[USE OF COUNTERCURRENT EXTRACTION IN THE ISOLATION OF STRYCHNINE FROM BIOLOGICAL MATERIAL].
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Cadaver↗
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The renal concentrating mechanism: countercurrent multiplier system.
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Countercurrent Distribution↗
Separation of flavonoids from the seeds of Vernonia anthelmintica Willd by high-speed counter-current chromatography.
Several flavonoids including 2',3,4,4'-tetrahydroxychalcone, 5,6,7,4'-tetrahydroxyflavone and butin, were separated from the seeds of Vernonia anthelmintica Willd by high-speed counter-current chromatography using a two-step operation. Two different types of solvent systems were used: chloroform-dichloromethane-methanol-water (2:2:3:2, v/v) and 1,2 dichloroethane-methanol-acetonitrile-water (4:1.1:0.25:2, v/v). From 1 kg of seeds of Vernonia anthelmintica Willd the method yielded about 45 mg of 2',3,4,4'-tetrahydroxychalcone, 40mg of 5,6,7,4'-tetrahydroxyflavone, and 55 mg of butin. Each isolated component showed 95-97% purity as determined by high-performance liquid chromatography analysis. These purified compounds were characterized by MS and NMR.
Chromatography, High Pressure Liquid↗
[Purification of ovalbumin from hen egg white by high-speed counter-current aqueous two-phase chromatography].
High-speed counte-recurrent chromatography (HSCCC) is a continuous liquid-liquid partition chromatography without solid matrix, which has the significant features of high resolution and high recovery. The separation of bio-macromolecule in aqueous two-phase systems (ATPs) with HSCCC is still under research, and the establishment of high-speed counter-current aqueous two-phase chromatography (HSCCC-ATP) relies on the improvement of equipment structure and optimization of operation parameters. By using a multi-column high-speed counter-current chromatograph, the separation of protein mixture and the purification of ovalbumin from hen egg white were studied. The effects of pH and PEG concentration on the partition coefficients of proteins were tested in PEG1000-phosphate ATPs, and distinct differences among partition coefficients of proteins were found at pH 9.2 and 15.0% (W/W) PEG concentration in said system. The separation of protein mixture, consisting of cytochrome C, lysozyme and myoglobin was successfully performed in 15.0% (W/W) PEG1000-17.0% (W/W) potassium phosphate ATPs at pH 9.2 with high-speed counter-current chromatograph at rotation speed of 850r/min and flow rate of 0.8mL/min, using upper phase as stationary phase. pH and PEG concentration also had distinct effects on the partition coefficients of the major protein components in hen egg white, including ovaltransferrin, ovalbumin and lysozyme. The optimal pH value and PEG concentration for the purification of ovalbumin by HSCCC-ATP were found to be 9.2 and 16.0% (W/W) respectively. Ovalbumin was successfully purified to homogeneity from the hen egg white sample in 16.0% (W/W) PEG1000-17.0% (W/W) potassium phosphate ATPs at pH 9.2 with high-speed counter-current chromatograph at rotation speed of 850r/min and flow rate of 1.8mL/min, using upper phase as stationary phase. The purification recovery of ovalbumin was around 95%.
Animals↗
[Separation of proteins in aqueous two-phase systems with high-speed counter-current chromatography].
High-speed counter-current chromatography (HSCCC) is a continuous liquid-liquid partition chromatography, with remarkable advantages of high separation efficiency and no adsorption or denaturation by solid phase. The retention of stationary phase and the separation of proteins in polyethylene glycol 1000 (PEG1000)-phosphate aqueous two-phase system (ATPs) were studied with a multi-column high speed-counter-current chromatograph. The flow direction and speed of the mobile phase, and the rotation direction and speed of the apparatus showed different effects on the retention of the stationary phase, which reached the maximum at 33.3% with a flow rate of 0.6 mL/min and a rotation speed of 900 r/min in 14.0% PEG1000-16.0% phosphate ATPs. Distinct differences in partition coefficients among cytochrome C, lysozyme and hemoglobin were found at pH 9.2 and these three proteins were successfully separated in 14.0% PEG1000-16.0% phosphate ATPs at pH 9.2 by HSCCC with the apparatus rotating at 850 r/min and the mobile phase flow rate of 1.0 mL/min. The major protein components in hen egg white, including ovaltransferrin, ovalbumin and lysozyme also show distinct differences of partition coefficients in PEG1000-phosphate ATPs at pH 9.2. Ovalbumin and lysozyme were successfully purified to homogeneity and ovaltransferrin to ca 60% purity from the hen egg white sample with yields over 90% in 15.0% PEG1000-17.0% phosphate ATPs at pH 9.2 with the apparatus rotating at 850 r/min and mobile phase flow rate of 1.0 mL/min.
