High-performance Liquid Chromatography Data Research Articles

Novel Double Fusion (βδβ) Hemoglobin Variant with Comparison to Lepore Hemoglobins

Lepore hemoglobins (Hbs) such as Hb Lepore-Boston-Washington [δ(1-87) β(116-146)], Hb Lepore-Baltimore [δ(1-50) β(86-146)], Hb Lepore-Hollandia [δ(1-22) β(50-146)] are δβ gene fusion/deletion variants (ε- Gγ- Aγ-[δβ] v )(Figure 1). They are controlled by the weak δ-globin gene promoter and synthesized at approximately 10-15% of hemoglobin fractions. This results in a similar phenotype as β +-thalassemia (microcytosis and hypochromia ± mild anemia). Anti-Lepore hemoglobins such as Hb P-Nilotic (β(1-22) δ(50-146) are βδ gene fusion/duplication variants (ε- Gγ- Aγ-δ-[βδ] v-β) associated with normal CBC values. Herein we describe a novel βδβ double fusion variant (ε- Gγ- Aγ-δ-[βδβ] v). Due to high homology between the δ and β genes, this double fusion event encodes an identical protein to Hb Lepore-Hollandia, albeit linked to the β-promoter and therefore expressed at ~40% of hemoglobin fractions, which would be expected to enhance any intrinsic properties of the variant. This novel hemoglobin variant is named Hb Lepore Rochester-MN. Two cases have been identified (Table 1). Case 1 was a 10-month-old Caucasian/Native American infant referred for an abnormal newborn screen and a reported family history of Hb G-Coushatta (β22, GAA>GCA, Glu>Ala; HGVS: c.68A>C, p.E23A). Case 2 was a 3-month-old infant referred for an abnormal newborn screen. CBCs were normal for age including MCV. Cation-exchange high performance liquid chromatography (HPLC) showed a shouldered peak in the Hb A 2 window typical for Hb Lepore (elution time: 3.41-3.46 mins), although the variant percentage was higher than expected, mimicking Hb G-Coushatta (expected elution time: 3.33 mins). Capillary electrophoresis identified a peak in the D zone (Z(D) zone), which is expected for Hb Lepore or Hb G-Coushatta. Intact mass spectrometry was consistent with Hb Lepore-Hollandia at 15836 amu, differing from Hb G-Coushatta (Glu>Ala, 15809 amu). Multiplex ligand probe amplification (MLPA) was normal because the highly variable fusion regions are unsuitable for probe placement. Sanger sequencing of the β-globin gene identified multiple single base substitutions which were challenging to interpret but matched the sequence expected for a complex double crossover event (NM_000518.4:c.28_68delins41 (p.Ser10_Glu23delins14TAVNALWGKVNVDA). The first crossover is located between c.-29 and c.9, the second crossover is located between c.69 and c.92+17. Long-read sequencing was performed (Sequel, Pacific Biosciences) which detected the multiple missense mutations in cis conformation, as was corroborated by the mass spectrometry data. In summary, Hb Lepore Rochester-MN represents a novel double fusion [βδβ] variant class that will pose a diagnostic challenge for laboratories. Although it is likely a result of sequential crossover events (deletion and duplication) between Lepore and anti-Lepore variants, MLPA analysis is normal due to technical constraints and high homology. DNA sequencing (both Sanger and long read methods) yielded complex results that require sophisticated analysis and correlation with protein data including intact mass spectrometry. HPLC and CE data including variant percentage are very similar to Hb G-Coushatta. In addition, although resulting in an identical protein, the variant should not be mistaken for Hb Lepore-Hollandia, which is produced at lower levels and causes a thalassemic phenotype. Hb Lepore Rochester-MN is controlled by the β-promoter and therefore is associated with approximately 40% variant and apparently normal CBC values in the two infants.

