Sephadex LH-20 Column Research Articles

Evaluation the hypolipidemic and antioxidant efficacy of diosgenin extracted from fenugreek seeds in experimentally induced oxidative stressed male rats

This study was conducted to assess the antioxidant status and hypolipidemic role of purified diosgenin extracted from fenugreek seeds in hydrogen peroxide (H2O2) and atherogenic diet experimentally induced oxidative stress male rats. Hexane and chloroform were used for the extraction of diosgenin from ground fenugreek seeds. Purification of extracted fraction was carried out on Sephadex LH-20 column using ethyl acetate as eluent, homogenous symmetrical peak was obtained, and thin layer chromatography on silica gel confirmed that purified fraction is diosgenin in compression to standard diosgenin and the calculated Rf values. The antioxidant scavenging activity was carried out using 2, 2, 1 diphenyl- 1- picrylhydrazyl (DPPH) assay, the result showed that purified diosgenin has an antioxidant activity 13.68 μg/ml while the corresponding values for vitamin C and butylated hydroxyl toluene were 8.43 and 7.26 μg/ml respectively. Fifteen adult Albino male rats were randomly and equally divided into three groups, and treated for six weeks as follows; G1 group served as control, G2: Animals of this group were received drinking water containing 0.75% H2O2 and atherogenic diet, Group 3; Rats of this group were given drinking water containing 0.75% H2O2 and atherogenic diet plus 40 mg purified diosgenin/ kg B.W. dissolved in 0.5 ethyl acetate daily by gavages needle. Fasting blood samples were collected by cardiac puncture technique at 0, third and sixth weeks of experiment. The results revealed that rats in G2 exposed to oxidative stress (0.75% H2O2 plus atherogenic diet) showed a significant elevation (P<0.05) in serum total cholesterol (TC), triacylglycerol (TAG), low density lipoprotein- cholesterol (LDL-C) as well as in malondialdehyde (MDA), and significant suppression in serum high density lipoprotein- cholesterol (HDL-C) and glutathione (GSH) concentrations in comparison to the animals in the first group (G1 group). Intubation of 40 mg/kg B.W. of purified diosgenin was caused a significant alteration in the lipid profile of the treated group (G3), manifested by significant reduction (P<0.05) in serum TC, TAG, LDL-C and significant elevation in HDL-C. Antioxidant status also exhibits significant changes manifested by an elevation of serum GSH and reduction in MDA concentration in G3 group animals. This study has shown the significant role of diosgenin as hypolipidemic agent and its role in restores of the oxidative stress.

Influence of Catechin Fraction and High Molecular Fraction from Green Tea Extract on Lactobacillus, Bifidobacterium and Streptococcus Strains

Green tea is known for its health-promoting properties. It is a complex mixture of polyphenols, and as interest in finding new possibilities for plant extracts as food additives grows, there is a need to investigate the potential interactions of these additives with food ingredients such as microorganisms. Lactic acid bacteria are widely used in food technology. The catechin fraction (CF) and high molecular weight fraction (HMW) from green tea extracts were obtained from Sephadex LH-20 column chromatography and characterized by SE-HPLC-DAD, with CF also characterized by RP-HPLC-DAD. To investigate the antimicrobial properties of green tea fractions, the dilution plating method was performed. SE-HPLC demonstrated clear differences between two of the fractions. The addition of the fractions to growth medium demonstrated that the HMW was a less selective anti-microbial agent than CF, which unequally modulated the growth of the tested strains. Growth of Lactobacillus bulgaricus 151 was promoted after the addition of both fractions. Bifidobacterium animalis 25527 was identified as the most sensitive strain.

