Clinical Pharmacokinetics of Approved RNA Therapeutics.

RNA-mediated drugs are a rapidly growing class of therapeutics. Over the last five years, the list of FDA-approved RNA therapeutics has expanded owing to their unique targets and prolonged pharmacological effects. Their absorption, distribution, metabolism, and excretion (ADME) have important clinical im-plications, but their pharmacokinetic properties have not been fully understood. Most RNA therapeutics have structural modifications to prevent rapid elimination from the plasma and are administered intravenously or subcutaneously, with some exceptions, for effective distribution to target organs. Distribution of drugs into tissues depends on the addition of a moiety that can be transported to the target and RNA therapeutics show a low volume of distribution because of their molecular size and negatively-charged backbone. Nucleases metabolize RNA therapeutics to a shortened chain, but their metabolic ratio is relatively low. Therefore, most RNA therapeutics are excreted in their intact form. This review covers not only ADME features but also clinical pharmacology data of the RNA therapeutics such as drug-drug interaction or population pharmacokinetic analyses. As the market of RNA therapeutics is expected to rapidly expand, comprehensive knowledge will contribute to interpreting and evaluating the pharmacological properties.

Pharmacokinetics and Tissue Distribution of Bee Venom-Derived Phospholipase A2 Using a Sandwich ELISA after Subcutaneous Injection of New Composition Bee Venom in Rats.

Bee venom is a traditional drug used to treat the nervous system, musculoskeletal system, and autoimmune diseases. A previous study found that bee venom and one of its components, phospholipase A2, can protect the brain by suppressing neuroinflammation and can also be used to treat Alzheimer's disease. Thus, new composition bee venom (NCBV), which has an increased phospholipase A2 content of up to 76.2%, was developed as a treatment agent for Alzheimer's disease by INISTst (Republic of Korea). The aim of this study was to characterize the pharmacokinetic profiles of phospholipase A2 contained in NCBV in rats. Single subcutaneous administration of NCBV at doses ranging from 0.2 mg/kg to 5 mg/kg was conducted, and pharmacokinetic parameters of bee venom-derived phospholipase A2 (bvPLA2) increased in a dose-dependent manner. Additionally, no accumulation was observed following multiple dosings (0.5 mg/kg/week), and other constituents of NCBV did not affect the pharmacokinetic profile of bvPLA2. After subcutaneous injection of NCBV, the tissue-to-plasma ratios of bvPLA2 for the tested nine tissues were all <1.0, indicating a limited distribution of the bvPLA2 within the tissues. The findings of this study may help understand the pharmacokinetic characteristics of bvPLA2 and provide useful information for the clinical application of NCBV.

Analytical Method Development of Benzisothiazolinone, a Biocide, Using LC-MS/MS and a Pharmacokinetic Application in Rat Biological Matrices.

Benzisothiazolinone (BIT), a biocide widely used as a preservative in household cleaning and personal care products, is cytotoxic to lung cells and a known skin allergen in humans, which highlights the importance of assessing its toxicity and pharmacokinetics. In this study, a simple, sensitive, and accurate LC−MS/MS method for the quantification of BIT in rat plasma, urine, or tissue homogenates (50 µL) using phenacetin as an internal standard was developed and validated. Samples were extracted with ethyl acetate and separated using a Kinetex phenyl−hexyl column (100 × 2.1 mm, 2.6 µm) with isocratic 0.1% formic acid in methanol and distilled water over a run time of 6 min. Positive electrospray ionization with multiple reaction monitoring transitions of m/z 152.2 > 134.1 for BIT and 180.2 > 110.1 for phenacetin was used for quantification. This assay achieved good linearity in the calibration ranges of 2−2000 ng/mL (plasma and urine) and 10−1000 ng/mL (tissue homogenates), with r ≥ 0.9929. All validation parameters met the acceptance criteria. BIT pharmacokinetics was evaluated via an intravenous and dermal application. This is the first study that evaluated BIT pharmacokinetics in rats, providing insights into the relationship between BIT exposure and toxicity and a basis for future risk assessment studies in humans.

Hispidulin Inhibits the Vascular Inflammation Triggered by Porphyromonas gingivalis Lipopolysaccharide.