Global estimation of phytoplankton pigment concentrations from satellite data using a deep-learning-based model

Based on a global matchup between satellite observations and high performance liquid chromatography (HPLC) measurements, we developed a deep-learning-based model (DL-PPCE model) for globally estimating concentrations of 17 different phytoplankton pigments. The model adopted a fusion architecture of residual and pyramid networks to achieve robust estimation performance. The model inputs include three different data types: essential ocean color parameters, satellite-derived environmental parameters, and the slope of above-surface remote-sensing reflectance (Rrs). We compared the model performances with various input parameters to determine the most effective inputs. The results showed that Rrs in the essential ocean color parameters and sea surface temperature (SST) in the environmental parameters were the most critical input parameters. The estimation of phytoplankton pigment concentrations was validated against HPLC data using the leave-one-out cross-validation method. Except for three pigments, 19′-butanoyloxy-fucoxanthin, prasinoxanthin, and lutein, the estimated pigment concentrations and in-situ observations were strongly correlated for all other pigments (an average relative root-mean-square error of 0.59, R2≥0.60, and regression slopes close to 1). In addition, a time series analysis was performed on the MODIS retrieved global pigment concentrations during 2003–2021 using the established DL-PPCE model to explore the relationship between the distribution of phytoplankton groups and El Niño in the western equatorial Pacific. Our findings revealed that the prokaryotes-dominated area extended eastward from180°E to 150°W during the 2015/2016 El Niño event. From 2003 to 2021, prokaryotic abundance was positively correlated with El Niño intensity (R=0.65,P≪0.01) but negatively correlated with the abundance of the entire phytoplankton community (R=−0.53,P≪0.01). These results demonstrate that the DL-PPCE model presents a novel approach for estimating the concentration of 17 pigments worldwide, and the estimated pigment concentrations are advantageous for analyzing the phytoplankton community dynamics on a large spatiotemporal scale.

Improving Chuanxiong Rhizoma quality standards using an effect-constituent index based bioassay

Chuanxiong Rhizoma is a traditional Chinese medicine (TCM) that is used to promote blood circulation. We set out to improve Chuanxiong Rhizoma quality standards using a bioassay-based Effect-constituent Index (ECI). We performed high performance liquid chromatography (HPLC) analysis to determine the chemical constituents of 10 Chuanxiong Rhizoma samples from different locations. We then constructed a direct bioassay method to investigate each sample’s antiplatelet aggregation effects. To screen for active ingredients that promote antiplatelet aggregation, we carried out Pearson correlation analyses between biopotency and compounds identified in the HPLC data. We developed an ECI of platelet aggregation inhibition using a multi-indicator synthetic evaluation method based on the integration of biopotency and active constituents. To further assess the biopotency-based Chuanxiong Rhizoma quality evaluation result accuracy, we compared the ECI with the chemical indicator’ method. Eight common chemical fingerprints peaks indicated notable content variation among samples. Biological evaluation showed that all 10 samples could inhibit platelet aggregation, although they had significantly different biological potencies. Using spectrum-effect relationships, we determined that Ligustilide was the significant active constituent responsible for antiplatelet aggregation. Using correlation analysis, we found that ECI correlated with the Chuanxiong Rhizoma extract’s platelet aggregation inhibitory effect. Additionally, ECI proved to be a good indicator of Chuanxiong Rhizoma quality, whereas chemical indicators failed to distinguish and predict the biopotency-based quality grade. This work indicates that ECI is a useful tool for associating sample quality with chemical markers linked to TCM clinical effects. ECI also provides a paradigm for improving the quality control of other TCMs that invigorate blood circulation.

Carotenoids from female Grapsus albolineatus as potential anti-ageing compounds

Introduction: Grapsus albolineatus is thought to have a high concentration of carotenoid pigments. Although studies on male G. albolineatus have been conducted, no studies on pigment extraction from female G. albolineatus have been carried out. Carotenoids have a high ability to fight free radicals. Previous research has shown that carotenoids can fight free radicals that cause premature skin-aging. Aim: The purpose of this research was to find out what kinds of carotenoids are found in G. albolineatus and whether they can act as natural inhibitors of proteins that cause premature aging, such as glucogenase, elastase, and hyaluronidase enzymes. Methods: Carotenoids were extracted from G. albolineatus using column chromatography and high-performance liquid chromatography (HPLC); molecular docking and visualization were done with Autodock 4.2 and Discovery Studio/Biovia, respectively. Results: According to HPLC data, there are carotenoid pigments such as didehydroastaxanthin, tetrahydroastaxanthin, dihydroastaxanthin, diatoxanthin, astaxanthin, and adonixanthin. According to molecular docking experiments, pigment carotenoids from G. albolineatus are efficient inhibitors of protein elastase and hyaluronidase with binding energy range -7.58 kcal/mol - -9.03 kcal/mol and -6.16 kcal/mol - -7.71 kcal/mol, respectively. Conclusions: G. albolineatus carotenoids have the potential to be anti-aging since they are more effective as protein elastase and hyaluronidase inhibitors than their native inhibitors.