Animals↗
[Purification and fingerprinting development of Salvia miltiorrhiza Bunge by high-speed counter-current chromatography].
In an attempt to apply high-speed counter-current chromatography HSCCC for TCM fingerprints, the separation and purification of the Chinese medicinal plant Salvia miltiorrhiza Bunge of different localities was realized using the technique. The equipments used include a HSCCC (TBE-300) of Shenzhen Tauto Biotech containing three connected preparative coils (diameter of tube = 2.6mm, total volume = 300mL) and a 20mL sample loop and a HPLC from Shimadzu of Japan with a Ultrasphere C18 column (150 x 4.6mm ID, 5microm) and a 20microL sample loop. Salvia miltiorrhiza Bunge samples from 3 locations were separated by HSCCC in a Step-wise elution program with solvent systems A (hexane:ethanol: water = 10:5.5:4.5) and B (hexane:ethanol: water = 10:7:3) at a speed of 900 r/min and a flow-rate of 2mL/min. All the 12 peak fractions were eluted within 13 hours. The contents of each component varied greatly in different samples, which confirmed previous observation that the locations and climates have a great impact on the TCM quality and also indicated a quality control system is necessary to safeguard the quality of the herb. The retention times of the 12 peak fractions from crude extracts of the samples were collected by HPLC and the absorption spectrums of the corresponding peaks were identified. The 12 components of the three crude samples were readily distinguishable and can be used as fingerprints of S. miltiorrhiza Bunge. The relative standard deviation of the HSCCC retention times was less than 3%, which satisfies the requirement of the national standard reference index. The components 7, 8 and 11 from the standards were identified to be crypototanshinone, tanshinone I and tanshinone II A respectively. This study demonstrates that if it is possible to apply HSCCC for TCM fingerprinting, especially with samples of high viscosity and highly absorptive components. The precision and the run time of fingerprinting can be further improved if larger volume and a temperature control system is used. With these and other improvements, HSCCC is expected to play an important role in TCM development.
Abietanes↗
[Preparative isolation and purification of calycosin and formononetin from Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao by high-speed counter-current chromatography].
Preparative high-speed counter-current chromatography (HSCCC) was used for the separation of calycosin and formononetin from the ethyl acetate extract of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao. Calycosin was obtained by HSCCC with a binary phase solvent system composed of n-hexane-chloroform-methanol-water (1.5:3:3:2, v/v). Then, formononetin was obtained with a system composed of n-hexane-chloroform-methanol-water (4:4:5:4, v/v). The mobile phase was the lower phase and operated at a flow-rate of 2.0 mL/min, while the apparatus rotated at 800 r/min. High performance liquid chromatographic analysis of calycosin and formononetin revealed that their purities were over 95%. HSCCC is a useful method for the separation of natural products.
Astragalus propinquus↗
[Isolation and identification of hetisine-type alkaloids from Aconitum coreanum by high speed countercurrent chromatography].
AIM: To search for more bioactive compounds from the roots of Aconitum coreanum (Lèvl.) Rapaics. METHODS: High speed countercurrent chromatography was successfully applied to the separation of alkaloids from Aconitum coreanum. The structures were elucidated by their physicochemical properties and spectroscopic analysis. RESULTS: Two-phase solvent system composed of CHCl3-CH3OH-0.2 mol x L(-1) HCl (10:3:3, volume ratio) was used in this experiment, eight alkaloids were obtained from the roots of Aconitum coreanum, which were identified as: 2alpha-propionyl-11alpha,13beta-diacetyl-14-hydroxyhetisine (I), Guanfu base P (II), Guanfu base G (III), Guanfu base F (IV), Guanfu base Z (V), Guanfu base O (VI), Guanfu base A (VII), Guanfu base B (VIII). CONCLUSION: Compound I is a new alkaloid, named Guanfu base R.
