OPTIMIZATION OF FLAVONOID EXTRACTION CONDITIONS FROM A PLANT OF THE GENUS SYMPHYOTRICHUM NOVI-BELGII

d-erythro -Sphingosine-1-phosphate (2), an intermediate in sphingosine metabolism, shows a diversity of biological activities. Comparable roles might be anticipated for d-ribo -phytosphingosine-1-phosphate (1). We describe an efficient three-step chemical synthesis of 1 from d-ribo -phytosphingosine. Our approach is based on standard phosphoramidite methodology and on the finding of Boumendjel and Miller ( J. Lipid Res. 1994. 35: 2305-2311) that sphingosine can be monophosphorylated at the 1-hydroxyl without protection of the 3-hydroxyl. However, we were unable to duplicate their reported synthesis of 2 without important modifications in reagents and reaction conditions. Under the reported conditions for preparing 2, we obtained a cyclic carbamate (14), which we have isolated and identified. The structures of 1 and the cyclic carbamate 14 were elucidated by a combination of mass spectrometry and 1D and 2D nuclear magnetic resonance spectroscopy.

The article presents the results of the amino and fatty acid analysis of the aerial parts of Zinnia elegans and Symphyotrichum novi-belgii, the Asteraceae family, collected during the fruiting period in September 2021 in the East Kazakhstan region (Altai region). The relevance of the work lies in the extraction of biologically active substances and the production of medicines from plants growing in East Kazakhstan.The study determined the volumetric composition of various amino and fatty acids in the aerial part of Zinnia elegans and Symphyotrichum novi-belgii. According to the results of the study, in the aerial part, in terms of the amount of nutrients from amino acids, the following dominate: glutamic and aspartic acids; from fatty acids - linoleic, oleic, stearic and palmitic acids. The aerial part has been determined to be the source of many essential compounds. By using paper chromatography were detected biology active compounds as phenolic acids, flavonoids and carbohydrates from plants. The results of the study showed that the plants of two species Zinnia elegans and Symphyotrichum novi-belgii have a sufficient content of biologically active substances, which can in the future expand the range of effective domestic herbal medicines available in medicine and agriculture of the Republic of Kazakhstan.

Many researchers have employed column and thin layer chromatography for the separation of lipids. Qualitative thin layer chro-matographic separation of cholesterol esters, triglycerides, fatty acids, cholesterol, di- and monoglycerides, and phospholipids on silica gel plates of normal plasma and of patients with lipid disorders has been reported(1). Also, separation of 1,3- and 1,2-diglycerides and α- and β-monoglycerides by thin layer chromatography, after pancreatic lipase digestion of fats, has been demonstrated(2). Silicic acid column chromatography was used for a similar purpose by Borgstrom(3), Fillerup and Mead(4), and Hirsch and Ahr-ens(5). In this report, the digestion products of the action of Clearing Factor on coconut oil were studied. The presence of α-, β-mono-glycerides and 1,2- and 1,3-diglycerides intermediates were indicated by various thin layer and column chromatographic procedures. The free fatty acids were the major end product of hydrolysis. Among several separation methods of complex lipid mixtures, column chromatography in combination with thin layer chromatography produced the best information and is recommended. Materials and methods. Post-heparin Clearing Factor was prepared as reported previously (10). Tripalmitin, triolein, and cholesterol were purchased from Mann Biochemical Laboratories. Monoolein, monopal-mitin, 1,2- and 1,3-dipalmitin were supplied by Dr. J. Hirsch. Conditions of hydrolysis of Ediol with Clearing Factor was the same as previously reported(9,10). Five ml aliquots were taken at zero time, 1 hr, 3 hr, and overnight from the incubation mixtures. Each aliquot was extracted with 50 ml of Bloor solution (3 parts ethanol:1 part diethyl ether). Thin layer plates were (20 × 20 cm glass) coated with a 225-250 layer of silica gel G and air dried(6). The plates were then activated for one hour at 160° and placed in a desiccator.

