Chemical Analysis of SU-Eohyeol Pharmacopuncture and Its In Vitro Biological Activities.

Introduction: Pharmacopuncture (PA) is widely used in traditional Korean medicine to treat various diseases, including abdominal obesity, nervous system diseases, and musculoskeletal disorders. In the present study, we attempted to identify the chemical components of SU-Eohyeol PA (SUEHP), comprising extracts of eight medicinal herbs and Cervi Parvum Cornu, using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) and evaluated the in vitro anti-inflammatory and antioxidant activities of SUEHP. Methods: Volatile components of SUEHP were identified by GC-MS analysis of the n-hexane, dichloromethane (DCM), and distilled water-acetonitrile (DW-CAN) solvent fractions. LC-MS was performed to identify small metabolites of SUEHP using a water-methanol solvent fraction. In vitro anti-inflammatory and antioxidant potential of SUEHP was evaluated using cell-free biochemical assays and a BV2 microglial cell culture system. Results: GC-MS of SUEHP using the n-hexane, DCM, and DW-ACN solvent fractions detected 32 components. The major components (>3%) were 9-octadecenamide (25.9%) and eicosane (5.4%) in n-hexane fraction, 9-octadecenamide (29.1%) and cis-11-octadecenoic acid (3.1%) in DCM fraction, and 9-octadecenamide (31.1%) and 9-octadecenoic acid (17.9%) in DW-CAN fraction. LC-MS of SUEHP using a water-methanol solvent fraction detected 36 primary metabolites, including (a)symmetric dimethylarginine, L-pyroglutamic acid, n,n-dimethylglycine, n-acetyl-L-aspartic acid, and amino acids. Enrichment analysis and subsequent network analysis of the primary metabolites suggested their association with neurodegenerative diseases, including Alzheimer's disease and schizophrenia. Cell-free biochemical assays and molecular signaling studies of lipopolysaccharide-stimulated BV2 murine microglial cells demonstrated the anti-inflammatory and antioxidant activities of SUEHP. Conclusion: The present study identified the biochemically active components of SUEHP and suggested their therapeutic potential against diseases related to inflammatory and oxidative stress.

Effect of Poria cocos Terpenes: Verifying Modes of Action Using Molecular Docking, Drug-Induced Transcriptomes, and Diffusion Network Analyses.

We characterized the therapeutic biological modes of action of several terpenes in Poria cocos F.A Wolf (PC) and proposed a broad therapeutic mode of action for PC. Molecular docking and drug-induced transcriptome analysis were performed to confirm the pharmacological mechanism of PC terpene, and a new analysis method, namely diffusion network analysis, was proposed to verify the mechanism of action against Alzheimer's disease. We confirmed that the compound that exists only in PC has a unique mechanism through statistical-based docking analysis. Also, docking and transcriptomic analysis results could reflect results in clinical practice when used complementarily. The detailed pharmacological mechanism of PC was confirmed by constructing and analyzing the Alzheimer's disease diffusion network, and the antioxidant activity based on microglial cells was verified. In this study, we used two bioinformatics approaches to reveal PC's broad mode of action while also using diffusion networks to identify its detailed pharmacological mechanisms of action. The results of this study provide evidence that future pharmacological mechanism analysis should simultaneously consider complementary docking and transcriptomics and suggest diffusion network analysis, a new method to derive pharmacological mechanisms based on natural complex compounds.

Toxicological assessments of an ethanol extract complex of Descurainia sophia and Peucedanum praeruptorum: Subacute oral toxicity and genotoxicity studies.

An ethanol extract complex of Descurainia sophia seeds and Peucedanum praeruptorum roots, called BP10A, has antitumor potential against colorectal cancer. In the present study, we evaluated the 28-day oral toxicity and the genotoxicity of BP10A. The subacute toxicity test was done through oral administration to mice. ICR mice (n = 10) received daily oral BP10A doses of 0, 500, 1000 and 2000 mg/kg for 28 consecutive days. During administration, general clinical signs, food consumption, organ weights, and hematologic, biochemical and histopathological parameters in male and female mice were assessed. No significant adverse effects up to the highest dose (2000 mg/kg) were found. The genotoxicity was evaluated using a battery of tests, including an in vitro bacterial reverse mutation (Ames) test, an in vivo micronucleus test using bone marrow cells in ICR mice and a chromosomal aberration test using CHL/IU cells. BP10A did not show any genotoxic signs in the Ames (up to 5000 µg/plate), micronucleus (up to 5000 mg/kg) and the chromosomal aberration tests (550-1750 µg/mL). Therefore, BP10A was considered safe based on the subacute toxicity and genotoxicity results, indicating that it is a useful pharmaceutical material with no adverse toxicity.

