Desarrollo de nuevas metodologías multianalíticas para la obtención de extractos bioactivos y detección de fraudes en complementos alimenticios

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Química Física Aplicada. Fecha de Lectura: 22-03-2023Esta tesis tiene embargado el acceso al texto completo hasta el 22-09-2024Esta Tesis ha sido realizada en el Instituto de Química Orgánica General (CSIC) y en el Departamento de Calidad de la empresa Pharmactive Biotech Products SLU. Este trabajo de investigación ha sido financiado mediante la ayuda para contratos para la formación de doctores en empresas “Doctorados Industriales” 2019 (DIN2019-010849/AEI/10.13039/501100011033) del Ministerio de Ciencias e Innovación. Este programa permite la colaboración efectiva entre universidades y empresas, otorgando financiación para el desarrollo del proyecto de investigación en la universidad y para contratar al doctorando en la empres

A Combined Gas and Liquid Chromatographic Approach for Quality Evaluation of Saffron-Based Food Supplements

Considering the interest in the bioactive properties of saffron (Crocus sativus L.), as well as its limited production and high price, saffron-based food supplements (SFS) are highly susceptible to adulteration. However, their complex composition and the wide variety of potential fraudulent practices make the comprehensive assessment of SFS quality a challenging task that has been scarcely addressed. To that aim, a new multianalytical strategy based on gas chromatography coupled to mass spectrometry (GC-MS) and liquid chromatography with diode array detection coupled to mass spectrometry (HPLC-DAD-MS) was developed and validated in order to detect different frauds affecting SFS. Dried saffron stigmas and a commercial standardized saffron extract (affron®) were selected as reference samples (RS) to obtain an authenticity profile, which was further used to evaluate the quality of 17 SFS. Up to 17 crocins and crocetins, 5 kaempferol glycosides, picrocrocin (determined for the first time by GC-MS), safranal, furanone and isophorone-related compounds were determined in RS. Safranal and crocins were identified in all SFS except for one sample. However, discrepancies with the content declared were detected in 65% of the cases. Moreover, this multianalytical methodology also allowed identifying undeclared additives and the non-declared addition of vegetable sources other than saffron

A Combined Gas and Liquid Chromatographic Approach for Quality Evaluation of Saffron-Based Food Supplements

Considering the interest in the bioactive properties of saffron (Crocus sativus L.), as well as its limited production and high price, saffron-based food supplements (SFS) are highly susceptible to adulteration. However, their complex composition and the wide variety of potential fraudulent practices make the comprehensive assessment of SFS quality a challenging task that has been scarcely addressed. To that aim, a new multianalytical strategy based on gas chromatography coupled to mass spectrometry (GC-MS) and liquid chromatography with diode array detection coupled to mass spectrometry (HPLC-DAD-MS) was developed and validated in order to detect different frauds affecting SFS. Dried saffron stigmas and a commercial standardized saffron extract (affron®) were selected as reference samples (RS) to obtain an authenticity profile, which was further used to evaluate the quality of 17 SFS. Up to 17 crocins and crocetins, 5 kaempferol glycosides, picrocrocin (determined for the first time by GC-MS), safranal, furanone and isophorone-related compounds were determined in RS. Safranal and crocins were identified in all SFS except for one sample. However, discrepancies with the content declared were detected in 65% of the cases. Moreover, this multianalytical methodology also allowed identifying undeclared additives and the non-declared addition of vegetable sources other than saffron

Development of a new method using gas chromatography coupled to mass spectrometry to determine (−)-hydroxycitric acid in Garcinia gummi-gutta food supplements

