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The authors, led by artist Naoko Tosa, discuss their collaboration on the video artwork Sound of Ikebana, made by applying sound vibration to fluid and shooting it with a high-speed camera. To study the fluid’s shape under zero gravity, the authors experimented with generating the artwork under weightlessness achieved through parabolic flight. The authors confirmed that a new shape significantly different from the one created under normal gravity is created. A three-dimensional artwork was also generated by shooting the phenomenon from multiple viewpoints.

Supplementary Figure 2 from Sodium Orthovanadate Inhibits p53-Mediated Apoptosis

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Supplementary Methods, Figure Legends 1-6 from Sodium Orthovanadate Inhibits p53-Mediated Apoptosis

Supplementary Methods, Figures 1-6 from Sodium Orthovanadate Inhibits p53-Mediated Apoptosis

Supplementary Figure 6 from Sodium Orthovanadate Inhibits p53-Mediated Apoptosis

Supplementary Figure 3 from Sodium Orthovanadate Inhibits p53-Mediated Apoptosis

Supplementary Figure 4 from Sodium Orthovanadate Inhibits p53-Mediated Apoptosis

Sodium orthovanadate (vanadate) inhibits the DNA-binding activity of p53, but its precise effects on p53 function have not been examined. Here, we show that vanadate exerts a potent antiapoptotic activity through both transcription-dependent and transcription-independent mechanisms relative to other p53 inhibitors, including pifithrin (PFT) α. We compared the effects of vanadate to PFTα and PFTμ, an inhibitor of transcription-independent apoptosis by p53. Vanadate suppressed p53-associated apoptotic events at the mitochondria, including the loss of mitochondrial membrane potential, the conformational change of Bax and Bak, the mitochondrial translocation of p53, and the interaction of p53 with Bcl-2. Similarly, vanadate suppressed the apoptosis-inducing activity of a mitochondrially targeted temperature-sensitive p53 in stable transfectants of SaOS-2 cells. In radioprotection assays, which rely on p53, vanadate completely protected mice from a sublethal dose of 8 Gy and partially from a lethal dose of 12 Gy. Together, our findings indicated that vanadate effectively suppresses p53-mediated apoptosis by both transcription-dependent and transcription-independent pathways, and suggested that both pathways must be inhibited to completely block p53-mediated apoptosis. Cancer Res; 70(1); 257–65

Supplementary Figure 1 from Sodium Orthovanadate Inhibits p53-Mediated Apoptosis

Calpain activation induces retinal ganglion cell (RGC) death, while calpain inhibition suppresses RGC death, in animal studies. However, the role of calpain in human retinal disease is unclear. This study investigated a new strategy to study the role of calpain based on real-time imaging. We synthesized a novel fluorescent probe for calpain, acetyl-l-leucyl-l-methionine-hydroxymethyl rhodamine green (Ac-LM-HMRG) and used it for real-time imaging of calpain activation. The toxicity of Ac-LM-HMRG was evaluated with a lactate dehydrogenase cytotoxicity assay, retinal sections, and electroretinograms. Here, we performed real-time imaging of calpain activation in a rat model. First, we administered N-methyl-d-aspartate (NMDA) to induce retinal injury. Twenty minutes later, we administered an intravitreal injection of Ac-LM-HMRG. Real-time imaging was then completed with a noninvasive confocal scanning laser ophthalmoscope. The inhibitory effect of SNJ-1945 against calpain activation was also examined with the same real-time imaging method. Ac-LM-HMRG had no toxic effects. The number of Ac-LM-HMRG-positive cells in real-time imaging significantly increased after NMDA injury, and SNJ-1945 significantly lowered the number of Ac-LM-HMRG-positive cells. Real-time imaging with Ac-LM-HMRG was able to quickly quantify the NMDA-induced activation of calpain and the inhibitory effect of SNJ-1945. This technique, used as a companion diagnostic system, may aid research into the development of new neuroprotective therapies.

