Chromatographic Evaluation and Characterization of Constituents of Sunflower Seed Extract Used as Food Additives.

Sunflower seed extract, an antioxidant agent registered on the List of Existing Food Additives in Japan, was evaluated using HPLC, and three common constituents were detected. These peaks were identified as monocaffeoylquinic acids (3-O-caffeoylquinic acid, 4-O-caffeoylquinic acid, and 5-O-caffeoylquinic acid [chlorogenic acid]). Upon scrutinizing other components, dicaffeoylquinic acids (isochlorogenic acids; 3,4-di-O-caffeoylquinic, 3,5-di-O-caffeoylquinic, and 4,5-di-O-caffeoylquinic acids) were also identified. Structures of two newly isolated compounds were determined to be 3-O-(3S-2-oxo-3-hydroxy-indole-3-acetyl)-5-O-caffeoylquinic and 4-O-(3S-2-oxo-3-hydroxy-indole-3-acetyl)-5-O-caffeoylquinic acids. To identify the components that contribute to the antioxidant activity of sunflower seed extract, we fractionated the food additive sample solution and examined the active fractions for 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity. Monocaffeoylquinic and dicaffeoylquinic acids showed high DPPH activity, including their contribution to the antioxidant activity of this food additive. DPPH radical scavenging activity of the new compounds showed almost the same value as that of the positive control, Trolox. Therefore, the contribution of these compounds was also considered.

[Proposal for Guidelines on the Notation and Investigation of Scientific Names for the Source of Natural Food Additives in Japanese Standards].

The official specifications for food additives from natural sources list the species according to their scientific and Japanese names, thereby providing a unique identifier for the species. This helps to prevent the use of nonprescribed species, which might cause unexpected or unintended health hazards. However, there are cases in which the names of the source species listed in the official specifications differ from the accepted scientific names based on the latest taxonomic research. In this paper, we argue that it is more important to define scientific and Japanese names with an emphasis on traceability in order to control the range of food additive ingredients in a rational and sustainable manner. Therefore, we proposed a method for ensuring traceability as well as a specific notation procedure for scientific and Japanese names. Using this method, we examined the source species for three food additives. In some cases, the range of sources species expanded with the change in scientific names. Ensuring traceability is extremely important, but it is also necessary to confirm whether unexpected species are included when names are changed.

Study on the Synthesis of Methylated Reference and Their Application in the Quantity of Curcuminoids Using Single Reference Liquid Chromatography Based on Relative Molar Sensitivity.

We report on the recommendation of the simple and versatility of methylated reference (MR) to improve applications in the single reference (SR)-LC based on relative molar sensitivity (RMS). Three curcuminoids (Curs) such as curcumin, demethoxycurcumin and bisdemethoxycurcumin in turmeric products were determined using authentic standards and methylated curcumin. In addition, high-speed countercurrent chromatography (HSCCC) purification is necessary to separate Curs for indicating the RMS. For HSCCC separation, a biphasic solvent system was used to obtain these fractions, which were then subjected to 1H quantitative NMR to determine their contents in each test solution. Using these solutions, the RMS of Curs are calculated from slopes ratios of calibration curves (three ranges from 0-100 µmol/L, r2 > 0.998). The averaged RMS of Curs were 8.92 (relative standard deviation (RSD), 1.17%), 8.97 (2.18%), and 9.61 (0.77%), respectively. Cur concentrations in turmeric products can be determined using RMS, peak area, and MR content added in these samples. This proposed method, which is based on chemical methylation and the SR-LC assay has been successfully applied for the simple and reliable estimation of Curs in turmeric products.

Determination of Mogroside V in Luohanguo Extract for Daily Quality Control Operation Using Relative Molar Sensitivity to Single-Reference Caffeine.