Sustainable utilization of dairy waste paneer whey by Pediococcus pentosaceus NCDC 273 for lactic acid production

The current study was performed to test the ability of the Pediococcus pentosaceus NCDC 273 strain for rapid utilization of lactose from paneer whey. We optimized the lactose utilization by monitoring the constitutive expression of the β-gal gene using real-time RT-PCR and also quantified lactic acid production using high-performance liquid chromatography (HPLC). Screening of β-gal gene for lactose utilization efficiency in P. pentosaceus NCDC 273 strain efficiency showed a significant (P < 0.0001) expression of the β-gal gene in the supplemented paneer whey medium (SPWM; lactose rich) compared to commercially available Deman-Rogosa-Sharpe (MRS; d-glucose rich) broth medium. In addition, with the growth of P. pentosaceus NCDC 273 in SPWM, the expression of the β-gal gene was found significantly (P < 0.001) higher at 6h of fermentation during the exponential growth phase and declining no significantly at stationary phase. After 48 h of incubation, HPLC data revealed a high lactose conversion rate (42.12%) to lactic acid production (14.50 ± 0.48 g L−1). Moreover, pediocin PA-1 activity in fermented SPWM showed almost contamination-free lactic acid production. Hence, our results showed the sustainable utilization of dairy waste paneer whey for lactic acid production by P. pentosaceus NCDC 273 at a lower disposal cost.

Carotenoids from female Grapsus albolineatus as potential anti-ageing compounds

Introduction: Grapsus albolineatus is thought to have a high concentration of carotenoid pigments. Although studies on male G. albolineatus have been conducted, no studies on pigment extraction from female G. albolineatus have been carried out. Carotenoids have a high ability to fight free radicals. Previous research has shown that carotenoids can fight free radicals that cause premature skin-aging. The purpose of this research was to find out what kinds of carotenoids are found in G. albolineatus and whether they can act as natural inhibitors of proteins that cause premature aging, such as glucogenase, elastase, and hyaluronidase enzymes. Methods: Carotenoids were extracted from G. albolineatus using column chromatography and high-performance liquid chromatography (HPLC); molecular docking and visualization were done with Autodock 4.2 and Discovery Studio/Biovia, respectively. Results: According to HPLC data, there are carotenoid pigments such as didehydroastaxanthin, tetrahydroastaxanthin, dihydroastaxanthin, diatoxanthin, astaxanthin, and adonixanthin. According to molecular docking experiments, pigment carotenoids from G. albolineatus are efficient inhibitors of protein elastase and hyaluronidase. Conclusions: G. albolineatus carotenoids have the potential to be anti-aging since they are more effective as protein elastase and hyaluronidase inhibitors than their native inhibitors.

Application of MIP-sensors to the determination of preservatives in non-alcoholic drinks

The piezosensors modified with a molecularly imprinted polymer (MIP) with potassium sorbate (MIP-E202) and sodium benzoate (MIP-E211) imprints are tested and implemented in the determination of preservatives in soft drinks. Molecularly imprinted polymers were synthesized by noncovalent imprinting on the base of copolymer of 1,2,4,5-benzene tetracarboxylic acid dianhydride and 4,4’-diaminodiphenyl oxide in N,N-dimethylformamide (DMF) in the presence of templates. Piezoelectric sensors based on MIP and non-imprinted polymer (polyimide) were compared. High values of the imprinting factor (IF) and selectivity coefficient (k) obtained for MIP-E202 (IF = 5.4) and MIP-E211 (IF = 6.0) sensors indicated better selectivity and ability of MIP-based sensors to recognize target molecules than piezosensors modified with a reference polymer. The detectable concentrations range within 5 – 500 mg/liter, the detection limits for potassium sorbate and sodium benzoate are 1.6 and 2.0 mg/liter, respectively. Correctness of the preservative determination in model solutions was verified using the spike test. MIP-based sensors appeared sensitive to the preservative determination and insensitive to interfering substances. The matrix composition of the non-alcoholic drinks did not affect the value of the analytical signal of the piezoelectric sensor. High performance liquid chromatography (HPLC) was used as a reference method. The results of potassium sorbate and sodium benzoate determination in non-alcoholic drinks using piezosensors match the HPLC data rather well, their content in the studied soft drinks being 130 – 176 and 129 – 146 mg/liter, respectively.

