Mg Of Fraction Research Articles

Targeted isolation of 1,1-diphenyl-2-picrylhydrazyl inhibitors from Saxifraga atrata using medium- and high- pressure liquid chromatography combined with online high performance liquid chromatography–1,1-diphenyl-2- picrylhydrazyl detection

Traditional Tibetan medicine (TTM) is a valuable source of novel therapeutic lead molecules inspired by natural products (NPs). The health benefits of Saxifraga atrata are well documented in TTM, but reports on its chemical composition are limited, most likely due to the complicated purification process. Herein, target separation and identification of 4 main radical scavenging compounds from the methanolic extract of S. atrata was were performed using medium- and high-pressure liquid chromatography coupled with online HPLC-DPPH detection. The sample was pretreated using medium pressure liquid chromatography with MCI GELⓇ CHP20P styrene-divinylbenzene beads as a stationary phase, yielding 1.4 g of the target DPPH inhibitors (Fr4, 11.9% recovery). The compounds were further purified and isolated using HPLC on RP-C18 (ReproSil-Pur C18 AQ) followed by HILIC (Click XIon) column separation, resulting in 2.8 mg of fraction Fr4-1-1, 6.8 mg of fraction Fr4-2, 244.9 mg of the Fr4-3-1 sample, and 38.3 mg of Fr4-4-1. The structure and purity of the target compounds were determined, and four compounds (ethyl gallate, 11-O-galloylbergenin, rutin and isoquercitrin) were isolated with >95% purity. The developed methodology is efficient for targeted isolation of high-purity radical scavengers from NP extracts and could be used for rapid identification and isolation of DPPH inhibitors from various NPs.

Untargeted Characterization of Chestnut (Castanea sativa Mill.) Shell Polyphenol Extract: A Valued Bioresource for Prostate Cancer Cell Growth Inhibition

Chestnut seeds are used for fresh consumption and for the industrial preparation of derivatives, such as chestnut flour. During industrial processing, large amounts of by-products are generally produced, such as leaves, flowers, shells and burs. In the present study, chestnut shells were extracted by boiling water in order to obtain polyphenol-rich extracts. Moreover, for the removal or non-phenolic compounds, a separation by preparative reverse phase chromatography in ten fractions was carried out. The richest fractions in terms of phenolic content were characterized by means of untargeted high-resolution mass spectrometric analysis together with a dedicated and customized data processing workflow. A total of 243 flavonoids, phenolic acids, proanthocyanidins and ellagitannins were tentatively identified in the five richest fractions. Due its high phenolic content (450.03 µg GAE per mg of fraction), one tumor cell line (DU 145) and one normal prostate epithelial cell line (PNT2) were exposed to increasing concentration of fraction 3 dry extract for 24, 48 and 72 h. Moreover, for DU 145 cell lines, increase of apoptotic cells and perturbation of cell cycle was demonstrated for the same extract. Those outcomes suggest that chestnut industrial by-products could be potentially employed as a source of bioresources.

In vitro and In vivo Evaluation of Anticancer Properties of Clerodendrum indicum (L.) Kuntze in Colon Cancer

Cancer has become a global burden currently. Although various treatment options exist today, there is still an urgent need for screening newer drugs owing to the fact that the chemotherapeutic agents have a huge and debilitating side effect profile and a risk of resistance to the existing drugs. The present study was aimed at evaluating the anticancer properties of Clerodendrum indicum both in vitro and in vivo. Root extract of the plant was prepared by soxhlet extraction method using ethanol after overnight maceration and then partitioned with petroleum ether (PEF) and ethyl acetate (EAF). Total flavonoid content was estimated in the fractions using quercetin as standard followed by HPLC characterization. PEF contained 300.37μg and EAF contained 235.14μg flavonoid equivalent to quercetin per mg of fraction. MTT assay was performed to evaluate the in vitro antiproliferative activity and the IC50 values of EAF and PEF were found to be 72.83μg/ml and 31.33μg/ml respectively on HCT116 cell line. Apoptotic index was calculated to check the mode of cell death in the treated groups and the PEF fraction showed significant apoptotic index. Cell cycle analysis was carried out using flow cytometer to check the cell cycle specificity and the inhibition was specific to G2/M phase. Dimethyl hydrazine induced in vivo model of colon cancer was used to investigate the activity of the fractions in vivo on male Wistar rats. The fractions showed no significant change in the hematological parameters. The study showed that the fractions exhibited cytotoxic properties both in vitro and in vivo

Systematic separation and purification of iridoid glycosides and crocetin derivatives from Gardenia jasminoides Ellis by high-speed counter-current chromatography.

