Diaion HP-20 Research Articles

풋사과(홍로) 폴리페놀 분획물의 항산화성 및 항균 활성

Unripe apples are discarded due to thinning out or falling in orchards, however they are abundant in functional ingredients. In this study, polyphenols were efficiently enzymatically extracted and isolated from unripe apples. Enzymatic extraction of polyphenols over 12 hours followed using Viscozyme L with an enzyme concentration of 2%(v/w) based on the total phenolic content. In order to isolate the polyphenols, extracts of unripe apples were eluted with water, 25, 50, 75 and 100% ethanol using Diaion HP-20 gel column chromatography. Polyphenol compounds of each fraction were analyzed by high performance liquid chromatography (HPLC), with the 50% ethanol fraction containing the most polyphenols (387.88 μg/mL of caffeic acid, 226.34 μg/mL of p-coumaric acid, and 187.33 μg/mL of phloridzin). The 50% ethanol fraction also contained the most antioxidants (total penolic and flavonoid content; p<0.05). Antioxidant assays using 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical scavenging activity and ferric ion reducing antioxidant power (FRAP) activity exhibited the highest activities in the 50% ethanol fraction (p<0.05). Antimicrobial activities were also assessed as being highest in 50% ethanol fraction (p<0.05). The 50% ethanol fraction of unripe apple enzyme extracts can be used as a functional polyphenol material to improve the utilization of unripe apple.

Enzymatic Synthesis of Glucosyl Rebaudioside A and its Characterization as a Sweetener.

Rebaudioside A was modified via glucosylation by recombinant dextransucrase of Leuconostoc lactis EG001 in Escherichia coli BL21 (DE3), forming single O-α-D-glucosyl-(1″→6') rebaudioside A with yield of 86%. O-α-D-glucosyl-(1″→6') rebaudioside A was purified using HPLC and Diaion HP-20 and its properties were characterized for possible use as a food ingredient. Almost 98% of O-α-D-glucosyl-(1″→6') rebaudioside A was dissolved after 15 days of storage at room temperature, compared to only 11% for rebaudioside A. Compared to rebaudioside A, O-α-D-glucosyl-(1″→6') rebaudioside A showed similar or improved acidic or thermal stability in commercial drinks. Thus, O-α-D-glucosyl-(1″→6') rebaudioside A could be used as a highly pure and improved sweetener with high stability in commercial drinks. PRACTICAL APPLICATION: The proposed method can be used to generate glucosyl rebaudioside A by enzymatic glucosylation. Simple glucosyl rebaudioside A exhibited high acid/thermal stability and improved sweetener in commercialized drinks. This method can be applied to obtain high value-added bioactive compounds by enzymatic modification.

Improved Production and In Situ Recovery of Sesquiterpene (+)-Zizaene from Metabolically-Engineered E. coli.

The sesquiterpene (+)-zizaene is the direct precursor of khusimol, the main fragrant compound of the vetiver essential oil from Chrysopogon zizanioides and used in nearly 20% of men’s fine perfumery. The biotechnological production of such fragrant sesquiterpenes is a promising alternative towards sustainability; nevertheless, product recovery from fermentation is one of the main constraints. In an effort to improve the (+)-zizaene recovery from a metabolically-engineered Escherichia coli, we developed an integrated bioprocess by coupling fermentation and (+)-zizaene recovery using adsorber extractants. Initially, (+)-zizaene volatilization was confirmed from cultivations with no extractants but application of liquid–liquid phase partitioning cultivation (LLPPC) improved (+)-zizaene recovery nearly 4-fold. Furthermore, solid–liquid phase partitioning cultivation (SLPPC) was evaluated by screening polymeric adsorbers, where Diaion HP20 reached the highest recovery. Bioprocess was scaled up to 2 L bioreactors and in situ recovery configurations integrated to fermentation were evaluated. External recovery configuration was performed with an expanded bed adsorption column and improved (+)-zizaene titers 2.5-fold higher than LLPPC. Moreover, internal recovery configuration (IRC) further enhanced the (+)-zizaene titers 2.2-fold, whereas adsorption velocity was determined as critical parameter for recovery efficiency. Consequently, IRC improved the (+)-zizaene titer 8.4-fold and productivity 3-fold from our last report, achieving a (+)-zizaene titer of 211.13 mg L−1 and productivity of 3.2 mg L−1 h−1. This study provides further knowledge for integration of terpene bioprocesses by in situ product recovery, which could be applied for many terpene studies towards the industrialization of fragrant molecules.

