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.

Extract of Aster koraiensis Nakai Leaf Ameliorates Memory Dysfunction via Anti-inflammatory Action.

Aster koraiensis Nakai (AK) leaf reportedly ameliorates health problems, such as diabetes. However, the effects of AK on cognitive dysfunction or memory impairment remain unclear. This study investigated whether AK leaf extract could attenuate cognitive impairment. We found that AK extract reduced the production of nitric oxide (NO), tumour necrosis factor (TNF)-α, phosphorylated-tau (p-tau), and the expression of inflammatory proteins in lipopolysaccharide- or amyloid-ß-treated cells. AK extract exhibited inhibitory activity of control specific binding on N-methyl-D-aspartate (NMDA) receptors. Scopolamine-induced AD models were used chronically in rats and acutely in mice. Relative to negative controls (NC), hippocampal choline acetyltransferase (ChAT) and B-cell lymphoma 2 (Bcl2) activity was increased in rats chronically treated with scopolamine and fed an AK extract-containing diet. In the Y-maze test, spontaneous alterations were increased in the AK extract-fed groups compared to NC. Rats administered AK extract showed increased escape latency in the passive avoidance test. In the hippocampus of rats fed a high-AK extract diet (AKH), the expression of neuroactive ligand-receptor interaction-related genes, including Npy2r, Htr2c, and Rxfp1, was significantly altered. In the Morris water maze assay of mice acutely treated with scopolamine, the swimming times in the target quadrant of AK extract-treated groups increased significantly to the levels of the Donepezil and normal groups. We used Tg6799 Aß-overexpressing 5XFAD transgenic mice to investigate Aß accumulation in animals. In the AD model using 5XFAD, the administration of AK extract decreased amyloid-ß (Aß) accumulation and increased the number of NeuN antibody-reactive cells in the subiculum relative to the control group. In conclusion, AK extract ameliorated memory dysfunction by modulating ChAT activity and Bcl2-related anti-apoptotic pathways, affecting the expression of neuroactive ligand-receptor interaction-related genes and inhibiting Aß accumulation. Therefore, AK extract could be a functional material improving cognition and memory.

The complete mitochondrial genome of Panax ginseng (Apiales, Araliaceae): an important medicinal plant.

Ginseng (Panax ginseng C. A. Meyer) is a multifunctional medicinal herb used worldwide and is an economically important high-value crop in Korea. Here, we presented the mitochondrial genome of P. ginseng landrace 'Jakyung', which is one of the most common cultivars cultivated in Korean farms. The complete mitochondrial genome sequence was 464,661 bp in length and had a single circular form. The ginseng mitochondrial genome encoded 72 unique genes, including 45 protein-coding genes, 24 tRNA genes, and three rRNA genes. Nucleotide composition analysis revealed a GC content of 45.1%, with a slightly higher A + T bias (A, 27.1%; T, 27.8%; G, 22.5%; C, 22.6%). Phylogenetic analysis showed that P. ginseng was closely related to Daucus carota in the Apiales. This complete mitochondrial genome sequence of P. ginseng provides valuable genetic information for further studies of this important medicinal plant.

Aster ageratoides Turcz. extract attenuates Alzheimer's disease-associated cognitive deficits and vascular dementia-associated neuronal death.

Dementia is the common neurodegenerative disorder affecting the elderly, with a progressive cognitive decline and memory loss. Since Alzheimer's disease (AD) and vascular dementia (VD) share key pathologies including oxidative damage, oral supplement of phytochemical medicines, which are well-known for their antioxidant properties, can be a viable therapy for both types of dementia. In this study, the therapeutic potential of the Aster ageratoides extract (AAE), an oriental drug with multiple medicinal properties, was tested on experimental rat models of AD and VD. After confirming the in vitro attenuation of neuronal excitotoxicity by AAE, rats were orally administered with AAE for 7 days and subsequently tested under 2 different experimental paradigms: efficacy screening against #1 AD and #2 VD. For paradigm #1, the rats received intraperitoneal scopolamine and subsequently underwent 3 different behavior tests i.e., the Y-maze, novel object recognition, and passive avoidance tests. For paradigm #2, the rats were operated with the 2-vessel occlusion and hypovolemia (2VO/H) technique, and at postoperative day 7, their hippocampal neuronal viability and the neuroinflammatory changes were quantified. The results showed that the scopolamine-induced impairment of memory performance was significantly improved by AAE intake. Furthermore, while the 2VO/H operation induced marked hippocampal neuronal death and microglial activation, both these effects were significantly attenuated by AAE supplements. Some of the aforementioned effects of AAE intake were dose-dependent. These results provided evidence that AAE supplements can exert anti-AD and -VD efficacies and suggested that AAE might be used as an edible phytotherapeutic for the 2 major types of dementia.