Iridoid glycosides and crocetin derivatives are the main bioactive components of Gardenia. The processes of separation of these compounds reported in much of the literature are tedious, time consuming and require multiple chromatographic steps, which results in lower recovery and higher costs. To develop a high-speed counter-current chromatography (HSCCC) method for the systematic separation and purification of iridoid glycosides and crocetin derivatives on a preparative scale from Gardenia. After fractionation using HPD100 column chromatography, n-butanol:ethanol:water (10:1:10, v/v) was selected to purify gardenoside, 6β-hydroxy geniposide and geniposidic acid from fraction A; ethyl acetate:n-butanol:water (2:1.5:3, v/v) was used to isolate geniposide from fraction B; crocin-1, crocin-2, crocin-3 and crocin-4 were purified by hexane:ethyl acetate:n-butanol:water (1:2:1:5, v/v) from fraction C. The head-to-tail elution mode was used with a flow rate of 8.0 mL/min and a rotary speed of 600 rpm. After HSCCC isolation, 151.1 mg of gardenoside, 52.2 mg of 6β-hydroxy geniposide and 24.5 mg of geniposidic acid were obtained from 800 mg of fraction A; 587.2 mg of geniposide was obtained from 800 mg of Fraction B; 246.2 mg of crocin-1, 34.2 mg of crocin-2, 24.4 mg of crocin-3 and 24.7 mg of crocin-4 were obtained from 1000mg of fraction C. Their purities were found by UPLC analysis to be 91.7%, 93.4%, 92.5%, 98.2%, 94.1%, 96.3%, 94.1% and 98.9% respectively. The present results demonstrates that the main iridoid glycosides and crocetin derivatives in Gardenia can be obtained efficiently from extracts using HSCCC.

Application of High-Speed Counter-Current Chromatography for Isolation and Purification of Paclitaxel and Related Taxanes from Taxus chinensis Cell Culture

Six taxanes were isolated and purified from Taxus chinensis cell culture extract using high-speed counter-current chromatography. The crude cell culture extract was first treated with Al2O3 column chromatography and then divided into two parts: fraction 1 and fraction 2. 52 mg of taxuyunnanine C and 11 mg of sinenxane C were yielded from 150 mg of fraction 1. 5 mg of 9-dihydrobaccatin III, 12 mg of baccatin III, 12 mg of paclitaxel, and 17 mg baccatin VI were yielded from 150 mg of fraction 2. The purities of the six compounds were 93.51%, 98.02%, 98.61%, 97.55%, 98.93%, and 98.76%.

Separation and Purification of Arctiin, Arctigenin, Matairesinol, and Lappaol F from Fructus Arctii by High-Speed Counter-Current Chromatography

Arctiin (I), arctigenin (II), matairesinol (III), and lappaol F (IV) were isolated and purified from the traditional Chinese medicine Fructus Arctii by high-speed counter-current chromatography (HSCCC). The crude extracts from Fructus Arctii were treated with D101 macroporous resin first and divided into two parts: fraction 1 and fraction 2. Fraction 1 was separated by ethyl acetate-n-butanol-water (4:0.5:5, v/v/v) and yielded 164 mg of I from 250 mg of fraction 1. Fraction 2 was separated by n-hexane-ethyl acetate-methanol-water (2:3:2:3, v/v/v/v) and yielded 27 mg of II, 5 mg of III, and 3 mg of IV from 150 mg of fraction 2. The purities of the four compounds were 99.64%, 98.48%, 96.16%, and 91.41%, respectively, as determined by HPLC-DAD. The chemical structures of the isolated compounds were identified by MS, UV, 1H NMR, and 13C NMR analysis.