Dotinurad, a novel selective urate reabsorption inhibitor (SURI), has potent inhibitory effects at low doses on the uptake of urate by urate transporter 1 (URAT1, solute carrier family 22 member 12 [SLC22A12]), localized at the apical membrane of renal proximal tubular cells. This study sought to clarify the pharmacokinetic (PK) profile of dotinurad. In rats, monkeys, and humans, the apparent distribution volume (0.257, 0.205, and 0.182 L/kg, respectively) and oral clearance (0.054, 0.037, and 0.013 L·h−1·kg−1, respectively) of dotinurad were very low, whereas plasma and luminal concentrations were adequately maintained at high levels. In addition, species differences were scarcely observed with plasma protein binding of 99.4%. The main metabolite was dotinurad glucuronide (no specific metabolites in humans), and percentage excretion of unchanged dotinurad was low in all the investigated species. The risk of drug interaction with dotinurad was expected to be low, because it weakly inhibits metabolic enzymes such as cytochrome P450 (CYP). In conclusion, low-dose dotinurad exhibited excellent pharmacological effects as well as ideal PK properties as a SURI.

Natural hybridization often occurs between insular and widespread species, and investigating these hybridization patterns is important in improving our understanding of the evolution of insular plant species. To this end, we studied the floral morphology and genetic structure of the widespread species Calanthe discolor, the insular closely related species C. izu-insularis and their putative natural hybrids on Honshu Island (mainland) and the Izu Islands (oceanic islands), Japan, using microsatellite markers. Natural hybridization probably occurred on all the investigated Izu Islands in Japan, although C. izu-insularis was morphologically and genetically different from C. discolor. Bumblebees and other relatively large bees are not distributed in the Izu Islands and therefore natural hybridization would have occurred via pollination by relatively small solitary bees that are pollinators of both Calanthe spp. We also showed that in the islands more remote from the mainland, the proportions of insular C. izu-insularis increased and the floral morphologies of both species changed. The results suggested that insular C. izu-insularis evolved on the Izu Islands and the widespread C. discolor adapted to the poor pollinator fauna on oceanic islands.

Microsatellite markers were developed for the endangered orchid Calanthe izu-insularis (Orchidaceae). This species is unique to the Izu Islands in Japan. Unfortunately, its population size has decreased because of excessive collection for horticultural purposes. In addition, although natural hybridization between C. izu-insularis and C. discolor var. discolor has been reported, morphological differences between C. izu-insularis and the hybridized individuals remain unclear. Using next-generation sequencing, 11 polymorphic microsatellite markers were developed. All developed markers could amplify C. aristulifera and nine markers could amplify C. d. var. discolor, two other orchid species that are also endangered in Japan. The number of alleles and expected heterozygosity at each locus were 1-6 (mean, 2.35) and 0.00-0.79 (mean, 0.30), respectively. These microsatellite markers will help conservation geneticists in their investigation of the proportion of pure C. izu-insularis individuals in the Izu Islands.

Calanthe izuinsularis is a rare, beautiful and fragrant orchid endemic to the Izu archipelago. Although it is known that mainland populations of closely related Calanthe species are pollinated by medium- to large-sized bees, it is likely that C. izuinsularis has been forced to alter its floral biology to attract alternative pollinators, as large-sized bees are rarely found on the Izu Islands. Indeed, the unusual floral characteristics of C. izuinsularis, which produces pale flowers that emit a strong and fragrant scent during the night, are considered the adaptation to a moth-pollinator syndrome. As expected, our time-lapse photography using a digital camera revealed that the inflorescences of C. izuinsularis were most frequently visited by nocturnal moths such as Noctuidae, Geometridae and Crambidae, which accounted for 50% of all insect visits (both in terms of the number of frames with captured visitors and number of times visited). However, our study could not provide evidence of a pollinator shift toward moths because none of the moth species was observed with pollinaria. On the other hand, our study found that the pollinaria of C. izuinsularis 1 could become attached to the mesothorax of the small-sized sweat bee Lasioglossum occidens. Therefore, our study suggested that C. izuinsularis is still somewhat dependent on bee pollination, while the orchid has changed its floral coloration and scent. The occurrence of bee-pollination in C. izuinsularis would provide an explanation for how the natural hybridization between C. aristulifera, C. discolor and C. izuinsularis can occur.

