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(A) 1 H NMR of Astragalus boeticus extract registered in phosphate buffer in D 2 O (pH 6.0) and methanol d 4 (1:1). (B). 1 H NMR of Trigonella esculenta registered in phosphate buffer in D 2 O (pH 6.0) and methanol d 4 (1:1). ABBREVIATIONS: aa, aspartic acid; al, alanine; c, cycloartane; fl, flavonoids; f, fructose; g, glucose; h, 4-hydroxyisoleucine; k, kaempferol; malic acid; q, quercetin; st, standard; s, sugar; t, trigonelline.
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The discovery of bioactive compounds from natural sources entails an extremely lengthy process due to the timescale and complexity of traditional methodologies. In our study, we used a rapid NMR based metabolomic approach as tool to identify secondary metabolites with anti-proliferative activity against a panel of human colorectal cancer cell lines...
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... showed the presence of three main secondary metabolites. The first two molecules were identified as Figure 1. HCA dendrogram (A) and PCA (B) of cell growth percentage from control of colon cancer cell lines treated with the selected plant extracts over 48 hours. Based on these analyses the species are classified in three subsets. Groups I included the active species (black), II those active only at the highest tested doses (grey) and III those that have no-significant effect (white). quercetin and kaempferol (Table S1), two flavonols widely distributed in the plant kingdom, while the third metabolite was a cycloartane triterpene. Beyond the methylene H-19 protons at δ H 0.37 and 0.57, seven methyl singlets were clearly detectable in the 1 H NMR spectrum (Fig. 3A). In detail, signals at δ H 1.01 (δ C 15.5) and 1.06 (δ C 25.7) were assigned to the H-28 and H-29 methyls, based on HMBC heterocorrelations ( Fig. 4A) with C-5 methine (δ C 49.5), carbon that was found to bind the proton at δ H 1.66. This proton (H-5) correlated with the H-19 methylene and H-6 proton in the COSY experiment; this resonated at δ H 4.75 suggesting the presence of an oxygen atom binding the C-6 carbon. Furthermore, both C-28 and C-29 carbons correlated with the H-5 methine and H-3 proton at δ H 4.44 (δ C 88.4). Besides, the H-19 protons displayed heterocorrelations with the C-8 methine carbon (δ C 45.0), which in turn correlates with the H-30 methyl that resonated at δ H 0.99 (δ C 19.2). Furthermore, the signal at δ H 1.24 (δ C 20.3), attributed to the 18 methyl, and the peak at δ H 1.26 (δ C 27.2), related to the H-21 methyl, correlated with the C-17 carbon at δ C 57.7. In the COSY spectrum, the proton bound to this carbon revealed homocorrelations with the H-16 proton at δ H 4.64; this chemical shift value was well in agreement with the presence of an hydroxyl group in this position. A series of HMBC correlations, which were found between the H-21 proton and the C-20 carbon (δ C 87.6); the H-26 methyl (δ H 1.24) and the C-25 carbinol (δ C 72.0); the H-27 methyl (δ H 1.34) and the C-24 carbinol (δ C 81.2) allowed us to hypothesise the presence of a substituted tetrahydrofuran moiety in the side chain of the triterpene. All these data were in agreement with the presence of a cycloastragenol 25,26 . The downfield shift of the H-3 and C-3 values justified the presence of a sugar moiety, indi- cating a presumable site of glycosylation at position 3. This hypothesis was validated with an HMBC experiment, in which the C-3 carbon clearly heterocorrelated with the anomeric proton at δ H 4.44 (δ C 107.0). Finally, the H-6 proton showed cross peak with a carbonyl at δ C 172.5, which in turn correlated with the methyl at δ H 2.04 (δ C 21.2). These data demonstrated the presence of an acetyl group, which formed an ester bond with the hydroxyl group located at C-6 ...
