Mass spectrometry-based ginsenoside profiling: Recent applications, limitations, and perspectives.

Ginseng, the roots of Panax species, is an important medicinal herb used as a tonic. As ginsenosides are key bioactive components of ginseng, holistic chemical profiling of them has provided many insights into understanding ginseng. Mass spectrometry has been a major methodology for profiling, which has been applied to realize numerous goals in ginseng research, such as the discrimination of different species, geographical origins, and ages, and the monitoring of processing and biotransformation. This review summarizes the various applications of ginsenoside profiling in ginseng research over the last three decades that have contributed to expanding our understanding of ginseng. However, we also note that most of the studies overlooked a crucial factor that influences the levels of ginsenosides: genetic variation. To highlight the effects of genetic variation on the chemical contents, we present our results of untargeted and targeted ginsenoside profiling of different genotypes cultivated under identical conditions, in addition to data regarding genome-level genetic diversity. Additionally, we analyze the other limitations of previous studies, such as imperfect variable control, deficient metadata, and lack of additional effort to validate causation. We conclude that the values of ginsenoside profiling studies can be enhanced by overcoming such limitations, as well as by integrating with other -omics techniques.

Comparative transcriptome and metabolome analyses of four Panax species explore the dynamics of metabolite biosynthesis.

Background: The genus Panax in the Araliaceae family has been used as traditional medicinal plants worldwide and is known to biosynthesize ginsenosides and phytosterols. However, genetic variation between Panax species has influenced their biosynthetic pathways is not fully understood. Methods: Simultaneous analysis of transcriptomes and metabolomes obtained from adventitious roots of two tetraploid species (Panax ginseng and P. quinquefolius) and two diploid species (P. notoginseng and P. vietnamensis) revealed the diversity of their metabolites and related gene expression profiles. Results: The transcriptome analysis showed that 2,3-OXIDOSQUALENE CYCLASEs (OSCs) involved in phytosterol biosynthesis are upregulated in the diploid species, while the expression of OSCs contributing to ginsenoside biosynthesis is higher in the tetraploid species. In agreement with these results, the contents of dammarenediol-type ginsenosides were higher in the tetraploid species relative to the diploid species. Conclusion: These results suggest that a whole-genome duplication event has influenced the triterpene biosynthesis pathway in tetraploid Panax species during their evolution or ecological adaptation. This study provides a basis for further efforts to explore the genetic variation of the Panax genus.

Genetic and chemical markers for authentication of three Artemisia species: A. capillaris, A. gmelinii, and A. fukudo.

The genus Artemisia is an important source of medicines in both traditional and modern pharmaceutics, particularly in East Asia. Despite the great benefits of herbal medicine, quality assessment methods for these medicinal herbs are lacking. The young leaves from Artemisia species are generally used, and most of the species have similar morphology, which often leads to adulteration and misuse. This study assembled five complete chloroplast genomes of three Artemisia species, two accessions of A. gmelinii and A. capillaris, and one A. fukudo. Through comparative analysis, we revealed genomic variations and phylogenetic relationships between these species and developed seven InDel-based barcode markers which discriminated the tested species from each other. Additionally, we analyzed specialized metabolites from the species using LC-MS and suggested chemical markers for the identification and authentication of these herbs. We expect that this integrated and complementary authentication method would aid in reducing the misuse of Artemisia species.

Glucose Uptake-Stimulating Galloyl Ester Triterpenoids from Castanopsis sieboldii.

Using molecular networking-guided isolation, three new galloyl ester triterpenoids (1-3), two new hexahydroxydiphenic acid-conjugated triterpenoids (6 and 7), and four known compounds (4, 5, 8, and 9) were isolated from the fruits and leaves of Castanopsis sieboldii. The chemical structures of 1-3, 6, and 7 were elucidated on the basis of interpreting their NMR, HRESIMS, and ECD spectra. All compounds (1-9) were evaluated for their glucose uptake-stimulating activities in differentiated adipocytes using 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-d-glucose as a fluorescent-tagged glucose probe. Compounds 2 and 9 resulted in a 1.5-fold increase in glucose uptake. Among them, compound 2 from the fruits showed an upregulation of p-AMPK/AMPK ratio in differentiated C2C12 myoblasts to support the mechanism proposed of glucose uptake stimulation.