Cloning and function analysis of chalcone isomerase gene and chalcone synthase gene in Lonicera macranthoides

In order to explore the functions of genes of key rate-limiting enzymes chalcone isomerase(CHI) and chalcone synthase(CHS) in the biosynthesis of flavonoids in Lonicera macranthoides, this study screened and cloned the cDNA sequences of CHI and CHS genes from the transcriptome data of conventional variety and 'Xianglei' of L. macranthoides. Online bioinformatics analysis software was used to analyze the characteristics of the encoded proteins, and quantitative reverse-transcription polymerase chain reaction(qRT-PCR) to detect the expression of CHI and CHS in different parts of the varieties at different flowering stages. The content of luteo-loside was determined by high performance liquid chromatography(HPLC) and the correlation with the expression of the two genes was analyzed. The results showed that the CHI and CHS of the two varieties contained a 627 bp and 1170 bp open reading frame(ORF), respectively, and the CHI protein and CHS protein were stable, hydrophilic, and non-secretory. qRT-PCR results demonstrated that CHI and CHS of the two varieties were differentially expressed in stems and leaves at different flowering stages, particularly the key stages. Based on HPLC data, luteoloside content was in negative correlation with the relative expression of the genes. Thus, CHI and CHS might regulate the accumulation of flavonoids in L. macranthoides, and the specific functions should be further studied. This study cloned CHI and CHS in L. macranthoides and analyzed their expression for the first time, which laid a basis for investigating the molecular mechanism of the differences in flavonoids such as luteoloside in L. macranthoides and variety breeding.

High quality standard of Scutellariae Radix based on plant metabolomics and index components

Scutellariae Radix(SR), derived from the dried root of Scutellaria baicalensis in the family Lamiaceae, commonly serves as Chinese medicinal material. Affected by producing areas, growing years, and harvesting periods, the quality of SR fluctuates in the market. However, baicalin≥9% in SR required in the Chinese Pharmacopoeia(2020 edition) can only determine the qualified SR but cannot identify high-quality SR. To improve the quality control methods of SR, the present study analyzed the accumulation of metabolites in SR of different growth years by plant metabolomics, and identified 28 metabolites increasing with growth years(1-3 years). Subsequently, 14 main metabolites were quantitatively analyzed by ultra-high performance liquid chromatography-tandem triple quadrupole mass spectrometry(UPLC-QQQ-MS). Among them, baicalin, wogonoside, baicalein, and wogonin with high content and good activity were selected as the index components of SR for quality evaluation. A high-performance liquid chromatography(HPLC) method was established to determine the content of four index components in 32 batches of SR from different producing areas, harvesting perio-ds, and growth years. The results showed that the growth years could greatly affect the content of index components. The total content of four index components in 2-year SR was the highest, followed by the 3-/4-year SR and 1-year SR. Based on HPLC data and verification results by enterprises, baicalin ≥12.0%, wogonoside ≥2.3%, baicalein ≥0.1%, and wogonin ≥0.03% were proposed as the evaluation criteria for the high-quality SR. The findings of this study are expected to provide a basis for improving the quality of SR.

Absorbance-Transmittance Excitation Emission Matrix Method for Quantification of Major Cannabinoids and Corresponding Acids: A Rapid Alternative to Chromatography for Rapid Chemotype Discrimination of Cannabis sativa Varieties.

Background: Phytocannabinoids naturally occur in the cannabis plant (Cannabis sativa), and Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) predominate. There is a need for rapid inexpensive methods to quantify total THC (for statutory definition) and THC-CBD ratio (for classification into three chemotypes). This study explores the capabilities of a spectroscopic technique that combines ultraviolet-visible and fluorescence, absorbance-transmittance excitation emission matrix (A-TEEM). Methods: The A-TEEM technique classifies 49 dry flower extracts into three C. sativa chemotypes, and quantifies the total THC-CBD ratio, using validated gas chromatography (GC)-flame ionization (FID) and High-Performance Liquid Chromatography (HPLC) methods for reference. Multivariate methods used are principal components analysis for a chemotype classification, extreme gradient boost (XGB) discriminant analysis (DA) to classify unknown samples by chemotype, and XGB regression to quantify total THC and CBD content using GC-FID and HPLC data on the same samples. Results: The A-TEEM technique provides robust classification of C. sativa samples, predicting chemotype classification, defined by THC-CBD content, of unknown samples with 100% accuracy. In addition, A-TEEM can quantify total THC and CBD levels relevant to statutory determination, with limit of quantifications (LOQs) of 0.061% (THC) and 0.059% (CBD), and high cross-validation (>0.99) and prediction (>0.99), using a GC-FID method for reference data; and LOQs of 0.026% (THC) and 0.080% (CBD) with high cross-validation (>0.98) and prediction (>0.98), using an HPLC method for reference data. A-TEEM is highly predictive in separately quantifying acid and neutral forms of THC and CBD with HPLC reference data. Conclusions: The A-TEEM technique provides a sensitive method for the qualitative and quantitative characterization of the major cannabinoids in solution, with LOQs comparable with GC-FID and HPLC, and high values of cross-validation and prediction. As a spectroscopic technique, it is rapid, with data acquisition <45 sec per measurement; sample preparation is simple, requiring only solvent extraction. A-TEEM has the sensitivity to resolve and quantify cannabinoids in solution based on their unique spectral characteristics. Discrimination of legal and illegal chemotypes can be rapidly verified using XGB DA, and quantitation of statutory levels of total THC and total CBD comparable with GC-FID and HPLC can be obtained using XBD regression.