A new method to prepare and redefine black tea thearubigins

Thearubigins (TRs) are the major components of black tea, which are formed during the fermentation reactions. Although anti-inflammatory and anti-cancer activities of TRs have been reported, the prepared TRs according to the literature methods still contain many floating peaks. It is puzzling whether the observed activities are from TRs or these floating peaks. Thus, it is urgent to develop a method to prepare pure TRs and redefine them. In the present study, we developed a new method, the combination of caffeine precipitation and Sephadex LH-20 column chromatography, to prepare pure TRs. The floating peaks on the hump of the crude TRs were removed, and pure TRs were prepared. The chemical profile of the floating peaks was established using LC/MS, and the major compounds in this fraction were identified as apigenin glycosides, quercetin glycosides, kaempferol glycosides, theaflavins, theasinensin, and galloylglucoses based on the analysis of their tandem mass spectra and in comparison with literature data. This study will pave the way to further study the chemistry and biological activities of TRs and the health effects of black tea consumption.

Terminalia laxiflora and Terminalia brownii contain a broad spectrum of antimycobacterial compounds including ellagitannins, ellagic acid derivatives, triterpenes, fatty acids and fatty alcohols

Ethnopharmacological relevanceTerminalia laxiflora Engl. & Diels, (Sudanese Arabic name: Darout الدروت) and Terminalia brownii Fresen (Sudanese Arabic name: Alshaf ألشاف) (Combretaceae) are used in Sudanese traditional folk medicine and in other African countries for treatment of infectious diseases, TB and its symptoms, such as cough, bronchitis and chest pain. Aim of studyBecause of the frequent use of T. laxiflora and T. brownii in African traditional medicine and due to the absence of studies regarding their antimycobacterial potential there was a need to screen extracts of T. laxiflora and T. brownii for their growth inhibitory potential and to study the chemical composition and compounds in growth inhibitory extracts. Materials and methodsThe plant species were collected in Sudan (Blue Nile Forest, Ed Damazin Forestry areas) and selected according to their uses in traditional medicine for the treatment of bacterial infections, including TB. Eighty extracts and fractions of the stem bark, stem wood, roots, leaves and fruits of T. laxiflora and T. brownii and nine pure compounds present in the active extracts were screened against Mycobacterium smegmatis ATCC 14468 using agar diffusion and microplate dilution methods. Inhibition zones and MIC values were estimated and compared to rifampicin. HPLC-UV/DAD, GC/MS and UHPLC/Q-TOF MS were employed to identify the compounds in the growth inhibitory extracts. ResultsThe roots of T. laxiflora and T. brownii gave the best antimycobacterial effects (IZ 22–27 mm) against Mycobacterium smegmatis. The lowest MIC of 625 µg/ml was observed for an acetone extract of the root of T. laxiflora followed by methanol and ethyl acetate extracts, both giving MIC values of 1250 µg/ml. Sephadex LH-20 column chromatography purification of T. brownii roots resulted in low MIC values of 62.5 µg/ml and 125 µg/ml for acetone and ethanol fractions, respectively, compared to 5000 µg/ml for the crude methanol extract. Methyl (S)-flavogallonate is suggested to be the main active compound in the Sephadex LH- 20 acetone fraction, while ellagic acid xyloside and methyl ellagic acid xyloside are suggested to give good antimycobacterial activity in the Sephadex LH-20 ethanol fraction. RP-18 TLC purifications of an ethyl acetate extract of T. laxiflora roots resulted in the enrichment of punicalagin in one of the fractions (Fr5). This fraction gave a five times smaller MIC (500 µg/ml) than the crude ethyl acetate extract (2500 µg/ml) and this improved activity is suggested to be mostly due to punicalagin. 1,18-octadec-9-ene-dioate, stigmast-4-en-3-one, 5α-stigmastan-3,6-dione, triacontanol, sitostenone and β-sitosterol were found in antimycobacterial hexane extracts of the stem bark of both studied species. Of these compounds, 1,18-octadec-9-ene-dioate, stigmast-4-en-3-one, 5α-stigmastan-3,6-dione, triacontanol, sitostenone have not been previously identified in T. brownii and T. laxiflora. Moreover, both plant species contained friedelin, betulinic acid, β-amyrine and two unknown oleanane-type triterpenoids. Of the listed compounds, friedelin, triacontanol and sitostenone gave a MIC of 250 µg/ml against M. smegmatis, whereas stigmasterol and β-sitosterol gave MIC values of 500 µg/ml. ConclusionsOur results show that T. laxiflora and T. brownii contain antimycobacterial compounds of diverse polarities and support the traditional uses of various parts of T. laxiflora and T.brownii as decoctions for treatment of tuberculosis. Further investigations are warranted to explore additional (new) antimycobacterial compounds in the active extracts of T. laxiflora and T. brownii.