Ameliorative effects of aqueous extract of Forsythiae suspensa fruits on oxaliplatin-induced neurotoxicity in vitro and in vivo.

BACKGROUND: The dried fruits of Forsythia suspensa has generally been used to clear heat and detoxify in traditional Korean and Chinese medicine. Oxaliplatin is a first-line treatment chemotherapeutic agent for advanced colorectal cancer, but it induces peripheral neuropathy as an adverse side effect affecting the treatment regimen and the patient's quality of life. The present study was conducted to evaluate the neuroprotective effects of an aqueous extract of F. suspensa fruits (EFSF) on oxaliplatin-induced peripheral neuropathy. METHODS: The chemical components from EFSF were characterized and quantified using the ultra-high performance liquid chromatography-diode array detector system. The cytotoxicities of anticancer drugs in cancer cells and PC12 cells were assessed by the Ez-Cytox viability assay. To measure the in vitro neurotoxicity, the neurite outgrowth was analyzed in the primary dorsal root ganglion (DRG) cells, and neural PC12 cells that were differentiated with nerve growth factor. To evaluate the in vivo neuroprotective activity, the von Frey test was performed in six-week-old male mice (C57BL/6) receiving EFSF (60-600 mg/kg) in the presence of 20-30 mg/kg cumulative doses of oxaliplatin. Thereafter, the mice were euthanized for immunohistochemical staining analysis with an antibody against PGP9.5. RESULTS: EFSF attenuated the cytotoxic activities of the various anticancer drugs in neural PC12 cells, but did not affect the anticancer activity of oxaliplatin in human cancer cells. Oxaliplatin remarkably induced neurotoxicities including cytotoxicity and the inhibited neurite outgrowth of DRG and neural PC12 cells. However, the co-treatment of EFSF (100 µg/ml) with oxaliplatin completely reversed the oxaliplatin-induced neurotoxicity. Forsythoside A, the major component of EFSF, also exerted remarkable neuroprotective effects against the oxaliplatin-induced neurotoxicity. In addition, EFSF (60-200 mg/kg) significantly alleviated the oxaliplatin-induced mechanical allodynia and loss of intra-epidermal nerve fiber to the levels of the vehicle control in the mouse peripheral neuropathy model. CONCLUSIONS: EFSF could be considered a useful herbal medicine for the treatment of peripheral neuropathy in cancer patients receiving chemotherapy with oxaliplatin.

Neuroprotective Effects of an Aqueous Extract of Forsythia viridissima and Its Major Constituents on Oxaliplatin-Induced Peripheral Neuropathy.

The dried fruits of Forsythia viridissima have been prescribed to relive fever, pain, vomiting, and nausea in traditional medicine. Oxaliplatin (LOHP) is used to treat advanced colorectal cancer; however, it frequently induces peripheral neuropathies. This study was done to evaluate the neuroprotective effects of an aqueous extract of Forsythia viridissima fruits (EFVF) and its major constituents. Chemical constituents from EFVF were characterized and quantified with the UHPLC-diode array detector method, and three major constituents were identified as arctiin, matairesinol, and arctigenin. The in vitro cytotoxicity was measured by the Ez-cytox viability assay, and the in vivo neuroprotection activity was evaluated by a von Frey test in two rodent animal models that were administered LOHP. EFVF significantly alleviated the LOHP-induced mechanical hypersensitivity in the induction model. EFVF also prevented the induction of mechanical hyperalgesia by LOHP in the pre- and co-treatment of LOHP and EFVF. Consistently, EFVF exerted protective effects against LOHP-induced neurotoxicity as well as inhibited neurite outgrowths in PC12 and dorsal root ganglion cells. Among the major components of EFVF, arctigenin and matairesinol exerted protective effects against LOHP-induced neurotoxicity. Therefore, EFVF may be useful for relieving or preventing LOHP-induced peripheral neuropathy in cancer patients undergoing chemotherapy with LOHP.