The Potential Use of Herbal Fingerprints by Means of HPLC and TLC for Characterization and Identification of Herbal Extracts and the Distinction of Latvian Native Medicinal Plants.

The growing market of herbal medicines, the increase in international trade in Latvia, and the lack of adequate analytical methods have raised the question of the potential use of herbal fingerprinting methods. In this study, high-performance liquid chromatography (HPLC) and thin layer chromatography (TLC) methods were developed for obtaining chromatographic fingerprints of four taxonomically and evolutionary different medicinal plants (Hibiscus sabdariffa L., Calendula officinalis L., Matricaria recutita L., Achillea millefolium L.). Retention time shifting, principal component analysis (PCA), hierarchical cluster analysis (HCA), and orthogonal projections to latent structures (OPLS) analysis were used to improve and analyze the obtained fingerprints. HPLC data detection at 270 nm was determined superior to 360 nm for the distinction of medicinal plants and used data alignment method significantly increased similarity between samples. Analyzed medicinal plant extracts formed separate, compact clusters in PCA, and the results of HCA correlated with the evolutionary relationships of the analyzed medicinal plants. Herbal fingerprinting using chromatographic analysis coupled with multivariate analysis has a great potential for the identification of medicinal plants as well as for the distinction of Latvian native medicinal plants.

Elucidation of Chemical Interactions between Crude Drugs Using Quantitative Thin-Layer Chromatography Analysis.

To elucidate the interactions between crude drugs in Kampo medicines (traditional Japanese medicines), it is important to determine the content of the constituents in a cost-effective and simple manner. In this study, we quantified the constituents in crude drug extracts using thin-layer chromatography (TLC), an inexpensive and simple analytical method, to elucidate the chemical interactions between crude drugs. We focused on five crude drugs, for which quantitative high-performance liquid chromatography (HPLC) methods are stipulated in the Japanese Pharmacopoeia XVIII (JP XVIII) and compared the analytical data of HPLC and TLC, confirming that the TLC results corresponded with the HPLC data and satisfied the criteria of JP XVIII. (Z)-ligustilide, a major constituent in Japanese Angelica Root, for which a method of quantification has not been stipulated in JP XVIII, was also quantitatively analyzed using HPLC and TLC. Furthermore, Japanese Angelica Root was combined with 26 crude drugs to observe the variation in the (Z)-ligustilide content from each combination by TLC. The results revealed that combinations with Phellodendron Bark, Citrus Unshiu Peel, Scutellaria Root, Coptis Rhizome, Gardenia Fruit, and Peony Root increased the (Z)-ligustilide content. Quantifying the constituents in crude drug extracts using the inexpensive and simple TLC method can contribute to elucidating interactions between crude drugs in Kampo medicines, as proposed by the herbal-pair theory.

Moment Theory of Chromatography for the Analysis of Reaction Kinetics of Intermolecular Interactions.