Antioxidant and Cosmeceutical Activities of Agarum cribrosum Phlorotannin Extracted by Ultrasound Treatment

An ultrasonic process for the effective extraction of phlorotannins from Agarum cribrosum with high total phlorotannin content (TPC) was optimized using response surface methodology (RSM). Optimal conditions for ultrasound extraction consisted of temperature: 30°C; time: 5.75 h; ratio of solvent to solid: 35 mL/g, where the TPC was 19.59 mg PGE/g. The crude extract was further partitioned using hexane, dichloromethane, ethyl acetate (EtOAc), and water, in that order. The EtOAc fraction with the highest TPC exhibited the strongest radical-scavenging activity against DPPH, ABTS, hydrogen peroxide, and reducing power, which was similar to that of ascorbic acid. Furthermore, the IC50 values of the EtOAc fraction against collagenase, elastase, and tyrosinase inhibitory activity were 29.63, 16.13, and 6.17 μg/mL, respectively, whereas epigallocatechin gallate (EGCG), EGCG, and kojic acid, the counterpart positive controls, were 68.90, 23.34, and 43.29 μg/mL, respectively. The EtOAc fraction was further fractionated using Sephadex LH-20 column chromatography, which yielded two major and four minor subfractions. The structure of the SF5 and SF6 subfractions were further determined through HPLC-DAD-QMS analyses and 1H and 13C NMR, in which only trifuhalol A was identified. The SF5 and SF6 subfractions exhibited significantly higher radical-scavenging activity against DPPH and reducing power than the EtOAc fraction, but no statistically significant differences were detected for ABTS, hydrogen peroxide, collagenase, elastase, and tyrosinase inhibitory activity. Based on the results, we propose that A. cribrosum phlorotannins could be a good source for natural antioxidants and cosmeceutical.

Studies on alkaloids from Ervatamia pandacaqui

To inverstigate the alkaloids from the twigs and leaves of Ervatamia pandacaqui, eleven known alkaloids were isolated by silica gel, Sephadex LH-20, and ODS column chromatography, as well as RP-HPLC. Their structures were elucidated by UV, IR, MS, and NMR spectral data as coronaridine (1), 3-oxocoronaridine (2), 19S-heyneanine (3), 19R-heyneanine (4), voacangine (5), 3-oxovoacangine (6), voacristine (7), 19-epi-voacristine (8), iso-voacangine (9), coronaridine 7-hydroxyindolenine (10), and voacangine 7-hydroxyindolenine (11). Compounds 1-11 were isolated from E. pandacaqui for the first time.

New Lignans from the Flower of Forsythia koreana and Their Suppression Effect on VCAM-1 Expression in MOVAS Cells.

Six lignans including two new lignans were obtained as the principal components of the Forsythia koreana flowers via silica gel (SiO2 ), octadecyl SiO2 (ODS) as well as Sephadex LH-20 column chromatography. In addition to two new lignans, named koreanaside A ((7R,8S,7'R,8'S)-7,7'-diepoxy-5'-hydroxy-3,3'-dimethoxylignan 4-O-β-d-glucopyranoside) and koreanaside B ((7R,8S,7'S,8'R)-7,9'-epoxy-9,5',7'-trihydroxy-3,3'-dimethoxylignan 4-O-β-d-glucopyranoside), four known lignans were identified to be (+)-phylligenin, (-)-epipinoresinol, pinoresinol, and tinosposide A. The structures and absolute configurations of koreanasides A and B were established by means of analysis of spectroscopic data (NMR, IR, FAB-MS, and CD), whereas the structures of known lignans were identified by comparison their NMR and MS values with those in the reported literature. Their chemical structures including configuration were established by means of analysis of spectroscopic data (NMR, IR, FAB-MS, and CD) but also comparison of their NMR and MS values with those in the reported literature. This is the first article for isolation of six lignans of F.koreana flowers. Koreanasides A and B showed high radical scavenging activity with oxygen radical absorbance capacity (ORAC) values of 0.97±0.01 and 1.02±0.01, respectively. Koreanaside A also prohibited expressing VCAM-1 in MOVAS cells with 80.5% at 25mg/mL.