Garcinia gummi-gutta based food supplements (GFS) are widely commercialized due to their antiobesity activity, which has been mainly attributed to their high content in (−)-hydroxycitric acid [(−)-HCA]. In order to overcome the limitations related to selectivity and sensitivity of the HPLC-UV methods usually employed for the analysis of this compound, a GC–MS method was fully developed for the first time and validated. Water with 0.1 % formic acid was used for the solubilization of (−)-HCA stabilized in the form of salt in GFS. After a drying step, different derivatization solvents were tested and only trifluoroacetic acid (TFA) allowed the complete dissolution of (−)-HCA. Regarding silylation, the best results were obtained by cold dropwise addition of hexamethyldisilazane and subsequent heating at 45 °C for 90 min. After optimizing the chromatographic conditions, the stability of derivatives for 1 month was also assessed. This new methodology was also applied to determine the (−)-HCA content of 12 commercial GFS. Experimental values ranged from 0.86 to 61%. Forty-two percent of the analyzed samples did not meet the (−)-HCA content declared on the label and 2 GFS showed HCA concentrations lower than 10% (0.86 and 6.0%), which puts into question their effectiveness in regulating obesity. One advantage of this new methodology is that it allows the qualitative and quantitative analysis of (−)-HCA in GFS without coelutions with other interfering compounds and it avoids the solubility problems presented by HCA salts. Moreover, the coupling of GC to MS provides more reliable identification of compounds present in GFS.This work is part of the I + D + I projects PID2019-106405 GB-I00 financed by MCIN/ AEI/10.13039/501100011033 and AGL2016- 80475-R, AEI/FEDER, UE, financed by MINECO. The authors would like to thank the Community of Madrid and European funding from FSE and FEDER programs for financial support (project S2018/BAA-4393, AVANSECAL-II-CM). A. Mena-García thanks the Spanish Ministry of Science, Innovation and Universities for an Industrial Doctorate grant (DIN2019-010849/AEI/10.13039/501100011033) awarded to Phar- mactive Biotech Products S.L.U

Fingerprint of Characteristic Saffron Compounds as Novel Standardization of Commercial Crocus sativus Extracts

Food supplements based on saffron (Crocus sativus L.) dried stigma extracts are widely consumed due to their multiple bioactive properties. Saffron extract (SE) standardization is of crucial importance, as it determines the reproducibility of the product quality and is essential for the evaluation of its bioactive effect and safety. Although SEs are commonly standardized considering their safranal content, the lack of specificity of the official methods may give inaccurate measurements. In addition to the development of more precise methodologies, the evaluation of alternative saffron components, such as crocins and picrocrocin, for standardization purposes would also be of interest. Thus, in this study, qualitative and quantitative information regarding picrocrocin and crocin isomers of different commercial saffron extracts was first obtained by a validated methodology using liquid chromatography (HPLC) coupled to diode array (DAD) and mass spectrometer (MS) detectors. Principal component analysis (PCA) was applied to gain insight into the compositional variability and natural grouping of SE. These studies suggested the potential use of the relative content of crocin isomers and trans-/cis-crocins and trans-4 GG/picrocrocin ratios as novel criteria for SE standardization. Their reproducibility and stability under controlled storage conditions for 36 months was demonstrated in a commercial standardized SE (affron®)

Development of a microwave-assisted extraction method for the recovery of bioactive inositols from lettuce (Lactuca sativa) byproducts