Hispidulin is a natural bioactive flavonoid that has been studied for its potential therapeutic properties, including its anti-inflammatory, antioxidant, and neuroprotective effects. The aim of this study was to explore whether hispidulin could inhibit the endothelial inflammation triggered by Porphyromonas gingivalis (P. gingivalis) lipopolysaccharide (LPS). The adhesion of monocytes to the vascular endothelium was evaluated through in vitro and ex vivo monocyte adhesion assays. We analyzed the migration of monocytes across the endothelial layer using a transmigration assay. The results showed that treatment with hispidulin decreased the P. gingivalis LPS-induced adhesion of monocytes to endothelial cells and their migration by suppressing the P. gingivalis LPS-triggered expression of intercellular adhesion molecule-1 (ICAM-1) through downregulating nuclear factor-қB (NF-қB). In addition, hispidulin inhibited P. gingivalis LPS-induced mitogen-activated protein kinases (MAPKs) and AKT in endothelial cells. Altogether, the results indicate that hispidulin suppresses the vascular inflammation induced by P. gingivalis LPS. Mechanistically, it prevents the adhesion of monocytes to the vascular endothelium and migration and inhibits NF-қB, MAPKs, and AKT signaling in endothelial cells.

Odontogenic Differentiation-Induced Tooth Regeneration by Psoralea corylifolia L.

Psoralea corylifolia L. (P. corylifolia) has been used as an oriental phytomedicine to treat coldness of hands and feet in bone marrow injury. Hydroxyapatite is usually used for tooth regeneration. In this study, the role of P. corylifolia and bakuchiol, a compound originated from P. corylifolia as differentiation-inducing substances for tooth regeneration, was determined by monitoring odontogenic differentiation in human dental pulp stem cells (hDPSCs). We confirmed that P. corylifolia extracts and bakuchiol increased the odontogenic differentiation of hDPSCs. In addition, the expression of the odontogenic differentiation marker genes alkaline phosphatase (APL), Runt-related transcription factor 2 (RUNX-2), osteocalcin (OC), and dentin matrix acidic phosphoprotein-1 (DMP-1) was proved by real-time polymerase chain reaction, and protein expression of dentin matrix acidic phosphoprotein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) was proved by western blotting. Further, by confirming the increase in small mothers against decapentaplegia (SMAD) 1/5/8 phosphorylation, the SMAD signaling pathway was found to increase the differentiation of odontoblasts. This study confirmed that P. corylifolia L. extracts and bakuchiol alone promote odontogenic differentiation in hDPSCs. These results suggest that bakuchiol from P. corylifolia is responsible for odontogenic differentiation, and they encourage future in vivo studies on dentin regeneration.

Proteomics Analysis of Antitumor Activity of Agrimonia pilosa Ledeb. in Human Oral Squamous Cell Carcinoma Cells.

Oral cancer is a malignant neoplasm of oral cavity. It accounts for approximately 5% of all malignant tumors. Approximately 97% of all oral cancers are squamous cell carcinomas, followed by adenocarcinomas, and rarely malignant melanomas. It occurs particularly in males (twice as common in males than in females) of middle age (above 40 years). Agrimonia pilosa Ledeb. has traditionally been known for its effective antitumor activity and is currently used in China for cancer therapy. A. pilosa Ledeb. has been traditionally used for the treatment of abdominal pain, sore throat, headache, blood discharge, parasitic infections, and eczema in Korea and other Asian countries. Most studies on A. pilosa Ledeb. are related to the leaves and a few investigated the roots of the plant. However, detailed mechanisms of antitumor activity of A. pilosa Ledeb. have not been fully elucidated. Furthermore, to date, there have been no reports on the antitumor effect of A. pilosa Ledeb. in oral squamous cells. In this study, we used proteomic technology to observe changes in proteins related to anticancer activity of A. pilosa Ledeb. and identified target proteins among altered proteins to reveal the underlying mechanism of action.

The Relationship between the Gut Microbiome and Metformin as a Key for Treating Type 2 Diabetes Mellitus.

Metformin is the first-line pharmacotherapy for treating type 2 diabetes mellitus (T2DM); however, its mechanism of modulating glucose metabolism is elusive. Recent advances have identified the gut as a potential target of metformin. As patients with metabolic disorders exhibit dysbiosis, the gut microbiome has garnered interest as a potential target for metabolic disease. Henceforth, studies have focused on unraveling the relationship of metabolic disorders with the human gut microbiome. According to various metagenome studies, gut dysbiosis is evident in T2DM patients. Besides this, alterations in the gut microbiome were also observed in the metformin-treated T2DM patients compared to the non-treated T2DM patients. Thus, several studies on rodents have suggested potential mechanisms interacting with the gut microbiome, including regulation of glucose metabolism, an increase in short-chain fatty acids, strengthening intestinal permeability against lipopolysaccharides, modulating the immune response, and interaction with bile acids. Furthermore, human studies have demonstrated evidence substantiating the hypotheses based on rodent studies. This review discusses the current knowledge of how metformin modulates T2DM with respect to the gut microbiome and discusses the prospect of harnessing this mechanism in treating T2DM.