Aims Evaluation of the retinal microcirculation is key to understanding retinal vasculopathies, such as diabetic retinopathy. Laser speckle flowgraphy (LSFG) has recently enabled us to directly evaluate the vascular resistance in both retinal vessels and capillaries, non-invasively. We therefore assessed whether retinal vessel blood flow and/or the capillary microcirculation are associated with blood flow in the cervical arteries in diabetic patients without severe retinopathy. Methods We enrolled 110 type 2 diabetes patients, with no or mild non-proliferative diabetic retinopathy, in this prospective cross-sectional study. We measured the resistivity indices (RIs) of the retinal vessel and capillaries by LSFG and those of cervical arteries by Doppler ultrasonography, followed by analyzing associations. Results The RIs of not only the carotid but also vertebral arteries were associated with those of retinal vessel blood flow and the retinal capillary microcirculation. Multiple regression analyses revealed these associations to be independent of other explanatory variables including age and diabetes duration. Conclusions We obtained novel and direct evidence demonstrating a close association between the retinal microcirculation and cervical artery hemodynamics in diabetic patients. These findings suggest shared mechanisms to underlie micro- and macro-angiopathies. Thus, high vascular resistance of cervical arteries may be a risk of developing retinopathy.

PurposeTo investigate the clinical significance of color visual acuity (CVA) in preperimetric glaucoma (PPG) and open-angle glaucoma (OAG).MethodsA total of 123 eyes of 73 subjects (22 normal eyes, 14 PPG eyes, and 87 OAG eyes; mean age: 44.9 ± 10.1 years, age range: 21-64 years) were enrolled. CVA was tested for red, green-yellow, blue-green and blue-purple with a newly developed test.ResultsThere was no statistical difference in clinical background factors, including age, sex, intraocular pressure, or spherical equivalent between the three groups. Red VA and blue-green VA were significantly worse in the OAG eyes than in the normal eyes (P = 0.008 and P = 0.015, respectively), although green-yellow VA and blue-purple VA were not significantly worse. Furthermore, red VA and blue-green VA were significantly correlated with MD in a group of eyes with either PPG or OAG (r = -0.23, P = 0.023; r = -0.25, P = 0.012, respectively), but green-yellow VA and blue-purple VA were not.ConclusionRed VA and blue-green VA were detectably worse in eyes with OAG, in close association with the degree of functional loss. This suggests that measuring CVA with the new color test described here may be a promising supplement to existing methods of detecting glaucoma and evaluating its severity.

The evaluation of retinal ganglion cell (RGC) death is a key part of retinal disease care. Previously, we used a Sytox Orange (SO)-based real-time imaging method to assess the RGCs in mice that underwent optic nerve crush. Here, we used N-methyl-D-aspartate (NMDA) injury in rats to confirm our model and assess the effect of neuroprotective agents on RGCs. The rats received NMDA injury and the intravitreal injection of SO, a cell-impermeant dyeing compound that targets nucleic acid. After ten minutes, non-invasive confocal scanning laser ophthalmoscopy visualized damaged or dying cells. Finally, the retinas were flat-mounted for histological confirmation of RGC death, with retrograde Fluorogold labeling and Alexa Fluor 488 Annexin V-conjugate (Annexin V) staining. This also revealed the time course of retinal cell death and the neuroprotective effect of SNJ-1945. Real-time imaging showed that SO-positive cells significantly increased starting 2 h after NMDA injection and reached an approximate plateau at 3 h. SO-positive cells were positive for Fluorogold and Annexin V in the isolated retinas. Moreover, the number of SO-positive retinal cells was significantly lower after treatment with SNJ-1945, compared to carboxymethyl cellulose. These results were confirmed in the isolated retinas. Thus, real-time imaging with SO allows the quick quantification of NMDA-induced RGC damage and death, and evaluation of neuroprotective agents. This technique may aid research into the development of new neuroprotective therapies.

Microsatellite markers were developed for the endangered orchid Calanthe izu-insularis (Orchidaceae). This species is unique to the Izu Islands in Japan. Unfortunately, its population size has decreased because of excessive collection for horticultural purposes. In addition, although natural hybridization between C. izu-insularis and C. discolor var. discolor has been reported, morphological differences between C. izu-insularis and the hybridized individuals remain unclear. Using next-generation sequencing, 11 polymorphic microsatellite markers were developed. All developed markers could amplify C. aristulifera and nine markers could amplify C. d. var. discolor, two other orchid species that are also endangered in Japan. The number of alleles and expected heterozygosity at each locus were 1-6 (mean, 2.35) and 0.00-0.79 (mean, 0.30), respectively. These microsatellite markers will help conservation geneticists in their investigation of the proportion of pure C. izu-insularis individuals in the Izu Islands.