Erigeron annuus Protects PC12 Neuronal Cells from Oxidative Stress Induced by ROS-Mediated Apoptosis.

Reactive oxygen species (ROS), associated with oxidative stress, are involved in many biological processes such as apoptosis, necrosis, and autophagy. Oxidative stress might induce neuronal damage via ROS generation, causing neurodegenerative diseases. Erigeron annuus (EA) has antioxidant properties and could protect neurons from oxidative stress. In this study, we investigated the protective effect of the aerial parts (EAA) and flowers (EAF) from EA on ROS-mediated apoptosis in pheochromocytoma 12 cells. We quantified 18 types of phenolic compounds using high-performance liquid chromatography. Pretreatment of the cells with EAA and EAF attenuated ROS generation and induced the expression of antioxidant enzymes such as superoxide dismutase 2, catalase, and glutathione peroxidase. In addition, EAF reduced the expression of apoptotic proteins such as Bax/Bcl-xL, caspase-3, and caspase-8 to a greater extent than that with EAA. These results suggested that the protective effect of EAF against oxidative stress-induced apoptosis might be due to the prevention of ROS generation mediated by oxidative enzymes.

Differences in anti-inflammatory effect of immature and mature of Rubus coreanus fruits on LPS-induced RAW 264.7 macrophages via NF-κB signal pathways.

BACKGROUND: Rubus coreanus fruit (RF) has been used as a traditional medicine formulation to treat various diseases including diarrhea, asthma, and cancer in East Asia (Korea, China, and Japan). RF, which is native to Korea, has a larger fruit size than that of exotic species. In this study, we aimed to compare the anti-inflammatory activities of immature and mature RF extracted with different solvents. METHODS: Mature and immature RF (MRF and IRF) were extracted with 30% ethanol, 70% ethanol and water at room temperature. The antioxidant activity was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assays. Anti-inflammatory activity was determined by measuring nitric oxide (NO) production, expression of inflammatory proteins (inducible NO synthase [iNOS], cyclooxygenase [COX]-2, nuclear factor [NF]-κB, and inhibitor of NF-κB [IκB]), and inflammatory cytokines using polymerase chain reaction in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. RESULTS: The IRF 30% ethanol extract showed higher radical scavenging activity in DPPH and ABTS assays (half-maximal inhibitory concentration [IC50] 16.0 ± 0.5 and 15.9 ± 0.4) than MRF did. In addition, the IRF 30% ethanol extract (200 µg/mL) significantly reduced the production of the inflammatory mediator NO by approximately 80% and inhibited iNOS, COX-2, phosphorylated (p)-IκB, and p-NF-κB activation compared with MRF. Moreover, IRF extract decreased the inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1ß, and IL-6 compared with the MRF extract. CONCLUSIONS: This study revealed that IRF showed more beneficial effects than MRF did in LPS-stimulated RAW 264.7 macrophages, suggesting that IRF may be a useful anti-inflammatory agent.

In vitro assessments of bone microcomputed tomography in an aged male rat model supplemented with Panax ginseng.

Recent research has confirmed that Panax ginseng (P. ginseng) has effect on cultured osteoblast of the mouse. In this study we aim to validate the usefulness of tibia quantification by correlating micro-computed tomographic (microCT) images with histology analysis in the aged male rats. A total of thirty - old male WISTAR rats were used and divided into ten 8 weeks rats and ten 112 weeks aged rats with vehicle and ten 112 weeks aged rats with P. ginseng (300 mg/kg/day). Daily oral administration of P. ginseng lasted for 8 weeks. Bone histomorphometric parameters and the trabecular bone microarchitectural properties of tibia were determined by microCT scan. MicroCT analysis showed significantly lower bone mineral density (BMD) and trabecular bone number in the aged group. Ginseng prevented total BMD decrease in the tibia induced by natural aging, which was accompanied by a significant decrease in skeletal remodeling. Furthermore, the aged group with ginseng was found to have a significantly higher osteoblast. In the blood biochemistry results, serum phosphorus, calcium, osteocalcin, T3, and T4 remained unchanged. The present study indicated that P. ginseng might be a potential alternative medicine for the prevention and treatment of natural aging-induced osteoporosis in human.

