Biochemometric Approach Research Articles

Biochemometric-guided isolation of new Isosteroidal alkaloids from Fritillaria cirrhosa D.Don (Liliaceae, syn. Fritillaria roylei Hook) as acetylcholinesterase inhibitors

Globally, Alzheimer's disease is an urgent public health concern with the ageing population in developing nations. Recent studies have identified isosteroidal alkaloids as promising therapeutic agents for Alzheimer's treatment. Fritillaria species are well-known rich sources of steroidal and isosteroidal alkaloids. In this context, the current study focuses on the biochemometric-guided isolation of three previously undescribed and two known isosteroidal alkaloids as acetylcholinesterase (AChE) inhibitors from the bulbs of Fritillaria cirrhosa D.Don. The isolated molecules were characterized by NMR, HR-ESI-MS, FT-IR, and DP4+ analysis. Subsequently, all isolates were evaluated for AChE inhibitory activity using Ellman's method. Among the evaluated molecules, 1 (IC50: 33.0 ± 4.4 μM) and 5 (IC50: 24.7 ± 4.5 μM) showed promising AChE inhibition in vitro. Enzyme kinetic studies of isolated molecules revealed mixed inhibition kinetics with Ki varying from 1.3 to 24.4 μM. Moreover, the in silico studies showed excellent binding affinities of isolated molecules with the target protein and good drug-like ADMET properties. The present study identified new isosteroidal alkaloids as promising AChE inhibitors from F. cirrhosa bulbs via a biochemometric approach and advocated their further exploration for treating neurodegenerative disorders.

Unveiling the metabolites underlying the skin anti-ageing properties of Cytinus hypocistis (L.) L. through a biochemometric approach

The genus Cytinus, recognised as one of the most enigmatic in the plant kingdom, has garnered attention for its bioactive potential, particularly its skin anti-ageing properties. Despite this recognition, much remains to be accomplished regarding deciphering and isolating its most active compounds. This study aimed to identify the compounds responsible for C. hypocistis skin anti-ageing potential. Using multivariate analysis, a biochemometric approach was applied to identify the discriminant metabolites by integrating extracts' chemical profile (Liquid Chromatography-High-Resolution Mass Spectrometry, LCHRMS) and bioactive properties. The identified bioactive metabolite was structurally elucidated by 1D and 2D Nuclear Magnetic Resonance (NMR). Among the studied bioactivities, the anti-elastase results exhibited a significant variation among the samples from different years. After the biochemometric analysis, the compound 2,3:4,6-bis(hexahydroxydiphenoyl)glucose, with a molecular mass of 784.075 Da, was structurally elucidated as the discriminant feature responsible for the outstanding human neutrophil elastase inhibition. Remarkably, the subfraction containing this compound exhibited a tenfold improvement in neutrophil elastase inhibition efficacy compared to the crude extract; its effectiveness fell within the same range as SPCK, a potent irreversible neutrophil elastase inhibitor. Moreover, this subfraction displayed no cytotoxicity or phototoxicity and excellent efficacy for the tested anti-ageing properties. Hydrolysable tannins were confirmed as the metabolites behind C. hypocistis skin anti-ageing properties, effectively mitigating critical molecular mechanisms that influence the phenotypically distinct ageing clinical manifestations. Pedunculagin was particularly effective in inhibiting neutrophil elastase, considered one of the most destructive enzymes in skin ageing.

Biochemometry identifies ostruthin as pluripotent antimicrobial and anthelmintic agent from masterwort

The root extract of Peucedanum ostruthium (PO-E) was identified as a promising antibacterial source from a screening of 158 extracts against Staphylococcus aureus. It has also recently been shown to significantly decrease the survival of the nematode Caenorhabditis elegans. We used the biochemometric approach ELINA to investigate the phytochemical characteristics of the multicomponent mixture PO-E to identify the anti-infective constituent(s) targeting S.aureus and C.elegans.1H NMR spectra of PO-E-derived microfractions were correlated with their respective bioactivity data. Heterocovariance analyses unambiguously identified ostruthin as an anti-staphylococcal constituent, which potently also inhibited Enterococcus spp.. ELINA demonstrated that anthelmintic activity was due to a combinatorial effect of ostruthin and isoimperatorin. A C.elegans-based survival and motility assay confirmed that isoimperatorin, imperatorin, and verapamil modulated the susceptibility of ostruthin. The combinatorial effect of these natural products was shown in larvae studies to be related to the function of the nematodes' efflux pump.