Aqueous extract of Phragmitis rhizoma ameliorates myelotoxicity of docetaxel in vitro and in vivo.

BACKGROUND: A variety of anticancer chemotherapeutics induce adverse side effects including myelotoxicity. Dried roots of Phragmites communis Trinius, Phragmitis rhizoma, have been clinically used in traditional folk medicine to relieve various symptoms like fever. In this study, we evaluated the protective effect of the aqueous extract of Phragmitis rhizoma (EPR) against docetaxel-induced myelotoxicity in vitro and in vivo. METHODS: The in vitro myelo-protective effect of EPR was evaluated using the colony forming unit (CFU) assay with hematopoietic progenitor cells. The in vivo efficacy of EPR was evaluated in myelosuppressed C57BL/6 male mice which were induced by repeated intraperitoneal injections of 30 mg/kg docetaxel for 3 times. EPR was orally administered for 4 days to docetaxel-induced myelosuppressed C57BL/6 male mice which were induced by intraperitoneal injection of 30 mg/kg docetaxel for 3 times: Group 1 (vehicle control, n = 10), Group 2 (docetaxel plus vehicle, n = 10), Group 3 (docetaxel plus EPR 30 mg/kg, n = 10), Group 4 (docetaxel plus EPR 100 mg/kg, n = 10) and Group 5 (docetaxel plus EPR 300 mg/kg, n = 10). Whole blood counts were measured automatically, and immune organs were histologically examined. Expression of immunomodulatory cytokines was measured by quantitative real-time polymerase chain reaction or enzyme-linked immunosorbent assay. The toxicity of EPR itself was evaluated in normal human cell lines including IMR-90, foreskin fibroblast and human umbilical vein endothelial cells. The hepatotoxicity of EPR was predicted by multi-parametric assays involving cell viability, caspase 3/7 activity, GSH contents and LDH leakage using the HepaRG hepatic cell line. RESULTS: Co-treatment of EPR or its major component, p-hydroxycinnamic acid, increased the numbers of hematopoietic CFU counts in the docetaxel-induced in vitro myelotoxicity assay system. The in vitro protective effect of EPR against docetaxel toxicity was replicated in a myelosuppressed animal model: white blood cells, neutrophils, lymphocytes and red blood cells rebounded; bone marrow niche and structural integrity of the thymus were preserved; and the expression of immune-stimulating cytokines including IL3, IL6, SCF and GM-CSF was enhanced. Furthermore, EPR and p-hydroxycinnamic acid promoted the proliferation of primary splenocytes and thymocytes. In the toxicity assays, no remarkable signs related with toxicity were observed in all tested normal human cells and HepaRG. CONCLUSIONS: EPR has the potential to ameliorate docetaxel-mediated myelotoxicity in both in vitro and in vivo models. However, the identification of the responsible active components and the precise underlying myelo-protective mechanism of EPR need to be elucidated before novel drug development using EPR can precede.

Ethanolic extract of Descurainia sophia seeds sensitizes A549 human lung cancer cells to TRAIL cytotoxicity by upregulating death receptors.

BACKGROUND: Our previous genome-wide gene expression analysis revealed that tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptors 4 (DR4) and 5 (DR5) are markedly upregulated by the ethanolic extract of D. sohia seeds (EEDS) in A549 TRAIL-refractory cancer cells. In the present study, we investigated whether the EEDS-mediated upregulation of TRAIL death receptors was associated with increased TRAIL-mediated toxicity in A549 cells in vitro. METHODS: Cell proliferation and viability were determined by an automatic cell counter. Gene silencing was performed by introducing small interfering RNA into cells. Expression changes of cellular proteins were determined by western blot analysis. Apoptotic cell death was monitored by western blot analysis. Analysis of variance followed by the post-hoc Dunnett's test was used to compare the data. RESULTS: EEDS treatment increased both mRNA and protein levels of DR4 and DR5 in the TRAIL refractory A549 cells. Co-treatment of A549 cells with sub-lethal dose of EEDS and recombinant TRAIL increased the apoptotic cell death. Upregulation of DR5 by EEDS was mediated by an endoplasmic reticulum stress-induced transcription factor, CCAAT/enhancer-binding protein homologous protein (CHOP), and knockdown of CHOP expression inhibited EEDS-induced DR5 upregulation and abolished the EEDS-associated increase in TRAIL toxicity in A549 cells. CONCLUSIONS: EEDS can sensitize A549 cells to TRAIL cytotoxicity by upregulation of TRAIL death receptors. Our findings suggested that EEDS is a good initial herbal source for the development of an anticancer supplement for anticancer therapeutics associated with TRAIL.

