Determination of Absolute Purities of Hygroscopic Substances by Quantitative NMR Analysis for the Standardization of Quantitative Reagents in the Japanese Pharmacopoeia (Part 2).

As a new absolute quantitation method for low-molecular compounds, quantitative NMR (qNMR) has emerged. In the Japanese Pharmacopoeia (JP), 15 compounds evaluated by qNMR are listed as reagents used as the HPLC reference standards in the assay of crude drug section of the JP. In a previous study, we revealed that humidity affects purity values of hygroscopic reagents and that (i) humidity control before and during weighing is important for a reproducible preparation and (ii) indication of the absolute amount (not purity value), which is not affected by water content, is important for hygroscopic products determined by qNMR. In this study, typical and optimal conditions that affect the determination of the purity of ginsenoside Rb1 (GRB1), saikosaponin a (SSA), and barbaloin (BB) (i.e., hygroscopic reagents) by qNMR were examined. First, the effect of humidity before and during weighing on the purity of commercial GRB1, with a purity value determined by qNMR, was examined. The results showed the importance afore-mentioned. The results of SSA, which is relatively unstable in the dissolved state, suggested that the standardization of humidity control before and during weighing for a specific time provides a practical approach for hygroscopic products. In regard to BB, its humidity control for a specific time, only before weighing, is enough for a reproducible purity determination.

Visualization of Aspalathin in Rooibos (Aspalathus linearis) Plant and Herbal Tea Extracts Using Thin-Layer Chromatography.

Aspalathin, the main polyphenol of rooibos (Aspalathus linearis), is associated with diverse health promoting properties of the tea. During fermentation, aspalathin is oxidized and concentrations are significantly reduced. Standardized methods for quality control of rooibos products do not investigate aspalathin, since current techniques of aspalathin detection require expensive equipment and expertise. Here, we describe a simple and fast thin-layer chromatography (TLC) method that can reproducibly visualize aspalathin in rooibos herbal tea and plant extracts at a limit of detection (LOD) equal to 178.7 ng and a limit of quantification (LOQ) equal to 541.6 ng. Aspalathin is a rare compound, so far only found in A. linearis and its (rare) sister species A. pendula. Therefore, aspalathin could serve as a marker compound for authentication and quality control of rooibos products, and the described TLC method represents a cost-effective approach for high-throughput screening of plant and herbal tea extracts.

Ultra-high performance liquid chromatography-tandem mass spectrometry assay for simultaneous determination of 22 marker compounds in traditional herbal medicine Ojeoksan.

Traditional herbal medicines (THMs) have long been in use worldwide and are considered safe for use as tonics or complementary treatments for many diseases. Advanced quality control methods for THMs are required in the regulatory framework of modern medicines. In this study, an ultra-high performance liquid chromatography-tandem mass spectrometry assay was established for the simultaneous determination of 22 marker compounds in Ojeoksan (OJS), which is composed of 15 herbal substances. All marker compounds were analyzed within 20 min and successfully identified via scheduled multiple reaction monitoring. The method validation revealed excellent performance characteristics of the method such as specificity, linearity, sensitivity, precision, and accuracy, demonstrating its suitability for intended use. The developed method was applied to samples of commercial OJS tablet and soft-extract dosage forms. The 14 marker compounds corresponding to 12 component herbal substances were determined in the samples; ephedirine, albiflorin, paeoniflorin, ferulic acid, hesperidine, neohesperidin, cinnamic acid, platycodin D, 6-gingerol, atractylenolide III, glycyrrhizin, honokiol, decursin, and magnolol. A fast and easy assay method with sufficient discrimination power was established. As a novel assay, this method can contribute to the quality control of OJS products.

Lipidomics combined with random forest machine learning algorithms to reveal freshness markers for duck eggs during storage in different rearing systems.

The differences in lipids in duck eggs between the 2 rearing systems during storage have not been fully studied. Herein, we propose untargeted lipidomics combined with a random forest (RF) algorithm to identify potential marker lipids based on ultra-performance liquid chromatography‒mass spectrometry (UPLPC-MS/MS). A total of 106 and 16 differential lipids (DL) were screened in egg yolk and white, respectively. In yolk, metabolic pathway analysis of DLs revealed that glycerophospholipid metabolism and sphingolipid metabolism were the key metabolic pathways in the traditional free-range system (TFS) during storage, glycosylphosphatidylinositol-anchored biosynthesis and glyceride metabolism were the key pathways in the floor-rearing system (FRS). In egg white, the key pathway in both systems is the biosynthesis of unsaturated fatty acids. Combined with RF algorithm, 12 marker lipids were screened during storage. Therefore, this study elucidates the changes in lipids in duck eggs during storage in 2 rearing systems and provides new ideas for screening marker lipids during storage. This approach is highly important for evaluating the quality of egg and egg products and provides guidance for duck egg production.

Study on Betaine and Growth Characteristics of Lycium chinense Mill. in Different Cultivation Environments in South Korea.