Biochemometric approach to reveal Terminalia bentzoe cytotoxic effect against HepG2 cells in relation to its different organs and extraction solvents as analysed via UPLC-MS

Biochemometric approach to reveal Terminalia bentzoe cytotoxic effect against HepG2 cells in relation to its different organs and extraction solvents as analysed via UPLC-MS

Antiplasmodial activity of the bulbs of Fritillaria cirrhosa D.Don (Syn: Fritillaria roylei Hook.): UPLC-IM-Q-TOF-MS/MS-based biochemometric approach for the identification of marker compounds.

Fritillaria cirrhosaD.Don (Syn: Fritillaria roylei Hook) (Hindi name: Kshirakakoli) is a critically endangered Himalayan medicinal plant, well documented in Ayurveda for its therapeutic uses against various disorders such as jvara (fever), kasa (respiratory tract disease) etc. Its bulbs are also used as Szechuan-Pei-Mu for their antipyretic properties in the traditional Chinese medicine. However, despite its ethnomedicinal usage, the therapeutic use of F. cirrhosa bulbs for jvara (fever) related conditions such as malaria has remained unexplored. Hence in the context of increasing global concerns about drug-resistant malaria, it is important to investigate the antiplasmodial activity of F. cirrhosa bulbs for novel antimalarial agents. To investigate the antiplasmodial effects of the extracts/fractions of F. cirrhosa bulbs by the biochemometric approach and to rationalize its ethnopharmacological usage for jvara (fever) related conditions such as malaria. This study involves the UHPLC-MS-based plant material selection, preparation, quantification, and assessment of F. cirrhosa bulb extracts against CQ-sensitive Pf 3D7 & CQ-resistant Pf INDO strains. Further, UPLC-IM-Q-TOF-MS-based biochemometric approach has been applied for the identification of marker compounds responsible for the observed antiplasmodial effects. The identified marker compounds were also assessed for their in silico ADMET properties and binding efficacy with the drug transporter Pf CRT . Different F. cirrhosa bulb extracts/fractions showed promising antiplasmodial activity with IC50 values 2.71-19.77 μg/mL for CQ-resistant Pf INDO strain and 1.76-21.52 μg/mL for CQ-sensitive Pf 3D7 strain. UPLC-IM-Q-TOF-MS-based biochemometric analysis revealed four marker compounds i.e., peimine (m/z 432.3448), peimisine (m/z 428.3504), puqiedinone (m/z 414.3379), and puqiedine (m/z 416.3509) responsible for the observed antiplasmodial activity. The identified marker compounds showed excellent binding efficacy with Pf CRT and suitable drug-like properties in silico. The study demonstrated promising antiplasmodial activity of the chloroform and alkaloid enriched fractions of F. cirrhosa bulbs and further identified the four marker compounds responsible for the promising antiplasmodial activity. These marker compounds i.e., peimine, peimisine, puqiedinone and puqiedine were identified by the biochemometric analysis as the putative antiplasmodial constituents of the F. cirrhosa bulbs. Further, in silico studies indicated the good binding affinity of the marker compounds with Pf CRT along with suitable ADMET properties. Overall, the study elucidates the antiplasmodial activity of F. cirrhosa bulbs from the western Himalayan region and provides nascent scientific evidence for their ethnopharmacological usage in jvara (fever) related conditions such as malaria.

Biochemometry identifies suppressors of pro-inflammatory gene expression in Pterocarpus santalinus heartwood

