Physical and chemical properties of nabak (Zizyphus spina-christi) seed kernel and sweet pepper (Capsicum annuum L.) seed oils.

BACKGROUND: Nabak seed kernels and sweet pepper seeds, which are separated from the fruits and discarded as waste after processing or consumption, contain high levels of oils (30.19% and 19.57%, respectively). The chemical and thermal characteristics of nabak seed kernel oil (NSO) and sweet pepper seed oil (PSO) were investigated in this study. RESULTS: The NSO and PSO contained high levels of unsaturated fatty acids (84.1% and 86.5%, respectively), and the major fatty acid was oleic acid (57.3%) in NSO, but it was linoleic acid (69.4%) in PSO. The triacylglycerol (TAG) profiles show that NSO contained ten TAG species, three of which represented 87.1%, namely C54:3, C52:2 and C54:4, and triolein was the dominant (OOO, 47.0%). Pepper seed oil contained nine TAG molecular species, four of which represented 93.6%, namely C54:6, C52:4, C54:4 and C52:5, and trilinolein was dominant (LLL, 44.0%). The differential scanning calorimetry (DSC) analysis of NSO revealed that three exothermal peaks were detected during cooling, two endothermal peaks were detected during melting, and the major peak occurred at a low temperature. For PSO, three exothermal peaks were detected during cooling, three peaks were detected (one of them was exothermal) during melting, and the major peaks were observed at low temperatures. Fourier transform infrared (FTIR) spectra indicated that NSO and PSO did not contain peroxides or trans fatty acids, but they did contain low concentrations of free fatty acids. CONCLUSION: This study offers a scientific basis for the use of NSO and PSO as new sources of edible oils for food applications. © 2021 Society of Chemical Industry.

Identification of the Effects of Chondroitin Sulfate on Inhibiting CDKs in Colorectal Cancer Based on Bioinformatic Analysis and Experimental Validation.

With a high occurrence rate and high mortality, the treatment of colorectal cancer (CRC) is increasingly attracting the attention of scholars. Hub genes that determine the phenotypes of CRC become essential for targeted therapy. In the present study, the importance of cyclin-dependent kinases (CDKs) on the occurrence of CRC was identified by data mining of The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The results showed that the gene expression levels of CDK1, CDK4, and CDK6 were obviously changed in different stages of CRC. Among the CDKs, CDK4 was suggested as an independent risk factor for CRC based on Cox analysis. Furthermore, chondroitin sulfate (CS), a kind of dietary supplement to treat osteoarthritis, was predicted to treat CRC based on its chemical structure and GEO datasets. Cell assay experiments with the human CRC cell line HCT-116 also verified this prediction. CS inhibited the gene and protein expression levels of CDKs and increased the ratios of apoptotic or dead HCT-116 cells by regulating mitogen-activated protein (MAP) kinase pathways. Our data highlight the essential roles of CDKs in CRC carcinogenesis and the effects of CS on treating CRC, both of which will contribute to the future CRC treatment.

Evaluation of spice and herb as phyto-derived selective modulators of human retinaldehyde dehydrogenases using a simple in vitro method.

Selective modulation of retinaldehyde dehydrogenases (RALDHs)-the main aldehyde dehydrogenase (ALDH) enzymes converting retinal into retinoic acid (RA), is very important not only in the RA signaling pathway but also for the potential regulatory effects on RALDH isozyme-specific processes and RALDH-related cancers. However, very few selective modulators for RALDHs have been identified, partly due to variable overexpression protocols of RALDHs and insensitive activity assay that needs to be addressed. In the present study, deletion of the N-terminal disordered regions is found to enable simple preparation of all RALDHs and their closest paralog ALDH2 using a single protocol. Fluorescence-based activity assay was employed for enzymatic activity investigation and screening for RALDH-specific modulators from extracts of various spices and herbs that are well-known for containing many phyto-derived anti-cancer constituents. Under the established conditions, spice and herb extracts exhibited differential regulatory effects on RALDHs/ALDH2 with several extracts showing potential selective inhibition of the activity of RALDHs. In addition, the presence of magnesium ions was shown to significantly increase the activity for the natural substrate retinal of RALDH3 but not the others, while His-tag cleavage considerably increased the activity of ALDH2 for the non-specific substrate retinal. Altogether we propose a readily reproducible workflow to find selective modulators for RALDHs and suggest potential sources of selective modulators from spices and herbs.

Metabolic profiling of natural and cultured Cordyceps by NMR spectroscopy.