RAPID SEPARATION OF FLAVONOIDS FROM HYDROLYSIS PRODUCTS OF Epimedium koreanum

A successful method of preparative high-speed counter-current chromatography (HSCCC) was established for direct isolation and purification of hydrolysis products of Epimedium koreanum Nakai to obtain the secondary glycosides and aglycons of flavonoids in E. koreanum. The two-phase solvent systems of HSCCC composed n-hexane-ethyl acetate-methanol-water at the ratios of 6:3.5:6:5 (v/v) and 6:3.5:8:5 (v/v) were used in a stepwise elution. Totally, 60.1 mg of Fraction I that would be isolated and purified by preparative HPLC, 9.4 mg of icartin (4), 3.5 mg of desmethylicartin (5), 4.7 mg of 3′′-ethyoxyl-icartin (6), 7.3 mg of anhydroicaritin (7), and 10.1 mg of β-anhydroicaritin (8) were obtained in one-step separation from 100 mg of the crude materials with purities of 97.7%, 96.2%, 99.2%, 99.5%, and 91.0%, respectively. And Fraction I of HSCCC was purified by the preparative HPLC to obtain 12.7 mg of pletypetaloside (I-1), 6.8 mg of icariside I (I-2) and 16.3 mg of 3′′-ethyoxyl-pletypetaloside (I-3) with purities of 90.8%, 93.4%, and 99.2%. All of the chemical structures were identified on the basis of widely spectral technology including 2D-NMR. Among them, compounds I-3 and 6 are novel.

SEPARATION AND PURIFICATION OF FLAVONOIDS FROM BLACK CURRANT LEAVES BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY AND PREPARATIVE HPLC

High-speed countercurrent chromatography (HSCCC) has been successfully used for the preparative isolation of flavonoids from the ethyl acetate extracts of black currant leaves. The HSCCC separation was performed with a two-phase solvent system composed of n-hexane/EtOAc/MeOH/H2O (1:10:1:10, v/v) at a flow rate of 1.5 mL/min. When the flow rate was increased from 1.0 to 3.0 mL/min, the retention of stationary phase decreased from 60.3% to 39.7% resulting in a loss of peak resolution, while the stationary phase retention is stable with an increase in sample size from 25 to 200 mg. From 100 mg of the crude sample, HSCCC separation yielded 4.0 mg of kaempferol 3-O-galactoside, 6.0 mg of kaempferol 3-O-glucoside, and 9.0 mg of fraction I containing a mixture of hyperoside and isoquercitrin. Then, from 18 mg of fraction I, 3.0 mg of hyperoside and 11.0 mg isoquercitrin were separated by preparative HPLC by successive sample injection at every 100 min interval. Chemical structures of all these compounds were confirmed by MS and NMR.

Investigation of fractions present in the stem bark of Annickia kummeriae on their P-glycoprotein inhibitory pump activity.

Using MCF-7R cells and rhodamine 6G as the fluorescent probe, a bioassay-targeted purification process was pursued in order to isolate the active P-gp inhibitory fractions from Annickia kummeriae. Of 24 fractions obtained in the first preparative liquid chromatography (p-LC) run, only fraction 1 exhibited activity. Further p-LC fractionation led to the separation of fraction 1 into fractions 1.1-1.8. Fractions 1.4, 1.5 and 1.6 proved to be active by inducing a significant accumulation of rhodamine 6G by 3.3-, 4.5- and 4.9-fold at 10 microg/mL, and by 5.3-, 6.3- and 6.8-fold at 100 microg/mL, respectively. Fraction 1.6 was separated into several fractions by using an analytical liquid chromatography (a-LC) system. Fractions 1.6.18, 1.6.19 and 1.6.20 were active and they induced an accumulation of rhodamine 6G by 3.0-, 1.8- and 3.5-fold at 1x microg/mL and by 4.8-, 6.7- and 6.8-fold at 10x microg/mL, respectively. Afterwards, 28.3 mg of fraction 1.6 was processed by a-LC, and fractions 1.6.18, 1.6.19 and 1.6.20 were collected separately and dried. The amounts of materials recovered were 6.2, 7.4 and <1 mg, corresponding to 21.9%, 26.1% and <3.5% of fraction 1.6, respectively. From the total amount injected and the relative masses represented by these fractions, it can be calculated that the 1x microg/mL level corresponded to ca. 35, 42 and <5 microg/mL, respectively. Fluorescence microscopy revealed that incubation of the cells with rhodamine 6G alone did not show any fluorescence, whereas cells which were incubated in medium containing rhodamine 6G together with fraction 1.4, 1.6 or reserpine, clearly indicated accumulation of the dye intracellularly. This is an indication that the active compounds effected high intracellular fluorescence by inducing accumulation of the dye in the cells through inhibition of the P-gp pump.