Moment theory was applied to the kinetic study of intermolecular interactions. The association equilibrium constant (KA) and association (ka) and dissociation (kd) rate constants of chemical reactions were analytically determined on the basis of the moment theory from elution peak profiles measured by high-performance liquid chromatography (HPLC). The HPLC data were measured under the conditions that neither immobilization nor fluorescence labeling of solute and ligand molecules is required. These are the advantages of the moment analysis method for determining accurate values of KA, ka, and kd. Moment equations were developed on the basis of the Einstein equation for diffusion, the random walk model, and the general rate model of chromatography. The moment analysis method was applied to the inclusion complex formation system between dibenzo-18-crown-6 or dibenzo-15-crown-5 and alkali metal cations. It was demonstrated that the values of KA, ka, and kd can be determined on the assumption that the stoichiometry between crown ethers and cations is 1:1 or 2:1. The influence of the difference in the size between the inner cavity of crown ethers and cations on the association and dissociation of the inclusion complex was considered. The moment analysis method using HPLC is effective for analyzing intermolecular interactions from various perspectives because it is based on the separation technique and has different characteristics from other methods such as spectroscopy. The results of this study contribute to the dissemination of an opportunity for studying intermolecular interactions from equilibrium and kinetic points of view to many researchers because HPLC is widespread.

UV spectroscopy and HPLC combined with chemometrics for rapid discrimination and quantification of Curcumae Rhizoma from three botanical origins

Curcumae Rhizoma (Ezhu in Chinese) is a multi-origin herbal medicine with excellent clinical efficacy. For fast discrimination and quantification analysis of Ezhu from three botanical origins (Curcuma kwangsiensis, Curcuma phaeocaulis, and Curcuma wenyujin), ultra-violet (UV) spectroscopy and high performance liquid chromatography (HPLC) combined with chemometric tools were employed in this study. Firstly, the analysis method for the simultaneous determination of eleven compounds in Ezhu was developed by HPLC, and the UV spectra of thirty-eight batches of Ezhu were acquired. Then, principal component analysis (PCA), an unsupervised pattern recognition method, was applied on the HPLC and UV spectral data. PCA did not show a clear separation between C. phaeocaulis and C. wenyujin samples with HPLC data. By contrast, the supervised techniques, decision tree (DT) and linear discriminant analysis (LDA), achieved the complete discrimination for the three species of Ezhu with 100 % correct classification rate (CCR), showing excellent performance. Based on UV spectral data, PCA presented good performance for discriminating the three species of Ezhu. LDA, support vector machine (SVM) and k-nearest neighbors (KNN) models provided 96.3 % CCR for the calibration set and 100 % CCR for the validation set. Moreover, the partial least squares (PLS) and back propagation-artificial neural network (BP-ANN) quantitative models established on UV spectral data were satisfactory in predicting the contents of zederone, curdione and 3,5-dihydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-heptane. The residual predictive deviation (RPD) for zederone, curdione and 3,5-dihydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-heptane of PLS models were 3.169, 1.502 and 1.735, and that of BP-ANN models were 3.467, 2.481 and 2.370, respectively. The present work proposed a rapid and reliable method for the discrimination of Ezhu from three botanical origins and the prediction of zederone, curdione and 3,5-dihydroxy-1-(3,4-dihydroxyphenyl)-7-(4-hydroxyphenyl)-heptane contents in Ezhu, which will help a lot in the quality control of Ezhu and other multi-origin herbal medicines.

4-Vinylguaiacol, an Active Metabolite of Ferulic Acid by Enteric Microbiota and Probiotics, Possesses Significant Activities against Drug-Resistant Human Colorectal Cancer Cells.

Ferulic acid, a hydroxycinnamic acid, is abundant in vegetables, grains, and medicinal plants. Emerging evidence suggests that ferulic acid may exert beneficial effects against colorectal cancer. However, the anticancer activity of ferulic acid is relatively low, and its metabolism after oral administration is largely unknown. In this study, mimicking the enteric environment, human intestinal microflora and commercial probiotics were used to metabolize ferulic acid to its metabolites, and their anticancer activities were evaluated. Ferulic acid can be biotransformed to 4-vinylguaiacol (2-methoxy-4-vinylphenol), and the contents of ferulic acid and 4-vinylguaiacol in bio-transformed extracts were determined by high-performance liquid chromatography (HPLC). Using the chemotherapy-sensitive cell line HCT-116 and the chemo-resistant cell line HT-29, the cell proliferation was determined by the modified trichrome stain assay. The cell cycle and induction of apoptosis were assayed using flow cytometry. HPLC data showed that there was a marked transformation from ferulic acid to 4-vinylguaiacol, and the conversion rates of intestinal microflora and four probiotics were from 1.3 to 36.8%. Both ferulic acid and 4-vinylguaiacol possessed dose- and time-related anticancer activities on the two cell lines, while 4-vinylguaiacol showed more potent effects than ferulic acid. Interestingly, 4-vinylguaiacol exhibited significantly higher antiproliferative effects on the HT-29 cell line than that on HCT-116. The IC50 of the metabolite 4-vinylguaiacol on HT-29 cells was 350 μM, 3.7-fold higher than its parent compound. The potential of cancer cell growth inhibition of 4-vinylguaiacol was mediated by cell cycle arrest at the G1 phase and induction of apoptosis. Data from this study indicate that the oral administration of ferulic acid offers a promising approach to increase its anticancer activity through gut microbial conversion to 4-vinylguaiacol, and the biotransformation could also be achieved by selected commercial probiotics. 4-Vinylguaiacol is a potential anticancer metabolite from ferulic acid for chemotherapy-resistant colon cancer cells.