Cordyceps, a type of Chinese herbal medicine that exhibits anti-angiogenesis and tumor growth suppression effects, has recently gained increasing popularity. However, high-quality, natural Cordyceps, such as Ophiocordyceps sinensis, is very rare and difficult to obtain in large amounts. Cordyceps is cultured instead of harvested from natural sources, but the quality with respect to the ingredients has not been fully studied. In this study, we performed an NMR metabolic profiling of aqueous extracts of Cordyceps without any sample treatment to evaluate the proper species and medium and influence of two different disinfection methods. It was discovered that Cordyceps militaris fungus and silkworm chrysalis medium were suitable for cultivation of Cordyceps. Furthermore, cordycepin, a Cordyceps-specific functional compound, was produced at different growth stages during different cultivation processes, even at the mycelial stage, and was found at three times higher concentrations in cultured C. militaris compared to that in naturally occurring C. militaris.

Comprehensive NMR analysis of two kinds of post-fermented tea and their anti-glycation activities in vitro.

Post-fermented tea (dark tea) is produced from enzyme-inactivated fresh tea leaves by microbial fermentation. Batabata tea and Awaban tea are two major dark teas fermented under aerobic and anaerobic conditions, respectively. However, how their chemical compositions and functionalities change during different post-fermentation processes remains unclear. Nuclear magnetic resonance (NMR)-based analyses showed that (-)-epigallocatechin gallate (EGCG), (-)-epigallocatechin (EGC) and (-)-epicatechin (EC) decreased in Batabata tea during post-fermentation with aerobic molds. In contrast, EGC and EC increased, and pyrogallol was produced in Awaban tea during post-fermentation with lactic acid bacteria (LAB). The anti-glycation activities of two dark teas were investigated using an in vitro assay system with human serum albumin (HSA). The anti-glycation activity decreased in Batabata tea, but it was retained in Awaban tea during post-fermentation. Our results showed that post-fermentation with LAB was an efficient way to enhance phenol content and that pyrogallol contributed to anti-glycation activity of Awaban tea.

Quantification of terpene trilactones in Ginkgo biloba with a 1H NMR method.

Ginkgo biloba L. has been used as a herbal medicine in the traditional treatment of insufficient blood flow, memory deficits, and cerebral insufficiency. The terpene trilactone components, the bioactive agents of Ginkgo biloba L., have also been reported to exhibit useful functionality such as anti-inflammatory and neuroprotective effects. Therefore, in the present research, we attempted to analyze quantitatively the terpene trilactone components in Ginkgo biloba leaf extract, with quantitative 1H NMR (qNMR) and obtained almost identical results to data reported using HPLC. Application of the qNMR method for the analysis of the terpene trilactone contents in commercial Ginkgo extract products, such as soft gel capsules and tablets, produced the same levels noted in package labels. Thus, qNMR is an alternative method for quantification of the terpene trilactone components in commercial Ginkgo extract products.

Quantitation of Minor Components in Mango Juice with Band-Selective Excitation NMR Spectroscopy.

1H NMR-based metabolic analysis of foods has been widely applied. However, dynamic range problems frequently impede its application because foodstuffs are composed of various organic compounds in a wide range of concentrations. Band-selective excitation 1H NMR spectroscopy has been found to be a useful tool for observing the minor components in foods. Because quantitative information is important for metabolic analysis of foods and complete metabolome data, quantitation with the band-selective excitation 1H NMR method was carefully investigated in the present study. As a result, the concentrations of minor components in mango juice of the "Carabao" cultivar were successfully quantitated by band-selective excitation 1H NMR using standard curves that exhibited good linearity. The band-selective excitation 1H NMR technique was therefore effective for determining the concentrations of minor components in foods.

NMR-based analysis of the chemical composition of Japanese persimmon aqueous extracts.

Japanese persimmon (Diospyros kaki L.) is recognized as an outstanding source of biologically active compounds relating to many health benefits. In the present study, NMR spectroscopy provided a comprehensive metabolic overview of Japanese persimmon juice. Detailed signal assignments of Japanese persimmon juice were carried out using various 2D NMR techniques incorporated with broadband water suppression enhanced through T1 effects (BB-WET) or WET sequences, and 26 components, including minor components, were identified. In addition, most components were quantitatively evaluated by the integration of signals using conventional (1) H NMR and BB-WET NMR. This is the first detailed analysis combined with quantitative characterization of chemical components using NMR for Japanese persimmon. Copyright © 2015 John Wiley & Sons, Ltd.