The Comprehensive "Omics" Approach from Metabolomics to Advanced Omics for Development of Immune Checkpoint Inhibitors: Potential Strategies for Next Generation of Cancer Immunotherapy.

In the past decade, immunotherapies have been emerging as an effective way to treat cancer. Among several categories of immunotherapies, immune checkpoint inhibitors (ICIs) are the most well-known and widely used options for cancer treatment. Although several studies continue, this treatment option has yet to be developed into a precise application in the clinical setting. Recently, omics as a high-throughput technique for understanding the genome, transcriptome, proteome, and metabolome has revolutionized medical research and led to integrative interpretation to advance our understanding of biological systems. Advanced omics techniques, such as multi-omics, single-cell omics, and typical omics approaches, have been adopted to investigate various cancer immunotherapies. In this review, we highlight metabolomic studies regarding the development of ICIs involved in the discovery of targets or mechanisms of action and assessment of clinical outcomes, including drug response and resistance and propose biomarkers. Furthermore, we also discuss the genomics, proteomics, and advanced omics studies providing insights and comprehensive or novel approaches for ICI development. The overview of ICI studies suggests potential strategies for the development of other cancer immunotherapies using omics techniques in future studies.

Nonpolymeric pH-Sensitive Carbon Dots for Treatment of Tumor.

Nonpolymer, pH-sensitive carbon dots (pSCDs) were developed to overcome the disadvantages of pH-sensitive polymers such as inevitable synthesis, wide distribution of molecular weight, uncontrolled loading and release rate of drugs, and toxicity by biodegradation. The pSCDs were synthesized via one spot synthesis for 3 min using citric acid (CA) and 1-(3-aminopropyl) imidazole (API). Imidazole groups were present on pSCD surfaces and facilitated DOX loading via hydrophobic interactions (loading efficiency: 78.55%). The DOX-loaded pSCDs collapsed at tumoral pH (pH ∼ 6.5) due to protonation of the imidazole groups, and DOX was released about 7 times higher than the control group. The therapeutic effect was confirmed in vitro using HCT-116 (human colon cancer), PANC-1 (human pancreatic cancer), and SKBR-3 (human breast cancer) cells. Additionally, the DOX-loaded pSCDs successfully inhibited tumor growth in an HCT-116-bearing mouse model and did not show toxicity. These results indicate that a nonpolymeric pSCDs platform has the potential to be used as a cancer targeting therapeutic material.

Pentraxin-3 Modulates Osteogenic/Odontogenic Differentiation and Migration of Human Dental Pulp Stem Cells.

Pentraxin-3 (PTX3) is recognized as a modulator of inflammation and a mediator of tissue repair. In this study, we characterized the role of PTX3 on some biological functions of human dental pulp stem cells (HDPSCs). The expression level of PTX3 significantly increased during osteogenic/odontogenic differentiation of HDPSCs, whereas the knockdown of PTX3 decreased this differentiation. Silencing of PTX3 in HDPSCs inhibited their migration and C-X-C chemokine receptor type 4 (CXCR4) expression. Our present study indicates that PTX3 is involved in osteogenic/odontogenic differentiation and migration of HDPSCs, and may contribute to the therapeutic potential of HDPSCs for regeneration and repair.

Involvement of Gastrin-Releasing Peptide Receptor in the Regulation of Adipocyte Differentiation in 3T3-L1 Cells.

Gastrin-releasing peptide (GRP), a member of bombesin-like peptides, and its receptor (GRP-R) play an important role in various physiological and pathological conditions. In this work, we investigated the role of GRP-R on adipogenesis in 3T3-L1 adipocytes. The expression of GRP-R was significantly increased during the adipocyte differentiation of 3T3-L1 cells. The inhibition of GRP-R by the antagonist RC-3095 affected adipogenesis in 3T3-L1 cells, which reduced lipid accumulation and regulated the expression of adipogenic genes. Moreover, cyclic AMP response element-binding protein (CREB) directly bound to the GRP-R promoter upon exposure to adipogenic stimuli. The down-regulation of GRP-R by the knockdown of CREB inhibited adipocyte differentiation of 3T3-L1 cells. Together these results suggest that the regulation of GRP-R activity or expression has an influence on adipogenesis through regulating adipogenic related genes.

