A method is proposed for isolating the main components (melittin, apamin, and phospholipase A 2 from the venom of the bee Apis mellifera using HPLC, and the synthesis of two sorbents for fractionating these peptides is described.Bee venom is widely used as a medicament in various diseases. [1]. However, to create highly specific and effective agents it is desirable to use the components of the venom in a highly purified state. In view of this, researchers are faced with the task of developing the simplest, fastest, and cheapest methods of fractionating the venom [2].It is known that the venom of the bee Apis mellifera is a complex mixture of proteins and peptides exhibiting the most diverse biological activities. A number of proteins and peptides have been studied in detail, and the primary structures of the phospholipase A 2, melittin, apamin, and the MCD peptide have been determined [2][3][4][5][6][7][8][9][10][11]. More recently, new methods have been developed for isolating the components of bee venom, since these components are of interest for pharmacology [1]. Purification of the majority of the components is complicated by high surface activity, low molecular mass, and the presence of a large amount of complex substances [3,[11][12][13][14]. All this is leading to the use of different and incompatible methods of isolation by different groups of researchers.Our aim was to improve the methods of fractionation, with a substantial increase in the yields of tlie components of the venom, and a decrease in the number of stages of the isolation process.According to the results of HPLC (Fig. 1), whole bee venom contains substances differing greatly in hydrophobic properties. In the first stage, therefore, we used reversed-phase adsorbents based on silica gel. To check these presuppositions, we carried out the separation of bee venom on the sorbent LiChroprep RP-8 in a stepwise gradient of isopropanol (Fig. 2).Five fractions were obtained which were characterized by the use of HPLC on a Nucleosil 100-5 C18 column. A disadvantage of this sorbent is that the low-molecular-mass colored substances of bee venom are irreversibly sorbed on the gel, which decreases the capacity and impairs the hydrodynamic properties of the column. This sorbent enables products to be obtained in a highly purified state, but a large amount of sorbent with a def'mite capacity stable in various buffer systems is necessary for semipreparative separation. For this purpose we used Polikhrom-1, but the capacity and hydrodynamic parameters of this gel proved to be inadequate for these purposes. We then synthesized a reversed-phase sorbent based on Silokhrom-80 by the direct modification of the silanol groups with octadecyltrichlorosilane in acetone at 50°C for 18 h.The fractionation of the venom of the bee Apis mellifera with the aid of this sorbent gave fractions containing large amounts of impurities. In all probability, this was due to the fact that only 50% of the silanol groups had been modified by the octadecylsilane. The unmodified silanol g...
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Cistanche tubulosa (Schrenk) Wight (Rou Cong Rong in Chinese) is a kind of perennial phanerogamic parasitic plant (Orobanchaeceae) attached underground to the roots of Tamarix talamakanensis, T. vamosissima, T. hohenackeri, T. arceuthoide, etc., which is widely cultivated in desert areas such as the southern edge of the Tarim Basin in Xinjiang of China, and which grows by absorbing nutrients from the host plant [1][2][3]. The dried succulent stem of C. tubulosa is a precious traditional Chinese herb and has been recorded in the Chinese Pharmacopoeia (2010).C. tubulosa is used to nourish the kidney, supplement essence and blood, and relieve constipation as a laxative [4]. A great number of investigations have proved its medicinal activities in scavenging free oxygen radicals [5][6][7]. Its stem extracts are used as an anti-decrepitude [8], to improve learning-memory ability [9], for neuroprotection [10], and for immunoregulation [11]. The major active compounds in Cistanche species are phenylethanoid glycosides such as echinacoside, acteoside, isoacteoside, and tubuloside A, which are usually chosen as marker compounds to assess the quality of C. tubulosa, and species sources of C. tubulosa var C. deserticola, C. salsa, and C. sinensis are distinguished based only on these compounds [12,13].In the course of further studies on active compounds from C. tubulosa, we have sitimultaneous isolated and purified echinacoside (1), cistanoside A (2), cistantubuloside A (3), acteoside (4), isoacteoside (5), 2c-acetylacteoside (6), and tubuloside A (7) from C. tubulosa by prep-HPLC, each at over 96.3% purity as determined by HPLC. The compounds were identified by their retention time, and confirmed by ESI-MS, and NMR experiments.In in vitro assays, the mouse skin melanoma action of phenylethanoid glycoside compounds on cell line KML was studied. Antitumor activities of one extract and five compounds were found for KML, such as Fr. 2 (31%), echinacoside (75%), cistanoside A (33%), cistantubuloside A (83%), acteoside (81%), and 2c-acetylacteoside (93%), expressed as percent inhibition of tumor growth.Plant Material. The stems of Cistanche tubulosa were collected from Keriya country, Xinjiang of China in September 2007 and was identified by Chief Pharmacist Sulayman Khalik at the Xinjiang Uyghur Autonomous Regional Institute for Food and Drug Control. A voucher specimen was deposited in the Xinjiang Technical Institute of Physics and Chemistry.Sample Preparation. The dried, powdered fresh stems (3.0 kg) of Cistanche tubulosa were soaked twice with 80% aqueous methanol (1.5 L, 1.2 L) for 78 h at room temperature, twice filtered, combined, and concentrated under reduced pressure before being suspended in water, then partitioned consecutively with petroleum-ether, CH 2 Cl 2 , and n-butanol. The n-butanol fraction (17.0 g) was separated by solid phase-extraction (SPE), and the stationary phase of the normal-phase silica gel was eluted by a CHCl 3 -CH 3 OH gradient (100:0; 75:25) and aqueous methanol (95% to 80%) to give three fract...
It has been shown that the yeast S. cerevisiae has the ability to synthesize nicotinic acid and NAD from 3-methylpyridine. Fractionation of intracellular yeast proteins established that the fraction of molecular weight 65-90 kDa had the highest activity for transformation of pyridine derivatives into nicotinate. HPLC showed that, in addition to nicotinic acid, nicotinamide was also present in the intermediates during transformation of 3-methylpyridine. The results were consistent with the presence in yeast cells of an enzymatic system that transforms 3-methylpyridine into vitamin PP.Nicotinic acid (NA) is known to be produced chemically from the pyridine derivatives β-picoline (3-methylpyridine, 3-MP) or 3-acetylpyridine (3-AP) [1]. The question of NA production in microbiological conversions was first raised in the 1970s by researchers [2-4] who isolated more than 100 cultures of Mycobacterium, Nocardia, Corynebacterium, and Arthrobacter and others that not only decomposed the pyridine ring but also oxidized alkyl substituents on it without destroying it. Also, the ability to oxidize the methyl on 3-MP was first demonstrated at that time. However, the enzymatic aspects of the microbiological transformation of pyridine derivatives have been poorly studied despite the other successes.We observed previously that local strains of the yeast Saccharomyces cerevisiae 913a-1, which were selected as producers of nicotinamideadeninedinucleotide (NAD), accumulate high concentrations of the coenzyme in the presence of 3-MP and 3-AP [5]. This suggests that enzymatic transformation of these compounds into nicotinate and its subsequent participation in NAD biosynthesis is possible. Therefore, our goal was to find intracellular proteins that are involved in the bioconversion of 3-MP into NA by S. cerevisiae 913a-1.Fermentation of S. cerevisiae 913a-1 biomass in the presence of 3-MP is accompanied by more than a doubling of the intracellular NAD concentration (from 35.6 to 68.2 µg/mL) whereas the free NA content did not change (8.6 µg/mL).However, it should be considered that intracellular NA is an intermediate in many exchange transformations of pyridinenucleotides whereas the intracellular content of free NA is low compared with that of nicotinamide or NAD. Thus, the ability of yeast cells to accumulate high concentrations of NAD indicates that biosynthesis can supply the required amount of NA. The increased intracellular pool of NAD observed in yeast in the presence of 3-MP may be due to its preliminary transformation into nicotinate due to its enzymatic transformation in cytoplasm. According to the literature, various microorganism groups can not only completely degrade pyridine bases but also partially transform them. Thus, the ability of the bacteria Nocardia and Arthrobacter to oxidize under co-oxidation conditions alkylpyridines to the corresponding acids, 3-and 2-MP to nicotinic and picolinic acids, has been demonstrated. Transformation of pyridine into nicotinamide also occurs through NA or other intermediates such as ...
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