Mogroside V is one of the characteristic and effective components of luohanguo extract, a food additive used as a sweetener in Japan as per Japan's Standards and Specifications for Food Additives (JSFA; 9th ed.). JSFA stipulates that the quantitative determination for mogroside V content in luohanguo extract applies HPLC using analytical standard mogroside V. However, no mogroside V reagents with proven purities are commercially available. Therefore the current JSFA determination method is not particularly suited for daily quality control operations involving luohanguo extract. In this study, we applied an alternative quantitative method using a single reference with relative molar sensitivity (RMS). It was possible to calculate the accurate RMS by an offline combination of 1H-quantitative NMR spectroscopy (1H-qNMR) and an HPLC/variable-wavelength detector (VWD). Using the RMS of mogroside V to a commercial certified reference material grade caffeine, the mogroside V contents in luohanguo extracts could be determined using HPLC/VWD without analytical standard mogroside V. There was no significant difference between the mogroside V contents in luohanguo extracts determined using the method employing single-reference caffeine with the RMS and using the JSFA method. The absolute calibration curve for the latter was prepared using an analytical standard mogroside V whose purity was determined by 1H-qNMR. These results demonstrate that our proposed method using a single reference with RMS is suitable for quantitative determination of mogroside V in luohanguo extract and can be used as an alternative method to the current assay method in JSFA.

Quantification of tea-derived catechins without the requirement for respective calibration curves by single reference liquid chromatography based on relative molar sensitivity.

BACKGROUND: Many studies report the monitoring of catechins in tea samples by chromatographic techniques. Unfortunately, only a small number of screening assays for catechins exist as a result of the complexity of authentic standards for the respective calibration curves. In the present study, a single reference (SR) exhaustive assay for the simultaneous quantification of tea-derived catechins by liquid chromatography (LC) with photodiode array and fluorescence detectors based on relative molar sensitivity (RMS) was developed as a screening assay of common tea samples without respective calibration curves using authentic standards. RESULTS: Three original SR standards were proposed based on flavonoid structures, evaluated by quantitative 1 H-NMR based on an indirect standard (1,4-bis(trimethylsilyl) benzene-d4 ) and successfully separated in a LC chromatogram. In tea samples with these added SR calculated based on RMS, the concentrations of eight tea-derived catechins could be measured with a relative SD of < 8.5% by a single LC run. CONCLUSION: This LC screening assay based on RMS allows reliable quantification without the requirement for respective calibration curves using authentic standards. © 2020 Society of Chemical Industry.

[Determination of Allyl Isothiocyanate in Mustard and Horseradish Extracts by Single Reference GC and HPLC Based on Relative Molar Sensitivities].

The main component of the Mustard and Horseradish extracts, which are used as natural food additives in Japan, is allyl isothiocyanate (AITC). The determination of AITC using GC-FID is the official method employed in the quality control assessments for these products. In this method, a commercially available AITC reagent is used as a calibrant. However, 1H-quantitative NMR (qNMR) analysis revealed that the AITC reagents contain impurity. Therefore, we examined the GC-FID and HPLC-refractive index detector (LC-RID) method based on relative molar sensitivities (RMSs) to high-purity single reference (SR). The RMSs of AITC/SR under the GC-FID and LC-RID conditions were accurately determined using qNMR. The AITC in two types of food additives was quantified using qNMR, SR GC-FID, and SR LC-RID methods. Both SR GC-FID and SR LC-RID showed good agreement within 2% with the AITC content determined by direct qNMR.

A 90-day repeated oral dose toxicity study of four stereoisomers of 2,4-dimethyl-4-phenyltetrahydrofuran, a synthetic flavoring substance, in F344 rats.