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... esculenta. T. esculenta metabolome presented little variability in terms of secondary metabolites; indeed, the 1 H spectrum was dominated by of 4-hydroxyisoleucine signals (Fig. 3B). In an attempt to see beyond these primary metabolites, the crude extract was purified on a Sep-pak C-18 cartridge and the resulting metha- nol eluate was analysed by NMR. In the up-field region of the spectrum, peculiar doublets and singlets between 4.2 and 5.2 ppm suggested the presence of triterpenoid saponins. In particular, two singlets at δ H 0.83 and 1.04, alongside two doublets at δ H 0.94 and 1.01 suggested the presence of a steroid moiety 27 . In the HMBC experi- ment (Fig. 4B), the doublet at δ H 0.94 (δ C 17.6) correlated either with the methine C-24 at δ C 35.2 or a methylene carbinol C-28 at δ C 75.9 (δ H 3.70 and 3.40). This signal showed cross peaks with the doublet at δ H 4.73 (δ C 104.8), indicating a glycosylation site at position 28. Moreover, the doublet at δ H 1.01 (δ C 15.7) heterocorrelated with two methines at δ C 40.8 and 64.6, and with the acetal carbon at δ C 113.9. These data are consistent with the presence of a furostanol moiety in the molecule. A second site of glycosylation was identified at position 3; indeed, the carbinol carbon at δ C 79.2 correlated with the anomeric proton at δ H 4.40. Moreover, overlapped doublets between 1.20 and 1.30 ppm correlated in the HSQC experiment with carbons at δ C 18.4, 18.1 and 17.9. Finally, the correla- tions of these protons with carbinol carbons supported the presence of three deoxy ...
Citations
... Several studies have highlighted the therapeutic significance of helminths and helminth-derived excretory/secretory products (ESP) [168] in modulating host immune responses and protecting against inflammatory conditions (Table 2). Helminth-induced immune modulation is multifactorial and often involves the potent stimulation of Th2 responses in conjunction with IL10-and transforming growth factor-β (TGFβ)-dependent immune regulation [169][170][171] and decreased type 1 and 17 T helper (Th1/Th17) inflammation [172][173][174][175]. Helminths are also potent drivers of regulatory cell responses, including Foxp3+ regulatory T cells (Tregs) [171,175,176], IL10-producing type 1 regulatory T (Tr1) cells [177], type 2 innate lymphoid cells [178], alternatively activated macrophages [179] and regulatory dendritic cells [180,181]. ...
... To expedite the discovery of bioactive NPs in extracts, metabolomics data have been subjected to chemometric methods like multivariate data analysis, which correlate measured activity with nuclear magnetic resonance (NMR) and MS spectra signals, facilitating the tracking of active compounds in complex mixtures without additional bioassays [168,220]. Recent advancements in analytical technologies, particularly higher-field NMR instruments and probe technology, have allowed for precise structure determination of NPs even from limited quantities (<10 µg) [221]. Microcrystal electron diffraction, a cryoelectron-microscopy-based technique, is being increasingly applied for unambiguous structure determination of SMs in NP research [222]. ...
Research is increasingly revealing that inflammation significantly contributes to various diseases, particularly inflammatory bowel disease (IBD). IBD is a major medical challenge due to its chronic nature, affecting at least one in a thousand individuals in many Western countries, with rising incidence in developing nations. Historically, indigenous people have used natural products to treat ailments, including IBD. Ethnobotanically guided studies have shown that plant-derived extracts and compounds effectively modulate immune responses and reduce inflammation. Similarly, helminths and their products offer unique mechanisms to modulate host immunity and alleviate inflammatory responses. This review explored the pharmaceutical potential of Aboriginal remedial plants and helminths for treating IBD, emphasizing recent advances in discovering anti-inflammatory small-molecule drug leads. The literature from Scopus, MEDLINE Ovid, PubMed, Google Scholar, and Web of Science was retrieved using keywords such as natural product, small molecule, cytokines, remedial plants, and helminths. This review identified 55 important Aboriginal medicinal plants and 9 helminth species that have been studied for their anti-inflammatory properties using animal models and in vitro cell assays. For example, curcumin, berberine, and triptolide, which have been isolated from plants; and the excretory-secretory products and their protein, which have been collected from helminths, have demonstrated anti-inflammatory activity with lower toxicity and fewer side effects. High-throughput screening, molecular docking, artificial intelligence, and machine learning have been engaged in compound identification, while clustered regularly interspaced short palindromic repeats (CRISPR) gene editing and RNA sequencing have been employed to understand molecular interactions and regulations. While there is potential for pharmaceutical application of Aboriginal medicinal plants and gastrointestinal parasites in treating IBD, there is an urgent need to qualify these plant and helminth therapies through reproducible clinical and mechanistic studies.