Antitumor effect of a peptide-glucan preparation extracted from Agaricus blazei in a double-grafted tumor system in mice.

The antitumor effect of extracts obtained from the fruit body of Agaricus blazei Murill was examined in a double-grafted tumor system, in which BALB/c mice received simultaneous intradermal injections of Meth-A tumor cells in both the right (10(6) cells) and left flank (2 x 10(5) cells), and were then injected with 5 mg of extracts of A. blazei in the right tumor on days 3, 4 and 5. Intratumoral administration of ethanol-soluble (Fraction 1), water-ethanol-soluble (Fraction 2), ammonium oxalate-soluble (Fraction 3) and ammonium oxalate-insoluble (Fraction 4) fractions resulted in inhibition of tumor growth, with Fraction 3 showing the most tumoricidal activity, producing regression of the right tumor and inhibition of growth of the left, non-injected tumor. The maximum effect was obtained using 0.5 mg of Fraction 3 and this amount was used in subsequent experiments. The antitumor effect of intratumorally administered Fraction 3 was enhanced by oral ad lib administration of feed containing 0.083% of Fraction 3. When immunized spleen cells from mice that had been cured by intratumoral administration of 0.5 mg of Fraction 3 were directly injected (2 x 10(7) cells/mouse) into the Meth-A tumor, tumor growth was inhibited. The tumor cells on day 7 from the Fraction 3-treated right tumor and from the left tumor were cultured for 24 h and their culture supernatants were assayed for neutrophil or macrophage chemotactic activity. Significant macrophage chemotactic factor activity was detected in the culture media from the left tumor tissue. Serum levels of immunosuppressive acidic protein (IAP), produced by activated macrophages and neutrophils, increased transiently soon after intradermal injection of 0.5 mg of Fraction 3. These results suggest that regression of the left non-injected tumor was due to an immune reaction, involving induction of cytotoxic cells in the spleen, and the release of chemotactic factors in the distant tumor.

46] Structure of poly(ADP-ribose)

Publisher Summary This chapter focuses on structure of poly(ADP-ribose). A calf thymus nuclear preparation is used as a source of poly(ADP-Rib) [poly(ADP-ribose)] polymerase for preparation and purification of poly(ADP-ribose). Fraction III obtained from purification of poly(ADP-Rib) is most easily obtained in a pure state. The specific radioactivity, radioactivity/A 260 unit, of Fraction III is 22% higher than the expected specific radioactivity of ADP-Rib, due to the hypochromicity of poly(ADP-Rib), resulting from its secondary structure. The ultraviolet absorption spectrum of Fraction III is similar to that of poly(A), with a peak in the ultraviolet region at 258 nm at pH 7.0 and a molar absorbance, E (2P), of 13,000 at pH 7.0. The melting (decomposition) point of Fraction III of poly(ADP-Rib) is at 219-220°. For the preparation of the monomer unit of Poly(ADP-Rib), Ado(P)-Rib-P, about 150 mg of fraction III (or II) of poly(ADP-Rib) is dissolved in 2.4 ml of distilled water and mixed with 24.6 units of snake venom phosphodiesterase, which has been purified by the method of Oka et al . and final concentrations of 1 m M MgCl 2 and at 0.02% NaN 3 . The mixture is incubated at 37° overnight.

Isolation and characterization of immunoreactive somatostatin from fish pancreatic islets.