Population Pharmacokinetics and Dose Optimization of Teicoplanin during Venoarterial Extracorporeal Membrane Oxygenation.

The pharmacokinetics (PK) of drugs are known to be significantly altered in patients receiving extracorporeal membrane oxygenation (ECMO). However, clinical studies of the PK of drugs administered during ECMO are scarce, and the proper dosing adjustment has yet to be established. We developed a population PK model for teicoplanin, investigated covariates influencing teicoplanin exposure, and suggested an optimal dosing regimen for ECMO patients. Samples for PK analysis were collected from 10 adult patients, and a population PK analysis and simulations were performed to identify an optimal teicoplanin dose needed to provide a >50% probability of target attainment at 72 h using a trough concentration target of >10 µg/ml for mild to moderate infections and a trough concentration target of >15 µg/ml for severe infections. Teicoplanin was well described by a two-compartment PK model with first-order elimination. The presence of ECMO was associated with a lower central volume of distribution, and continuous renal replacement therapy (CRRT) was associated with a higher peripheral volume of distribution. For mild to moderate infections, an optimal dose was a loading dose (LD) of 600 mg and a maintenance dose (MD) of 400 mg for ECMO patients not receiving CRRT and an LD of 800 mg and an MD of 600 mg for those receiving CRRT. For severe infections, an optimal dose was an LD of 1,000 mg and an MD of 800 mg for ECMO patients not receiving CRRT and an LD of 1,200 mg and an MD of 1,000 mg for those receiving CRRT. In conclusion, doses higher than the standard doses are needed to achieve fast and appropriate teicoplanin exposure during ECMO. (This study has been registered at ClinicalTrials.gov under identifier NCT02581280.).

Gastrin-releasing peptide induces monocyte adhesion to vascular endothelium by upregulating endothelial adhesion molecules.

Gastrin-releasing peptide (GRP) is a neuropeptide that plays roles in various pathophysiological conditions including inflammatory diseases in peripheral tissues; however, little is known about whether GRP can directly regulate endothelial inflammatory processes. In this study, we showed that GRP promotes the adhesion of leukocytes to human umbilical vein endothelial cells (HUVECs) and the aortic endothelium. GRP increased the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by activating nuclear factor-κB (NF-κB) in endothelial cells. In addition, GRP activated extracellular signal-regulated kinase 1/2 (ERK1/2), p38MAPK, and AKT, and the inhibition of these signaling pathways significantly reduced GRP-induced monocyte adhesion to the endothelium. Overall, our results suggested that GRP may cause endothelial dysfunction, which could be of particular relevance in the development of vascular inflammatory disorders.

Role of the focal adhesion protein TRIM15 in colon cancer development.

The tripartite motif containing (TRIM) proteins are a large family of proteins that have been implicated in many biological processes including cell differentiation, apoptosis, transcriptional regulation, and signaling pathways. Here, we show that TRIM15 co-localized to focal adhesions through homo-dimerization and significantly suppressed cell migration. Domain mapping analysis indicated that B-box2 and PRY domains were essential for TRIM15 localization to focal adhesions and inhibition of cell migration. Our protein-protein interaction screen of TRIM15 with the integrin adhesome identified several TRIM15 interacting proteins including coronin 1B, cortactin, filamin binding LIM protein1, and vasodilator-stimulated phosphoprotein, which are involved in actin cytoskeleton dynamics. TRIM15 expression was tissue-restricted and downregulated in colon cancer. Level of TRIM15 expression was associated with colon cancer cell migration, as well as both in vitro and in vivo tumor growth. These data provide novel insights into the role of TRIM15 as an additional component of the integrin adhesome, regulating cell migration, and suggest that TRIM15 may function as a tumor suppressor of colon cancer.

Simultaneous determination of ß-sitosterol, campesterol, and stigmasterol in rat plasma by using LC-APCI-MS/MS: Application in a pharmacokinetic study of a titrated extract of the unsaponifiable fraction of Zea mays L.

