibition of Aspergillus flavus growth and aflatoxin production.

Occurrence of aflatoxin in Madhuca indica Gmel. seeds was determined by competitive ELISA. Eighty percent of mahua seed samples were found to be contaminated with aflatoxin. Total aflatoxin content ranged from 115.35 to 400.54ppb whereas the concentration of AFB(1) was in the range of 86.43 to 382.45ppb. Mahua oil was extracted by cold press expeller and analysed for contamination of aflatoxin in both the oil and cake samples. Total aflatoxin and aflatoxin B(1) were 220.66 and 201.57ppb in oil as compared to that in cake samples where it was 87.55 and 74.35ppb, respectively. Various individual and combined plant extracts were evaluated for their efficacy against growth of Aspergillus flavus and aflatoxin production in vitro. Combination of botanicals were found to be more effective in controlling fungal growth and aflatoxin production than individual extracts. Results of the present study suggests that synergistic effect of plant extracts can be used for control of fungal growth and aflatoxin production. These natural plant products may successfully replace synthetic chemicals and provide an alternative method to protect mahua as well as other agricultural commodities of nutritional significance from toxigenic fungi such as A. flavus and aflatoxin production.