Antialgal compounds with antialgal activity against the common red tide microalgae from a green algae Ulva pertusa

Nine antialgal active compounds, (i.e. trehalose (1), twenty-two methyl carbonate (2), (-)-dihydromenisdaurilide (3), 3,7,11,15-tetramethyl-2-hexadecen-1-ol (4), isophytol (5), 8-hexadecenol (6), 17-hydroxyheptadecanoic acid (7), trans-asarone (8) and 2-amino-3-mercaptopropanoic acid (9)) were isolated from Ulva pertusa for the first time by sephadex LH-20 column chromatography, silica gel column chromatography and repeated preparative TLC. Except for compound 4, all compounds represented novel isolated molecules from marine macroalgae. Further, antialgal activities of these compounds against Amphidinium carterae, Heterosigma akashiwo, Karenia mikimitoi, Phaeocystis globosa, Prorocentrum donghaiense and Skeletonema costatum were investigated for the first time. Results showed these nine compounds have selectivity antialgal effects on all test red tide microalgae, and antialgal activities against red tide microalgae obviously enhanced with the increase of concentration of antialgal compounds. Based on this, EC50–96 h values of these nine compounds for six red tide microalgae were obtained for the first time. By analyzing and comparing EC50–96 h values, it has been determined that seven compounds (1, 3, 4, 6, 7, 8 and 9) showed the superior application potential than potassium dichromate or gossonorol and other six compounds as a characteristic antialgal agent against Heterosigma akashiwo, Karenia mikimitoi and Prorocentrum donghaiense. Overall this study has suggested that green algae Ulva pertusa is a new source of bioactive compounds with antialgal activity.

Novel Theaflavin-Type Chlorogenic Acid Derivatives Identified in Black Tea.

Consumption of black tea contributed to many health benefits including the prevention of heart disease and certain types of cancer. However, the chemical composition of black tea has not been fully explored. Most studies have examined different interactions between the four major tea catechins, and limited studies have investigated the interaction between catechins and other components in tea. In the present study, we tested our hypothesis that the ortho-dihydroxyl structure of chlorogenic acid (CGA) could react with the vic-trihydroxy structure of (-)-epigallocatechin 3-gallate (EGCG) and (-)-epigallocatechin (EGC) to generate theaflavin-type of compounds during black tea fermentation. The reaction between CGA and EGCG or EGC was catalyzed by horseradish peroxidase (POD) in the presence of H2O2. Two theaflavin-type compounds EGCG-CGA and EGC-CGA were purified using a Sephadex LH-20 column. Their structures were elucidated on the basis of the analysis of their MS and 1D- and 2D-NMR spectroscopic data. Furthermore, the existence of these two novel compounds was characterized by LC/MS/MS analysis. We also found that EGCG-CGA and EGC-CGA had very similar inhibitory effects on the growth of human colon cancer cells with that of theaflavin 3,3'-digallate. These findings shed light on the interactions between the major bioactive compounds, catechins, and other minor compounds in tea. The confirmation of the presence of this type of reaction in black tea may provide more understanding of the complexity of black tea chemistry.

