Metabolomics approach in pharmacognosy

This chapter introduces readers to metabolomics as a new tool in pharmacognosy research. The escalating cost of medicine and health care services warrant the development of new ways and approaches in remedying the problem. Application of metabolomics approach in various aspects of pharmacognosy research may assist in reducing the cost of drug discovery and development.Metabolomics is a holistic approach in understanding biological processes at a system level. It incorporates an extensive use of instrumentation (especially spectroscopy) and statistical methods. The tool has been successfully tested in solving numerous problems from diverse fields, and offers good promises of its benefits and potential use. This chapter discusses to the basic understanding and procedures in metabolomics, including sample selection, collection, data acquisition, and data analysis. Relevant topics to pharmacognosy are also discussed to expose readers to some examples of the investigations involving metabolomics

Discrimination and nitric oxide inhibitory activity correlation of ajwa dates from different grades and origin

This study was aimed at examining the variations in the metabolite constituents of the different Ajwa grades and farm origins. It is also targeted at establishing the correlations between the metabolite contents and the grades and further to the nitric oxide (NO) inhibitory activity. Identification of the metabolites was generated using ¹H-NMR spectroscopy metabolomics analyses utilizing multivariate methods. The NO inhibitory activity was determined using a Griess assay. Multivariate data analysis, for both supervised and unsupervised approaches, showed clusters among different grades of Ajwa dates obtained from different farms. The compounds that contribute towards the observed separation between Ajwa samples were suggested to be phenolic compounds, ascorbic acid and phenylalanine. Ajwa dates were shown to have different metabolite compositions and exhibited a wide range of NO inhibitory activity. It is also revealed that Ajwa Grade 1 from the al-Aliah farm exhibited more than 90% NO inhibitory activity compared to the other grades and origins. Phenolic compounds were among the compounds that played a role towards the greater capacity of NO inhibitory activity shown by Ajwa Grade 1 from the al-Aliah farm

Metabolite characterization of different palm date varieties and the correlation with their NO inhibitory activity, texture and sweetness

The aim of this study was to examine the variation in metabolite constituents of five commercial varieties of date fruits; Ajwa, Safawi and Ambar which originated from Madinah, the Iranian Bam and Tunisian Deglet Noor. The differences of metabolome were inves-tigated using proton nuclear magnetic resonance (1HNMR) spectroscopy combined with multivariate data analysis (MVDA). Principal Component Analysis (PCA) revealed clear separation between the date varieties. The Tunisian Deglet Noor demonstrated distinct cluster from the rest of the palm date samples based on the metabolite composition as shown by the pattern observed in Hierarchical Clustering Analysis (HCA) and PCA. Deglet Noor exhibited a significant higher level of sucrose (d5.40) and fructose (d4.16) in comparison with the other four varieties which can be associated with the distinctive sweet taste of this variety.Dates originated from Madinah and Tunisia exhibited a contrast manner in the amount of xylose and moisture content. These two aspects may contribute towards the soft texture of Tunisian dates. All Madinah dates were found to contain phenolic compounds which were well established as great antioxidant and anti-inflammatory agent. Ajwa dates exerted greater effect in inhibiting the generation of nitric oxide (NO) from the stimulated RAW264.7 cells at 95.37% inhibition. Succinic acid was suggested to have the most significant correlation with the trend of NO inhibitory shown by the selected date palm varieties

Chemical profile of xanthine oxidase inhibitor fraction of Persicaria hydropiper

Persicaria hydropiper, locally known as kesum, is an herb belongs to the family Polygonaceae. It has been used widely in many countries as food flavoring and possesses a wide range of medicinal values. The total phenolic content and xanthine oxidase inhibitory activity of the methanolic extract of P. hydropiper and fractions were determined spectrophotometrically. The butanol fraction was found to contain high phenolic content and was able to inhibit xanthine oxidase activity. Online profiling using liquid chromatography coupled with electrospray ionisation spectrometry (LC-ESIMS/MS) has revealed ten constituents in this active fraction. The major components were flavonoid derivatives and flavonoid sulphates, which were confirmed by comparison with an authentic standards as well as their MS/MS fragmentation patterns and UV spectra

Multivariate modelling analysis for prediction of glycidyl esters and 3-monochloropropane-1,2-diol (3-MCPD) formation in periodically heated palm oil