2,4-Dimethyl-4-phenyltetrahydrofuran (CAS no. 82461-14-1) is a food additive used as a synthetic flavoring substance. To investigate the toxicological properties and determine the no-observed-adverse-effect level (NOAEL), a 90-day repeated oral dose toxicity study of 2,4-dimethyl-4-phenyltetrahydrofuran containing four stereoisomers was conducted in F344 rats at doses of 0, 6, 24, and 96 mg/kg body weight (BW)/day. No mortality or abnormal clinical signs related to treatment in any group was observed. At a dose of 96 mg/kg BW, fluctuated serum total protein and total cholesterol and increased absolute and relative liver weights and relative kidney weights were observed in both sexes. Increased serum albumin in males and decreased Na and Cl in females were also observed. On histopathological assessment, at a dose of 96 mg/kg BW, diffuse hepatocellular hypertrophy in the liver in both sexes and tubular regeneration with scattered proximal tubular degeneration and/or necrosis throughout the cortex in the kidney in males were detected. Based on these findings, the NOAEL for 2,4-dimethyl-4-phenyltetrahydrofuran used in the current study was found to be 24 mg/kg BW/day for both sexes.

[Quantitative Analysis of Piperine and the Derivatives in Long Pepper Extract by HPLC Using Relative Molar Sensitivity].

A novel method was developed for quantification of five major piperine derivatives (piperanine, piperine, chavicine, isopiperine, and isochavicine) in a hot water extract of long pepper fruit (LPE) using the relative molar sensitivity (RMS) based on the combination of HPLC/UV and 1H- quantitative NMR (1H-qNMR). The RMSs of piperanine, chavicine, isopiperine, and isochavicine to piperine of which the absolute purity was determined by 1H-qNMR were calculated to be 0.3693, 1.138, 0.9164, and 1.277, respectively. The total amount of piperine derivatives in LPE was quantified by both 1H-qNMR and HPLC/UV based on the RMS using piperine as a single-reference material (RMS method). The relative difference in quantitation values of 1H-qNMR and calibration curve method from the RMS method was 2.01% or less. The relative difference of the total cis-trans piperine isomers content between before and after photoirradiation in piperine solution was quantified to be 2.84% by the RMS method. In addition, the interlaboratory difference of the RMS method was confirmed in the range of 0.600 to 4.00 µg/g when analysis was performed on piperine derivatives in LPE containing tablets, while the total amount of piperine derivatives in the tablets was quantified at 606 µg/g. Our proposed method is a reliable tool for determining the contents of piperine and the derivatives in LPE and processed foods containing LPE.

Preliminary Quality Evaluation and Characterization of Phenolic Constituents in Cynanchi Wilfordii Radix.

A new phenolic compound, 2-O-ß-laminaribiosyl-4-hydroxyacetophenone (1), was isolated from Cynanchi Wilfordii Radix (CWR, the root of Cynanchum wilfordii Hemsley), along with 10 known aromatic compounds, including cynandione A (2), bungeisides-C (7) and -D (8), p-hydroxyacetophenone (9), 2',5'-dihydroxyacetophenone (10), and 2',4'-dihydroxyacetophenone (11). The structure of the new compound (1) was elucidated using spectroscopic methods and chemical methods. The structure of cynandione A (2), including a linkage mode of the biphenyl parts that remained uncertain, was unambiguously confirmed using the 2D 13C-13C incredible natural abundance double quantum transfer experiment (INADEQUATE) spectrum. Additionally, health issues related to the use of Cynanchi Auriculati Radix (CAR, the root of Cynanchum auriculatum Royle ex Wight) instead of CWR have emerged. Therefore, constituents present in methanolic extracts of commercially available CWRs and CARs were examined using UV-sensitive high-performance liquid chromatography (HPLC), resulting in common detection of three major peaks ascribed to cynandione A (2), p-hydroxyacetophenone (9), and 2',4'-dihydroxyacetophenone (11). Thus, to distinguish between these ingredients, a thin-layer chromatography (TLC) method, combined with only UV irradiation detection, focusing on wilfosides C1N (12) and K1N (13) as marker compounds characteristic of CAR, was performed. Furthermore, we propose this method as a simple and convenient strategy for the preliminary distinction of CWR and CAR to ensure the quality and safety of their crude drugs.

Survey on the Original Plant Species of Crude Drugs Distributed as Cynanchi Wilfordii Radix and Its Related Crude Drugs in the Korean and Chinese Markets.