Characterizations of the endogenous starch hydrolase inhibitors in acorns of Quercus fabri Hance

The acorns of Quercus fabri Hance tree, or the mandarin duck fruits have been consumed by locals in the form of noodle, yet there is little scientific study. We found that they have much lower digestibility compared with that of rice flour. Solvent extraction using hexane, dichloromethane, and acetone-ethanol-water (AEW) mixture yielded three fractions but the starch hydrolase inhibitors were only found in the AEW fraction. Using assay-guided fractionation of the extracts, we were able to further separate the active compounds by using Sephadex LH-20 column and characterize the inhibitor chemical identities by LC-MS(n). The main active compounds are ellagitannins including pedunculagin, punicalagin, castalagin, and eucalbanin. Our results indicate that the acorns from Quercus fabri Hance have potential for preparation of low glycemic index foods due to the endogenous starch hydrolase inhibitors.

Characteristics of proanthocyanidins in leaves of Chamaecyparis obtusa var. formosana as strong α-glucosidase inhibitors.

In recent decades, there has been a growing demand for natural products with a view to using them as α-glucosidase inhibitors for reducing postprandial hyperglycemia. In this study, the hot water extract (HWE) from Chamaecyparis obtusa var. formosana (Hayata) Rehder (Cupressaceae) leaves and its soluble fractions were screened for α-glucosidase inhibition properties. The n-butanol-soluble fraction of HWE was further fractionated into 14 subfractions (B1-B14) using a Sephadex LH-20 column. The α-glucosidase-inhibitory activities and proanthocyanidin contents of all subfractions were determined. The structural characteristics of proanthocyanidins in proanthocyanidin-rich fractions were also elucidated. HWE produced a dose-dependent inhibition of α-glucosidase at low dose. Its IC50 value was 1.4 µg mL-1 , showing high inhibitory activity. Subfractions B7-B14 displayed powerful α-glucosidase-inhibitory activities with IC50 values ranging between 1 and 0.015 µg mL-1 and contained abundant proanthocyanidins exceeding 300 mg g-1 . The proanthocyanidins with higher mean degree of polymerization (mDP), higher proportions of procyanidin dimer (A1 or A2) and (epi)afzelechin of extension units and a lower proportion of epicatechin of terminal units displayed high α-glucosidase-inhibitory activities. Proanthocyanidins in HWE were viewed as potential natural α-glucosidase inhibitors for decreasing postprandial hyperglycemia. The results indicated that specific structural characteristics of proanthocyanidins would be required for α-glucosidase-inhibitory activity. © 2018 Society of Chemical Industry.

Studies on liposoluble constituents from Momordicae Semen

The liposoluble constituents in Momordicae Semen were investigated in the present study. By silica gel, Sephadex LH-20 column chromatography, and semi-preparative HPLC, 22 compounds were isolated and purified from dichloromethane and ethyl acetate fraction. Based on NMR and MS spectra analyses, these compounds were identified as lupeol (1), 5-(1'-hydroxypentyl)-5H-furan-2-one (2), palmitic acid (3), viscumamide (4), clavatustide C (5), laxanol (6), threo-1-(4-hydroxyphenyl)-2-{4-[2-formyl-(E)-vinyl]-2-methoxyphenoxyl}-propane-1, 3-diol (7), α-spinasterol-3-O-β-D-glucoside (8), chushizisin F (9), ehletianol C (10), tanegool (11), (7R, 8R, 8'R)-4'-guaiacylglyceryl-evofolin B (12), ligballinone (13), (7R, 8S, 8'R)- 4, 4', 9-trihydroxy- 7, 9'-epoxy- 8, 8'-lignan (14), chushizisin I (15), chushizisin A (16), chushizisin G (17), p-coumaraldehyde (18), α-spinasterol (19), p-hydroxybenzoic acid (20), chushizisin E (21), and 3-[2-(4-hydroxyphenyl)-3-hydroxyphenyl-2, 3-dihydro-1-benzofuran-5-yl] propane-1-ol (22), respectively. Compounds 1-17 were isolated from Momordica cochinchinensis for the first time. Compound 2 was a new natural product while compounds 4 and 5 were first found in the terrestrial organism.