Aqueous extract of Lithospermi radix attenuates oxaliplatin-induced neurotoxicity in both in vitro and in vivo models.

BACKGROUND: Oxaliplatin can induce peripheral neuropathy (OXIPN) as an adverse side effect in cancer patients. Until now, no effective preventive or therapeutic drug has been developed; therefore, the dose-limiting factor of OXIPN is still an obstacle in the use of oxaliplatin to treat cancer patients. In the present study, we report for the first time that the aqueous extract of Lithospermi radix (WLR) can attenuate the OXIPN in both in vitro and in vivo neuropathic models. METHODS: The protective effect of WLR on OXIPN was evaluated in vitro by quantifying nerve growth factor (NGF)-stimulated neurite outgrowth in PC12 cells treated with a combination of oxaliplatin and WLR. The neuroprotective potential of WLR was further confirmed by measuring the changes in nociceptive sensitivities to external mechanical stimuli in neuropathic animals induced by oxaliplatin. Histological and immunohistochemical studies were further done to examine the effect of WLR in mouse spinal cords and footpads. RESULTS: Oxaliplatin-induced neurotoxicity in NGF-stimulated PC12 cells. It could reduce the lengths and branching numbers of neuritis in NGF-stimulated PC12 cells. Co-treatment of WLR rescued the differentiated PC12 cells from the neurotoxicity of oxaliplatin. In a chronic OXIPN animal model, administration of oxaliplatin i.p. induced enhanced nociceptive sensitivity to mechanical stimuli (25.0 to 72.5 % of response rate) along with spinal activation of microglias and astrocytes and loss of intraepidermal nerve fibers in footpads, which is remarkably suppressed by oral administration of WLR (67.5 to 35 % of response rate at the end of experiment). Cytotoxicity of oxaliplatin determined in human cancer cells was not affected irrespective of the presence of WLR. CONCLUSIONS: In conclusion, we demonstrated that WLR can attenuate OXIPN in both in vitro and in vivo experimental models, which may be in part attributed to its anti-inflammatory activity in the spinal cord and its neuroprotective potential in the peripheral nerve system without affecting the anti-tumor potential of oxaliplatin. Therefore, WLR could be considered as a good starting material to develop a novel therapeutic agent targeting OXIPN. However, further studies should be done to elucidate the underlying mechanism such as molecular targets and active constituent(s) in WLR with neuroprotective potential.

Helveticoside is a biologically active component of the seed extract of Descurainia sophia and induces reciprocal gene regulation in A549 human lung cancer cells.

BACKGROUND: Although the pharmacological activities of the seed extract of Descurainia sophia have been proven to be useful against cough, asthma, and edema, the biologically active components, particularly at the molecular level, remain elusive. Therefore, we aimed to identify the active component of an ethanol extract of D. sophia seeds (EEDS) by applying a systematic genomic approach. RESULTS: After treatment with EEDS, the dose-dependently expressed genes in A549 cells were used to query the Connectivity map to determine which small molecules could closely mimic EEDS in terms of whole gene expression. Gene ontology and pathway analyses were also performed to identify the functional involvement of the drug responsive genes. In addition, interaction network and enrichment map assays were implemented to measure the functional network structure of the drug-responsive genes. A Connectivity map analysis of differentially expressed genes resulted in the discovery of helveticoside as a candidate drug that induces a similar gene expression pattern to EEDS. We identified the presence of helveticoside in EEDS and determined that helveticoside was responsible for the dose-dependent gene expression induced by EEDS. Gene ontology and pathway analyses revealed that the metabolism and signaling processes in A549 cells were reciprocally regulated by helveticoside and inter-connected as functional modules. Additionally, in an ontological network analysis, diverse cancer type-related genes were found to be associated with the biological functions regulated by helveticoside. CONCLUSIONS: Using bioinformatic analyses, we confirmed that helveticoside is a biologically active component of EEDS that induces reciprocal regulation of metabolism and signaling processes. Our approach may provide novel insights to the herbal research field for identifying biologically active components from extracts.