Assessing specialized metabolite diversity of Alnus species by a digitized LC-MS/MS data analysis workflow.

Alnus spp. (Betulaceae) have been used for treatments of hemorrhage, burn injuries, antipyretic fever, diarrhea, and alcoholism in traditional medicines. In this study, a digitized LC-MS/MS data analysis workflow was applied to provide an overview on chemical diversity of 15 Alnus extracts prepared from bark, twigs, leaves, and fruits of A. japonica, A. firma, A. hirsuta, and A. hirsuta var. sibirica. Most of the MS/MS spectra could be putatively annotated based on library matching, in silico fragmentation, and substructural topic modeling. The putative annotation allowed us to discriminate the extracts into three chemotypes based on dominant chemical scaffolds: diarylheptanoids, flavonoids or tannins. This high-throughput chemical annotation was correlated with α-glucosidase inhibition data of extracts, and it allowed us to identify gallic acid as the major active compound of A. firma.

Exploring novel secondary metabolites from natural products using pre-processed mass spectral data.

Many natural product chemists are working to identify a wide variety of novel secondary metabolites from natural materials and are eager to avoid repeatedly discovering known compounds. Here, we developed liquid chromatography/mass spectrometry (LC/MS) data-processing protocols for assessing high-throughput spectral data from natural sources and scoring the novelty of unknown metabolites from natural products. This approach automatically produces representative MS spectra (RMSs) corresponding to single secondary metabolites in natural sources. In this study, we used the RMSs of Agrimonia pilosa roots and aerial parts as models to reveal the structural similarities of their secondary metabolites and identify novel compounds, as well as isolation of three types of nine new compounds including three pilosanidin- and four pilosanol-type molecules and two 3-hydroxy-3-methylglutaryl (HMG)-conjugated chromones. Furthermore, we devised a new scoring system, the Fresh Compound Index (FCI), which grades the novelty of single secondary metabolites from a natural material using an in-house database constructed from 466 representative medicinal plants from East Asian countries. We expect that the FCIs of RMSs in a sample will help natural product chemists to discover other compounds of interest with similar chemical scaffolds or novel compounds and will provide insights relevant to the structural diversity and novelty of secondary metabolites in natural products.

Identification of candidate UDP-glycosyltransferases involved in protopanaxadiol-type ginsenoside biosynthesis in Panax ginseng.

Ginsenosides are dammarane-type or triterpenoidal saponins that contribute to the various pharmacological activities of the medicinal herb Panax ginseng. The putative biosynthetic pathway for ginsenoside biosynthesis is known in P. ginseng, as are some of the transcripts and enzyme-encoding genes. However, few genes related to the UDP-glycosyltransferases (UGTs), enzymes that mediate glycosylation processes in final saponin biosynthesis, have been identified. Here, we generated three replicated Illumina RNA-Seq datasets from the adventitious roots of P. ginseng cultivar Cheongsun (CS) after 0, 12, 24, and 48 h of treatment with methyl jasmonate (MeJA). Using the same CS cultivar, metabolomic data were also generated at 0 h and every 12-24 h thereafter until 120 h of MeJA treatment. Differential gene expression, phylogenetic analysis, and metabolic profiling were used to identify candidate UGTs. Eleven candidate UGTs likely to be involved in ginsenoside glycosylation were identified. Eight of these were considered novel UGTs, newly identified in this study, and three were matched to previously characterized UGTs in P. ginseng. Phylogenetic analysis further asserted their association with ginsenoside biosynthesis. Additionally, metabolomic analysis revealed that the newly identified UGTs might be involved in the elongation of glycosyl chains of ginsenosides, especially of protopanaxadiol (PPD)-type ginsenosides.