The heartwood extract of the Ayurvedic medicinal plant Pterocarpus santalinus L. f. has previously been shown to significantly suppress the expression of CX3CL1 and other pro-inflammatory molecules in IL-1-stimulated human endothelial cells. Here, we identify the pigment-depleted extract PSD as the most promising yet still complex source of metabolites acting as an inhibitor of CX3CL1 gene expression. For the target-oriented identification of the constituents contributing to the observed in vitro anti-inflammatory effect of PSD, the biochemometric approach ELINA (Eliciting Nature's Activities) was applied. ELINA relies on the deconvolution of complex mixtures by generating microfractions with quantitative variances of constituents over several consecutive fractions. Therefore, PSD was separated into 35 microfractions by means of flash chromatography. Their 1H NMR data and bioactivity data were correlated by heterocovariance analysis. Complemented by LC-MS-ELSD data, ELINA differentiated between constituents with positive and detrimental effects towards activity and allowed for the prioritization of compounds to be isolated in the early steps of phytochemical investigation. A hyphenated high-performance counter-current chromatographic device (HPCCC+) was employed for efficient and targeted isolation of bioactive constituents. A total of 15 metabolites were isolated, including four previously unreported constituents and nine that have never been described before from red sandalwood. Nine isolates were probed for their inhibitory effects on CX3CL1 gene expression, of which four isoflavonoids, namely pterosonin A (1), santal (6), 7,3′-dimethylorobol (12) and the previously unreported compound pterosantalin A (2), were identified as pronounced inhibitors of CX3CL1 gene expression in vitro.

Assessing phytoequivalency of four Zingiberaceae spices (galangals, turmeric and ginger) using a biochemometric approach: A case study

The Zingiberaceae family (the ginger family) encompasses featured candidates used extensively in perfume, dyes, medicine as well as food industries. This study provides NMR metabolite fingerprinting, quantification and biological evaluation of four major plants viz. lesser and greater galangal, curcuma and ginger. 1H NMR derived metabolite profiles of the four spices were modeled via orthogonal partial least squares discriminate analysis (OPLS-DA). The cytotoxic activity of the four plants against colon and prostate cancer cell lines and in-vitro cyclooxygenase-1 inhibition activity were also evaluated. A correlation between the plants’ bioactivities and their 1H NMR metabolite profiles was established using OPLS and revealed putative metabolites responsible for these bioactivities. The most active cytotoxic plant was greater galangal due to its enrichment in galangal acetate. In contrast, lesser galangal exhibited the strongest anti-inflammatory action being enriched in 5-hydroxy-7-(4ꞌ-hydroxy-3 ꞌ-methoxyphenyl)-1-phenyl-3-heptanone and kaempferide. This study provides insight on the potential medicinal merit of genus Alpinia comparable to the more explored drugs turmeric and ginger within the Zingiberaceae family.

Antibacterial Screening, Biochemometric and Bioautographic Evaluation of the Non-Volatile Bioactive Components of Three Indigenous South African Salvia Species.

Salvia africana-lutea L., S. lanceolata L., and S. chamelaeagnea L. are used in South Africa as traditional medicines to treat infections. This paper describes an in-depth investigation into their antibacterial activities to identify bioactive compounds. Methanol extracts from 81 samples were screened against seven bacterial pathogens, using the microdilution assay. Biochemometric models were constructed using data derived from minimum inhibitory concentration (MIC) and ultra-performance liquid chromatography-mass spectrometry data. Active molecules in selected extracts were tentatively identified using high-performance thin layer chromatography (HPTLC), combined with bioautography, and finally, by analysis of active zone eluates by mass spectrometry (MS) via a dedicated interface. Salvia chamelaeagnea displayed notable activity towards all seven pathogens, and the activity, reflected by MICs, was superior to that of the other two species, as confirmed through ANOVA. Biochemometric models highlighted potentially bioactive compounds, including rosmanol methyl ether, epiisorosmanol methyl ether and carnosic acid. Bioautography assays revealed inhibition zones against A. baumannii, an increasingly multidrug-resistant pathogen. Mass spectral data of the eluted zones correlated to those revealed through biochemometric analysis. The study demonstrates the application of a biochemometric approach, bioautography, and direct MS analysis as useful tools for the rapid identification of bioactive constituents in plant extracts.