De novo transcriptome assembly and the identification of gene-associated single-nucleotide polymorphism markers in Asian and American ginseng roots.

We performed de novo transcriptome sequencing for Panax ginseng and Panax quinquefolius accessions using the 454 GS FLX Titanium System and discovered annotation-based genome-wide single-nucleotide polymorphism (SNPs) using next-generation ginseng transcriptome data without reference genome sequence. The comprehensive transcriptome characterization with the mature roots of four ginseng accessions generated 297,170 reads for 'Cheonryang' cultivar, 305,673 reads for 'Yunpoong' cultivar, 311,861 reads for the G03080 breeding line, and 308,313 reads for P. quinquefolius. In transcriptome assembly, the lengths of the sample read were 156.42 Mb for 'Cheonryang', 161.95 Mb for 'Yunpoong', 165.07 Mb for G03080 breeding line, and 166.48 Mb for P. quinquefolius. A total of 97 primer pairs were designed with the homozygous SNP presented in all four accessions. SNP genotyping using high-resolution melting (HRM) analysis was performed to validate the putative SNP markers of 97 primer pairs. Out of the 73 primer pairs, 73 primer pairs amplified the target sequence and 34 primer pairs showed polymorphic melting curves in samples from 11 P. ginseng cultivars and one P. quinquefolius accession. Among the 34 polymorphic HRM-SNP primers, four primers were useful to distinguish ginseng cultivars. In the present study, we demonstrated that de novo transcriptome assembly and mapping analyses are useful in providing four HRM-SNP primer pairs that reliably show a high degree of polymorphism among ginseng cultivars.

Dietary effect of Rubus coreanus ethanolic extract on immune gene expression in white leg shrimp, Penaeus vannamei.

The objective of this study was to evaluate the effect of dietary supplementation of a Rubus coreanus ethanolic extract on immunostimulatory response in white leg shrimp Penaeus vannamei. Shrimps with an average initial weight of 0.5 ± 0.04 g were collected and acclimatized for 10 days. Four experimental diets including a control diet, a probiotic diet and 0.25 and 0.5% of R. coreanus ethanolic extract (RcEE) diets were used to feed the shrimps. After 8 weeks of culture, shrimp fed with probiotic and 0.25% RcEE diet had showed significant enhancement in the growth while shrimp fed with 0.5% RcEE diet showed significantly increased expression of immune genes and antioxidant enzymes activities. One week of challenge experiments for all the four diets fed shrimps showed decreased cumulative mortality in the 0.5% RcEE diets fed shrimps, when compared with the probiotic and 0.25% RcEE diet fed shrimp groups. The results indicates that R. coreanus ethanolic extract could be used as a herbal immunostimulant for shrimps to increase its immunity and disease resistance against the bacterial pathogen, Vibrio alginolyticus.

Non-specific immune response and disease resistance induced by Siegesbeckia glabrescens against Vibrio parahaemolyticus in Epinephelus bruneus.

The immunomodulatory effect of Siegesbeckia glabrescens extract-supplementation diets on innate immune response and disease resistance of kelp grouper, Epinephelus bruneus against Vibrio parahaemolyticus at weeks 1, 2, and 4 is reported. The serum lysozyme activity was significantly enhanced with any enriched diet from weeks 1-4 when compared to control diet (0%). The alternative complement haemolytic activities significantly were enhanced with all enriched diets on weeks 2 and 4 whereas the cellular reactive oxygen species (ROS) was significantly enhanced only with 1.0% and 2.0% diets. The reactive nitrogen intermediate (RNI) value was significantly enhanced with any enriched diet on weeks 2 and 4, but on first week it did not differ from control. The myeloperoxidase (MPO) production significantly increased with 1.0% and 2.0% diets from second week onwards; with other enriched diets the increase manifested on fourth week; but during first week it did not vary from that of the control with any enriched diet. The protection in terms of cumulative mortality was the least being 25% and 20% when fed with 1.0% and 2.0% diets. The present results indicate that feeding kelp grouper with S. glabrescens extract enriched diet at 1.0% and 2.0% levels significantly enhance the immunological parameters, increase the disease resistance and minimize the cumulative mortality in E. bruneus against V. parahaemolyticus.

Effect of dietary supplementation with Suaeda maritima on blood physiology, innate immune response, and disease resistance in olive flounder against Miamiensis avidus.