Dynamics of the deep chlorophyll maximum in the Black Sea as depicted by BGC-Argo floats

Abstract. The deep chlorophyll maximum (DCM) is a well-known feature of the global ocean. However, its description and the study of its formation are a challenge, especially in the peculiar environment that is the Black Sea. The retrieval of chlorophyll a (chl a) from fluorescence (Fluo) profiles recorded by Biogeochemical Argo (BGC-Argo) floats is not trivial in the Black Sea, due to the very high content of coloured dissolved organic matter (CDOM) which contributes to the fluorescence signal and produces an apparent increase in the chl a concentration with depth. Here, we revised Fluo correction protocols for the Black Sea context using co-located in situ high-performance liquid chromatography (HPLC) and BGC-Argo measurements. The processed set of chl a data (2014–2019) is then used to provide a systematic description of the seasonal DCM dynamics in the Black Sea and to explore different hypotheses concerning the mechanisms underlying its development. Our results show that the corrections applied to the chl a profiles are consistent with HPLC data. In the Black Sea, the DCM begins to form in March, throughout the basin, at a density level set by the previous winter mixed layer. During a first phase (April–May), the DCM remains attached to this particular layer. The spatial homogeneity of this feature suggests a hysteresis mechanism, i.e. that the DCM structure locally influences environmental conditions rather than adapting instantaneously to external factors. In a second phase (July–September), the DCM migrates upward, where there is higher irradiance, which suggests the interplay of biotic factors. Overall, the DCM concentrates around 45 % to 65 % of the total chlorophyll content within a 10 m layer centred around a depth of 30 to 40 m, which stresses the importance of considering DCM dynamics when evaluating phytoplankton productivity at basin scale.

Development of near‐infrared reflectance spectroscopy calibration for sugar content in ground soybean seed using Perten DA7250 analyzer

AbstractThere is growing interest in developing calibrations for soybean [Glycine max (L.) Merr.] seed sugars on near‐infrared reflectance spectroscopy (NIRS) instruments to increase the efficiency of sugar profiling. In this study, a set of 253 samples from Virginia Tech's soybean germplasm with a wide range of sugar content were used to create prediction models for sucrose, raffinose, and stachyose in ground soybean seed on the Perten DA7250 NIRS instrument. Following acquisition of spectral data, seed sugars were extracted from ground samples and analyzed using high‐performance liquid chromatography (HPLC) to obtain reference data. Spectral and HPLC data were modeled using partial least squares regression (PLSR) on CAMO Unscrambler X software and internally cross‐validated using the same software. Resulting calibrations showed high quantitative accuracy with the coefficient of determination of calibration (R2C) = .901, the coefficient of determination of cross‐validation (R2CV) = .869, root mean squared error of calibration (RMSEC) = .516, and root mean squared error of cross‐validation (RMSECV) of .596 for sucrose and R2C = .911, R2CV = .891, RMSEC = .361, and RMSECV of .405 for stachyose. These calibrations appear suitable for use in breeding operations. Meanwhile, performance of the raffinose calibration remained poor with R2C = .568, R2CV = .476, RMSEC = .129, and RMSECV = .143. Alternative methods for more accurate and rapid quantification of raffinose concentration in soybean seed should be investigated.

Biogenic Silver and Zero-Valent Iron Nanoparticles by Feijoa: Biosynthesis, Characterization, Cytotoxic, Antibacterial and Antioxidant Activities.