Biochemometric approach combined with 1D CSSF-TOCSY for the identification of sensitization agents in Curcuma longa L. and prediction of their action mechanisms

Herbal medicines are valuable resources of clinical anticancer drugs. However, due to the complicated composition of the medicines, the process of searching for anticancer components is laborious. In current study, we proved the sensitization effects of total extract of Curcuma longa L. on 5-FU against HCT8/FU, a 5-FU resistant colorectal cancer cell line. A biochemometric analytical method using orthogonal partial least squares-discriminant analysis (OPLS-DA) model was established to identify the potential sensitization agents in Curcuma longa L. and predict their possible action mechanisms simultaneously before the agents were obtained. By correlating the 1H NMR-based metabolic profiles of Curcuma longa L. extract with its specific biological functions, we screened out the proton signals being responsible for the effects. According to the 1D CSSF-TOCSY spectra, the proton signals were attributed to three compounds, including curcumin, demethoxycurcumin, and bisdemethoxycurcumin, respectively. Moreover, two mechanisms, including reversal of drug resistance and disruption in lactate excretion, were revealed to be possible explanations for the activity enhancement. To validate the method, the three compounds were obtained and evaluated for their combined effects with 5-FU against HCT8/FU cells. The results confirmed that all the compounds significantly enhanced the sensitivity of HCT8/FU cells to 5-FU treatment. Furthermore, downregulation of the drug resistance related transporters ABCG2, MRP1 and P-gp, and lactate membrane transporter MCT1 in the cells following combination treatments, corroborated the previous predictions about the action mechanisms obtained from the methods. This work may provide a method for the fast identification of active agents in the mixture, and, meanwhile, prediction of their possible action mechanisms before the process of isolation.

Biochemometry-Based Discovery of Phenylpropanoids from Azadirachta indica Fruits as Inhibitors of In Vitro Osteoclast Formation.

(1) Background: Inhibition of osteoclast differentiation is the key approach in treating osteoporosis. However, using state-of-the-art treatments such as bisphosphonates and estrogen-based therapy is usually accompanied by many side effects. As opposed to this, the use of natural products as an osteoporotic remedy delivers promising outcomes with minimal side effects. (2) Methods: In the present study, we implemented a biochemometric workflow comprising (i) chemometric approaches using NMR and mass spectrometry and (ii) cell biological approaches using an osteoclast cytochemical marker (TRAP). The workflow serves as a screening tool to pursue potential in vitro osteoclast inhibitors. (3) Results: The workflow allowed for the selective isolation of two phenylpropanoids (coniferyl alcohol and sinapyl alcohol) from the fruits of neem tree (Azadirachta indica). These two isolated phenylpropanoids showed a very promising dose-dependent inhibition of osteoclast differentiation with negligible effects in terms of cell viability. (4) Conclusion: The presented workflow is an effective tool in the discovery of potential candidates for osteoclast inhibition from complex extracts. The used biochemometric approach saves time, effort and costs while delivering precise hints to selectively isolate bioactive constituents.

Biochemometric Approach Combined with 1d Cssf-Tocsy for the Identification of Sensitization Agents in Curcuma Longa L. And Prediction of Their Action Mechanisms

Biochemometric Approach Combined with 1d Cssf-Tocsy for the Identification of Sensitization Agents in Curcuma Longa L. And Prediction of Their Action Mechanisms

Phoenix dactylifera L. date tree pollen fertility effects on female rats in relation to its UPLC-MS profile via a biochemometric approach

Date palm (Phoenix dactylifera L.) is among the most ancient cultivated crops, of special value owing to its fruits high nutritive and economic benefits. Asides, date palm pollen is a high energy material that has been used traditionally used for fertility enhancement. In this study, effects of date palm pollen crude extract and its fractions viz., petroleum ether, methylene chloride, ethyl acetate and n-butanol on the female reproductive system were evaluated for the first time in relation to its metabolite fingerprint.Fertility activity was evaluated in immature female rats by assessing their FSH-, LH- and estrogen- activities. To pinpoint active hormonal agents in crude pollen extract and fractions, UPLC- MS analysis was employed for metabolites profiling, and in correlation to extract/fraction bioassays using multivariate OPLS analysis. Results revealed that both polar n-butanol and non-polar petroleum ether fractions exhibited the strongest activities; with a significant increase in FSH (25.7 mIU/ml in n-butanol group), estradiol (414.7 pg/ml in petroleum ether group) and progesterone levels (122.4 pg/ml in n-butanol group). Correlation between UPLC-MS and fraction bioassays was attempted using multivariate OPLS analysis to reveal for bioactive hits in these fractions. This study provides the first report on the fertility effect of date palm pollen in female rats and in relation to its metabolite fingerprint.