Secondary Metabolites with Anti-complementary Activity from the Stem Barks of Juglans mandshurica Maxim

Juglans mandshurica is a fast growing hard species, which is a tree in family of Juglandaceae and has a wide distribution in China, Korea and eastern Russia. Plant materials from J. mandshurica have extensively been used in folk medicines to prevent or cure gastric, esophageal, lung and cardiac cancer. As one chain of our searching for anticomplementary agents from natural sources, two epimeric ellagitannins, [2,3-O-4,4′,5,5′,6,6′,-hexahydroxydiphenoyl (HHDP))-(α,β)-D-glucose] (I) and pedunculagin (II) were purified from 70% acetone extracts of the stem barks of J. mandshurica by Thin Layer Chromatography and Sephadex LH-20 column chromatography approaches. The chemical structures of the isolated compounds were characterized by MS, NMR, and a careful comparation with published literatures. The epimeric ellagitannins I and II exhibited inhibitory properties against a classical pathway of complementary system with 50 % inhibitory concentrations (IC50) values of 65.3 and 47.7 μM, respectively, comparing with riliroside (IC50=104 μM) and rosmarinic acid (IC50=182 μM), which were used as positive controls. Thus, the work indicated both the two secondary metabolites possess excellent inhibitory activity and might be developed as potential anticomplementary chemicals.

Fractionation, antioxidant and inhibitory activity of Thai mango seed kernel extracts

Mango seed kernel extracts (MSKE) from Kaew and Choke-Anan mango cultivars were fractionated using Sephadex LH-20 column chromatography. Antioxidant activity and the inhibitory effects on tyrosinase, 5-lipoxygenase, hyaluronidase and α-glucosidase of MSKE fractions were evaluated. The key components of polyphenol, found in the most active fraction, were identified using LC-ESI-MS. Three major isolates were obtained from both cultivars of the MSKE with the absorb­ance being higher for Choke-Anan than Kaew. Fraction 3 of MSKE from both cultivars showed significantly (P ≤ 0.05) higher antioxidant activity and 5-lipoxygenase, hyaluronidase and α-glucosidase inhibitory activity with Choke-Anan more effective than Kaew. Six different polyphenols were found in fraction 3 of both cultivars. These were tri-o-galloyl-glucoside, tetra-o-galloyl-glucoside, maclurin tri-o-galloyl-glucoside, penta-o-galloyl-glucoside, hexa-o-galloyl-glucoside, and hepta-o-galloyl-glucoside. In the MSKE from Choke-Anan, hexa-o-galloyl-glucoside, and tetra-o-galloyl-glucoside were the two major components, whereas in the MSKE from Kaew tetra-o-galloyl-glucoside was the only major component. The results indicate that MSKE is a suitable by-product that could be utilised for adding value to the mango processing industry and could represent a valuable input into functional foods and pharmaceutical production.

Separation, Identification, and Bioactivities of the Main Gallotannins of Red Sword Bean (Canavalia gladiata) Coats.

The red sword bean (Canavalia gladiata) is an underutilized edible bean cultivated in China. It was previously found to have the highest content of antioxidant polyphenols among 42 edible beans, mainly gallic acid, and gallotannins in its red bean coat, an apparently unique characteristic among edible beans. In this study, the main phenolic compounds in red sword bean coats were further separated by Sephadex LH-20 column chromatography, and identified by LC-MS/MS. Furthermore, the FRAP and ABTS antioxidant activities and antibacterial activity (diameter of inhibition zone, DIZ) of main gallotannin-rich fractions were tested. Our results showed that gallotannins of red sword bean coats were mainly comprised of monogalloyl to hexagalloyl hexosides. Interestingly, tetragalloyl, pentagalloyl, and hexagalloyl hexosides were identified as the possible candidates responsible for the red color of the coats. On the other hand, gallotannin-rich fractions exhibited diverse antioxidant and antibacterial activities, and tetragalloyl hexoside overall had the highest free radical scavenging and antibacterial activities. The degree of galloylation did not completely explain the structure-function relationship of gallotannins isolated from red sword bean coats, as there should exist other factors affecting their bioactivities. In conclusion, red sword bean coats are excellent natural sources of gallotannins, and their gallotannin-rich extracts can be utilized as natural antioxidant and antibacterial agents with potential health benefits as well as application in food industry.