Green nanotechnology is an interesting method for the synthesis of functional nanoparticles. Because of their wide application, they have set up great attention in recent years. The present research examines the green synthesis of Ag and zero-valent iron nanoparticles (AgNPs, ZVINPs) by Feijoa sellowiana fruit extract. In this synthesis, no stabilizers or surfactants were applied. Eco-friendly synthesis of Iron and biogenic synthesis of Ag nanoparticles were accomplished by controlling critical parameters such as concentration, incubation period and temperature. Scanning Electron Microscopy (SEM), Transmission Electron Microscope (TEM), Energy-Dispersive X-ray Spectroscopy (EDS), Fourier-Transform Infrared (FT-IR) spectroscopy, X-ray Diffraction analysis (XRD), Dynamic Light Scattering (DLS) and UV-Vis were applied to characterize NPs. The cytotoxicity of NPs was investigated in two cell lines, MCF-7 (breast cancer) and AGS (human gastric carcinoma). A high-performance liquid chromatography (HPLC) analysis was also performed for characterization of phenolic acids in the extract. Both NPs displayed powerful anticancer activities against two tumor cell lines with little effect on BEAS-2B normal cells. Synthesized AgNPs and ZVINPs inhibited the growth of all selected bacteria. Pseudomonas aeruginosa, Proteus mirabilis, Klebsiella pneumonia, Staphylococcus aureus, Enterococcus faecalis, Acinetobacter baumannii and Escherichia coli have been studied in two stages. We initially examined the ATCCs followed by clinical strain isolation. Based on the results from resistant strains, we showed that nanoparticles were superior to conventional antibiotics. DPPH (diphenyl-1-picrylhydrazyl) free radical scavenging assay and iron chelating activity were used for the determination of antioxidant properties. Results showed a high antioxidant activity of scavenging free radicals for ZVINPs and powerful iron-chelating activity for AgNPs. Based on the HPLC data, catechin was the major phenolic compound in the extract. Our synthesized nanoparticles displayed potent cytotoxic, antibacterial and antioxidant activities.

Degradation of different pesticides in water by microplasma: the roles of individual radicals and degradation pathways.

Pesticides are emergent toxins often identified in aquatic environments. In the present study, microplasma was employed to reduce the pesticide content in water. The degradation efficacy, rate, and pathways of standard organophosphorus pesticides (namely, chlorpyrifos, chlorpyrifos oxone, and diazinone) and an organochlorine pesticide (namely, DDT solution) were evaluated using microplasma. High-performance liquid chromatography (HPLC) analysis was performed to elucidate the degradation efficiency of pesticides as a function of plasma-produced substances that originally contributed to the main reduction procedure. Microplasma produces several types of radicals or reactive substances, for instance dissolved ozone (O3), nitrogen oxides, hydroxyl radicals (OH radicals), and hydrogen peroxide (H2O2). The removal potential differs due to the existence or absence of varieties of plasma-produced substances. The functions of major plasma-produced species on pesticide removal were determined by a passive technique. Nitrogen oxides showed a key role in organophosphorus pesticide removal, whereas dissolved ozone and OH radicals played major roles in DDT degradation. HPLC data showed that plasma-induced pesticide removal showed first-order reaction kinetics. The pesticide removal pathways through microplasma were validated by investigating the achieved data from LC-MS and GC-MS.

Lemon Exosome-like Nanoparticles-Manipulated Probiotics Protect Mice from C.d iff Infection.

SummaryClostridioides difficile (C. diff) is the leading cause of antibiotic-associated colitis. Here, we report that lemon exosome-like nanoparticles (LELNs) manipulated probiotics to inhibit C. diff infection (CDI). LELN-manipulated Lactobacillus rhamnosus GG (LGG) and Streptococcus thermophilus ST-21 (STH) (LELN-LS) decrease CDI mortality via an LELN-mediated increase in bile resistance and gut survivability. LELN-LS treatment increases the AhR ligands indole-3-lactic acid (I3LA) and indole-3-carboxaldehyde (I3Ald), leading to induction of IL-22, and increases lactic acid leading to a decrease of C. diff fecal shedding by inhibiting C. diff growth and indole biosynthesis. A synergistic effect between STH and LGG was identified. The STH metabolites inhibit gluconeogenesis of LGG and allow fructose-1,6-bisphosphate (FBP) to accumulate in LGG; accumulated FBP then activates lactate dehydrogenase of LGG (LGG-LDH) and enhances production of lactic acid and the AhR ligand. Our findings provide a new strategy for CDI prevention and treatment with a new type of prebiotics.