... Moreover, the application of metabolomics in the screening of natural product extracts via hyphenated techniques allows detailed structural characterization [40]. It also permits implementing databases that can be used to discover new bioactive natural products [41]. ...
Malaria kills more than 500,000 people yearly, mainly affecting Africa and Southeast Asia. The disease is caused by the protozoan parasite from the genus Plasmodium, with Plasmodium vivax and Plasmodium falciparum being the main species that cause the disease in humans. Although substantial progress has been observed in malaria research in the last years, the threat of the spread of Plasmodium parasites persists. Artemisinin-resistant strains of this parasite have been reported mainly in Southeast Asia, highlighting the urgent need to develop more effective and safe antimalarial drugs. In this context, natural sources, mainly from flora, remain underexplored antimalarial spaces. The present mini-review explores this space focusing on plant extracts and some of their isolated natural products with at least in vitro antiplasmodial effects reported in the literature comprising the last five years (2018-2022).
... There are various chemical components in C. morifolium, such as flavonoids, sesquiterpenes, essential oils, and triterpenes [13]. The wide range of chemical variability and the huge number of metabolites in plants make the processes of detection and characterization challenging to perform [14]. Metabolomics is a powerful tool for identifying and analyzing metabolites for biological properties. ...
The current therapeutic approach for gout is through the inhibition of the xanthine oxidase (XO) enzyme. Allopurinol, a clinically used XO inhibitor, causes many side effects. This study aimed to investigate the interaction between XO and inhibitors identified from Chrysanthemum morifolium by using computational simulation and multispectroscopic methods. The crude extract, petroleum ether, ethyl acetate (EtOAc), and residual fractions were subjected to an XO inhibitory assay and 1H NMR analysis. The EtOAc fraction was shown to be strongly correlated to the XO inhibitory activity by using PLS biplot regression analysis. Kaempferol, apigenin, homovanillic acid, and trans-cinnamic acid were suggested to contribute to the XO inhibitory activity. Molecular docking showed that kaempferol and apigenin bound to the active site of XO with their benzopyran moiety sandwiched between Phe914 and Phe1009, interacting with Thr1010 and Arg880 by hydrogen bonding. Kaempferol showed the lowest binding energy in molecular dynamic simulation. The residues that contributed to the binding energy were Glu802, Arg880, Phe 914, and Phe 1009. A fluorescence quenching study showed a combination of static and dynamic quenching for all four inhibitors binding to XO. Circular dichroism spectroscopy revealed that there was no major change in XO conformation after binding with each inhibitor.
... NMR-based identification is extremely powerful for rapid identification of natural products with relative concentration determination. Furthermore, relating metabolites with bioactivities is feasible by integrating metabolite profiling with bioassay results [17,18]. In this study, we combined an NMR-based identification approach with an anti-inflammatory assay to select natural products with inhibitory effects on NO production in RAW 264.7 cells induced by poly I:C, a synthetic analog of dsRNA known to be produced by viral replication and presented in viral infection. ...