A microwave-assisted extraction (MAE) method was developed for the extraction of bioactive inositols (D-chiro- and myo-inositols) from lettuce (Lactuca sativa) leaves as a strategy for the revalorization of these agrofood residues. Gas chromatography-mass spectrometry was selected for the simultaneous determination of inositols and sugars (glucose, fructose, and sucrose) in these samples. A Box–Behnken experimental design was used to maximize the extraction of inositols based on the results of single factor tests. Optimal conditions of the extraction process were as follows: liquid-to-solid ratio of 100:1 v/w, 40°C, 30 min extraction time, 20:80 ethanol:water (v/v), and one extraction cycle. When compared with conventional solid-liquid extraction (SLE), MAE was found to be more effective for the extraction of target bioactive carbohydrates (MAE 5.42 mg/g dry sample versus SLE 4.01 mg/g dry sample). Then, MAE methodology was applied to the extraction of inositols from L. sativa leaves of different varieties (var. longifolia, var. capitata and var. crispa). D-chiro- and myo-inositol contents varied between 0.57–7.15 and 0.83–3.48 mg/g dry sample, respectively. Interfering sugars were removed from the extracts using a biotechnological procedure based on the use of Saccharomyces cerevisiae for 24 h. The developed methodology was a good alternative to classical procedures to obtain extracts enriched in inositols from lettuce residues, which could be of interest for the agrofood industry.This work has been funded by Ministerio de Economía, Industria y Competitividad (project AGL2016-80475-R, AEI/FEDER, UE), by CSIC (project 202080E216), by the Comunidad of Madrid and European funding from FSE and FEDER programs (project S2018/BAA-4393, AVANSECALII- CM), and by Universidad del Cauca (501100005682) of Colombia (project ID-4486)

Quality Evaluation of Dietary Supplements for Weight Loss Based on Garcinia cambogia

Food supplements of plant origin for weight control are increasingly being demanded by consumers as a way to promote good health. Among them, those based on Garcinia cambogia (GCFS) are widely commercialized considering their bioactive properties, mainly due to (-)-hydroxycitric acid ((-)-HCA). However, recently, controversy has arisen over their safety; thus, further research and continuous monitoring of their composition is required. Hence, in this work, a multi-analytical approach was followed to determine not only (-)-HCA but also other constituents of 18 GCFS, which could be used as quality markers to detect fraudulent practices in these samples. Discrepancies between the declared (-)-HCA content and that experimentally determined were detected by LC–UV in 33% of the samples. Moreover, GC–MS analyses of GCFS allowed the detection of different compounds not present in G. cambogia fruits and not declared on supplement labels, probably related to heat exposure or to the addition of excipients or other extracts. This multi-analytical methodology is shown to be advantageous to address different fraudulent practices affecting the quality of these supplements.This work is part of the I + D + I projects AGL2016-80475-R funded by the Spanish MINECO/AEI/FEDER, UE, and PID2019-106405GB-I00 financed by MCIN/AEI/10.13039/ 501100011033. The authors thank the Comunidad de Madrid and European funding from FSE and FEDER programs (project S2018/BAA-4393, AVANSECAL-II-CM) and the Universidad del Cauca (501100005682) of Colombia (project ID-4806) for their financial support. A. Mena-García was funded by a predoctoral contract (DIN2019-010849/AEI/10.13039/501100011033) supported by the Spanish Ministry of Science, Innovation and Universitie

Microwave assisted extraction of inositols for the valorization of legume by-products

Currently there is great interest in exploitation of agrofood by-products, such as those from legumes, as source of bioactive ingredients. To that aim, characterization of cyclitol and sugar composition of pods and seeds from different legume species has been carried out. A green Microwave Assisted Extraction (MAE) method was optimized for extraction of bioactive inositols, and both pod and seed yields were compared with those obtained by conventional reflux extraction. A subsequent Saccharomyces cerevisiae treatment was also evaluated for removal of interfering coextracted sugars. myo-Inositol was present in all legume pods and seeds and galactinol in most seeds. chiro-Inositol was found in soybean and basul, whereas outstanding pinitol concentrations were only detected in soybean. The optimized MAE method provided extracts rich in pinitol from soybean pods (41.5–58.0 mg g) and seeds (5.3–7.5 mg g). Removal of 97% of interfering sugars in MAE soybean pod extracts was achieved by yeast treatment.This work has been funded by Ministerio de Economía, Industria y Competitividad (project AGL2016-80475-R, AEI/FEDER, UE), by the Comunidad of Madrid and European funding from FSE and FEDER programs (project S2018/BAA-4393, AVANSECAL–II–CM) and by Universidad del Cauca of Colombia (projects ID-4486 and ID-4652