In Vivo Anti-tumor Effects of the Ethanol Extract of Gleditsia sinensis Thorns and Its Active Constituent, Cytochalasin H.

Angiogenesis is the process of new vessel formation from pre-existing blood vasculature and is critical for continuous tumor growth. We previously reported that an ethanolic extract of Gleditsia sinensis thorns (EEGS) and its active constituent, cytochalasin H, have anti-angiogenic activity in vitro and in vivo via suppression of endothelial cell functions. In the present study, EEGS and cytochalasin H were observed to efficiently inhibit tumor growth in an in ovo xenograft model without significant toxicity. We repeatedly observed the anti-tumor and anti-metastatic effects of EEGS in representative animal models. These results suggest that EEGS and its active constituent, cytochalasin H, are potential candidates for the development of anti-angiogenic cancer drugs.

An evaluation of the anti-angiogenic effect of the Korean medicinal formula "Sa-mi-yeon-geon-tang" in vitro and in ovo.

BACKGROUND: Angiogenesis is a general hallmark of cancer; therefore, the inhibition of tumor-derived angiogenesis is considered to be an attractive target in the development of anti-cancer agents. Sa-mi-yeon-geon-tang (SMYGT), a decoction that consists of four natural medicinal products, has been traditionally prescribed in Oriental medicine to treat diverse diseases, including cancer. In the present study, we investigated the anti-angiogenic potential of SMYGT in vitro and in ovo. METHODS: The anti-angiogenic potential of SMYGT was evaluated using conventional in vitro assays with human umbilical vein endothelial cells (HUVECs) and chorioallantoic membrane (CAM) assays with fertilized eggs. The expression changes of pro-angiogenic proteins and intracellular signaling in HUVECs following SMYGT treatment were determined by quantitative polymerase chain reaction, gelatinase zymography, and western blot analysis. RESULTS: SMYGT efficiently inhibited three-dimensional capillary-like tube formation by HUVECs on extracellular matrix supports, as well as new vessel formation on CAMs. SMYGT inhibited cell adhesion to the extracellular matrix and HUVEC cell invasion through Matrigel without affecting cell proliferation, viability, and motility. These anti-angiogenic effects of SMYGT in HUVECs were related to decreases in the phosphorylation of focal adhesion kinase and the expression of matrix metallopeptidase-2 activity. CONCLUSIONS: SMYGT exhibited an anti-angiogenic potential in both in vitro and in ovo experiments, which may partially contribute to its anti-tumor effect in clinical conditions. We suggest that SMYGT may be a promising source material for the development of anti-cancer chemotherapeutics that target angiogenesis.

Effect of an ethanol extract of Descurainia sophia seeds on Phase I and II drug metabolizing enzymes and P-glycoprotein activity in vitro.

BACKGROUND: Descurainia sophia seeds have a variety of pharmacological functions and been widely used in traditional folk medicine. However, their effects on human drug metabolizing enzyme (DME) activities have not been elucidated. The present study investigated the inhibitory effects of an ethanol extract of D. sophia seeds (EEDS) on human Phase I/II (DMEs) and P-glycoprotein (p-gp) in vitro. METHODS: The enzyme activities of human Phase I (cytochrome P450s, CYPs), Phase II (uridine diphosphate glucuronosyltransferases, UGTs) DMEs, and the drug transporter P-gp were determined in the presence of various concentrations of EEDS using commercially available luminogenic assay systems. The mode of enzyme inhibition and the inhibitory constant (Ki) value of EEDS were graphically determined with Lineweaver-Burk double reciprocal plots and secondary plots, respectively. RESULTS: The enzyme activity assays showed that EEDS moderately inhibited the CYP1A2, CYP2C9, and CYP2C19 isoforms with half maximal inhibitory concentrations (IC50) of 47.3, 25.8, and 38.7 µg/mL, respectively. Graphical analyses with Lineweaver-Burk double reciprocal plots and secondary plots indicated that EEDS competitively inhibited CYP2C9 with a Ki value of 19.8 µg/mL; however, it inhibited CYP2C9 and CYP2C19 in a mixed mode with Ki values of 5.2, and 11.9 µg/mL, respectively. Other Phase I (CYP2C8, CYP2D6, and CYP3A4) and Phase II (UGT1A1 and UGT2B7) enzymes as well as P-gp were weakly or negligibly affected by EEDS with concentrations up to 500 µg/mL. CONCLUSIONS: EEDS is a selective inhibitor of CYP1A2, CYP2C9, and CYP2C19 with moderate enzymatic inhibition. Clinically, full consideration should be given to a potential toxic adverse effect from a herb-drug interaction when drugs that are particularly susceptible to CYP1A2, CYP2C9, or CYP2C19-mediated metabolism are taken together with EEDS. Characterization of metabolic profiles of specific herbal drugs could help consumers and medical specialists to use them safely as a complementary and alternative medicine.