Aconitum↗
[Preparative separation of aloin diastereoisomers by high-speed countercurrent chromatography combined with silica gel column chromatography].
Aloin, naturally a mixture of two diastereoisomers, aloin A and aloin B, is the major anthraquinone in aloe, and now served as one of the important control constituents in most of the commercial aloe products. High-speed countercurrent chromatography (HSCCC) combined with silica gel column chromatography was developed for the preparative separation of the two individual aloins. Aloin A (98%) and aloin B (96%) were obtained. Fast atom bombardment mass spectrometry (FAB-MS), 1H nuclear magnetic resonance (1H NMR) and GOESY (gradient-enhanced nuclear Overhauser effect spectroscopy) were employed for the elucidation of their structure conformation. The developed method is of high preparative capacity and high efficiency in resolution.
Aloe↗
[Isolation and purification of the process impurity in losartan by reversed-phase column chromatography].
A process impurity (approximately 4%, w/w) was isolated from losartan crude by low-pressure reversed-phase column chromatography. At first the elution portions containing the target compound were collected. After evaporation and recrystallization, the purity of the target compound was found to be over 99% based on high performance liquid chromatography with ultraviolet absorption. The electrospray ionization-mass spectrometry result showed that it was an isomeric compound of losartan, which had same fragmentation pattern with its parent compound. And at last the compound was further characterized by nuclear magnetic resonance. This research demonstrates that the low-pressure reversed-phase column chromatography can raise separation efficiency and recovery comparing with other conventional column chromatography, and the technique can be applied as an effective means for the enrichment and extraction of impurities in pharmaceutical separation.
Adsorption↗
Ca2+ and pH dependence of hydrophobicity of alpha-lactalbumin: affinity partitioning of proteins in aqueous two-phase systems containing poly(ethylene glycol) esters of fatty acids.
Hydrophobic affinity partitioning in an aqueous two-phase system, composed of dextran and poly(ethylene glycol), has been used to study the hydrophobic binding capacity of bovine alpha-lactalbumin. The hydrophobicity of the poly(ethylene glycol)-containing phase was adjusted by including varying amounts of fatty acids bound to the polymer via an ester linkage. The change in the logarithmic partition coefficient of the protein in such systems was used as a measure of the hydrophobic binding. This value was strongly influenced by the amount of Ca2+ present as well as the pH value. The results are discussed in terms of the exposure of hydrophobic binding sites on alpha-lactalbumin and their relation to the conformational change in this protein due to Ca(2+)-binding, chelation of Ca2+ and pH dependence.
Animals↗
Disposition of cyproheptadine in rats, mice, and humans and identification of a stable epoxide metabolite.
Radioactivity was excreted in the urine and feces of rats, mice, and humans after a dose of 14C-cyproheptadine. The major metabolite in rat urine was unconjugated, but the majority of radioactive materials in mouse and human urine were conjugated with glucuronic acid. Identification of the rat urinary metabolite of cyproheptadine as an epoxide was accomplished with mass spectrometry and other methods. The rat metabolite was 10.11 -epoxydesmethylcyproheptadine and accounted for about 25% of a 45-mg dose of cyproheptadine per kg. Only a small amount of this epoxide was found in mouse urine, and none was apparent in the urine of two humans who received 5 mg of the drug. Dihydrodiols, which could arise by epoxide hydrase hydrolysis of possible 10.11-epoxy metabolites, were not found in the urine of any of the species studied. The spoxide found in rat urine appears to be unusually stable to in vivo hydrolysis. Possible implications of these results in the species-selective pancreotoxicity of cyproheptadine in the rat are presented.
Adult↗
The effect of liver disease in man on the disposition of phenobarbital.