Comprehensive NMR analysis of compositional changes of black garlic during thermal processing.

Black garlic is a processed food product obtained by subjecting whole raw garlic to thermal processing that causes chemical reactions, such as the Maillard reaction, which change the composition of the garlic. In this paper, we report a nuclear magnetic resonance (NMR)-based comprehensive analysis of raw garlic and black garlic extracts to determine the compositional changes resulting from thermal processing. (1)H NMR spectra with a detailed signal assignment showed that 38 components were altered by thermal processing of raw garlic. For example, the contents of 11 l-amino acids increased during the first step of thermal processing over 5 days and then decreased. Multivariate data analysis revealed changes in the contents of fructose, glucose, acetic acid, formic acid, pyroglutamic acid, cycloalliin, and 5-(hydroxymethyl)furfural (5-HMF). Our results provide comprehensive information on changes in NMR-detectable components during thermal processing of whole garlic.

A pilot study of NMR-based sensory prediction of roasted coffee bean extracts.

Nuclear magnetic resonance (NMR) spectroscopy can be considered a kind of "magnetic tongue" for the characterisation and prediction of the tastes of foods, since it provides a wealth of information in a nondestructive and nontargeted manner. In the present study, the chemical substances in roasted coffee bean extracts that could distinguish and predict the different sensations of coffee taste were identified by the combination of NMR-based metabolomics and human sensory test and the application of the multivariate projection method of orthogonal projection to latent structures (OPLS). In addition, the tastes of commercial coffee beans were successfully predicted based on their NMR metabolite profiles using our OPLS model, suggesting that NMR-based metabolomics accompanied with multiple statistical models is convenient, fast and accurate for the sensory evaluation of coffee.

Expression, high-pressure refolding and purification of human leukocyte cell-derived chemotaxin 2 (LECT2).

Human leukocyte cell-derived chemotaxin 2 (LECT2) is a chemotactic factor for neutrophils and a 16-kDa secreted protein consisting of 133 amino acids with three intramolecular disulfide bonds. Here, we propose an efficient method for the preparation of human LECT2 using a high hydrostatic pressure (HHP, 200 MPa) refolding technique. When LECT2 was over-expressed in Escherichia coli cells, most of LECT2 molecules became insoluble inclusion bodies (IBs). HHP was applied to the refolding of LECT2 from insoluble IBs, which dramatically improved the yield of the active LECT2. CD and NMR measurements demonstrated that the refolded LECT2 had a tertiary structure indistinguishable from the solubly expressed LECT2. In addition, both the refolded and solubly expressed LECT2 showed the same level of chemotactic activity.

Two-dimensional 1H-13C nuclear magnetic resonance (NMR)-based comprehensive analysis of roasted coffee bean extract.

Coffee was characterized by proton and carbon nuclear magnetic resonance (NMR) spectroscopy. To identify the coffee components, a detailed and approximately 90% signal assignment was carried out using various two-dimensional NMR spectra and a spiking method, in which authentic compounds were added to the roasted coffee bean extract (RCBE) sample. A total of 24 coffee components, including 5 polysaccharide units, 3 stereoisomers of chlorogenic acids, and 2 stereoisomers of quinic acids, were identified with the NMR spectra of RCBE. On the basis of the signal assignment, state analyses were further launched for the metal ion-citrate complexes and caffeine-chlorogenate complexes. On the basis of the signal integration, the coffee components were successfully quantified. This NMR methodology yielded detailed information on RCBE using only a single observation and provides a systemic approach for the analysis of other complex mixtures.

Isolation and Caenorhabditis elegans lifespan assay of flavonoids from onion.

The main flavonoids were isolated from three selected onion cultivars. Three phenolic compounds were obtained by reverse-phase HPLC, and their structures were elucidated by multiple NMR measurements. There were two known compounds, quercetin and quercetin 3'-O-ß-D-glucopyranoside (Q3'G), and one novel compound, quercetin 3-O-ß-D-glucopyranoside-(4→1)-ß-d-glucopyranoside (Q3M), which was identified in onion for the first time. These flavonoids were found to be more abundant in the onion peel than in the flesh or core. Their antioxidative activities were tested using the DPPH method, and their antiaging activities were evaluated using a Caenorhabditis elegans lifespan assay. No direct correlation was found between antioxidative activity and antiaging activity. Quercetin showed the highest antioxidative activity, whereas Q3M showed the strongest antiaging activity among these flavonoids, which might be related to its high hydrophilicity.