Targeted Isolation of Neuroprotective Dicoumaroyl Neolignans and Lignans from Sageretia theezans Using in Silico Molecular Network Annotation Propagation-Based Dereplication.

The integration of LC-MS/MS molecular networking and in silico MS/MS fragmentation is an emerging method for dereplication of natural products. In the present study, a targeted isolation of natural products using a new in silico-based annotation tool named Network Annotation Propagation (NAP) is described. NAP improves accuracy of in silico fragmentation analyses by reranking candidate structures based on the network topology from MS/MS-based molecular networking. Annotation for the MS/MS spectral network of the Sageratia theezans twig extract was performed using NAP, and most molecular families within the network, including the known triterpenoids 1-7, could be putatively annotated, without relying on any previous reports of molecules from this species. Based on the in silico dereplication results, molecules were prioritized for isolation. In total, six dicoumaroyl 8- O-4' neolignans (8-13) and three dicoumaroyl lignans (14-16) were isolated from the twigs of S. theezans and structurally characterized by spectroscopic analyses. Isolates were evaluated for their neuroprotective activity, and compounds 14-16 showed potent protective effects against glutamate-induced oxidative stress in mouse HT22 cells at a concentration of 12.5 µM.

Chemical and genomic diversity of six Lonicera species occurring in Korea.

Lonicera spp. (Caprifoliaceae) are important not only as a common medicinal herb in East Asia but also as one of the most problematic invasive species in North America. In the present study, we performed a systemic analysis of genomic and chemical diversity among six Lonicera species occurring in Korea, L. japonica, L. maackii, L. insularis, L. sachalinensis, L. praeflorens, and L. vesicaria, using chloroplast DNA whole genome shotgun (WGS) sequencing and LC-MS analyses. The phylogenetic and phylochemical relationships did not coincide with each other, but partial consistency could be found among them. InDel-based cDNA marker for authentication was developed based on the genome sequences. Flavonoids, iridoids, and organic acids were identified in the LC-MS analyses, and their inter-species distribution and localization were also revealed.

Terminalia chebula extract prevents scopolamine-induced amnesia via cholinergic modulation and anti-oxidative effects in mice.

BACKGROUND: Terminalia chebula Retz. (Combretaceae) is a traditional herbal medicine that is widely used in the treatment of diabetes, immunodeficiency diseases, and stomach ulcer in Asia. However, the anti-amnesic effect of T. chebula has not yet been investigated. The present study was designed to determine whether T. chebula extract (TCE) alleviates amnesia induced by scopolamine in mice. We also investigated possible mechanisms associated with cholinergic system and anti-oxidant effects. METHODS: TCE (100 or 200 mg/kg) was orally administered to mice for fourteen days (days 1-14), and scopolamine was intraperitoneally injected to induce memory impairment for seven days (days 8-14). Learning and memory status were evaluated using the Morris water maze. Hippocampal levels of acetylcholine (ACh), acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) were measured ex vivo. Levels of reactive oxygen species (ROS), nitric oxide (NO), and malondialdehyde (MDA) in the hippocampus were also examined. RESULTS: In the Morris water maze task, TCE treatment reversed scopolamine-induced learning and memory deficits in acquisition and retention. TCE reduced hippocampal AChE activities and increased ChAT and ACh levels in the scopolamine-induced model. Moreover, TCE treatment suppressed scopolamine-induced oxidative damage by ameliorating the increased levels of ROS, NO, and MDA. CONCLUSION: These findings suggest that TCE exerts potent anti-amnesic effects via cholinergic modulation and anti-oxidant activity, thus providing evidence for its potential as a cognitive enhancer for amnesia.

Genome and evolution of the shade-requiring medicinal herb Panax ginseng.