Antrodia camphorata is an endemic mushroom in Taiwan. This study was designed to screen anti-inflammatory compounds from the methanolic extract of the mycelium of A. camphorata on nitric oxide (NO) production in RAW 264.7 cells induced by polyinosinic-polycytidylic acid (poly I:C), a synthetic analog of double-stranded RNA (dsRNA) known to be present in viral infection. A combination of bioactivity-guided isolation with an NMR-based identification led to the isolation of 4-acetylantroquinonol B (1), along with seven compounds. The structure of new compounds (4 and 5) was elucidated by spectroscopic experiments, including MS, IR, and NMR analysis. The anti-inflammatory activity of all isolated compounds was assessed at non-cytotoxic concentrations. 4-Acetylantroquinonol B (1) was the most potent compound against poly I:C-induced NO production in RAW 264.7 cells with an IC50 value of 0.57 ± 0.06 μM.
... Furthermore, in some cell models, berberine has also been shown to promote cell death by autophagy, affecting the cycle cell arrest, or through epigenetic modulation [137,157]. Particularly, at the epigenetic level berberine has induced DNA methylation and histone alterations in HepG2, U266, and A549 cells directly affecting the cell proliferation, invasion, and metastasis in cancer development [161,162]. Thus, in the sight of the DNA damage and histone modifications observed with berberine treatment, complementary studies are needed to determine the selectivity index, safety, and toxicology profile of this alkaloid to establish the feasibility potential in cancer treatment. ...
Complex diseases such as neurodegenerative disorders and cancer constitute a growing public health problem due to the rising incidence and lack in effective therapies. Since pharmacotherapy based on a single target has been insufficient for drug development in complex diseases, the emerging multi-target approach is a promising strategy for the search of new drug candidates. Plant-derived isoquinoline alkaloids comprise a vast source of multimodal agents with unique structural diversity, and variated range of pharmacological activities. This review offers an exhaustive compilation of the pharmacological relevance and multi-target potential of natural isoquinolines, emphasizing their features and promising activity in complex diseases such as Alzheimer, Parkinson, and Cancer. Selected examples were discussed in depth to illustrate the most relevant structural motifs and their possible relationship with the multimodal activity offering a comprehensive baseline in the search and optimization of isoquinoline scaffolds with polypharmacological potential for complex diseases.
... The metabolites identified can be tested for its bioactivity (Graziani et al. 2018). Previous reports on halophilic bacteria Rhodobacter sphaeroides sp. ...
The present study was aimed at evaluating the cytotoxic potential of selected halophilic bacterial metabolites. The use of the metabolomics approach in identifying the unexplored bioactive metabolites from halophilic bacterial isolate reduces time and complex experiments. In our study, we used UV/Visible spectroscopy, LC-MS/MS, and NMR to identify the metabolites present in the methanolic extract of the halophilic bacterium Bacillus VITPS16. MTT assay revealed that metabolite fractions (S1-79.61% and S2-85.74%) possess cytotoxic activity. Colonogenic assay confirmed the cytotoxic potential of the fractions and apoptosis assays showed that 83.37% of the cells undergo apoptosis at 10 mg/mL concentration (MF-S2). The DNA binding studies revealed the metabolite fraction interacts with DNA resulting in cytotoxicity. The study states that MF- S2 induced an antiproliferative effect that led to apoptosis through DNA binding as one of the possible pathways. The toxicity analysis using zebrafish indicated that the metabolite fractions are non-toxic even at 10 mg/mL concentration. Fraction MF-S2 is found to contain phosphoethanolamines, glycerophospholipids, sphingolipids, apocarotenoid, enigmol and its analogue, ankaflavin and flavonoid type of metabolites, which have been previously reported to have anti-cancer activity.
Supplementary information:
The online version contains supplementary material available at 10.1007/s13205-021-02724-9.
... The rich chemical response that plants have to battle biotic and abiotic stress has drawn many plant scientists to study the plant metabolome. In the field of plant studies, metabolite profiling has been widely used not only to study stress response in plants but to assist with gene annotation [2], to perform plant phenotyping or chemotyping [3][4][5], to assist with disease detection or analysis [6] and to discover new medicinal compounds [7][8][9][10]. The chemicals identified through these studies include both primary and secondary plant metabolites. ...