A liquid chromatography with atmospheric pressure chemical ionization tandem mass spectrometry method was developed and validated to investigate the pharmacokinetic properties of ß-sitosterol, campesterol, and stigmasterol in rat plasma. Cholesterol-d6 was used as an internal standard. To avoid interference of the three phytosterols in rat plasma and minimize matrix effects, a small volume (10 µL) of 4% bovine serum albumin was used as a surrogate matrix for making calibrators and quality control samples. Rat plasma (10 µL) samples were extracted by liquid-liquid extraction with methyl tert-butyl ether and separated on a Kinetex C18 column. The detection was performed on a triple quadrupole tandem mass spectrometer in selected reaction monitoring mode using positive atmospheric pressure chemical ionization. This assay was linear over concentration ranges of 250-5000 ng/mL (ß-sitosterol), 250-5000 ng/mL (campesterol), and 50-2000 ng/mL (stigmasterol). Additionally, a second set of quality controls made in rat plasma was also evaluated against calibration curves made using the surrogate matrix. All the validation data, including the specificity, precision, accuracy, recovery, matrix effect, stability, and incurred sample reanalysis conformed to the acceptance requirements. Our method was successfully applied to study the pharmacokinetics of three phytosterols in rats.

In Vitro Inhibition of Human UDP-Glucuronosyl-Transferase (UGT) Isoforms by Astaxanthin, ß-Cryptoxanthin, Canthaxanthin, Lutein, and Zeaxanthin: Prediction of in Vivo Dietary Supplement-Drug Interactions.

Despite the widespread use of the five major xanthophylls astaxanthin, ß-cryptoxanthin, canthaxanthin, lutein, and zeaxanthin as dietary supplements, there have been no studies regarding their inhibitory effects on hepatic UDP-glucuronosyltransferases (UGTs). Here, we evaluated the inhibitory potential of these xanthophylls on the seven major human hepatic UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7 and UGT2B15) in vitro by LC-MS/MS using specific marker reactions in human liver microsomes (except UGT2B15) or recombinant supersomes (UGT2B15). We also predicted potential dietary supplement-drug interactions for ß-cryptoxanthin via UGT1A1 inhibition. We demonstrated that astaxanthin and zeaxanthin showed no apparent inhibition, while the remaining xanthophylls showed only weak inhibitory effects on the seven UGTs. ß-Cryptoxanthin mildly inhibited UGT1A1, UGT1A3, and UGT1A4, with IC50 values of 18.8 ± 2.07, 28.3 ± 4.40 and 34.9 ± 5.98 µM, respectively. Canthaxanthin weakly inhibited UGT1A1 and UGT1A3, with IC50 values of 38.5 ± 4.65 and 41.2 ± 3.14 µM, respectively; and lutein inhibited UGT1A1 and UGT1A4, with IC50 values of 45.5 ± 4.01 and 28.7 ± 3.79 µM, respectively. Among the tested xanthophyll-UGT pairs, ß-cryptoxanthin showed the strongest competitive inhibition of UGT1A1 (Ki, 12.2 ± 0.985 µM). In addition, we predicted the risk of UGT1A1 inhibition in vivo using the reported maximum plasma concentration after oral administration of ß-cryptoxanthin in humans. Our data suggests that these xanthophylls are unlikely to cause dietary supplement-drug interactions mediated by inhibition of the hepatic UGTs. These findings provide useful information for the safe clinical use of the tested xanthophylls.

Simultaneous determination of sarpogrelate and its active metabolite in human plasma by liquid chromatography with tandem mass spectrometry and its application to a pharmacokinetic study.

We established a rapid and simple liquid chromatography with tandem mass spectrometry method for the simultaneous determination of sarpogrelate and its active metabolite, M-1, in human plasma. Sarpogrelate, M-1, and the internal standard, ketanserin, were extracted from a 50 µL aliquot of human plasma by protein precipitation using acetonitrile. Chromatographic separation was performed on a Shim-pack GIS ODS C18 column (100 × 3.0 mm; 3 µm) with an isocratic mobile phase consisting of 10 mM ammonium acetate and acetonitrile (70:30, v/v) at a flow rate of 0.6 mL/min; the total run time was <2.5 min. Mass spectrometric detection was conducted in selected reaction-monitoring mode with positive electrospray ionization at m/z 430.35 → 135.10 for sarpogrelate, m/z 330.30 → 58.10 for M-1, and m/z 395.70 → 188.85 for ketanserin. The linear ranges of concentration for sarpogrelate and M-1 were 1-1000 and 0.5-500 ng/mL, respectively. The coefficient of variation for the assay's precision was ≤9.95%, and the accuracy was 90.6-107%. All analytes were stable under various storage and handling conditions, and no relevant crosstalk and matrix effect was observed. This method was successfully applied to a pharmacokinetic study after oral administration of a 100 mg sarpogrelate tablet to healthy male Korean volunteers.