Investigating antimicrobial compounds in South African Combretaceae species using a biochemometric approach

Ethnopharmacological relevanceMany species within the family Combretaceae are popular medicinal plants that are used traditionally to treat various conditions, of which many are related to bacterial infections. Global concerns regarding the increasing resistance of pathogens towards currently available antibiotics have encouraged researchers to find new drugs with antibacterial activity, particularly from plant sources. Aim of the studyThis study was aimed at exploring the broad-spectrum antibacterial potential of methanol extracts of species representing four genera of Combretaceae (Combretum, Pteleopsis, Quisqualis, Terminalia), indigenous to South Africa, using a biochemometric approach. Materials and methodsThe microdilution assay was used to determine the antibacterial activities, measured as minimum inhibitory concentrations (MICs), of the 51 methanol extracts representing 35 Combretaceae species, against nine species of pathogenic bacteria. Integrative biochemometric analysis was performed, thereby correlating the MIC values with the metabolomic data obtained from ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) analysis. Orthogonal projections to latent structures-discriminant analysis (OPLS-DA) models were constructed for six pathogens displaying variation in their susceptibility towards the extracts. ResultsEvaluation of the overall MIC values obtained indicated that extracts of species from the four genera displayed the highest activity towards Bacillus cereus ATCC 11778 (average MIC 0.52 mg/mL) and Salmonella typhimurium ATCC 14028 (average MIC 0.63 mg/mL). These bacteria were the most sensitive Gram-positive and Gram-negative bacteria, respectively. Extracts from Combretum acutifolium, Combretum imberbe and Combretum elaeagnoides were the most active, with average MIC values of 0.70 mg/mL, 0.52 mg/mL and 0.45 mg/mL, respectively. Five triterpenoid compounds were tentatively identified as biomarkers from the biochemometric analysis. ConclusionCorrelation of the phytochemistry of species from four genera in the Combretaceae family with antibacterial activity revealed that triterpenoids are responsible for the broad-spectrum antibacterial activity observed.

A Biochemometric Approach for the Identification of In Vitro Anti-Inflammatory Constituents in Masterwort.

Peucedanum ostruthium (L.) Koch, commonly known as masterwort, has a longstanding history as herbal remedy in the Alpine region of Austria, where the roots and rhizomes are traditionally used to treat disorders of the gastrointestinal and respiratory tract. Based on a significant NF-κB inhibitory activity of a P. ostruthium extract (PO-E), this study aimed to decipher those constituents contributing to the observed activity using a recently developed biochemometric approach named ELINA (Eliciting Nature’s Activities). This -omics tool relies on a deconvolution of the multicomponent mixture, which was employed by generating microfractions with quantitative variances of constituents over several consecutive fractions. Using an optimized and single high-performance counter-current chromatographic (HPCCC) fractionation step 31 microfractions of PO-E were obtained. 1H NMR data and bioactivity data from three in vitro cell-based assays, i.e., an NF-ĸB reporter-gene assay and two NF-κB target-gene assays (addressing the endothelial adhesion molecules E-selectin and VCAM-1) were collected for all microfractions. Applying heterocovariance analyses (HetCA) and statistical total correlation spectroscopy (STOCSY), quantitative variances of 1H NMR signals of neighboring fractions and their bioactivities were correlated. This revealed distinct chemical features crucial for the observed activities. Complemented by LC-MS-CAD data this biochemometric approach differentiated between active and inactive constituents of the complex mixture, which was confirmed by NF-κB reporter-gene testing of the isolates. In this way, four furanocoumarins (imperatorin, ostruthol, saxalin, and 2’-O-acetyloxypeucedanin), one coumarin (ostruthin), and one chromone (peucenin) were identified as NF-κB inhibiting constituents of PO-E contributing to the observed NF-ĸB inhibitory activity. Additionally, this approach also enabled the disclose of synergistic effects of the PO-E metabolites imperatorin and peucenin. In sum, prior to any isolation an early identification of even minor active constituents, e.g. peucenin and saxalin, ELINA enables the targeted isolation of bioactive constituents and, thus, to effectively accelerate the NP-based drug discovery process.