Metabolomics plays an important role in various fields from health to agriculture. However, the comprehensive quantitative metabolomic analysis of plants and plant metabolites has not been widely performed. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS)-based plant metabolomics offers the sensitivity and breadth of coverage for both phenotyping and disease diagnosis of plants. Here, we report a high-coverage and quantitative MS-based assay for plant metabolite analysis. The assay detects and quantifies 206 primary and secondary plant metabolites, including many key plant hormones. In total, it measures 28 amino acids and derivatives, 27 organic acids, 20 biogenic amines and derivatives, 40 acylcarnitines, 90 phospholipids and C-6 sugars. All the analysis methods in this assay are based on LC-MS/MS techniques using both positive and negative-mode multiple reaction monitoring (MRM). The recovery rates of spiked plant samples at three different concentration levels (low, medium and high) ranged from 80% to 120%, with satisfactory precision values of less than 20%. This targeted plant metabolomic assay has been successfully applied to the analysis of large numbers of pine and spruce needle samples, canola root samples, as well as cannabis samples. Moreover, the assay was specifically developed in a 96-well plate format, which enables automated, high-throughput sample analysis. This assay has already been used to analyze over 1500 crop plant samples in less than two months.
... In addition, the PARADISe-deconvoluted peak areas of the alkaloids were imported into the GENE-E online software (https:// software.broadinstitute.org/GENE-E/index.html) for pattern recognition and classification through Hierarchical Cluster Analysis (HCA) [14]. The result was presented in the form of a dendrogram with a heat map, which illustrates the dissimilarity relationship between the harvest time combined with the relative concentration of each alkaloid represented in the colors red (high intensity), white (intermediate intensity) and blue (low intensity). ...
Hippeastrum elegans is an Amaryllidaceae species producing alkaloids with pharmaceutical potential including lycorine and galanthamine. Herein, we developed a non-targeted metabolomic study associated to chemometrics and biological evaluations to identify the H. elegans constituents that were able to reduce the human neutrophils proinflammatory mechanisms. The alkaloid fractions were extracted from bulbs cultivated for 15 months (m) and harvested in six harvest periods (5, 7, 9, 11, 13, and 15 m). The GC-MS analysis allowed the detection of 41 alkaloids being 31 identified. All alkaloid components varied over the cultivation and most of them were lycorine-type skeletons. Principal Component Analysis (PCA) and Hierarchical Cluster Analysis (HCA) distinguished three groups according to the chemical profile (group I: 5, 7, and 9 m; group II: 11 m and group III: 13 and 15 m). Therefore, the biological assays were only performed with one of the representative samples of each group: 7 m, 11 m and 15 m. None of them was toxic to human neutrophils by LDH activity and MTT test. The 7 m and 15 m-alkaloid fractions showed anti-inflammatory effects by reducing human neutrophil degranulation. However, the former one was more effective in inhibiting the cell activation based on the reduction of myeloperoxidase (MPO) release and reactive oxygen species (ROS) production. Afterwards, Partial Least Squares analysis (PLS) indicated lycorine and 11,12-dehydro-2-methoxy-assoanine as the compounds responsible for the anti-inflammatory activity of the bioactive fraction. Thus, the 7 m-alkaloid fraction of H. elegans seems to be a promising anti-inflammatory drug that deserves additional research.
... Many major flavonoids glycosides dominated by apigenin, luteolin, chrysoeriol and tricin were identified in T. coerulescens and T. foenum-graecum and Medicago species (Saleh et al., 1982). Triterpenoid saponins and steroids moiety were also detected in T. esculenta by NMR spectroscopy (Graziani et al., 2018). ...