Application of high-performance countercurrent chromatography for the isolation of steroidal saponins from Liriope plathyphylla.

High-performance countercurrent chromatography (HPCCC) with electrospray light-scattering detection was applied for the first time to isolate a spirostanol and a novel furostanol saponin from Liriope platyphylla. Due to the large differences in KD values between the two compounds, a two-step HPCCC method was applied in this study. The primary HPCCC employed methylene chloride/methanol/isopropanol/water (9:6:1:4 v/v, 4 mL/min, normal-phase mode) conditions to yield a spirostanol saponin (1). After the primary HPCCC run, the solute retained in the stationary phase (SP extract) in HPCCC column was recovered and subjected to the second HPCCC on the n-hexane/n-butanol/water system (1:9:10 v/v, 5 mL/min, reversed-phase mode) to yield a novel furostanol saponin (2). The isolated spirostanol saponin was determined to be 25(S)-ruscogenin 1-O-ß-D-glucopyranosyl (1→2)-[ß-D-xylopyranosyl (1→3)]-ß-D-fucopyranoside (spicatoside A), and the novel furostanol saponin was elucidated to be 26-O-ß-D-glucopyranosyl-25(S)-furost-5(6)-ene-1ß-3ß-22α-26-tetraol-1-O-ß-D-glucopyranosyl (1→2)-[ß-D-xylopyranosyl-(1→3)]-ß-D-fucopyranoside (spicatoside D).

Development and validation of a liquid chromatography with tandem mass spectrometry method for the quantification of vitisin B in rat plasma and urine.

A new, rapid, and sensitive liquid chromatography with tandem mass spectrometry method was developed for the determination of vitisin B and validated in rat plasma and urine using carbamazepine as an internal standard. The plasma (0.05 mL) or urine (0.2 mL) samples were extracted by liquid-liquid extraction with ethyl acetate and separated on an Eclipse Plus C18 column (100 × 4.6 mm, 3.5 µm) with a mobile phase consisting of acetonitrile and 0.1% formic acid water (60:40, v/v) at a flow rate of 0.7 mL/min. Detection and quantification were performed by mass spectrometry in selected reaction-monitoring mode with positive electrospray ionization. The calibration curves were recovered over the concentration ranges of 10-5000 ng/mL (correlation coefficients, r≥0.9833) in plasma and 5-2500 ng/mL (r≥0.9977) in urine, respectively. All validation data, including the specificity, precision, accuracy, recovery, and stability, conformed to the acceptance requirements. No matrix effects were observed. The developed method was successfully applied to pharmacokinetic studies of vitisin B following intravenous administration of 0.5 and 1 mg/kg and intraperitoneal injection of 5, 10, and 25 mg/kg to rats. This is the first report on the pharmacokinetic properties of vitisin B. The results provide a meaningful basis to evaluate preclinical or clinical applications of vitisin B.

Rapid separation of cyanidin-3-glucoside and cyanidin-3-rutinoside from crude mulberry extract using high-performance countercurrent chromatography and establishment of a volumetric scale-up process.

This study describes the rapid separation of mulberry anthocyanins; namely, cyanidin-3-glucoside and cyanidin-3-rutinoside, using high-performance countercurrent chromatography, and the establishment of a volumetric scale-up process from semi-preparative to preparative-scale. To optimize the separation parameters, biphasic solvent systems composed of tert-butyl methyl ether/n-butanol/acetonitrile/0.01% trifluoroacetic acid, flow rate, sample amount and rotational speed were evaluated for the semi-preparative-scale high-performance countercurrent chromatography. The optimized semi-preparative-scale high-performance countercurrent chromatography parameters (tert-butyl methyl ether/n-butanol/acetonitrile/0.01% trifluoroacetic acid, 1:3:1:5, v/v; flow rate, 4.0 mL/min; sample amount, 200-1000 mg; rotational speed, 1600 rpm) were transferred directly to a preparative-scale (tert-butyl methyl ether/n-butanol/acetonitrile/0.01% trifluoroacetic acid, 1:3:1:5, v/v; flow rate, 28 mL/min; sample amount, 5.0-10.0 g; rotational speed, 1400 rpm) to achieve separation results identical to cyanidin-3-glucoside and cyanidin-3-rutinoside. The separation of mulberry anthocyanins using semi-preparative high-performance countercurrent chromatography and its volumetric scale-up to preparative-scale was addressed for the first time in this report.