Cytochalasin H, an active anti-angiogenic constituent of the ethanol extract of Gleditsia sinensis thorns.

Angiogenesis, the process of new vessel formation from the pre-existing blood vasculature, is critical for continuous tumor growth and is considered to be a validated antitumor target. The results of our previous study demonstrate the anti-angiogenic potential of an extract of Gleditsia sinensis thorns, which has been traditionally used in Korean medicine to remedy diverse diseases, including tumors. In the present study, we attempted to identify the active anti-angiogenic constituents of the ethanol extract of G. sinensis thorns (EEGS). By virtue of in vitro activity-guided fractionation using human umbilical vein endothelial cells (HUVEC) primary endothelial cells, chromatographic separation, and NMR spectral analyses, we isolated and identified the potent active constituent, cytochalasin H, a biologically active secondary metabolite of fungi. This unexpected active constituent may have originated from the endophytic fungi, Chaetomium globosum, which naturally populate G. sinensis, the identity of which was determined by analysis of fungal community. Cytochalasin H isolated from the EEGS showed in vitro anti-angiogenic activities such as suppressed cell growth and mobility in HUVEC, and inhibited the pro-angiogenic protein-induced formation of new blood vessels in vivo. The anti-angiogenic effect of cytochalasin H was in part due to reduced expression of pro-angiogenic factor, such as endothelin-1. This is the first report regarding the isolation and identification of cytochalasin H, as an active anti-angiogenic constituent of G. sinensis thorns.

Anti-angiogenic potential of an ethanol extract of Annona atemoya seeds in vitro and in vivo.

BACKGROUND: Angiogenesis, which is initiated by certain tumor micro-environmental conditions and diverse protein factors, plays a pivotal role during tumor development and metastasis. Therefore, many efforts have been made to develop effective anti-angiogenic agents as anticancer therapeutics. In the current study, we investigated the anti-angiogenic potential of an ethanol extract of Annona atemoya seeds (EEAA) in vitro and in vivo. METHODS: The anti-angiogenic potential of EEAA was evaluated using various in vitro/in vivo models, including cell proliferation, migration, and tube formation by human umbilical vascular endothelial cells (HUVECs); a Matrigel plug assay; and tumor-induced angiogenesis. The expression of hypoxia-inducible factors (HIFs) and vascular endothelial growth factor (VEGF) was investigated using reverse transcription-polymerase chain reaction, immunoassays, and western blotting. RESULTS: EEAA was able to significantly inhibit the angiogenic properties of HUVECs in vitro as well as angiogenic factor-induced blood vessel formation in vivo. EEAA down-regulated the expression of VEGF and HIF-1alpha/2alpha at the mRNA and protein levels, respectively, in cancer cells under hypoxic conditions. CONCLUSIONS: EEAA shows a strong anti-angiogenic potential in both in vitro and in vivo systems, and we suggest that EEAA may be a valuable herbal source for anticancer drug development.