The effect of Suaeda maritima enriched diet on blood physiology, innate immune response, and disease resistance in olive flounder Paralichythys olivaceus against Miamiensis avidus on weeks 1, 2, and 4 was investigated. Feeding with any enriched diet and then challenging with M. avidus significantly increased white blood cells (WBC) on weeks 2 and 4; the red blood cells (RBC) significantly increased with 0.1% and 1.0% enriched diets on week 4. The hemoglobin (Hb) and hematocrit (Ht) levels significantly increased when fed with 0.1% and 1.0% supplementation diets on weeks 2 and 4. The mean corpuscular volume (MCV) did not significantly vary with any diet and time; however the mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) significantly increased with 0.1% and 1.0% supplementation diets on weeks 2 and 4. The leukocytes such as lymphocytes (Lym), monocytes (Mon), neutrophils (Neu) and biochemical parameters such as total protein (TP), glucose (GLU), and calcium (CAL) levels significantly increased in 0.1% and 1.0% supplementation diet fed groups on weeks 2 and 4. The serum lysozyme activity was significantly enhanced in 0.1% and 1.0% supplementation diet fed groups from weeks 1 to 4 when compared to the control (0% herbal extract enriched diet). The scuticocidal activity and respiratory burst activity were significantly enhanced when fish were fed with 0.1% and 1.0% supplementation diets from weeks 2 and 4. The protective effect in terms of cumulative mortality (50% and 40%) was low in groups on being fed with 0.1% and 1.0% supplemented diet. Therefore the present study suggested that 0.1% and 1.0% S. maritime-supplemented diets protect the hematological and biochemical parameters, improving the innate immunity, affording protection disease from M. avidus infection in olive flounder.

Enhancement of Eriobotrya japonica extracts on non-specific immune response and disease resistance in kelp grouper Epinephelus bruneus against Vibrio carchariae.

The present study investigated the effect of Eriobotrya japonica extracts at 0%, 0.1%, 1.0%, and 2.0% doses supplementation with feed on non-specific immune response, hematological and biochemical profile, and disease resistance against Vibrio carchariae in kelp grouper Epinephelus bruneus at weeks 1, 2, and 4. The white blood cell (WBC) significantly increased in fish fed with 0.1%, 1.0%, and 2.0% diets on weeks 1 and 2 when compared to the control. However, the glucose always decreased from the control except on week 2 against pathogen. The serum total protein, albumin, and globulin significantly increased at week 2 but they did not changed significantly at weeks 1 and 4. The superoxide anion, lymphokines production index, and phogocytosis did not significantly increased in any diet on the first week whereas it was significantly enhanced in 1.0% and 2.0% supplementation diets on weeks 2 and 4 against V. carchariae when compared to control. All diets significantly enhanced the serum lysozyme activity, bactericidal activity, and haemolytic complement activity from weeks 1-4 as compared to control. The serum agglutinating antibody titre did not significantly enhance on the first week whereas it was significantly enhanced on weeks 2 and 4. Fish fed with 1.0% and 2.0% doses diets was found lower mortality than 0.1% diet. Thus, this study suggested that 1.0% and 2.0% doses supplementation diets could be advocated to enhance the immune response and production disease from V. carchariae in E. bruneus.

Construction of genomic DNA library of Korean ginseng (Panax ginseng C. A. MEYER) and development of sequence-tagged sites.

This study describes an efficient approach for developing sequence tagged sites (STS) for Panax ginseng C.A. MEYER, and their applications for line discrimination. By using the methylation filtering (MF) technique, a genomic library was constructed, in which clone inserts were derived from the hypomethylated regions of ginseng genome. A methylation unfiltered genomic library was also constructed and the clone inserts were compared to those from the MF library in terms of sequence characteristics. Sequence analysis revealed that MF efficiently enriched the protein coding region of P. ginseng, for which the repetitive DNA appeared to be as little as 2.5 fold lower than clones in the unfiltered library, and also indicated that the P. ginseng genome may contain a large fraction of methylated repetitive DNA elements. A total of 99 and 100 highly stringent STS primer sets were designed from the filtered and unfiltered library, respectively. Amplification products were tested for latent polymorphism across six cultivars of P. ginseng and other 2 Panax species using six endonucleases recognizing four-bases. STS primer sets described here will be useful for marker-assisted selection, genome mapping and line discrimination of P. ginseng or its cultivars from other Panax species.