S. Miyauchi, A. Ito, S. Takeuchi and T. Kajishima, "Fixed-mesh approach for different dimensional solids in fluid flows : application to biological mechanics", Journal of Mechanical Engineering and Sciences, Vol. 6, pp.818-844, UMP, 2014, For simulations of biological flows, our original immersed solid method and feedback immersed boundary method are coupled to deal with the interaction between a fluid and solids of different dimensional topologies. The mesh used in both methods is non-conforming with respect to the fluid-solid interface, and the coupled approach facilitates the handling of the fluid-structure interaction including large deformation of the solids. We show the validity of our method through simulations of static and dynamic benchmark problems. The results of the simulations confirmed that our method has a first order accuracy and shows good agreement with the strong coupling method reported in the literature. As for examples of biological flow, the expiratory flow through vocal cords and pulsative flow through an elastic channel with fibers are analyzed. For the flow through vocal cords, the vocal cords exhibit different vibration modes depending on the initial clearance of the vocal cords, regardless of the contacts of the vocal cords. For the flow through the elastic channel with fibers, fibers decrease the wall shear stress, and the elasticity of the wall enhances the reduction of the wall shear stress.

A series of benzo[b]furan derivatives having a five-membered heterocyclic substituent at the 2-position were prepared from 2-(1-chloro-2-formylvinyl)benzo[b]furans (2) and 2-(4-alkylcarbamoylbuta-1,3-dienyl)benzo[b]furans. These 2-heterocyclic benzo[b]furans were evaluated for their cysteinyl leukotriene receptor (cysLT1, cysLT2) inhibitory activity. Several compounds showed moderate inhibition of calcium mobilization in HEK 293T-cysLT2 or CHO-cysLT1 cells.

The effects of lysine (Lys), monosodium glutamate (GluNa), glycine (Gly) and alanine (Ala) on the viscosity, turbidity, morphological features, solubility of saccharides and molecular size distribution of the dissolved saccharides in retorted potato starch paste were investigated. Gly and Ala with zero net charge-containing pastes showed a large decrease in the viscosity due to collapse of the swollen starch granules by the retort treatment, whereas GluNa and Lys with a positive or negative net charge could inhibit such viscosity change and collapse of the starch granules. The turbidity of GluNa- and Lys-containing pastes evaluated by the absorbance at 500 nm was higher than that of Gly- and Ala-containing pastes. The dissolved saccharides for each retorted paste mainly consisted of low-molecular-weight molecules below Mr 600. GluNa and Lys could strongly suppress the increased viscosity and turbidity of the retorted paste during storage for 7 days at 5 and 55°C, although Gly and Ala had little effect. The charged amino acids could thus be applied to improving the viscosity and turbidity of the retorted starch paste by maintaining the swollen starch granules.

The characteristic effect of such amino acid couples as aspartic acid (Asp) and glutamic acid (Glu), Glu and lysine (Lys), and asparagine (Asn) and glutamine (Gln) selected by the difference in net charge on the gelatinization temperature (GT) and viscosity of potato starch granules was analyzed from the results evaluated by differential scanning calorimeter and rapid viscoanalyzer, respectively. These results were compared with those of the individual amino acids. Asp, Glu and Lys had a greater effect than Asn and Gln on increasing GT, and markedly reduced the peak viscosity (PV). The effect of the Asp+Glu couple, which both have a negative net charge, on GT was according to the sum of the increasing effect of the individual amino acids on GT, and that of the Glu+Lys couple, which respectively have a negative and positive net charge, was synergistically multiplied by those of the two. However, the effect of the Asn+Gln couple, which both have a zero net charge, on GT was hard to estimate due to little change in GT. The effect of the Asp+Glu, and Glu+Lys couples on PV both approached the sum of the decreasing effect of the respective two.

Alkylphenol polyethoxylates (APEOs) have been widely used as nonionic surfactants in a variety of industrial and commercial products. Typical compounds are nonylphenol polyethoxylates (NPEOs) and octylphenol polyethoxylates (OPEOs), which serve as precursors to nonylphenol (NP) and octylphenol (OP), respectively. NP and 4-t-OP are known to have endocrine disrupting effects on fish (medaka, Oryzias latipes), so it is important to know the concentrations of APEOs in the environment. Because the analytical characteristics of these compounds depend on the length of the ethoxy chain, it is necessary to use appropriate compounds as internal standards or surrogates. We synthesized two 13C-labeled surrogate compounds and used these compounds as internal standards to determine NPEOs and OPEOs by high-performance liquid chromatography (LC)-mass spectrometry. Method detection limits were 0.015 microg/L for NP (2)EO to 0.037 microg/L for NP(12)EO, and 0.011 microg/L for OP(3,6)EO to 0.024 microg/L for OP (4)EO. NPEO concentrations in water from a sewage treatment plant were less than 0.05-0.52 microg/L for final effluent and 1.2-15 microg/L for influent. OPEO concentrations were less than 0.05-0.15 microg/L for the final effluent and less than 0.05-1.1 microg/L for influent.