Biological network comparison identifies a novel synergistic mechanism of Ginseng Radix-Astragali Radix herb pair in cancer-related fatigue.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginseng Radix and Astragali Radix are commonly combined to tonify Qi and alleviate fatigue. Previous studies have employed biological networks to investigate the mechanisms of herb pairs in treating different diseases. However, these studies have only elucidated a single network for each herb pair, without emphasizing the superiority of the herb combination over individual herbs. AIM OF THE STUDY: This study proposes an approach of comparing biological networks to highlight the synergistic effect of the pair in treating cancer-related fatigue (CRF). METHODS: The compounds and targets of Ginseng Radix, Astragali Radix, and CRF diseases were collected and predicted using different databases. Subsequently, the overlapping targets between herbs and disease were imported into the STRING and DAVID tools to build protein-protein interaction (PPI) networks and analyze enriched KEGG pathways. The biological networks of Ginseng Radix and Astragali Radix were compared separately or together using the DyNet application. Molecular docking was used to verify the predicted results. Further, in vitro experiments were conducted to validate the synergistic pathways identified in in silico studies. RESULTS: In the PPI network comparison, the combination created 89 new interactions and an increased average degree (11.260) when compared to single herbs (10.296 and 9.394). The new interactions concentrated on HRAS, STAT3, JUN, and IL6. The topological analysis identified 20 core targets of the combination, including three Ginseng Radix-specific targets, three Astragali Radix-specific targets, and 14 shared targets. In KEGG enrichment analysis, the combination regulated additional signaling pathways (152) more than Ginseng Radix (146) and Astragali Radix (134) alone. The targets of the herb pair synergistically regulated cancer pathways, specifically hypoxia-inducible factor 1 (HIF-1) signaling pathway. In vitro experiments including enzyme-linked immunosorbent assay and Western blot demonstrated that two herbs combination could up-regulate HIF-1α signaling pathway at different combined concentrations compared to either single herb alone. CONCLUSION: The herb pair increased protein interactions and adjusted metabolic pathways more than single herbs. This study provides insights into the combination of Ginseng Radix and Astragali Radix in clinical practice.

The HPLC-PDA Method for Simultaneous Determination of Regalosides from Bulbs of Lilium lancifolium Thunb. and Their Antioxidant Effects.

Lilium lancifolium Thunb. is a herbal medicine that is widely used to treat inflammation and lung diseases. In this study, a simultaneous quantitative method was developed for the quality control of BLL using high-performance liquid chromatography coupled with a photodiode array detector (HPLC-PDA), and their antioxidant effects were evaluated. Eight regalosides (i.e., regaloside A, B, C, E, F, H, I, and K) were selected as marker substances and separated on a Gemini C18 reversed-phase analytical column by gradient elution with distilled water-acetonitrile mobile phase containing 0.1% (v/v) formic acid. The method was validated with respect to linearity, sensitivities (limit of detection (LOD) and limit of quantitation (LOQ)), accuracy, and precision. The antioxidant effects of the extract and each component were evaluated using the 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay and 2-2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) assay. The coefficients of determination values used as indicators of linearity for all components were ≥0.9999. LOD and LOQ concentrations were 0.10-0.66 µg/mL and 0.29-2.01 µg/mL, respectively. The recovery was 95.39-103.925% (relative standard deviation; RSD ≤ 2.55%), and precision RSD was <2.78%. The HPLC-PDA method was applied to real samples, and all components were detected at 1.12-29.76 mg/freeze-dried g. The evaluation of antioxidant effects showed that regalosides C, E, and K exhibited significant antioxidant effects. Our knowledge will be appropriately utilized in raw material management and conducting clinical and non-clinical studies on L. lancifolium or herbal medicine prescriptions containing L. lancifolium.

Jakyak-gamcho-tang, a decoction of Paeoniae Radix and Glycyrrhizae Radix et Rhizoma, ameliorates dexamethasone-induced muscle atrophy and muscle dysfunction.

BACKGROUND: Although chronic treatment with glucocorticoids, such as dexamethasone, is frequently associated with muscle atrophy, effective and safe therapeutics for treating muscle atrophy remain elusive. Jakyak-gamcho-tang (JGT), a decoction of Paeoniae Radix and Glycyrrhizae Radix et Rhizoma, has long been used to relieve muscle tension and control muscle cramp-related pain. However, the effects of JGT on glucocorticoid-induced muscle atrophy are yet to be comprehensively clarified. PURPOSE: The objective of the current study was to validate the protective effect of JGT in dexamethasone-induced muscle atrophy models and elucidate its underlying mechanism through integrated in silico - in vitro - in vivo studies. STUDY DESIGN AND METHODS: Differential gene expression was preliminarily analyzed using the RNA-seq data to determine the effects of JGT on C2C12 myotubes. The protective effects of JGT were further validated in dexamethasone-treated C2C12 myotubes by assessing cell viability, myotube integrity, and mitochondrial function or in C57BL/6 N male mice with dexamethasone-induced muscle atrophy by evaluating muscle mass and physical performance. Transcriptomic pathway analysis was also performed to elucidate the underlying mechanism. RESULTS: Based on preliminary gene set enrichment analysis using the RNA-seq data, JGT regulated various pathways related to muscle differentiation and regeneration. Dexamethasone-treated C2C12 myotubes and muscle tissues of atrophic mice displayed substantial muscle protein degradation and muscle loss, respectively, which was efficiently alleviated by JGT treatment. Importantly, JGT-mediated protective effects were associated with observations such as preservation of mitochondrial function, upregulation of myogenic signaling pathways, including protein kinase B/mammalian target of rapamycin/forkhead box O3, inhibition of ubiquitin-mediated muscle protein breakdown, and downregulation of inflammatory and apoptotic pathways induced by dexamethasone. CONCLUSION: To the best of our knowledge, this is the first report to demonstrate that JGT could be a potential pharmaceutical candidate to prevent muscle atrophy induced by chronic glucocorticoid treatment, highlighting its known effects for relieving muscle spasms and pain. Moreover, transcriptomic pathway analysis can be employed as an efficient in silico tool to predict novel pharmacological candidates and elucidate molecular mechanisms underlying the effects of herbal medications comprising diverse biologically active ingredients.