Chumacin-1 and Chumacin-2 from Pseudomonas aeruginosa strain CGK-KS-1 as novel quorum sensing signaling inhibitors for biocontrol of bacterial blight of rice

The in vitro inhibition of quorum sensing signal, xanthan gum secretion, biofilm formation in different Xanthomonas pathovars and biological control of bacterial blight of rice by the two bioactive extrolites produced by Pseudomonas aeruginosa strain CGK-KS-1 were explored. These extrolites were extracted from Diaion HP-20 resin with methanol and purified by preparative-thin layer chromatography. Further, spectroscopic structural elucidation revealed the tentative identity of these extrolites to be (R,3E,5E,9Z,11E)-13-((3S,5R)-5-acetyl-2,6-dimethylheptan-3-yl)-10-hydroxy-4-methyl-1,8-diazabicyclo[9.3.1]pentadeca-3,5,9,11(15),13-pentaen-2-one and (R,3E,5E,8E,11E)-13-((3S,5R)-5-acetyl-2,6-dimethylheptan-3-yl)-4-methyl-1,8-diazabicyclo[9.3.1]pentadeca-3,5,8,11(15),13-pentaene-2,10-dione, named as Chumacin-1 and Chumacin-2, respectively. Antimicrobial assay showed Chumacin-1 and Chumacin-2 exhibited a strong in vitro growth inhibition against various Xanthomonas pathovars. Quorum sensing overlay assay using a reporter strain Chromobacterium violaceum strain CV026 showed that Chumacin-1 and Chumacin-2 inhibited quorum sensing signaling. The mechanistic studies revealed that these extrolites inhibited the production of quorum sensing signaling factor, cis-11-methyl-2-dodecenoic acid; suppressed the xanthan gum secretion and also inhibited the biofilms formed by various Xanthomonas pathovars. Both Chumacin-1 and Chumacin-2 showed ROS generation in the test Xanthomonas strains, resulting in in vitro cell membrane damage was revealed through CSLM and FE-SEM micrographs. Further, greenhouse experiments using Samba Mashuri (BPT-5204) revealed that seed treatment with Chumacin-1 and Chumacin-2 along with foliar spray groups showed up to ˜80% reduction in bacterial blight disease in rice. To the best of our knowledge, this is the first report on new quorum sensing inhibitors, Chumacin-1 and Chumacin-2 produced by Pseudomonas aeruginosa strain CGK-KS-1 exhibiting DSF inhibition activity in Xanthomonas oryzae pv. oryzae.

Polyaromatic Resin HP-20 Induced Accumulation of Intermediate Azaphilones in Monascus purpureus ∆mppC and ∆mpp7 Strains.

Monascus purpureus recombinant mppC and mpp7 knockout strains were subjected to extractive fermentation in the context of azaphilone pigment production. Inclusion of Diaion HP-20 resin resulted in the selective production of unreduced azaphilone congeners, in addition to the early intermediate FK17-P2a, from ∆mppC and ∆mpp7 strains that would otherwise mainly produce reduced congeners. Structural determination of two novel unreduced azaphilones from the ∆mpp7 strain was accomplished. The unreduced azaphilone compound was converted into the cognate reduced congener in recombinant M. purpureus strains, demonstrating its intermediate role in azaphilone biosynthesis. This study demonstrates the possibility that extractive fermentation with Diaion HP-20 resin can be used to obtain cryptic azaphilone metabolites.