The retinal ganglion cells (RGCs) are the main source of therapeutic targets for neuroprotective glaucoma treatment, and evaluating RGCs is key for effective glaucoma care. Thus, we developed a minimally invasive, quick, real-time method to evaluate RGC death in mice. In this article we describe the details of our method, report new results obtained from C57BL/6J mice, and report that our method was usable in wild type (WT) and knockout (KO) mice lacking an RGC-death-suppressing gene. It used a non-invasive confocal scanning laser ophthalmoscope (cSLO) and a low molecular weight, photo-switching, cell-impermeant, fluorescent nucleic acid dyeing compound, SYTOX orange (SO). The RGCs were retrogradely labeled with Fluorogold (FG), the optic nerve was crushed (ONC), and SO was injected into the vitreous. After ten minutes, RGC death was visualized with cSLO in vivo. The retinas were then extracted and flat mounted for histological observation. SO-labeled RGCs were counted in vivo and FG-labeled RGCs were counted in retinal flat mounts. The time course of RGC death was examined in Calpastatin KO mice and wild type (WT) mice. Our in vivo imaging method revealed that SO-positive dead RGCs were mainly present from 4 to 6 days after ONC, and the peak of RGC death was after 5 days. Moreover, the number of SO-positive dead RGCs after 5 days differed significantly in the Calpastatin KO mice and the WT mice. Counting FG-labeled RGCs in isolated retinas confirmed these results. Thus, real-time imaging with SO was able to quickly quantify ONC-induced RGC death. This technique may aid research into RGC death and the development of new neuroprotective therapies for glaucoma.

The binding of an amino acid, glycine (Gly), alanine (Ala), epsilon-aminocaproic acid (-AC), monosodium glutamate (GluNa), or lysine (Lys), to starch was examined by a biomolecular interaction analyzer (IAsys). A starch sample (ATS) hydrolyzed to an extent of 1% hydrolysis rate with 15% sulfuric acid was used as a model starch for the binding examination. The reducing end of ATS was oxidized by the Somogyi reagent, and the conversion of the reducing end to the carboxyl group of ATS was confirmed by a carboxylic acid fluorescence labeling reagent. The oxidized ATS was immobilized to the amino group of a sensor cuvette by using water-soluble carbodiimide and N-hydroxysuccinimide through an amide bond. The IAsys examination showed that Gly, Ala, and epsilon-AC scarcely bound to the immobilized starch chains but that GluNa and Lys favorably bound with their increasing concentrations. The relative binding index (RBI) of each amino acid was defined by the ratio of the slope of the linear regression equation between the binding response and the concentration for each amino acid to that for Gly. Because the relationships between the RBI and the pasting characteristics (pasting temperature, peak viscosity, breakdown, and swelling index) could each be expressed by a linear regression equation with a high correlation coefficient, it is concluded that the regulation of the pasting behavior of starch with an amino acid is caused by binding of the amino acid to the starch chains.

Poly(e-lysine) (PL), lysine (Lys), monosodium glutamate (GluNa), glycine (Gly), alanine (Ala), and leucine (Leu) were used to regulate the characteristic gelatinization behavior of potato starch. As determined by differential scanning calorimetry, PL, Lys, and GluNa with positive or negative net charge elevated the gelatinization temperature with increasing amount added as compared with the small effect of Gly, Ala, and Leu with zero net charge. The peak viscosity evaluated by a Rapid Viscoanalyser was markedly decreased by adding PL, Lys, and GluNa, whereas Gly and Ala had no effect on the peak viscosity. The swelling was also decreased by added PL, Lys, and GluNa, whereas it was unchanged by added Gly, Ala, and Leu. Potato starch immersed in PL or amino acid solution released most of the retained PL and amino acids by the subsequent washing with water. The increased gelatinization temperature of the PL-treat­ed potato starch returned to the original value by washing with water. It is thus considered that the regulatory effects of PL and amino acids on the gelatinization behavior of potato starch were substantially reversible.