A biochemometric approach for the assessment ofPhyllanthus emblicafemale fertility effects as determinedviaUPLC-ESI-qTOF-MS and GC-MS

Phyllanthus emblica L. fruits have long been used in Ayurvedic medicine for their many health benefits. In this study, we present P. emblica fruit crude extract and fractions' effects on the female reproductive system by assessing its estrogenic and gonadotropic activities. Results revealed that the non-polar petroleum ether and chloroform fractions exhibited the strongest estrogenic and follicle-stimulating hormone-like [FSH] activity, while the n-butanol fraction exhibited a significant luteinizing hormone-like [LH] activity. The ethyl acetate fraction showed neither estrogenic nor gonadotropic activities and in contrast it may impair female fertility suggesting that different metabolite classes contribute to the plant's overall effect on female fertility. To pinpoint active agents in these fractions, UPLC/ESI-qTOF-MS- was employed for secondary metabolite profiling with 100 metabolites annotated including ellagitannins, gallic acid derivatives, terpeneoids, sterols, phthalates and fatty acids. Correlation between extracts/fraction bioassays and UPLC/MS data was attempted using orthogonal partial least squares-discriminant analysis (OPLS-DA) revealing that guaiane-type sesquiterpenes, phthalates, diterpenes and oxygenated fatty acids showed positive correlation with estrogenic and gonadotropic activities. In contrast, mucic acid gallates, gallic acid derivatives and ellagitannins correlated negatively. GC/MS analysis of the non-polar bioactive fractions viz. petroleum ether and chloroform was also attempted which revealed its enrichment in fatty acids/fatty acyl esters (34%) and phenolic compounds (19.6%). This study provides the first report on the estrogenic and gonadotropic activities of P. emblica fruits in relation to their metabolite fingerprint.

Identification of Intestinal UDP-Glucuronosyltransferase Inhibitors in Green Tea (Camellia sinensis) Using a Biochemometric Approach: Application to Raloxifene as a Test Drug via In Vitro to In Vivo Extrapolation

Green tea (Camellia sinensis) is a popular beverage worldwide, raising concern for adverse interactions when co-consumed with conventional drugs. Like many botanical natural products, green tea contains numerous polyphenolic constituents that undergo extensive glucuronidation. As such, the UDP-glucuronosyltransferases (UGTs), particularly intestinal UGTs, represent potential first-pass targets for green tea-drug interactions. Candidate intestinal UGT inhibitors were identified using a biochemometrics approach, which combines bioassay and chemometric data. Extracts and fractions prepared from four widely consumed teas were screened (20-180 μg/ml) as inhibitors of UGT activity (4-methylumbelliferone glucuronidation) in human intestinal microsomes; all demonstrated concentration-dependent inhibition. A biochemometrics-identified fraction rich in UGT inhibitors from a representative tea was purified further and subjected to second-stage biochemometric analysis. Five catechins were identified as major constituents in the bioactive subfractions and prioritized for further evaluation. Of these catechins, (-)-epicatechin gallate and (-)-epigallocatechin gallate showed concentration-dependent inhibition, with IC50 values (105 and 59 μM, respectively) near or below concentrations measured in a cup (240 ml) of tea (66 and 240 μM, respectively). Using the clinical intestinal UGT substrate raloxifene, the Ki values were ∼1.0 and 2.0 μM, respectively. Using estimated intestinal lumen and enterocyte inhibitor concentrations, a mechanistic static model predicted green tea to increase the raloxifene plasma area under the curve up to 6.1- and 1.3-fold, respectively. Application of this novel approach, which combines biochemometrics with in vitro-in vivo extrapolation, to other natural product-drug combinations will refine these procedures, informing the need for further evaluation via dynamic modeling and clinical testing.

Quantitative Purity–Activity Relationships of Natural Products: The Case of Anti-Tuberculosis Active Triterpenes from Oplopanax horridus

The present study provides an extension of the previously developed concept of purity-activity relationships (PARs) and enables the quantitative evaluation of the effects of multiple minor components on the bioactivity of residually complex natural products. The anti-tuberculosis active triterpenes from the Alaskan ethnobotanical Oplopanax horridus were selected as a case for the development of the quantitative PAR (QPAR) concept. The residual complexity of the purified triterpenes was initially evaluated by 1D- and 2D-NMR and identified as a combination of structurally related and unrelated impurities. Using a biochemometric approach, the qHNMR purity and anti-TB activity of successive chromatographic fractions of O. horridus triterpenes were correlated by linear regression analysis to generate a mathematical QPAR model. The results demonstrate that impurities, such as widely occurring monoglycerides, can have a profound impact on the observed antimycobacterial activity of triterpene-enriched fractions. The QPAR concept is shown to be capable of providing a quantitative assessment in situations where residually complex constitution contributes toward the biological activity of natural products.