Panax ginseng C. A. Meyer, reputed as the king of medicinal herbs, has slow growth, long generation time, low seed production and complicated genome structure that hamper its study. Here, we unveil the genomic architecture of tetraploid P. ginseng by de novo genome assembly, representing 2.98 Gbp with 59 352 annotated genes. Resequencing data indicated that diploid Panax species diverged in association with global warming in Southern Asia, and two North American species evolved via two intercontinental migrations. Two whole genome duplications (WGD) occurred in the family Araliaceae (including Panax) after divergence with the Apiaceae, the more recent one contributing to the ability of P. ginseng to overwinter, enabling it to spread broadly through the Northern Hemisphere. Functional and evolutionary analyses suggest that production of pharmacologically important dammarane-type ginsenosides originated in Panax and are produced largely in shoot tissues and transported to roots; that newly evolved P. ginseng fatty acid desaturases increase freezing tolerance; and that unprecedented retention of chlorophyll a/b binding protein genes enables efficient photosynthesis under low light. A genome-scale metabolic network provides a holistic view of Panax ginsenoside biosynthesis. This study provides valuable resources for improving medicinal values of ginseng either through genomics-assisted breeding or metabolic engineering.

Rhamnellosides A and B, ω-Phenylpentaene Fatty Acid Amide Diglycosides from the Fruits of Rhamnella franguloides.

Two new ω-phenylpentaene fatty acid amide diglycosides, rhamnellosides A (1) and B (2), were isolated from the fruits of Rhamnella franguloides (Rhamnaceae). These compounds were prioritized using LC-MS/MS molecular networking dereplication based on our previous discovery of 2-acetoxy-ω-phenylpentaene fatty acid triglycosides berchemiosides A-C from a phylogenetically related species, Berchemia berchemiifolia. The structures of the isolated compounds were elucidated by spectroscopic analyses in combination with chemical derivatization. The pentaene groups of 1 and 2 were found to have (6E, 8E, 10Z, 12Z, 14E)-geometry, which is the same as that found in berchemioside A.

Anti-inflammatory activity of mulberrofuran K isolated from the bark of Morus bombycis.

Morus bombycis Koidzumi, commonly known as silkworm mulberry, is a plant belonging to family Moraceae. It has been used in Asian countries as a traditional medicine for treating hypertension, diabetes, and inflammatory disorders. In this study, we isolated eleven compounds from the cortex of M. bombycis and evaluated their inhibitory effects on nitric oxide (NO) production as an indicator of their anti-inflammatory activities using lipopolysaccharide (LPS)-stimulated murine macrophages. Compound 4 showed the most potent inhibitory activity on NO production. It was identified as mulberrofuran K (MFK). Anti-inflammatory activity of MFK was then carried out using LPS-stimulated RAW264.7 cells. MFK suppressed the production of NO, reactive oxygen species (ROS), and proinflammatory cytokines (interleukin (IL)-1ß, IL-6 and tumor necrosis factor alpha (TNF-α)) in a concentration-dependent manner. Western blot analysis revealed that MFK treatment inhibited expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). MFK also inhibited transcriptional activation of nuclear factor-κB (NF-κB) and extracellular-regulated kinases (ERK) 1/2 in LPS-stimulated murine macrophages. These results suggest that MFK, an anti-inflammatory constituents of M. bombycis cortex, has potential as a therapeutic candidates for preventing and treating inflammatory diseases.

High-throughput and direct measurement of androgen levels using turbulent flow chromatography liquid chromatography-triple quadrupole mass spectrometry (TFC-LC-TQMS) to discover chemicals that modulate dihydrotestosterone production in human prostate cancer cells.

OBJECTIVES: To develop a high-throughput screening system to measure the conversion of testosterone to dihydrotestosterone (DHT) in cultured human prostate cancer cells using turbulent flow chromatography liquid chromatography-triple quadrupole mass spectrometry (TFC-LC-TQMS). RESULTS: After optimizing the cell reaction system, this method demonstrated a screening capability of 103 samples, including 78 single compounds and 25 extracts, in less than 12 h without manual sample preparation. Consequently, fucoxanthin, phenethyl caffeate, and Curcuma longa L. extract were validated as bioactive chemicals that inhibited DHT production in cultured DU145 cells. In addition, naringenin boosted DHT production in DU145 cells. CONCLUSION: The method can facilitate the discovery of bioactive chemicals that modulate the DHT production, and four phytochemicals are potential candidates of nutraceuticals to adjust DHT levels in male hormonal dysfunction.