Studies on chemical constituents of saponins from Panax ginseng flower buds

This project is to investigate the chemical constituents of ginsenosides from the flower buds of Panax ginseng. The compounds were isolated by using a variety of chromatographic methods including Diaion HP-20,silica gel,MCI gel and semi-preparative HPLC chromatography. Their structures were identified by NMR,and MS data. As a result,32 compounds were isolated from the extract of P. ginseng flower buds,and identified as ginsenoside Rk_3( 1),ginsenoside Rh_4( 2),ginsenoside Rh_8( 3),pseudoginsenoside Rc_1( 4),ginsenoside Rc( 5),ginsenoside Rb_2( 6),ginsenoside Rg_6( 7),20( E)-ginsenoside F_4( 8),ginsenoside Rb_1( 9),vinaginsenoside R_(16)( 10),ginsenoside Rh_6( 11),vinaginsenoside R_3( 12),5,6-didehydro-ginsenoside Rd( 13),vinaginsenoside R_4( 14),vinaginsenoside R_8( 15),ginsenoside Rf( 16),notoginsenoside E( 17),ginsenoside Ⅲ( 18),3-O-β-D-glucopyranosyl-3β,7β,12β,20 S-tetrahydroxydammar-5( 6),24-diene-20-O-β-D-glucopyranoside( 19),20( S)-ginsenoside Rg_2( 20),20( R)-ginsenoside Rg_2( 21),notoginsenoside R_2( 22),ginsenoside F_2( 23),quinquenoside I( 24),ginsenoside M_1( 25),quinquenoside L_(10)( 26),ginsenoside Rh_5( 27),ginsenoside Rg_5( 28),ginsenoside Rk_1( 29),20( R)-ginsenoside Rg_3( 30),oleanolic acid 3-O-β-D-glucopyranosyl-( 1→2)-β-D-( 6'-methyl ester)-glucuronopyranoside( 31) and ginsenoside MC( 32). Among them,compounds 10,12,13,15,19,22,24,31 and 32 were isolated from P. ginseng for the first time,and compound 19 was a genuine ginsenoside firstly obtained by separation and identification,with NMR data that were also reported. Compounds 1-3,7,8,23,25-30 were isolated from P. ginseng flower buds for the first time.

Complex Tannins Isolated from Jelly Fig Achenes Affect Pectin Gelation through Non-Specific Inhibitory Effect on Pectin Methylesterase.

Jelly fig (Ficus awkeotsang Makino) is used to prepare drinks and desserts in Asia, owing to the gelling capability of its pectin via endogenous pectin methylesterase (PE) catalyzation. Meanwhile, substances with PE inhibitory activity (SPEI) in jelly fig achenes (JFA) residue were noticed to be able to impede the gelation. In this study, we characterized and isolated SPEI from JFA by a series of PE inhibition-guided isolations. Crude aqueous extract of JFA residue was mixed with acetone, and 90% acetone-soluble matter was further fractionated by Diaion HP-20 chromatography. The retained fraction with dominant PE inhibitory activity was collected from 100% methanol eluate. Results from high-performance liquid chromatography mass spectrometry (HPLC/MS) and hydrolysis-induced chromogenic transition revealed the SPEI as complex tannins. Total tannins content was determined in each isolated fraction, and was closely related to PE inhibitory activity. In addition, SPEI in this study could inhibit activities of digestive enzymes in vitro and may, therefore, be assumed to act as non-specific protein binding agent.

Transglycosylation of Rebaudioside А by β-fructofuranosidase

Rebaudioside A (RebaA) was subjected to β-2.6 transglycosylation with β-fructofuranosidase from Arthrobacter sp. K-1 and sucrose as a source of fructose units. The yield of transglycosylation depends significantly on the concentration of the acceptor, the donor and the enzyme, as well as the reaction time. At the weight ratio of RebА to sucrose of 1: 1, the degree of transfructosylation in 24 hours was only 5.4%, while at a ratio of 1 : 5, it reaches to more than 23%. It was revealed that transfructosylation proceeds more efficiently in the concentrated solutions, the higher the total concentration of sucrose and RebaA, the greater the yield of fructosylated RebA. To determine the effect of рН on transfructosylation, β-fructofuranosidase was incubated with a solution of 1% RebА and 10% sucrose at 40°C for 15 hours at various pH values. It was also revealed that with an increase in the amount of the enzyme, the reaction accelerates. The most optimal were quantities of 50-100 units per 1 g of sucrose. The reaction of transfructosylation of stevioside is examined, and an organoleptic evaluation of fructosylated derivatives of fructosyl-RebА, fructosyl-stevioside and fructosyl-rubuzoside is also shown. Isolation and purification of fructosylated Reba was carried out by ethanol precipitation and purification on the columns filled with macroporous Diaion HP-20 resin. The resulting product is possessing improved sensory characteristics and can be used as low-calorie sweetener.