Unbiased evaluation of bioactive secondary metabolites in complex matrices

The majority of bioactive principles in a complex matrix such as natural products and botanical medicines are secondary rather than primary metabolites. In addition to being chemically diverse, the bioactivity of an ethnobotanical can comprise from one to several bioactive compounds, present in a complex mixture. Conventional discovery efforts utilize bioassay-guided fractionation (BGF) to isolate individual active compounds. When applied to complex natural products, BGF is often challenged by an apparent loss of activity during fractionation, resulting in weakly active isolated compounds. Metabolomic analysis can potentially complement existing the BGF paradigm by capturing the chemical complexity of the metabolites. The proposed biochemometric approach establishes a link between the chemistry of a secondary metabolome and a deserved health impact, using a high-throughput, high-resolution capable biological endpoint. The proof of principle is demonstrated for the anti-tuberculosis (TB) activity of the Alaskan ethnobotanical, Oplopanax horridus. Biochemometric analysis identified the 100 most active constituents from thousands of metabolites in the active extract by means of 2D orthogonal chromatography using countercurrent and GC–MS methods. Previously isolated O. horridus phytoconstituents were used as reference markers of known structure and bio (in)activity. Positive correlations allowed distinction of anti-TB actives from inactive compounds. A total of 29 bioactives from 3 main structural classes were assigned based on MS data. Biochemometric analysis is a new tool for the standardization of herbal medicines and ethnobotanicals, as well as for drug discovery from nature. The method can assign multiple active compounds in complex mixtures without their prior isolation or structure elucidation, while still providing an interface to structural information.

Natural Product Research in TB Drug Discovery

With 16 M active and 9 M new cases, and an estimated 2 M lethal infections per annum, TB is a leading global infectious threat. TB treatment is still difficult, mainly due to multi-drug resistance (MDR). Natural products play an important role in the interdisciplinary approach to TB drug discovery taken at the ITR. In order to generate lead structures, explore drug targets, and evaluate alternative medicine, TB-specific biological and chemical tools are developed as part of an integrated pharmacognostic tactic against the globally rising (MDR-)TB problem. In order to redefine the TB bioassay-guided fractionation process from the biological perspective, new HTS-capable bioassay tools are employed such as luminescence assays using luciferase recombinant H37Rv strains of Mycobacterium tuberculosis, and a reporter gene low oxygen recovery assay (LORA) that mimics TB dormancy conditions in vitro. On the natural product chemistry end, a new biochemometric approach that utilized high-resolution bio-chromatograms has been developed and used to pin down the active principle of anti-TB botanicals. Method development in countercurrent chromatography (CCC) and nuclear magnetic resonance (NMR) technology fosters the chemical characterization of potential drug lead structures, supports the characterization of well-defined natural products mixtures (qNMR) that can be further evaluated in vitro (macrophage assay) and even in vivo (TB infected mice). The interplay of mycobacteriological and chemical methods will be exemplified by experimental proof of anti-TB synergism of natural products and small molecule metabolomics of TB in different growth stages.

Vitamins C and E reverse effect of herbicide-induced toxicity on human epidermal cells HaCaT: a biochemometric approach

The purpose of this study was to investigate and compare the cytotoxicity of glyphosate alone or included in Roundup 3 plus ® modulated by the cytoprotective effects of additional antioxidants such as Vitamin C and Vitamin E on the human keratinocytes cell line HaCaT. An experimental design which allows to minimize the number of experiments was carried out to determine the optimal conditions for cytoprotection against herbicide-induced toxicity. It was shown that HaCaT cell line provides a useful model to study components with toxicity or antioxidant activity. Our results indicated that (i) glyphosate-based formulations can be responsible for oxidative damage to human epidermal cells, (ii) antioxidant compounds should be associated to herbicide formulations to decrease their deleterious effects on human skin. The use of an experimental design connected with the simplex method can be consider to be a fast technique to classify, with a limited number of experiments, the respective role of five parameters in the in vitro cytoprotection by antioxidants of herbicide-induced toxicity.