... Numerous primary and secondary metabolites were identified which included great chemo-diversity, either from primary metabolites such as amino, organic acids and sugars or secondary metabolites such as flavonoids derivatives, alkaloids (trigonelline) and sterols. As compared to other analytical techniques, the NMR spectroscopy has strong ability to detect a diverse range of metabolites in a single run (Choi et al., 2006;Verpoorte et al., 2008;Graziani et al., 2018). The importance of PCA as a technique of multivariate data analysis is attributed to its role in the reduction of the dimensionality of multivariate datasets. ...
The metabolomic profiling of Trigonella foenum-graecum and Trigonella hamosa seeds was performed using NMR spectroscopy combined with multivariate data analysis (MVDA). The database of the chemical shifts and the J-resolved two-dimensional NMR technique were used to identify the metabolites. Additionally, the identified metabolites were confirmed by comparison with previously identified compounds as well. Among the elucidated metabolites, choline, sterols, leucine, valine, flavonoids derivatives and trigonelline were found to contribute to distinguish between the two species of Trigonella in score scatter and loading plots of multivariate data analysis. Phytochemical analysis of plant seeds extracts for total carbohydrates, proteins, phenolics, flavonoids, saponins and anthocyanins was performed by spectrophotometer. The antioxidant potential of both species was evaluated by phosphomolybdenum method and DPPH radical scavenging activity. T. foenum-graecum was characterized by higher concentrations of trigonelline, sterols, protein, flavonoids derivatives, saponins and anthocyanins, and lower IC50 in DPPH radical scavenging activity assay than T. hamosa. It was observed that total antioxidant capacity was positively correlated with glucose, trigonelline, protein, flavonoids, saponins and anthocyanins. T. foenum-graecum revealed an interesting metabolomic pattern with antioxidant activity that assigns this plant as a promising candidate to explore its targeted and non-targeted metabolomics profile along with potential bioactivities.
... Several metabolomics studies have been reported using cancer cell lines including breast, 17 pancreatic, 18 ovarian, 19 gastric, 20 and CRC cell lines. 21 Metabolic profiling on CRC cell lines, SW 1116 and SW 480 using gas chromatography-mass spectrometry (GC-MS) 22 revealed the differences in metabolism between CRC and healthy cells. Additionally, metabolomics on CRC cell lines of HT 29 highlighted perturbed metabolic pathways like glycolysis, tricarboxylic acid cycle, and pentose phosphate pathways. ...
Introduction:
Metabolomic studies on various colorectal cancer (CRC) cell lines have improved our understanding of the biochemical events underlying the disease. However, the metabolic profile dynamics associated with different stages of CRC progression is still lacking. Such information can provide further insights into the pathophysiology and progression of the disease that will prove useful in identifying specific targets for drug designing and therapeutics. Thus, our study aims to characterize the metabolite profiles in the established cell lines corresponding to different stages of CRC.
Methods:
Metabolite profiling of normal colon cell lines (CCD 841 CoN) and CRC cell lines corresponding to different stages, i.e., SW 1116 (stage A), HT 29 and SW 480 (stage B), HCT 15 and DLD-1 (stage C), and HCT 116 (stage D), was carried out using liquid chromatography-mass spectrometry (LC-MS). Mass Profiler Professional and Metaboanalyst 4.0 software were used for statistical and pathway analysis. METLIN database was used for the identification of metabolites.
Results:
We identified 72 differential metabolites compared between CRC cell lines of all the stages and normal colon cells. Principle component analysis and partial least squares discriminant analysis score plot were used to segregate normal and CRC cells, as well as CRC cells in different stages of the disease. Variable importance in projection score identified unique differential metabolites in CRC cells of the different stages. We identified 7 differential metabolites unique to stage A, 3 in stage B, 5 in stage C, and 5 in stage D.
Conclusion:
This study highlights the differential metabolite profiling in CRC cell lines corresponding to different stages. The identification of the differential metabolites in CRC cells at individual stages will lead to a better understanding of the pathophysiology of CRC development and progression and, hence, its application in treatment strategies.