Cynanchi Wilfordii Radix (CWR) is used in Korea as a substitute for Polygoni Multiflori Radix (PMR), which is a crude drug traditionally used in East Asian countries. Recently, the use of Cynanchi Auriculati Radix (CAR) in place of PMR and CWR has emerged a major concern in the Korean market. In Japan, PMR is permitted to be distributed as a pharmaceutical regulated by the Japanese Pharmacopoeia 17th edition (JP17). Although CWR and CAR have not traditionally been used as medicines, CWR was recently introduced as a health food. The distribution of unfamiliar CWR-containing products could lead to the misuse of original species for PMR and CWR like in Korea. To prevent this situation, the original species of plant products distributed as PMR, CWR, and CAR in the Korean and Chinese markets were surveyed and identified by their genes and components. The results revealed that all two PMR in the Korean market were misapplied as CAR, and that CAR was incorrectly used in eight of thirteen products distributed as CWR in both markets. As PMR is strictly controlled by JP17, the risk of mistaking PMR for CWR and CAR would be low in Japan. In contrast, the less stringent regulation of health food products and the present situation of misidentification of CWR in the Korean and Chinese markets could lead to unexpected health hazards. To ensure the quality and safety of crude drugs, it is important to use the information about the genes and components of these crude drugs.

Membrane bound O-acyltransferases and their inhibitors.

Since the identification of the membrane-bound O-acyltransferase (MBOATs) protein family in the early 2000s, three distinct members [porcupine (PORCN), hedgehog (Hh) acyltransferase (HHAT) and ghrelin O-acyltransferase (GOAT)] have been shown to acylate specific proteins or peptides. In this review, topology determination, development of assays to measure enzymatic activities and discovery of small molecule inhibitors are compared and discussed for each of these enzymes.

Click chemistry armed enzyme-linked immunosorbent assay to measure palmitoylation by hedgehog acyltransferase.

Hedgehog signaling is critical for correct embryogenesis and tissue development. However, on maturation, signaling is also found to be aberrantly activated in many cancers. Palmitoylation of the secreted signaling protein sonic hedgehog (Shh) by the enzyme hedgehog acyltransferase (Hhat) is required for functional signaling. To quantify this important posttranslational modification, many in vitro Shh palmitoylation assays employ radiolabeled fatty acids, which have limitations in terms of cost and safety. Here we present a click chemistry armed enzyme-linked immunosorbent assay (click-ELISA) for assessment of Hhat activity through acylation of biotinylated Shh peptide with an alkyne-tagged palmitoyl-CoA (coenzyme A) analogue. Click chemistry functionalization of the alkyne tag with azido-FLAG peptide allows analysis through an ELISA protocol and colorimetric readout. This assay format identified the detergent n-dodecyl ß-d-maltopyranoside as an improved solubilizing agent for Hhat activity. Quantification of the potency of RU-SKI small molecule Hhat inhibitors by click-ELISA indicated IC50 values in the low- or sub-micromolar range. A stopped assay format was also employed that allows measurement of Hhat kinetic parameters where saturating substrate concentrations exceed the binding capacity of the streptavidin-coated plate. Therefore, click-ELISA represents a nonradioactive method for assessing protein palmitoylation in vitro that is readily expandable to other classes of protein lipidation.

Modulation of Amide Bond Rotamers in 5-Acyl-6,7-dihydrothieno[3,2-c]pyridines.

2-Substituted N-acyl-piperidine is a widespread and important structural motif, found in approximately 500 currently available structures, and present in nearly 30 pharmaceutically active compounds. Restricted rotation of the acyl substituent in such molecules can give rise to two distinct chemical environments. Here we demonstrate, using NMR studies and density functional theory modeling of the lowest energy structures of 5-acyl-6,7-dihydrothieno[3,2-c]pyridine derivatives, that the amide E:Z equilibrium is affected by non-covalent interactions between the amide oxygen and adjacent aromatic protons. Structural predictions were used to design molecules that promote either the E- or Z-amide conformation, enabling preparation of compounds with a tailored conformational ratio, as proven by NMR studies. Analysis of the available X-ray data of a variety of published N-acyl-piperidine-containing compounds further indicates that these molecules are also clustered in the two observed conformations. This finding emphasizes that directed conformational isomerism has significant implications for the design of both small molecules and larger amide-containing molecular architectures.