Collagenase inhibition by water-pepper (Polygonum hydropiper L.) sprout extract

Introduction: Collagenase plays an important role in the degradation of dermal matrix proteins leading to wrinkle formation. The objectives of this study were to evaluate the inhibitory effect of water-pepper (Polygonum hydropiper L.) sprout extract on the activity of collagenase and to identify the inhibitory compounds.Methods: Collagenase inhibitory activity was measured by spectrophotometric assay. Activity-guided fractionation was performed using liquid-liquid extraction of water and n-butanol and Diaion HP-20 column chromatography, followed by high-performance liquid chromatography (HPLC) fraction collection.Results: A methanolic extract of water-pepper sprout inhibited collagenase activity in a concentration-dependent manner with an IC50 value of 156.7 μg/mL. Collagenase inhibitory activity (IC50 = 23.5 μg/mL) was found in 50% methanol eluate from the HP-20 column chromatography of the n-butanol soluble fraction. The active compound (IC50 = 1.9 μg/mL) in the eluate was isolated by HPLC and identified as quercetin-3-O-galactoside (hyperoside) from comparing retention time, UV-Vis absorption, and mass spectra with those of the standard. Lineweaver-Burk plots revealed that hyperoside was an uncompetitive inhibitor against collagenase. Hyperoside was also the most abundant flavonoid present in the methanolic extract.Conclusion: These results suggest that water-pepper sprouts could be beneficial as a natural source of collagenase inhibitor which might be used for the treatment of skin aging.

Simultaneous Extraction and Determination of Allura Red (E129) and Brilliant Blue FCF (E133) in Foodstuffs by Column Solid-Phase Spectrophotometry.

Background: Allura Red (AR) and Brilliant Blue FCF (BB) are highly water-soluble synthetic food dyes used to color baked goods, beverages, candies, jellies, sausages, etc. Although AR and BB are not entirely toxic, they can lead to health problems in humans. Objective: The aim of the study was to develop a column solid-phase extraction (SPE) and preconcentration method based on the adsorption on a Diaion HP-20 polymeric resin for simultaneous spectrophotometric determination of AR and BB. Methods: The column SPE method was used, and the analytical parameters of the SPE method, such as pH, sample flow rate, sample volume, etc., were systematically investigated and optimized. Results: The detection limits of AR and BB ranged between 0.90 and 0.19 μg/L and quantification limits between 2.59 and 0.53 μg/L, respectively. Preconcentration factors were obtained at 80 and 100 for AR and BB, respectively. The RSDs of the method were lower than 4% for both dyes. The method was successfully applied to foodstuffs. AR and BB contents in foodstuffs were determined between 9.48-407.34 and 2.96-137.12 μg/g, respectively, for solid samples; 52.28 and 5.91 μg/mL of dye contents of liquid samples were determined for AR and BB, respectively. Conclusions: Satisfactory recoveries show that the method will be more useful for future food quality and control applications. Highlights: The developed method exhibited simplicity and reliable, simultaneous determination of AR and BB in foodstuffs.