The complete chloroplast genome sequence of Korean Lonicera japonica and intra-species diversity.

Lonicera japonica is a traditional medicinal plant well known for its anti-inflammatory effect. The complete chloroplast genome sequence of L. japonica collected from Korea was obtained by de novo assembly using whole genome sequence data. The chloroplast genome is 155,060 bp in length, containing 88,853 bp in a large single copy (LSC), 18,653 bp in a small single copy (SSC) and 23,777 bp in a pair of inverted repeats (IRs). A total of 112 genes including 78 protein-coding genes and 34 structural RNA genes were identified. The sequence comparison of two L. japonica collected from Korea and China revealed 48 single nucleotide polymorphisms (SNPs) and 45 insertions/deletions (InDels). In addition, phylogenetic analysis represented intraspecific diversity within L. japonica species collected in Korea and China.

Identification and quantification of flavonoids in yellow grain mutant of rice (Oryza sativa L.).

Flavonoids are naturally occurring phenolic compounds with potential health-promoting activities. Although anthocyanins and phenolic acids in coloured rice have been investigated, few studies have focused on flavonoids. Herein, we analysed flavonoids in a yellow grain rice mutant using UHPLC-DAD-ESI-Q-TOF-MS, and identified 19 flavonoids by comparing retention times and accurate mass measurements. Among them, six flavonoids, isoorientin, isoorientin 2″-O-glucoside, vitexin 2″-O-glucoside, isovitexin, isoscoparin 2″-O-glucoside and isoscoparin, were isolated and fully identified from the yellow grain rice mutant, and the levels were significantly higher than wild-type, with isoorientin particularly abundant in mutant embryo. Significant differences in total phenolic compounds and antioxidant activity were observed in mutant rice by DPPH, FRAP and TEAC assays. The results suggest that the representative six flavonoids may play an important role in colouration and antioxidant activity of embryo and endosperm tissue. The findings provide insight into flavonoid biosynthesis and the possibility of improving functionality in rice.

Nrf2-Mediated HO-1 Induction and Antineuroinflammatory Activities of Halleridone.

Nuclear factor E2-related factor 2 (Nrf2) is the master regulator of antioxidant enzymes and is known to act on the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling pathway. Few studies have examined the bioactivity of halleridone. Herein, we investigated whether halleridone, which was isolated from the stems of the plant Cornus walteri, could regulate Nrf2-mediated heme oxygenase (HO)-1 expression and prevent intramicroglial inflammation induced by amyloid beta (Aß)1-42 overexpression. Biochemical and molecular experiments, such as real-time polymerase chain reaction, Western blot analysis, immunocytochemistry, immunofluorescence, and luciferase reporter gene assays, were performed. The results demonstrated that halleridone promoted the upregulation of Nrf2 expression and its translocation to the nucleus, thereby activating antioxidant response element gene transcription and HO-1 expression in murine hippocampal HT22 cells. Additionally, halleridone removed intramicroglial Aß1-42 and suppressed the production of inflammatory mediators such as interleukin (IL)-1ß, IL-6, prostaglandin E2, and nitric oxide (NO) induced by artificially overexpressed Aß1-42 and decreased pNF-κB accumulation in the nucleus and the expression of inducible NO synthase and cyclooxygenase II in BV-2 cells. In conclusion, halleridone activated Nrf2-mediated HO-1 expression and inhibited Aß1-42-overexpressed microglial BV-2 cell activation. These observations suggest that halleridone may have therapeutic potential for targeting neurodegeneration through neuroinflammation.