Betaine is a useful compound that has various activities and is the marker compound of Lycium chinense fruit in Korean Pharmacopoeia. we seek to support the stable production of medicinal goji berries, which have significant potential in the pharmaceutical industry due to their high values, and to provide foundational data for consistent quality control. This study's purpose was to examine the correlation among betaine content, environmental variables, and the growth characteristics of L. chinense fruits. The fruits were collected from 25 cultivation sites across South Korea. We investigated five growth characteristics and betaine contents in L. chinense fruits and twelve soil physicochemical properties, and seven meteorological data at cultivation sites. The fruit's growth characteristics included a length of 15.62-26.49 mm, a width of 7.09-11.38 mm, a fresh weight of 0.73-1.62 g, and a sugar content of 11.10-19.62 Brix°. Its betaine content ranged from 0.54% to 0.97%. The betaine content was positively correlated with electrical conductivity (0.327 **), exchangeable potassium (0.314 **), and sodium (0.259 *) and negatively correlated with annual average minimum temperature (-0.256 *) and annual average temperature (-0.242 *). Also, betaine showed a positive correlation with the length of the fruit (0.294 *) and the fresh weight of the fruit (0.238 *). These results can be used to find the best cultivation method and to manage quality control for the highly economical L. chinense fruit.

Identification of marker compounds in fermented Benincasa hispida and validation of the method for its analysis.

Fermentation is a process that improves health functionality by inducing the production and increase of bioactive compounds. In this study, to standardize the fermentation process for Benincasa hispida, marker compounds that are increased or produced during fermentation were identified based on UPLC-QTOF-MS/MS. Analysis method verification and content analysis were conducted using HPLC-PDA. The marker compounds produced or increased in content were identified as 2-furoic acid, 2,3-dihydroxybenzoic acid, and rubinaphthin A by comparing their retention times, UV and MS spectra, and molecular formulas with those reported in previous studies. In addition, the increase in the content of the marker compounds by fermentation was confirmed, and the analytical method was validated by measuring its specificity, linearity, limit of detection and quantitation, precision, and accuracy. These results suggest that the developed fermentation process, marker compound identification, and verified analysis method can be applied to develop potential functional food ingredients from fermented B. hispida.

Metabolomics Reveals Distinctive Metabolic Profiles and Marker Compounds of Camellia (Camellia sinensis L.) Bee Pollen.

Camellia bee pollen (CBP) is a major kind of bee product which is collected by honeybees from tea tree (Camellia sinensis L.) flowers and agglutinated into pellets via oral secretion. Due to its special healthcare value, the authenticity of its botanical origin is of great interest. This study aimed at distinguishing CBP from other bee pollen, including rose, apricot, lotus, rape, and wuweizi bee pollen, based on a non-targeted metabolomics approach using ultra-high performance liquid chromatography-mass spectrometry. Among the bee pollen groups, 54 differential compounds were identified, including flavonol glycosides and flavone glycosides, catechins, amino acids, and organic acids. A clear separation between CBP and all other samples was observed in the score plots of the principal component analysis, indicating distinctive metabolic profiles of CBP. Notably, L-theanine (864.83-2204.26 mg/kg) and epicatechin gallate (94.08-401.82 mg/kg) were identified exclusively in all CBP and were proposed as marker compounds of CBP. Our study unravels the distinctive metabolic profiles of CBP and provides specific and quantified metabolite indicators for the assessment of authentic CBP.

Phytochemical Investigation of Marker Compounds from Indigenous Korean Salix Species and Their Antimicrobial Effects.

Salix species, including willow trees, are distributed in the temperate regions of Asian countries, including South Korea. Willow trees are used to treat pain and inflammatory diseases. Due to the medicinal properties of willow trees, pharmacological studies of other Salix spp. have gained attention; however, only a few studies have investigated the phytochemicals of these species. As part of our ongoing natural product research to identify bioactive phytochemicals and elucidate their chemical structures from natural resources, we investigated the marker compounds from indigenous Korean Salix species, namely, Salix triandra, S. chaenomeloides, S. gracilistyla, S. koriyanagi, S. koreensis, S. pseudolasiogyne, S. caprea, and S. rorida. The ethanolic extract of each Salix sp. was investigated using high-performance liquid chromatography combined with thin-layer chromatography and liquid chromatography−mass spectrometry-based analysis, and marker compounds of each Salix sp. were isolated. The chemical structures of the marker compounds (1−8), 3-(4-hydroxyphenyl)propyl ß-D-glucopyranoside (1), 2-O-acetylsalicin (2), 1-O-p-coumaroyl glucoside (3), picein (4), isograndidentatin B (5), 2'-O-acetylsalicortin (6), dihydromyricetin (7), and salicin (8) were elucidated via nuclear magnetic resonance spectroscopy and high-resolution liquid chromatography−mass spectrometry using ultrahigh-performance liquid chromatography coupled with a G6545B Q-TOF MS system with a dual electrospray ionization source. The identified marker compounds 1−8 were examined for their antimicrobial effects against plant pathogenic fungi and bacteria. Dihydromyricetin (7) exhibited antibacterial activity against Staphylococcus aureus, inducing 32.4% inhibition at a final concentration of 125 µg/mL with an MIC50 value of 250 µg/mL. Overall, this study isolated the marker compounds of S. triandra, S. chaenomeloides, S. gracilistyla, S. koriyanagi, S. koreensis, S. pseudolasiogyne, S. caprea, and S. rorida and identified the anti-Staphylococcus aureus bacterial compound dihydromyricetin.