Using a radioimmunoassay with labeled synthetic tetradecapeptide somatostatin, a large amount of immunoreactive somatostatin was found in the principal pancreatic islet of the channel catfish (Ictalurus punctata). The purpose of these experiments was to isolate and characterize the somatostatin-like material. Extracts of islets were chromatographed on a Bio-Gel P-30 column, and over 90% of the immunoreactive somatostatin migrated with proteins at least twice the size of synthetic tetradecapeptide somatostatin. This fraction was further purified by ion-exchange chromatography on carboxymethyl-cellulose and DEAE-cellulose columns. Two peptides were obtained with identical immunoreactivity, which was approximately 25% that of the synthetic somatostatin. Each peptide was judged to be >95% pure by thin-layer electrophoresis, polyacrylamide gel electrophoresis at pH 8.9, and highpressure liquid chromatography. Further criteria of purity included amino-terminal analysis of fraction IV yielding only aspartic acid. A total of 1.3 mg of fraction II, and 3.8 mg of fraction IV somatostatin-like peptides were obtained from 10 g of fresh frozen islets. Characterization of the two peptides revealed both peptides slightly more acidic than synthetic tetradecapeptide somatostatin. Fraction II had an isoelectric point of 8.0-8.3, and fraction IV 8.3-9.0. Molecular weight estimation by sodium dodecyl sulfate-urea polyacrylamide gel electrophoresis revealed similar mobility of both peptides, between pancreatic polypeptide (mol wt 4,500) and glucagon (mol wt 3,500). The mobility was not altered by reduction, and was approximately twice the size of synthetic tetradecapeptide somatostatin (mol wt 1,800). This confirmed that the peptides were single polypeptide chains and not aggregates, or somatostatin bound to larger proteins. Molecular weight determination by gel filtration chromatography on Bio-Gel P-6 in 8 M urea gave an estimated mol wt of 3,700. Amino acid analysis of the two immunoreactive somatostatins indicated that they were very similar in composition. Both pancreatic somatostatins (1 muM) had full biological activity relative to synthetic somatostatin measured as inhibition of growth hormone release from rat anterior pituitary cells.

Squamous lesions in lungs of rats exposed to tobacco-smoke-condensate fractions by repeated intratracheal instillation.

Twice-weekly intratracheal instillations in rats of up to 24 mg of Fraction (R + P)G suspended in either infusine (I) or buffered saline/gelatine (BS/G) gave rise to foci of squamous metaplasia of alveolar epithelium (SqM) and squamous neoplasms (SqN). Fraction (R + P)G, which is a fraction of cigarette-smoke condensate almost as tumorigenic for mouse skin as the nearly 30 × larger mass of condensate from which it is derived, could be given in this way for up to 40 weeks without excessive mortality or any marked effect on the rate of body-weight gain. By contrast, similar treatment with Fraction N(QG), a fraction having very low tumorigenic activity for mouse skin, induced no SqN and barely any excess of SqM over that induced by either vehicle alone.The effects of Fraction (R + P)G on the incidence of SqM and SqN were both time and dose related, the effect on SqM incidence being already evident after 10 weeks of treatment. No SqN seen were unequivocally malignant, though, due to the design of the experiment, only 5 rats exposed to Fraction (R + P)G were observed more than 60 weeks after the start of the experiment.Other changes in the lung, including aggregates of alveolar macrophages laden with golden-brown pigment (GBM) and foci of cuboidal/columnar metaplasia of alveolar epithelium (CCM), were frequently seen in response to both fractions. Fraction (R + P)G administered in I was more effective in causing SqM and SqN than the same fraction administered in BS/G. The implications of the findings are discussed, particularly the possibility that the intratracheal/instillation technique might be useful as a rapid bioassay for comparing the tumorigenicity of different cigarette-smoke condensates.

Immunosuppressive activity of submaxillary gland extracts of the mouse. I. Effect on antibody formation in response to sheep red blood cells.

Aqueous extracts of mouse submaxillary glands yield six peaks on Sephadex G-75 columns, each containing several molecular species. Two of these fractions (III and IV) have a profound effect on the immune response. When given a day before immunization with sheep erythrocytes, the number of antibody-producing cells is reduced ten-fold or more, and the switch from IgM to IgG is virtually absent. Administration two days before or on the day of immunization results in about 50% depression; outside this range there is no effect. The dose-response curve is of sigmoid shape, with the median at about 0.5 mg of fraction III/40 g mouse, reaching an asymptote after doses of greater than or equal to 5 mg at about 5% of the response of untreated mice. Depression of the primary response does not prevent the development of immunological memory: the secondary response is typical in the early and predominant appearance of IgG-producing cells, although their numbers are somewhat lower than in the controls which received no treatment before primary immunization. Treatment on the day before boosting abrogates the secondary response. The immunotranquilizer from mouse submaxillary glands acts equally well on syngeneic and allogeneic recipients, affecting the earliest stage of the immune response. The results are compatible with the induction of a temporary block in the development of helper T cells.