Isolation and characterization of isopiperitenol dehydrogenase from piperitenone-type Perilla.

Studying the biosynthesis of oil compounds in Perilla will help to elucidate regulatory systems for secondary metabolites and reaction mechanisms for natural product synthesis. In this study, two types of alcohol dehydrogenases, isopiperitenol dehydrogenases 1 and 2 (ISPD1 and ISPD2), which are thought to participate the oxidation of isopiperitenol in the biosynthesis of perilla, were isolated from three pure lines of perilla. Both ISPD1 and ISPD2 oxidized isopiperitenol into isopiperitenone with an oxidized form of nicotinamide adenine dinucleotide (NAD(+)) cofactor. ISPD1 used both isopiperitenol diastereomers, whereas ISPD2 used cis-isomer as a substrate. However, only ISPD2 was isolated from piperitenone-type perilla. These results suggests that in perilla, ISPD2 is related to the biosynthesis of piperitenone, which was formed via (-)-cis-isopiperitenol.

Novel compounds isolated from health food products containing beni-koji (red yeast rice) with adverse event reports.

Recently, health hazards, such as kidney damage, have been reported owing to the ingestion of a health food product, so-called "foods with functional claims (FFC)'', containing beni-koji (red yeast rice). Although not an expected compound in the FFC, the detection of puberulic acid has also been reported. Further investigations of these health food products, such as the identification of other unintended compounds and clarifying the health impacts of puberulic acid, are required. To clarify the causes of these health issues, we investigated the presence of unintended compounds in the FFC containing beni-koji using comprehensive instrumental analyses. Using differential analysis, novel compounds 1 and 2 were detected as unexpected components between the samples with and without adverse event reports. Although limited to the samples available for analyses in this study, both compounds 1 and 2 were detected in all the samples that also contained puberulic acid. Compounds 1 and 2, with molecular formulas of C23H34O7 and C28H42O8, respectively, may be lovastatin derivatives. Their structures were confirmed using NMR analyses and are novel natural compounds. For definitive confirmation, we are in the process of synthesizing compounds 1 and 2 from lovastatin. The route of contamination of these compounds are currently under investigation. The findings of this study could be used to address the growing health hazards associated with health food products.

Design, Synthesis, and Evaluation of Inhibitors of Hedgehog Acyltransferase.

Hedgehog signaling is involved in embryonic development and cancer growth. Functional activity of secreted Hedgehog signaling proteins is dependent on N-terminal palmitoylation, making the palmitoyl transferase Hedgehog acyltransferase (HHAT), a potential drug target and a series of 4,5,6,7-tetrahydrothieno[3,2-c]pyridines have been identified as HHAT inhibitors. Based on structural data, we designed and synthesized 37 new analogues which we profiled alongside 13 previously reported analogues in enzymatic and cellular assays. Our results show that a central amide linkage, a secondary amine, and (R)-configuration at the 4-position of the core are three key factors for inhibitory potency. Several potent analogues with low- or sub-µM IC50 against purified HHAT also inhibit Sonic Hedgehog (SHH) palmitoylation in cells and suppress the SHH signaling pathway. This work identifies IMP-1575 as the most potent cell-active chemical probe for HHAT function, alongside an inactive control enantiomer, providing tool compounds for validation of HHAT as a target in cellular assays.

Genetic identification of the original plant species for Mentha Herb listed in the Japanese Pharmacopoeia and analyses of their essential oil composition.