The disposition of phenobarbital (PB) was studied in normal individuals and in patients with cirrhosis or acute viral hepatitis to determine 1) if there is significant impairment of PB metabolism in hepatic disease and 2) to what extent such abnormal disposition of the drug affects its disappearance from blood. The diagnosis of liver disease was based on characteristic clinical findings, biochemical liver "function" tests and liver biopsy when necessary. All individuals had normal renal function and were free of other drug and alcohol intake for at least 3 weeks. With radiotracer methodology, PB and its principal metabolites, p-hydroxyphenobarbital (PBOH) and conjugated PBOH (PBOC), were monitored in blood and urine for 5 days after a single dose of 14-C-PBadministered intraduodenally. PB blood half-life (T1/2) in the control group was 86 plus or minus 3 hours (S.E.). In cirrhotics the T1/2 was prolonged to 130 plus or minus 15 hours (P less than .001) and this was accompanied by a 50% reduction in urinary PBOC excretion (P less than .05). Urinary excretion of PB and PBOH was unaltered by cirrhosis. In patients with acute viral hepatitis, PB T1/2 was not significantly prolonged and urinary excretion of PB and its metabolites was in the normal range (P greater than .05). No PBOH and only traces of PBOC were detected in the blood of either control individuals or patients with liver disease. Urinary excretion of unchanged PB was an important elimination pathway of the drug in all groups. As a result of this, PB T1/2 in cirrhosis was only moderately prolonged.
Adult↗
Physiological disposition and metabolism of timolol in man and laboratory animals.
Timolol [3-(3-tert.-butylamino-2-hydroxypropoxy)-4-morpholino-1,2,5-thiadiazole], was rapidly absorbed, metabolized, and effectively excreted in man, rats, and dogs. Peak plasma levels of timolol-14C were observed in these species 1-2 hr after oral administration. Generally, less than 20% of the radioactivity was present in the plasma in the unmetabolized form. The intact drug had a plasma half-life of 28 min in the rat, 48 min in the dog, and 5.5 hr in man. After oral administration of timolol-14C to humans approximately 72% of the dose was excreted in 84 hr, with 66% in the urine and 6% in the feces. In the rat, 58% of an oral dose was excreted in the urine and 26% in the feces. The dog excreted 68% of an oral dose in the urine and 19% in feces in 72 hr. Following intravenous administration, rats excreted 50% in the urine and 28% in the feces, which suggests that extensive biliary excretion occurred. Timolol was extensively metabolized. Approximately 50% of the radioactivity was identified in dog urine as the lactic acid metabolite. An additional metabolite was tentatively identified as the 3-oxomorpholino derivative of timolol. Approximately 20% of the dose in man was excreted in the urine unchanged. Two metabolites, resulting from cleavage of the morpholine ring, were identified as 1-tert-butylamino-3-[4-(2-hydroxyethylamino)-1,2,5-thiadiazol-3-xloxyl-2-propanol, accounting for 10% of the urine radioactivity, and t-tert-butylamino-[4-(N-2-hydroxyethylglycolamido)-1,2,5-thiadiazol-3-yloxy]-2-propanol, accounting for 30%. A minor metabolite, resulting from hydroxylation of a terminal methyl group, accounted for an additional 3% of the urine radioactivity.
Animals↗
Subfractionation of human peripheral blood lymphocytes by partitioning in two-polymer aqueous phase systems. Characteristics of a small subpopulation of cells with high partition coefficient.
Mixtures of aqueous solutions of dextran and poly(ethylene glycol) form immiscible 2-phase systems that are suitable for the separation of viable, functionally-competent cells on the basis of their surface properties. With the appropriate choice of ionic composition, these systems will separate cells on the basis of charge-related surface characteristics. We have previously shown that in charge-sensitive phase systems, human B lymphocytes have a low partition coefficient (K) and T cells have an intermediate K. The cell subpopulation with the highest K values contains large null lymphocytes that have most of the antibody-dependent and natural killer activities. The present study further characterizes this latter cell subpopulation. 8-33% of peripheral blood lymphocytes in normal persons had high K values. About 50% of these cells had typical Giemsa-staining granules characteristic of killer cells and one third had Fc receptors, but lacked complement receptors. Although 15-30% of the cells with high K values formed rosettes with sheep erythrocytes, there was virtually no response to T-cell mitogens such as PHA and Con A. Mixing cells having high K values with mitogen-responsive T cells from intermediate cell fractions revealed no evidence of suppressor-cell activity to explain the poor mitogen response. The studies indicate that a heterogeneous population of cells share charge-related surface properties resulting in a high partition coefficient. These cells comprise a significant proportion of human peripheral blood lymphocytes and include virtually all of the large granular lymphocytes as well as a large population without this morphology that shares the feature of unresponsiveness to T-cell mitogens.