The human T-cell leukemia cell line MOLT-4 is highly radiosensitive, and thus it is often used as a model of p53-dependent radiation-induced apoptosis. Two branches of the p53-mediated apoptotic pathway are reported: "transcription-dependent" and "transcription-independent." However, the relative contribution of each in different types of cells is not yet clearly defined. Moreover, recent studies have shown that the codon 72 polymorphic variants of p53 show different sensitivities to apoptosis signals. The Arg72 variant has a more potent apoptosis-inducing activity in mitochondria than the Pro72 variant. Here, we initially investigated the codon 72 polymorphism of p53 in MOLT-4 cells. Analysis of the p53 exon 4 genomic DNA sequence, which includes codon 72, revealed that MOLT-4 cells are homozygous for the allele encoding Arg72. We next investigated the involvement of the transcription-independent function of p53 using an RNA synthesis inhibitor, actinomycin D (ActD), and a protein synthesis inhibitor, cycloheximide (CHX), and found that the apoptosis was suppressed by CHX but not by ActD. We also revealed that the suppressive effect of CHX on apoptosis was specifically mediated by p53, using a p53-knockdown MOLT-4 transfectant. Furthermore, the suppressive effect of CHX on apoptosis was highly correlated with the suppression of p53 protein accumulation, and less correlated with the suppression of p53 target genes expression. These results indicated that p53 transactivation is not necessary to induce apoptosis, and that p53 protein accumulation itself is both necessary and sufficient to do so.

We recently reported a novel suppressive effect of sodium orthovanadate (vanadate) on the DNA-binding activity of p53. Here, we showed that vanadate had a more potent antiapoptotic activity than three other chemical p53 inhibitors, including pifithrin-alpha. Although the other agents inhibited p53's transcriptional activity, they did not suppress p53-dependent apoptosis in irradiated MOLT-4 cells. To investigate the cause for the different effects of vanadate and the other inhibitors, we chose PFT-alpha and PFT-mu (an inhibitor of the p53-mediated transcription-independent apoptotic pathway), as references, and determined their and vanadate's effect on p53-mediated apoptosis, with a focus on the transcription-independent pathway. We found that vanadate suppressed the p53-associated apoptotic events at the mitochondria, including the loss of mitochondrial membrane potential, the conformational change of Bax and Bak, the mitochondrial translocation of p53, and p53's interaction with Bcl-2. Vanadate also suppressed the apoptosis-inducing activity of a mitochondrially targeted temperature-sensitive p53 in stable transfectants of the SaOS-2 cell line. Finally, we tested vanadate's potential as a radioprotector. Vanadate completely protected mice from a sublethal dose of 8 Gy and partially from a lethal dose of 12 Gy. Our data demonstrate that vanadate can suppress both the transcription-dependent and the transcription-independent p53 pathways, and suggest that both pathways must be inhibited to completely block p53-mediated apoptosis.

The effects of lysine (Lys), monosodium glutamate (GluNa), glycine, alanine and epsilon-poly(L-lysine) (PL) with different degrees of polymerization on the gelatinization behavior of potato starch granules were investigated by DSC, viscosity and swelling measurements, microscopic observation, and measurement of the retained amino acid amount to clarify the contribution of the net charge to their regulatory effects on the gelatinization behavior. The amino acids and PL each contributed to an increase in the gelatinization temperature, and a decrease in the peak viscosity and swelling. These effects strongly depended on the absolute value of their net charge. The disappearance of a negative or positive net charge by adjusting the pH value weakened the contribution. The swelling index and size of the potato starch granules changed according to replacement of the swelling medium. The amino acids and PL were easily retained by the swollen potato starch granules according to replacement of the outer solution of the starch granules.

Apple procyanidins were separated by high-speed counter-current chromatography using a type-J multilayer coil planet centrifuge. Several two-phase solvent systems with a wide range of hydrophobicities from a non-polar hexane system to polar n-butanol systems were evaluated their performance in terms of the partition coefficient and the retention of the phase. The best separation of procyanidins B and C was achieved with a two-phase solvent system composed of n-butanol-methyl tert.-butyl ether-acetonitrile-0.1% trifluoroacetic acid (2:4:3:8) using the lower phase as a mobile at a flow-rate of 1.0 ml/min.