Identification of the antifungal activity of Trichoderma longibrachiatum T6 and assessment of bioactive substances in controlling phytopathgens

Biological control with microbial antagonists is considered an alternative approach for controlling plant diseases. Trichoderma species are one of the potential fungal biocontrol agents in suppression of soil-borne pathogens. However, the mechanism and characterization of Trichoderma spp. in inhibiting different phytopathogenic fungi are largely unknown. In this study, we investigated the antagonistic potential of the endophytic fungus Trichoderma longibrachiatum T6 as a biocontrol agent against different phytopathogenic fungi and the associated antagonistic mechanism with bioactive substances. We found that the fermentation and crude extract of T. longibrachiatum T6 had a broad spectrum and potent activity inhibiting the growth of eleven phytopathogens evaluated, and of which, the inhibitory rate against Valsa mali reached 95% at 5 days after incubation. Ten fractions and six sub-fractions of bioactive substances were obtained on silica gel G chromatography and Sephadex LH-20 columns. One of the sub-fractions (coded sub-Fr.4f) exhibited highest inhibition against the pathogen V. mail, with the inhibitory rate of 80.64% at Day 5 of the treatment. Four key chemical inhibitors were identified: (i) 1, 2-Benzenedicarboxylicacid, bis (2-methylpropyl) ester (DIBP) (C16H22O4); (ii) (Z)-octadec-9-enoic acid (C18H34O2); (iii) 1, 2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester (MEHP) (C16H22O4); and (iv) (Z)-13-Docosenamide (C22H43NO), using spectroscopic and nuclear magnetic resonance data. Two fungicidal compounds DIBP and MEHP provided significantly greater antifungal activities than the other compounds in the inhibition of the V. mail growth. There was a significant linear relationship between the monomer compounds MEPH or DIBP and the inhibitory rates of V. mail; at the concentration of 200 μg mL−1, the inhibitory rate reached over 86% or 78%. We conclude that the strain of T. longibrachiatum T6 can serve as an effective biocontrol agent against V. mali and the mechanism for this function was due to the secondary metabolites with effective bioactive substance.

Bio-guided isolation of Centaurea bruguierana subsp. belangerana cytotoxic components

Centaurea bruguierana subsp. belangerana was extracted by 80% ethanol. The total extract was then partitioned into four fractions including chloroform, ethyl acetate and methanol. Cytotoxic effect of fractions was examined by MTT assay in K562 (chronic myelogenous leukemia), AGS (gastric adenocarcinoma), MCF-7 (breast adenocarcinoma) and SW742 (colon adenocarcinoma) cell lines. The Chloroform fraction, with the lowest LC50 against K-562 cell lines, was partitioned into 14 subfractions and subjected to further purification by reversed-phase (C18) silica gel and sephadex LH-20 column chromatography. Three flavonoids including cirsimaritin, cirsilinelol and eupatilin were isolated for the first time from the species and the structures were confirmed by spectroscopic data. The high selectivity index of the purified flavonoids indicates valuable components with potential few side effects for normal cell lines. However, solubility tests for isolated components indicates the need for novel pharmaceutical dosage forms, in the case for using natural flavonoids as chemotherapeutic agents.

Solid state fermentation of pomegranate husk: Recovery of ellagic acid by SEC and identification of ellagitannins by HPLC/ESI/MS

Abstract 2, 3, 7, 8-Tetrahydroxy-chromeno [5, 4, 3-cde] chromene-5, 10-dione, commonly named ellagic acid was successfully separated by size-exclusion chromatography (SEC) from pomegranate husk ellagitannins purified by solid-state fermentation. Recovery of metabolites from fermented mash was carried out by the addition of ethanol and manual pressing. Culture extracts were loaded on a Sephadex LH-20 column, three fractions were separated, and second fraction was identified and evaluated by high performance liquid chromatography followed by electro spray ionization and mass spectrometry (HPLC/ESI/MS). It was possible to obtain a yield of 47 mg/g of pomegranate husk polyphenols. The analysis of HPLC/ESI/MS allowed to identify punicalagin (1082.97 m/z), punicalin (780.99 m/z) and ellagic acid (300.80 m/z). After the fermentation process, the main compound was ellagic acid. The present study describes a rapid and effective fermentation/chromatographic separation process for ellagic acid production.