Extractive fermentation of Monascus purpureus promotes the production of oxidized congeners of the pigment azaphilone

Monascus is a source of food colorant with high productivity of the pigment azaphilone. Monascus azaphilone (MAz) is biosynthesized through a single non-reducing polyketide pathway, the major components of which are ankaflavin (1), monascin (2), rubropunctatin (3) and monascorubrin (4); valuable biological activities have been reported for these compounds. Thus, various culture conditions were explored to reduce the cost of culture ingredients, enhance productivity and modulate compound composition. In the present study, we examined an extractive fermentation (EF) method with Diaion HP-20 resin (HP20) in direct comparison to a previously explored method involving Triton X-100 (TX100) to explore the modulated production of the major MAzs. We employed wild-type Monascus purpureus as well as two derivative recombinant strains (ΔmppG and ΔmppE) that are known to have differential MAz profiles as that of the wild-type strain. The HP20 resin was capable of modulating the MAz profile in favor of orange MAzs 3 and 4, oxidized congeners in this class, as was TX100-a phenomenon not previously observed for TX100 EF with Monascus anka. These finding substantiate that HP20 can be employed for the selective production of oxidized MAz and for diversifying the culture conditions used for Az production.

New tetrahydroquinoline and indoline compounds containing a hydroxy cyclopentenone, virantmycin B and C, produced by Streptomyces sp. AM-2504.

Two new antibiotics, designated virantmycin B (1) and C (2), were isolated from the cultured broth of Streptomyces sp. AM-2504. Compounds 1 and 2 were purified by Diaion HP-20, silica gel, and octadecylsilane chromatography, followed by high-performance liquid chromatography. The chemical structures of the new compounds, 1 and 2, were determined by nuclear magnetic resonance and mass spectrometry, as containing a terahydroquinoline and an indoline, respectively, each also containing a hydroxy cyclopentenone moiety. Both compounds demonstrated weak antimicrobial (both antibacterial and antifungal) activity and compound 1 also showed antiviral activity against the dengue virus, whereas compound 2 exhibited no antiviral properties.

Simpotentin, a new potentiator of amphotericin B activity against Candida albicans, produced by Simplicillium minatense FKI-4981.

Simpotentin, a new potentiator of amphotericin B activity against Candida albicans and Cryptococcus neoformans, was isolated from the culture broth of Simplicillium minatense FKI-4981 by Diaion HP-20 column chromatography, centrifugal partition chromatography, and preparative HPLC. The structure of simpotentin was elucidated by spectroscopic analyses including NMR and MS. The compound has a mannose core to which two medium-chain fatty acids are linked. Simpotentin was found to potentiate amphotericin B activity against C. albicans by the microdilution method.

A Novel Compound and Biological Evaluation of Phytoconstituents Isolated from Erythrina corallodendron L. Flowers

Objective: This study aimed at phytochemical investigation of the 70% alcoholic extract of Erythrina corallodendron L. flowers and biological evaluation of the isolated compounds for their activity as antiprotozoal drugs also evaluation the binding affinity to opioid and cannabinoid receptors as well as the inhibition activity against monoamine oxidase (MAO) enzymes. Method: The 70% alcoholic extract was subjected to successive column chromatographic (CC) separations using silica gel normal phase, reversed phase RP-18, Diaion HP-20, and Sephadex LH-20. The structural elucidation of the isolated compounds was achieved using HR-ESI-MS, UV, 1D and 2D NMR spectroscopic analysis. The isolated compounds were screened in vitro for the binding affinity to opioid and cannabinoid receptors using receptor binding assay as well as the inhibition activity against MAO enzymes using kynuramine deamination assay, while their antiprotozoal activity was evaluatedusing parasitelactate dehydrogenase serum assay (pLDH). Results: The phytochemical evaluation of the alcoholic extract of E.corallodendron flowers, afforded the isolation of an indole alkaloid Hypaphorine 1, a new flavonoid glucoside; Kaempferol-3-O-α-sophoroside 2 and three known flavonoid C-glycosides vis;, Neoschaftoside 3, Isoschaftoside 4 and Vicenin-II 5. Compounds 3 and 4 are reported for the first time from genus Erythrina. Compounds 4 and 5 showed significant antimalarial activity both with IC50 value 1.7µg/mL against (D6) strain and with IC50 1.4 and 1.1 µg/mL against (W2) strain, respectively. Compound 3 showed selective inhibition to MAO-B with IC50 value of 32.08 µM and selective index (SI) > 3.12. Conclusion: The significant antiplasmodial activity of compounds 4 and 5 correlated the known antimalarial activity of different Erythrina species to flavonoid C-glycosides, Also compounds 3 and 4 are position isomers but exhibited different response against MAO-B which gives indication about the selectivity pattern of the flavonoid C-glycosides with MAO-B receptor subtype.