KORE-Map 1.0: Korean medicine Omics Resource Extension Map on transcriptome data of tonifying herbal medicine.

Traditional herbal medicine, rooted in a long history of use in East Asia, combines several herbs to create treatments showing high efficacy with minimal side effects, for specific diseases. Such combination therapies represent a potential reservoir of new drugs for treating multifactorial and incurable chronic diseases. However, the complexity of their mechanisms of action due to the combination of multiple compounds, has limited their research integration into modern pharmacological science. To address this challenge, we constructed drug-induced transcriptome data for herbal medicines through systematic experiments, analyzed with the aid of various omics databases. We introduce KORE-Map 1.0 (Korean medicine Omics Resource Extension Map), the first comprehensive resource of drug-derived transcriptome data for representative tonifying herbal medicines, effective in enhancing the immune system. This dataset aims to provide novel insights into the combinatorial mechanisms of these herbal medicines and to aid in the discovery of new therapeutic targets and indications for various incurable diseases.

Magnolol suppresses vascular endothelial growth factor-induced angiogenesis by inhibiting Ras-dependent mitogen-activated protein kinase and phosphatidylinositol 3-kinase/Akt signaling pathways.

Magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, has been reported to possess anticancer activity. Recent studies have also demonstrated that magnolol inhibits cell growth and induces the apoptosis of cancer cells. However, the effects of magnolol on vascular endothelial growth factor (VEGF)-induced angiogenesis in endothelial cells have not been studied. In the present study, we have used human umbilical vein endothelial cells (HUVECs) to investigate the antiangiogenic effect and molecular mechanism of magnolol. Magnolol inhibited the VEGF-induced proliferation, chemotactic motility and tube formation of HUVECs in vitro as well as the vessel sprouting of the aorta ex vivo. Furthermore, magnolol inhibited VEGF-induced Ras activation and subsequently suppressed extracellular signal-regulated kinase (ERK), phosphatidylinositol-3-kinase (PI3K)/Akt and p38, but not Src and focal adhesion kinase (FAK). Interestingly, the knockdown of Ras by short interfering RNA produced inhibitory effects that were similar to the effects of magnolol on VEGF-induced angiogenic signaling events, such as ERK and Akt/eNOS activation, and resulted in the inhibition of proliferation, migration, and vessel sprouting in HUVECs. In combination, these results demonstrate that magnolol is an inhibitor of angiogenesis and suggest that this compound could be a potential candidate in the treatment of angiogenesis-related diseases.

Bioactive compounds from the roots of Asiasarum heterotropoides.

A new tetrahydrofuran lignan, (7S,8R,7'S,8'S)-3-methoxy-3',4'-methylenedioxy-7,9'-epoxylignane-4,7',9-triol (1), and 21 known compounds 2-22 were isolated from the roots of Asiasarum heterotropoides by chromatographic separation methods. The structures of all compounds 1-22 were elucidated by spectroscopic analysis including 1D- and 2D-NMR. Fourteen of these compounds (1-3, 7, 10, 12-17, 19, 21, and 22) were isolated from this species in this study for the first time. All of the isolates were evaluated for their anticancer activities using in vitro assays. Among the 22 tested compounds, two (compounds 5 and 7) induced the downregulation of NO production, FOXP3 expression, and HIF-1α transcriptional activity.