Palm oil is a vegetable oil that is widely used for cooking and deep-frying because of its affordability. However, repeatedly heated palm oil is also prone to oxidation due to its significant content of unsaturated fatty acids and other chemical toxicants such as glycidyl esters and 3-monochloropropane-1,2-diol (3-MCPD). Initially, the physicochemical properties such as colour, viscosity, peroxide, p-anisidine and total oxidation (TOTOX) of periodically heated palm oil were investigated. Chemical profiling and fingerprinting of six different brands of palm cooking oil during heating cycles between 90 and 360 min were conducted using Fourier transform infrared (FTIR) and 1H Nuclear Magnetic Resonance (NMR) metabolomics. In addition, the multivariate analysis was employed to evaluate the 1H NMR spectroscopic pattern of repeatedly heated palm oil with the corresponding physicochemical properties. The FTIR metabolomics showed significant different of the chemical fingerprinting subjected to heating duration, which in agreement with the result of 1H NMR metabolomics. Partial least squares (PLS) model revealed that most of the physicochemical properties of periodically heated palm oil are positively correlated (R2 values of 0.98–0.99) to their spectroscopic pattern. Based on the findings, the color of the oils darkened with increased heating time. The peroxide value (PV), p-anisidine value (p-AnV), and total oxidation (TOTOX) values increased significantly due to degradation of unsaturated compounds and oxidation products formed. We identified targeted metabolites (probable carcinogens) such as 3-monochloropropane-1,2-diol (3-MCPD) and glycidyl ester (GE), indicating the conversion of 3-MCPD to GE in repeatedly heated oils based on PCA and OPLSDA models. Our correlation analysis of NMR and physicochemical properties has shown that the conversion of 3-MCPD to GE was significantly increased from 180 to 360 min cooking time. The combination spectroscopic techniques with physicochemical properties are a reliable and robust methods to evaluate the characteristics, stability and chemical's structure changes of periodically heated palm oil, which may contribute to probable carcinogens development. This study has proven that combination of NMR and physicochemical analysis may predict the formation of the probable carcinogens of heated cooking oil over time which emphasizing the need to avoid certain heating cycles to mitigate formation of probable carcinogens during cooking process

Discrimination of Ipomoea aquatica cultivars and bioactivity correlations using NMR-based metabolomics approach

Ipomoea aquatica Forsk is a green leafy vegetable that is a rich source of minerals, proteins, vitamins, amino acids, and secondary metabolites. Different types of I. aquatica cultivars are grown for consumption but little is known about the metabolites variation. Proton nuclear magnetic resonance (1H NMR) spectroscopy combined with multivariate data analysis was applied for metabolic profiling of three I. aquatica cultivars including “broad leaf (K-25)”, “bamboo leaf (K-88)”, and “special pointed leaf (K-11)”. The orthogonal partial least squares discriminant analysis (OPLS-DA) indicated a clear separation among cultivars. The relative levels of various compounds, such as amino acids, organic acids, sugars, and phenolic compounds were specific to each cultivar. The K-11 cultivar was different from the other cultivars due to a high phenolic content. The content of sugars and some amino acids was higher in K-88 and K-25 possessed a higher content of organic acids. The in vitro study revealed that the I. aquatica cultivars exhibited potent antioxidant and α-glucosidase activities. The results of this study indicate that the K-11 cultivar was the most active due to the abundance of epicatechin, 4,5-dicaffeoylquinic, protocatechuic acid, and rutin

Metabolomic analysis and biochemical changes in the urine and serum of streptozotocin-induced normal- and obese-diabetic rats

Diabetes mellitus (DM) is a chronic disease that can affect metabolism of glucose and other metabolites. In this study, the normal-and obese-diabetic rats were compared to understand the diabetes disorders of type 1 and 2 diabetes mellitus. This was done byevaluating their urine metabolites using proton nuclear magnetic resonance (1H NMR)-based metabolomics and comparing withcontrols at different time points, considering the induction periods of obesity and diabetes. The biochemical parameters of theserum were also investigated. The obese-diabetic model was developed by feeding the rats a high-fat diet and inducing diabeticconditions with a low dose of streptozotocin (STZ) (25 mg/kg bw). However, the normal rats were induced by a high dose of STZ(55 mg/kg bw). A partial least squares discriminant analysis (PLS-DA) model showed the biomarkers of both DM typescompared to control. The synthesis and degradation of ketone bodies, tricarboxylic (TCA) cycles, and amino acid pathwayswere the ones most involved in the variation with the highest impact. The diabetic groups also exhibited a noticeable increase inthe plasma glucose level and lipid profile disorders compared to the control. There was also an increase in the plasma cholesteroland low-density lipoprotein (LDL) levels and a decline in the high-density lipoprotein (HDL) of diabetic rats. The normal-diabetic rats exhibited the highest effect of all parameters compared to the obese-diabetic rats in the advancement of the DMperiod. This finding can build a platform to understand the metabolic and biochemical complications of both types of DM and cangenerate ideas for finding targeted drugs