Polyphenols LC-MS2 profile of Ajwa date fruit (Phoenix dactylifera L.) and their microemulsion: Potential impact on hepatic fibrosis

Ajwa fruits are among the most nutritious and economically valuable fruits. In this study, the effect of different solvents on the extraction of polyphenols was investigated. Aqueous acetone (50%) extract contained the highest content of polyphenols and flavonoids. Filtration of the extract through a Diaion HP-20 column led to a polyphenols-enriched fraction (Fr 1) that had the highest antioxidant activity as measured with ORAC and DPPH assays. Metabolic profiling of the different extracts using HPLC-ESI-QTOF-MS-MS led to the tentative identification of 33 secondary metabolites: 3 phenolic acids, 19 flavonoid glycosides including 4 sulfated derivatives, 2 lignans, and 9 fatty acids. Fr 1 showed a promising hepatoprotective activity when tested in-vitro utilizing HepG2 cell lines and in-vivo against thioacetamide-induced liver fibrosis in mice. Ingestion of Fr 1 in the form of a microemulsion had significantly higher oral bioavailability than when ingested as a suspension in water.

Antioxidant Activity, α-Glucosidase and Lipase Inhibitory Activity in Rice Miso with Kidney Bean

Rice miso is a traditional Japanese seasoning food which are not only prolonging the consumption term, but also has good sensory characteristics. So thus, rice miso is very popular in Japan. In the present study, in order to improve the potential functionality, we processed rice miso adding with kidney bean (RMKB), red bean (RMRB) and buckwheat (RMBW), respectively. DPPH radical-scavenging activity, melanoidin and polyphenol content, and enzyme inhibitory activity were detected in rice miso products at different fermentation period (0, 3, 6, 24, 36 months), respectively. The results showed the DPPH radical-scavenging activity of each rice miso product was increased with prolonging the fermentation period, and RMKB showed the highest DPPH radical-scavenging activity in different rice miso products at 6, 24, 36 months. Moreover, DPPH radical-scavenging activity, melanoidin and polyphenol content of rice miso with kidney bean (RMKB) were significantly higher than those of rice miso (RM; as control) in methanol fraction purified by Diaion HP-20 column. RMKB-24M showed the highest antioxidant activity which was a better process to produce high antioxidant profiles. Furthermore, the melanoidin content in rice miso products was approximately 6-folds higher than polyphenol content. Comparing with polyphenol, melanoidin was the main antioxidant material in the rice miso products. In addition, RMKB-24M showed stronger α-glucosidase and lipase inhibitions. These results suggest rice miso with kidney bean had useful components, in particular, potential applications for antioxidant, and the treatments of diabetes and anti-obesity.

Simultaneous Preparation of Salidroside and p-Tyrosol from Rhodiola crenulata by DIAION HP-20 Macroporous Resin Chromatography Combined with Silica Gel Chromatography.

The Rhodiola species have a long history of utilization in traditional medicine and have been considered as a source of adaptation to environmental challenges; salidroside and p-tyrosol are the major responsible compounds. Here we propose a novel UPLC-guided two-step method consisting of a DIAION HP-20 adsorption and silica gel column chromatographies, which can simultaneously prepare high purities of salidroside and p-tyrosol with noticeable yields from the rhizome of Rhodiola crenulata. Results demonstrated that DIAION HP-20 could successfully remove all impurities except crenulatin during a gradient elution with 5–20% ethanol, which could achieve an optimal purification of salidroside and p-tyrosol with increasing rates of 29.19% and 33.44%, respectively. Furthermore, chloroform was selected as an ideal solvent for separating p-tyrosol with salidroside, and thus crenulatin was subsequently applied in the silica gel chromatography, and the separation of salidroside with crenulatin could be achieved using silica gel chromatography with a mixture of chloroform and methanol at a volume ratio of 4:1. High purity rates of 94.17% and 97.29% and overall yields of 39.09% and 43.73% for salidroside and p-tyrosol were simultaneously achieved. Our method provides a new way to simultaneously obtain salidroside and p-tyrosol from R. Crenulata, as well as other related plant species.

Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma.

Pomegranate (Punica granatum L.) fruit has been demonstrated to have the inhibitory activities to various tumors. In this study, we try to uncover the molecular mechanism underlying the inhibitory capability of Taiwanese local pomegranate fruit to urinary bladder urothelial carcinoma. The results collected from the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay indicated that the ethanol extract of pomegranate peel exhibited better inhibitory activity to human urinary bladder urothelial carcinoma T24 and J82 cells than that of pulp. Furthermore, the ethylacetate layer of peel ethanol extract was observed to have the best inhibitory activity against urinary bladder urothelial carcinoma cells. One of the eight fractions (PEPE2 fraction) collected from the ethylacetate layer with Diaion HP-20 column chromatography demonstrated the highest inhibitory activity in urinary bladder urothelial carcinoma cells. The results of the flow cytometry and apoptotic pathway studies suggested that the inhibitory activity of PEPE2 fraction were attributed to the UBUC cell apoptosis. To confirm the above results, our results of xenograft-induced bladder tumor in nude mice showed that the oral consumption of the ethylacetate layer (2, 5, 10 and 100 mg/kg) could decrease the volume and weight of T24 tumors and caused the apoptosis in the xenografted tumors, which was observed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling assay. This study provided the likelihood that the traditionally non-edible pomegranate peel waste is re-utilized to make an affordable and promising chemopreventive product to prevent UBUC incidence or recurrence.

Iridoid glucosides from <i>Vitex grandifolia</i> displayed Anti-inflammatory and Antileishmania Effects and Structure Activity Relationship

In this study, two iridoids i.e. agnuside (1) and bartioside (2) were isolated from Vitex grandifolia for the first time. These potent flavonoids were purified using fractionation and were separated by Sephadex LH 20, Silica gel 60, RP C-18 and Diaion HP-20 columns. Their structure was elucidated using FTIR, 1D and 2D NMR data, these were further evaluated for anti-inflammatory and antileishmania effects. Agnuside, isolated for the first time from V. grandifolia exhibited good activity against NF-kB assay with IC50 (ug/mL) of 8.9 while bartsioside showed an IC50 (ug/mL) of 12. Agnuside (1), an iridoid showed the highest IC50 (μg/mL) of 5.38 and corresponding IC90 (μg/mL) of 7.07 against L. donovani (intracellular amastigotes in THP1 cells). Positive standards were employed in these studies. The results allowed us to establish the relationship between the structure and the activities on the basis of the different patterns of substitution, particularly hydroxylation. This study gave credence to the view that there isa link between low incidence of some diseases and consumption of vegetables hence advantages of these vegetables are beyond nutritional gains.

Large-scale purification of unstable, water-soluble secologanic acid using centrifugal partition chromatography.

Secologanic acid, a major secoiridoid in the flower buds of Lonicera japonica, is a fragile, highly polar compound that readily changes to epivogeloside or vogeloside after being dissolved in methanol. Thus, it is very difficult to obtain secologanic acid on a large-scale. To develop a centrifugal partition chromatography (CPC) method for large-scale purification of secologanic acid with high purity from the flower buds of L. japonica. After fractionation with Diaion HP-20 macroporous resin, 30% methanol eluent was purified by CPC with a ternary biphasic solvent system with ethyl acetate/isopropanol/water (6:4:10, v/v/v). CPC was performed separately twice with the same solvent system, first in descending mode and second in ascending mode. After the first CPC operation, a secologanic acid enriched fraction (586mg) was obtained from 3g of crude extract, and secologanic acid (206mg) was isolated with a purity over 93% in the subsequent ascending mode with the same solvent system from a 586mg enriched fraction. In addition, it was confirmed that epivogeloside and vogeloside were reversely converted to secologanic acid in an aqueous acidic solution. These results demonstrate that CPC is a simple, effective, and rapid method for the purification of secologanic acid in the flower buds of L. japonica.