Mechanism of action of cytotoxic compounds from the seeds of Euphorbia lathyris

BackgroundThe seeds of Euphorbia lathyris are used in traditional Chinese medicines for the treatment of various medical conditions. E. lathyris contains many natural diterpenes with a lathyrane skeleton. Purpose and study designFive lathyrane-type diterpenoids named Euphorbia factors L1, L2, L3, L8, and L9 (1–5), were investigated for cytotoxicity against A549, MDA-MB-231, KB, and MCF-7 cancer cell lines and the KB-VIN multidrug resistant (MDR) cancer cell line. Also, a tetraol derivative (6) of Euphorbia factor L2 (2) was synthesized to assess the effect of hydroxy moieties. MethodsAn ethanolic extract of seeds of Euphorbia lathyris was prepared and separated into petroleum ether, EtOAc, n-butanol, and n-hexane extracts. The natural diterpenes were isolated by using silica gel and Sephadex LH-20 column chromatography as well as preparative thin-layer chromatography. Saponification of 2 gave tetraol derivative 6. Cytotoxic activity was determined by the sulforhodamine B (SRB) colorimetric assay. Mechanism of action studies focused on the impact of compounds on the cell cycle progression as well as cell morphology. ResultsCompound 5 exhibited the strongest cytotoxicity against all cell lines, while compound 2 showed selectivity against KB-VIN. In cells treated with 3 and 5, accumulation of G1 to early S phase cells was obvious, while no effect was seen on G2/M phase. ConclusionAnalysis of the screening data compared with compound structures suggested that the substitutions at C-3, C-5, C-7, and C-15 are critical for cytotoxicity, as well as cell type-selectivity. Furthermore, results of cytotoxic mechanism analysis demonstrated for the first time that compounds 3 and 5 disrupted normal cell cycle progression, whereas compounds 2‒5 induced obvious actin filament aggregation, as well as partial interference of the microtubule network.

B Type and Complex A/B Type Epicatechin Trimers Isolated from Litchi pericarp Aqueous Extract Show High Antioxidant and Anticancer Activity.

Litchi (Litchi chinensis Sonn.) fruit is known for its rich source of phenolics. Litchi pericarp contains high levels of epicatechin that may form oligomers of various lengths. Except for several A or B type epicatechin dimers, other soluble oligomers have rarely been identified in the pericarp. Here, bioassay-guided column fractionation was applied to isolate bioactive phenolics from aqueous pericarp extract. A fraction (S3) was obtained by two rounds of Sephadex LH-20 column chromatography, and showed higher antioxidant activity and inhibition on the proliferation of human lung cancer cells (A549) than Litchi anthocyanins. S3 was further separated to isolate fractions P1–P4, which all showed higher antioxidant activity than vitamin C. P3 showed 32.9% inhibition on A549 cells at 30 μg/mL, higher than other fractions and cis-Dichlorodiamineplatinum (DDP, 0.5 μg/mL), but not as high as the combination of the four fractions. Using HPLC-Q-TOF-MS/MS, one B-type and complex A/B type epicatechin trimers were identified in P3; another B-type and two A/B-type trimers were identified in P4. P1 and P2, containing epicatechin and proanthocyanidin B2, respectively, showed no cell inhibition at 30 μg/mL. It is the first time that the two B type trimers of epicatechins (Litchitannin B1 and B2), have been found in Litchi species. The identified proanthocyanidins were detected in the pericarp of the young fruit, and the levels of the compounds decreased as the fruit developed, correlating to the decreasing patterns of the expression of LcLAR and LcANR, two key genes in the catechin biosynthesis pathway.