Isolation and tyrosinase inhibitory effects of polyphenols from the leaves of persimmon, Diospyros kaki.

The main polyphenols were isolated from the leaves of six selected persimmon cultivars. Seven compounds were obtained by reverse-phase HPLC, and their structures were elucidated by multiple NMR measurements. These compounds are hyperoside, isoquercitrin, trifolin, astragalin, chrysontemin, quercetin-3-O-(2''-O-galloyl-ß-D-glucopyranoside) (QOG), and kaempferol-3-O-(2''-O-galloyl-ß-D-glucopyranoside) (KOG). Their inhibitory activity was tested against tyrosinase for the oxidation of L-DOPA, and only chrysontemin showed inhibitory activity. To investigate the differences of their inhibitory effects, the tyrosinase inhibitory activities of their aglycons, cyanidin, quercetin, and kaempferol, were also tested. As a result, it was confirmed that the most influential moiety for tyrosinase inhibition was the 3',4'-dihydroxy groups of the catechol moiety. Moreover, the tyrosinase inhibitory activity of chrysontemin, which was identified in persimmon leaves for the first time, is supported by a simulated model of chrysontemin docking into mushroom tyrosinase.

Complex mixture analysis of organic compounds in green coffee bean extract by two-dimensional NMR spectroscopy.

A complex mixture analysis by one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy was carried out for the first time for the identification and quantification of organic compounds in green coffee bean extract (GCBE). A combination of (1)H-(1)H DQF-COSY, (1)H-(13)C HSQC, and (1)H-(13)C CT-HMBC two-dimensional sequences was used, and 16 compounds were identified. In particular, three isomers of caffeoylquinic acid were identified in the complex mixture without any separation. In addition, GCBE components were quantified by the integration of carbon signals by use of a relaxation reagent and an inverse-gated decoupling method without a nuclear Overhauser effect. This NMR methodology provides detailed information about the kinds and amounts of GCBE components, and in our study, the chemical makeup of GCBE was clarified by the NMR results.

Absolute side-chain structure at position 13 is required for the inhibitory activity of bromein.

Bromelain isoinhibitor (bromein), a cysteine proteinase inhibitor from pineapple stem, has a unique double-chain structure. The bromein precursor protein includes three homologous inhibitor domains, each containing an interchain peptide between the light and heavy chains. The interchain peptide in the single-chain precursor is immediately processed by bromelain, a target proteinase. In the present study, to clarify the essential inhibitory site of bromein, we constructed 44 kinds of site-directed and deletion mutants and investigated the inhibitory activity of each toward bromelain. As a result, the complete chemical structure of Leu13 in the light chain was revealed to be essential for inhibition. Pro12 prior to the leucine residue was also involved in the inhibitory activity and would control the location of the leucine side chain by the fixed dihedral angle of proline. Furthermore, the five-residue length of the interchain peptide was strictly required for the inhibitory activity. On the other hand, no inhibitory activity against bromelain was observed by the substitution of proline for the N terminus residue Thr15 of the interchain peptide. In summary, these mutational analyses of bromein demonstrated that the appropriate position and conformation of Leu13 are absolutely crucial for bromelain inhibition.

Characterization of the acidic and basic limbs of a bell-shaped pH profile in the inhibitory activity of bromelain inhibitor VI.

Bromelain inhibitor VI (BI-VI) is a cysteine proteinase inhibitor from pineapple stem and a unique two-chain inhibitor composed of two distinct domains. BI-VI's inhibitory activity toward the target enzyme bromelain is maximal at pH 4 and shows a bell-shaped pH profile with pKa values of about 2.5 and 5.3. This pH profile is quite different from that of bromelain, which is optimally active around pH 7. In the present article, to characterize the acidic limb, we first expressed the recombinant inhibitors designed to lose two putative hydrogen bonds of Ser7(NH)-Asp28(beta-CO2H) and Lys38(NH)-Asp51(beta-CO2H) and confirmed the existence of the hydrogen bonds by two-dimensional nuclear magnetic resonance (NMR). Moreover, it was revealed that these hydrogen bonds are not the essential electrostatic factor and some ionizable groups would be responsible for the acidic limb in the pH-inhibition profile. On the other hand, to characterize the basic limb, we examined the pH-dependent inhibition using the cysteine proteinase papain, some of whose properties differ from those of bromelain, and compared the data with the corresponding data for bromelain. The result suggests that the basic limb would be affected by some electrostatic factors, probably some carboxyl groups in the target proteinase.