Pectolinarigenin, an aglycone of pectolinarin, has more potent inhibitory activities on melanogenesis than pectolinarin.

Pectolinarin and pectolinarigenin have been reported to be major compounds in Cirsium setidens. In the present study, we demonstrated inhibitory effects of pectolinarin and pectolinarigenin from C. setidens on melanogenesis. Melanin synthesis was decreased in both pectolinarin- and pectolinarigenin-treated melan-a cells and in a reconstructed human skin model. However, pectolinarigenin treatment showed more potent inhibitory activity of melanin synthesis than did pectolinarin treatment. The concentrations of pectolinarin and pectolinarigenin in C. setidens water extracts were determined by HPLC. Unfortunately, the amount of pectolinarigenin of C. setidens water extract was lower than that of pectolinarin. To increase the pectolinarigenin content in C. setidens water extract, several component conversion methods were studied. Consequently, we identified that microwave irradiation under 1% acetic acid was an optimum sugar elimination method.

Anti-Differentiation Effect of B, D-Seco limonoids of Swietenia mahogani.

BACKGROUND: Obesity is a pathological state caused by abnormal or excessive accumulation of fat. Swietenia mahogani JACQ., known as West Indian mahogany, is a medium-sized semi-evergreen tree belonging to Meliaceae. Their seeds are used in Indonesian folk medicine as a treatment for hypertension, diabetes, malaria, and it also has anti-feedant activities. The major components of S. mahogani are B, D-seco limonoids, a type of irregular triterpenes are well known. OBJECTIVE: We tried to find the bioactive components, which have the inhibitory activity on adipocyte differentiation from the seeds of S. mahogani. MATERIAL AND METHODS: 3T3-L1 cells, derived from mouse preadipocyte, are widely used in studying adipogenesis process. In this study, we used 3T3-L1 cells to find natural products with the inhibitory activity on adipocyte differentiation. S. mahogani seeds were dried and extracted with 100% MeOH. RESULTS: The methanolic extract was fractionated by bioassay-guided method to give nine B, D-seco limonoids (1-9) with slight structural modifications. Among nine compounds, compounds 4, 6 and 8 exhibited significant inhibitory effects of cell differentiation on 3T3-L1 cells. Those compounds have tigloyl residue at C-3 in common. Besides, compounds with no tigloyl residue at C-3 showed insignificant effect. Nevertheless, not all compounds with tigloyl residue at C-3 exerted significant inhibitory effect. CONCLUSION: These results suggested that tigloyl residue at C-3 may play a role in the anti-proliferative activity on a dipogenesis and the refined extract of S. mahogani may have a potential to be developed as a therapeutic agent to treat obesity. SUMMARY: Nine irregular seco-limonoids were isolated from Swietenia mahogani.Total extract and CHCl3 fraction of S. mahogani showed the significant inhibitory activities on 3T3-L1 cell differentiation.A tigloyl residue at C-3 in an aglycone may play a role in the anti-proliferative activity on adipogenesis.

Cytotoxic pterosins from Pteris multifida roots against HCT116 human colon cancer cells.

Two new pterosin glycosides, (2S,3S)-pterosin C 3-O-ß-d-(4'-(E)-caffeoyl)-glucopyranoside (1) and (2S,3S)-pterosin C 3-O-ß-d-(6'-(E)-p-coumaroyl)-glucopyranoside (2), were isolated from Pteris multifida (Pteridaceae) roots along with ten known pterosin compounds (3-12). The chemical structures of the isolated compounds were elucidated by extensive analysis of the 1D, 2D NMR, HRESIMS, and CD spectroscopic data. The cytotoxicities of 1-12 against HCT116 human colorectal cancer cell line were evaluated. Among the isolates, compound 1 showed moderate antiproliferative activity in HCT116 cells with an IC50 value of 8.0±1.7µM. Additionally, 1 induced the upregulation of the caspase-9 and procaspase-9 levels in Western blots and increased the annexin V/propidium iodide (PI)-positive cell population in flow cytometry.