Antibody-Dependent Cell Cytotoxicity↗
Plasma membrane purification from roots of sunflower by phase partitioning.
Plasma membrane vesicles were purified from the roots of sunflower (Helianthus annuus L. cv. Topflor) by aqueous polymer two-phase partitioning. The optimal conditions for separation were determined by systematic variation of the polymer concentration and salt composition. The phase system containing 6% (w/w) dextran T-500, 6% (w/w) polyethylene glycol 3350, 250 mM sucrose, 5 mM potassium phosphate, pH 7.8, without added salts proved to be the best. The ATPase activity had a pH optimum at 6.5 and it was stimulated by Mg2+, but not by Ca2+. The plasma membrane MgATPase activity was inhibited by vanadate but not by nitrate, an inhibitor of tonoplast ATPase. Only 10% of the microsomal protein was responsible for 36% of the total MgATPase activity. Moreover IDPase activity, a Golgi marker, appeared to be very low indicating the high purity of the preparation.
Ca(2+) Mg(2+)-ATPase↗
Counter current transfer in the female adnex.
The utero-ovarian veins and lymph vessels are intimately connected with the ovarian artery in the human female and in domestic animals, with the exception of the horse and the human female. A direct, local exchange of molecules from veins and lymph vessels to arteries (counter current transfer) has been documented for this anatomic structure. Countercurrent transfer of certain inert gases (133xenon, 85krypton), of prostaglandins (PGF2 alpha), of steroid hormones (e.g. progesterone, estradiol, testosterone), and of small peptide hormones (oxytocin, relaxin) has been shown to occur in laboratory and domestic animals as well as in the human female. The transfer of the inert gases takes place within seconds. The transfer of steroid hormones and peptides is detectable within minutes while the transfer of PGF2 alpha is delayed for 20 minutes. Red blood cells or albumin are not transferred. The existence of the local transfer is postulated to be of importance for: 1) the pregnancy/non-pregnancy signal from the uterus and tube to the ovary. The signal may be a combination of a luteotrophic signal from the embryo and lack of a "non-pregnant" luteolytic signal from the endometrium, the latter probably being PGF2 alpha in some species; 2) the unilateral influence of the ovarian hormones on the function of the ovarian, tubal, and possibly uterine tissues. An active corpus luteum may create in a mono-ovulatory animal a higher progesterone level in arterial blood supplying the ipsilateral tube and ovarian interstitial tissue than on equivalent contralateral organs.
Adnexa Uteri↗
Counter current transfer and back transport of 3H-PGF2 alpha in the cow's broad ligament vasculature ipsilateral and contralateral to the corpus luteum.
Eight cows of similar age (5-7 years) were chosen for the experiment. Isolated reproductive tract was supplied with autologous oxygenated and heated (40 degrees C) blood through the uterine artery and ovarian artery. 3H-PGF2 alpha in total dose of 2 MBq (10(7) cpm) was injected into each of the uterine lumen of isolated organ. Blood samples were collected at 5 min intervals during 120 min of experiment using cannulae inserted into the branches of uterine arteries about 1 cm below the horns and from ovarian arteries inserted 0.5 cm below the ovaries. The concentration of 3H-PGF2 alpha found in blood plasma taken from uterine artery or from ovarian artery on the side with active corpus luteum (CL) was significantly lower (p less than 0.001) compare with contralateral side to active CL. Radioactive PGF2 alpha found in branches of uterine arteries on both ipsilateral and contralateral side to CL was significantly higher (p less than 0.001) compare to ovarian artery of the same side. It is concluded that absorption of 3H-PGF2 alpha from uterine lumen into venous blood as well as its counter current transfer in area of broad ligament vasculature were reduced on the side of uterine horn with active CL probably as an effect of estrogen:progesterone ratio on vascular constriction in area of uterine vasculature.
Adnexa Uteri↗