Mentha arvensis Linné var. piperascens Malinvaud is an original plant species for "Mentha Herb (Hakka, ハッカ)" and "Mentha Oil (Hakka-yu, ハッカ)" listed in the Japanese Pharmacopoeia, whereas Mentha canadensis L. is that of "Mint oil, partly dementholised" listed in the European Pharmacopoeia. Although these two species are thought to be taxonomically identical, there are no data on whether the source plants of the Mentha Herb products distributed in the Japanese market are actually M. canadensis L. This is an important issue for international harmonization of the Japanese Pharmacopoeia and European Pharmacopoeia. In this study, 43 Mentha Herb products collected from the Japanese market and two plant samples of the original species of Japanese Mentha Herb harvested in China were identified by sequence analyses of the rpl16 regions in the chloroplast DNA, and the composition of their ether extracts was analyzed by GC-MS. Almost all samples were identified as M. canadensis L., and the main component of their ether extracts was menthol, although there were variations in their composition. However, there were some samples thought to be derived from other Mentha species, even though their main component was menthol. For quality control of Mentha Herb, it is important to be sure of not only the original plant species but also the composition of the essential oil and amount of menthol as the characteristic compound.

Application of the relative molar sensitivity method using GC-FID to quantify safranal in saffron (Crocus sativus L.).

Safranal is one flavor component of saffron, which is used as a spice, food additive, and crude drug. In ISO3632, safranal is defined as the compound that contributes to the quality of saffron, and many quantitative determination methods for safranal have been reported. However, safranal is volatile and degrades easily during storage, and an analytical standard with an exact known purity is not commercially available, making it difficult to quantify accurately the content of safranal in saffron. Here, we developed a method for quantifying safranal using relative molar sensitivity (RMS), called the RMS method, using a GC-flame ionization detector (GC-FID). We determined the RMS of safranal to 1,4-bis(trimethylsilyl)benzene-d4, a certified reference material commercially available, by a combination of quantitative NMR and chromatography. Using two GC-FID instruments made by different manufacturers to evaluate inter-instrument effect, the resultant RMS was 0.770, and the inter-instrument difference was 0.6%. The test solution, with a known safranal concentration, was measured by the RMS method, with an accuracy of 99.4-101%, repeatability of 0.81%, and reproducibility of 0.81-1.3%. Given the ease of degradation, high volatility, and uncertain purity of safranal reagents, the RMS method is a more accurate quantification approach compared to the calibration curve method and methods based on absorption spectrophotometry. Moreover, our findings revealed that the GC-FID makeup gas affected the RMS and quantitative values.

A new single-reference quantitative method using liquid chromatography with relative molar sensitivity based on 1H-qNMR for khellactone esters from Peucedanum japonicum root extract.

Khellactone ester (KLE) quantification using the absolute calibration method is difficult owing to the unavailability of standard reagents that can guarantee purity. Herein, a new method was developed to quantify KLEs from Peucedanum japonicum root extracts using liquid chromatography (LC) without utilizing standards. This method used relative molar sensitivity (RMS) and 7-ethoxy-4-methylcoumarin as a single-reference (SR) compound instead of KLE standards. RMS is the sensitivity ratio of SR to analytes, determined using an offline combination of quantitative NMR and LC. LC was performed using a triacontylsilyl silica gel column of superficially porous particles with a ternary mobile phase. The range of the method was 2.60-509 µmol/L. The accuracy and precision were reasonable. This is the first study to apply the RMS method to both conventional LC and ultra-high-performance liquid chromatography using the same mobile phase and column. This method may aid the quality assurance of foods containing KLEs.

Use of Relative Molar Sensitivity as a Specific Value for Evaluating Heptaoxyethylene Dodecyl Ether Concentrations in Methanol Solution.

Relative molar sensitivity (RMS) determined using quantitative 1H NMR and HPLC with a refractive index (RI) detector was applied as a specific value for quantifying the levels of heptaoxyethylene dodecyl ether (HOEDE), a typical non-ionic surfactant, in methanol solutions. RMS was robust against changes of the analytical conditions (i.e., RI cell temperature, acetonitrile content in the mobile phase, HPLC system). Furthermore, the obtained HOEDE concentrations using a previously evaluated RMS were comparable to those obtained using a reference method for over 1 year.