Phytochemical and bioactivity alterations of Curcuma species harvested at different growth stages by NMR-based metabolomics

Curcuma species is a popular traditional folk medicine in India and Southeast Asia. This study aims to profile the metabolites in four Curcuma species, including C. zedoaria, C. xanthorrhiza, C. aeruginosa and C. mangga at three developmental stages (seven, eight and nine months old) using nuclear magnetic resonance (NMR)-based metabolomics. Principal component analysis (PCA) showed that there are metabolites changes due to the month of harvest in each of the four species. A large quantity of curcumin and demethoxycurcumin contributed to the separation of C. xanthorrhiza, whereas the diterpenoids, such as curcumanggoside, (E)-labda-8(17),12-diene15,16-dial, calcaratarin A and zerumin B were responsible for the discrimination of C. mangga. Eight-month-old C. xanthorrhiza exhibited the highest nitric oxide (NO) inhibitory activity, while nine-month-old C. mangga exhibited the highest α-glucosidase inhibitory activity. The correlation among the bioactivities and phytochemical constituents was determined using partial least square (PLS) analysis. Curcumin, demethoxycurcumin, germacrone, zedoarol and xanthorrhizol were correlated with the NO inhibitory activity in C. xanthorrhiza, whereas curcumanggoside, labda-8(17),12-diene-15,16-dial and zerumin B were correlated with the α-glucosidase inhibitory activity in C. mangga. From the phytochemical markers and metabolic changes among growth stages of C. xanthorrhiza and C. mangga, the biosynthetic pathway was proposed to show the metabolites that might contribute to their health benefits

Identification of α-glucosidase inhibitory compounds from Curcuma mangga fractions

Curcuma mangga is a medicinal plant, and its rhizomes are often used to treat various conditions, such as fever, thorax pain, itching, stomachaches, skin diseases, gout, and asthma. Although C. mangga is commonly used, information on the relationship between its chemical constituents and the bioactivities of the rhizomes is still limited. The extraction solvents used have a strong effect on the metabolite profile and the bioactivity of the extract. A nuclear magnetic resonance (NMR)-based metabolomics approach was used to differentiate the metabolite profiles of hexane, chloroform, ethyl acetate, and methanol fractions of C. mangga rhizomes and to correlate the metabolites with α-glucosidase inhibitory activity. Primary and secondary metabolites were identified, including curcuminoids, carbohydrates, terpenoids, and amino acids. The ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) analysis of the most active fraction (ethyl acetate) revealed the identification of additional metabolites, such as zerumin A, epigallocatechin, p-hydroxycinnamic, and copallic acids. A partial least square (PLS) biplot demonstrated that the existence of curcumin, demethoxycurcumin, curcumanggoside, calcaratarin A, labda-8(17),12-diene-15,16-dial, zerumin B, and difurocumenonol in the ethyl acetate fraction could be responsible for the α-glucosidase inhibitory activity

NMR metabolomics for evaluating passage number and harvesting effects on mammalian cell metabolome

The variation in the extracellular metabolites of RAW 264.7 cells obtained from different passage numbers (passage 9, 12 and 14) was examined. The impact of different harvesting protocols (trypsinization and scraping) on recovery of intracellular metabolites was then assessed. The similarity and variation in the cell metabolome was investigated using 1H NMR metabolic profiling modeled using multivariate data analysis. The characterization and quantification of metabolites was performed to determine the passage-related and harvesting-dependent effects on impacted metabolic networks. The trypsinized RAW cells from lower passages gave higher intensities of most identified metabolites, including asparagine, serine and tryptophan. Principal component analysis revealed variation between cells from different passages and harvesting methods, as indicated by the formation of clusters in score plot. Analysis of S-plots revealed metabolites that acted as biomarkers in discriminating cells from different passages including acetate, serine, lactate and choline. Meanwhile lactate, glutamine and pyruvate served as biomarkers for differentiating trypsinized and scraped cells. In passage-dependent effects, glycolysis and TCA cycle were influential, whereas glycerophospholipid metabolism was affected by the harvesting method. Overall, it is proposed that typsinized RAW cells from lower passage numbers are more appropriate when conducting experiments related to NMR metabolomics