Evaluation of the Farming Potential of Echinacea Angustifolia DC. Accessions Grown in Italy by Root-Marker Compound Content and Morphological Trait Analyses.

The Echinacea genus includes a number of species that are commercially employed for the preparation of herbal products. Echinacea angustifolia DC. is one of these and is widely used, mainly for its immunomodulating properties, as it contains a wide range of compounds that belong to different chemical classes. In particular, echinacoside, cynarin and lipophylic alkylamides are the main specialized metabolites of the roots and can be considered to be marker compounds. In this work, 65 E. angustifolia accessions have been compared in a field trial in Italy, with the aim of investigating the variability/stability of the weight and chemical composition of their roots in order to identify the accessions that are most promising for future genetic-improvement programs. The morphological characteristics of the aerial parts have also been investigated. Seventeen samples were discarded due to germination or plantlet-development issues. Seven of the remaining accessions were identified as being different Echinacea species after a combined phytochemical and morphological evaluation. The morphological traits of the epigeal part, the root weight and the chemical composition data of the 41 confirmed E. angustifolia accessions were submitted to multivariate statistical analysis and a moderately homogenous sample distribution, with low selected-marker variability, was observed. Good echinacoside content was detected in almost all roots (>0.5%). However, two groups of accessions stood out because of their interesting features: One group possessed small roots, but had a high concentration of marker compounds, while another had highly developed roots and a good amount of marker compounds. These accessions can therefore be exploited for future selection work.

Studies on olive-and silicone-oils-based Janus macroemulsions containing ginger to manage primary dysmenorrheal pain.

Ginger (GIN) powder-loaded oil-in-water (o/w) macroemulsions were prepared based on olive-and silicone-oils. The dispersed oil droplets with paired-beans structure were evident and thus the final emulsion can be termed as Janus macroemulsions. The objectives of the present study are (1) to identify the marker compound present in GIN powder via HPLC analysis, (2) to process the GIN powder via anti-solvent precipitation technique, (3) to see the solubility of GIN powder in various single oils or oil combination, (4) to optimize the GIN-loaded o/w macroemulsions using the central composite design (CCD) with respect to mean particle size of dispersed oil droplets and highest percentage drug entrapment efficiency values (DEE) and (5) to evaluate the pain reducing activity of optimized GIN-loaded macroemulsion via in vivo primary dysmenorrhea (PD) mice model. Both predicted and obtained values of percentage DEE (76.29 Vs.76.09) and mean particle size (245.99 Vs. 272.51 µm) were almost the same indicating the CCD statistical design applicability. The optimized Janus macroemulsion was stable at 4 °C for over a period of 90 days. Using the PD mice model, the counting of writhing reaction produced by the tested GIN-loaded macroemulsions at low and high doses did not reveal significant difference in comparison to the positive control (aspirin treated). Only the high dose of GIN-loaded macroemulsion was able to restore the uterine tissue's normal histomorphological structure after the H & E staining. Nevertheless, the paired beans structure should be tested for entrapping the plant-derived drugs having dissimilar physicochemical characteristics but similar therapeutic activity.

Isolation and characterization of quinine from Polygonatum verticillatum: A new marker approach to identify substitution and adulteration.

Polygonatum verticillatum (Mahameda) is an important ingredient of Ashtawarga and Ayurvedic formulations. Nowadays, it comes under the category of endangered plants due to large scale and indiscriminate collection of wild material. To overcome the scarcity, substitutes of Mahameda are also commonly used in market. These additives are different from the authentic plant by Ayurvedic and pharmacological theory of drug action, thereby resulting in substitution/adulteration. Substitution is a critical issue in isolation and quantification of the therapeutically active ingredients that can be used as markers in the identification of substitution/adulteration. Methanolic extract of the rhizomes of P. verticillatum was subjected to preliminary phytochemical screening for the detection of phytoconstituents, followed by column chromatography for isolation of the marker. The column was first eluted with pure hexane, and polarity of the solvent was gradually increased. A total of 1180 fractions were collected and pooled on the basis of thin-layer chromatography profile. The single compound was isolated and confirmed by chemical test, melting point, spectral analysis, and comparison with literature. Phytochemical screening of the extract shows the presence of alkaloids, flavonoids, carbohydrates, terpenoids, and phenolics. A pure white crystalline powder was isolated by column chromatography which was characterized as (6-methoxyquinolin-4-yl-8-vinylquinuclidin-2-yl) methanol, i.e. Quinine. The isolated compound, Quinine, was identified as a novel compound in Mahameda as it has not been reported in the genus Polygonatum, till now. It can be used as a marker for the identification of substitution/adulteration and standardization of P. verticillatum.