Agarwood (Aquilaria sinensis), well known as an incense in Southeast Asia, has been used as a sedative, analgesic, and digestive aid in traditional medicine. Agarwood leaves are consumed as a healthy tea in Thailand and Taiwan. [1]. Previous phytochemical investigation on Chinese eaglewood revealed characteristic sesquiterpenes and chromone derivatives [2–7], but little is known about the chemical constituents of the healthy wood. By investigation of interrelated studies, agarwood has significant anticancer [8], analgesic and anti-inflammatory activities [9], and antidepression activities [10]. These observations provide useful information for potential chemopreventive drug design. The MeOH extract of its rhizomes was subjected to solvent partitioning and chromatographic separation to afford eight pure substances. The chemical constituents in the bark of A. sinensis were separated by column chromatography into eight compounds, including 5-hydroxy-4 ,7-dimethoxyflavone (1) [11], luteolin-7,3 ,4 -trimethyl ether (2) [12], 5,3 -dihydroxy-7,4 -dimethoxyflavone (3) [13], persicogenin (4) [14], genkwanin (5) [15], methylparaben (6) [16], -sitosterol (7), and (+)-syringaresinol (8) [17]. Compounds 1–8 were obtained for the first time from this plant. The bark (3.5 kg) of Aquilaria sinensis was chipped and air dried and extracted repeatedly with MeOH (5 L 4) at room temperature. The combined MeOH extracts (132.8 g) were then evaporated and further separated into three fractions by column chromatography on silica gel (4.7 kg, 70–230 mesh) with gradients of n-hexane–CH2Cl2–acetone–MeOH. Part of fraction 1 (57.3 g) was subjected to silica gel chromatography by eluting with n-hexane–acetone (60:1) enriched with acetone to furnish two further fractions (2-1–2-2). Fraction 2-1 (31.7 g) was further purified on a silica gel column using n-hexane– acetone mixtures to obtain 5-hydroxy-4 ,7-dimethoxyflavone (1) (30.4 mg). Fraction 2-2 (25.6 g) was further purified on a silica gel column using n-hexane–acetone mixtures to yielded luteolin-7,3 ,4 -trimethyl ether (2) (36.9 mg). Part of fraction 2 (64.2 g) was subjected to silica gel chromatography by eluting with n-hexane–acetone (40:1) enriched with acetone to furnish two further fractions (2-1–2-2). Fraction 2-1 (38.2 g) was further purified on a silica gel column using n-hexane–acetone mixtures to obtain methylparaben (6) (34.6 mg). Fraction 2-2 (26 g) was subjected to silica gel chromatography by eluting with CH2Cl2–MeOH (100:1) enriched gradually with MeOH to obtain two fractions (3-2-1–3-2-2). Fractions 3-2-2 (17.5 g) were subjected to further silica gel column chromatography and purified by preparative TLC (thin layer chromatography) to yield 5,3 -dihydroxy-7,4 -dimethoxyflavone (3) (35.2 mg). Part of fraction 3 (82.5 g) was subjected to silica gel chromatography by eluting with CH2Cl2–MeOH (80:1) enriched with MeOH to furnish two fractions (3-1–3-2). Fraction 3-1 (36.5 g) was further purified on a silica gel column using CH2Cl2–MeOH mixtures to obtain persicogenin (4) (28 mg) and genkwanin (5) (24 mg). Fraction 3-2 (46 g) was further purified on a silica gel column using CH2Cl2–MeOH mixtures to obtain -sitosterol (7) (36.2 mg) and (+)-syringaresinol (8) (34.1 mg).

Mitomycin C is a natural product with potent alkylating activity, and it is an important anticancer drug and antibiotic. mitN, one of three genes with high similarity to methyltransferases, is located within the mitomycin biosynthetic gene cluster. An inframe deletion in mitN of the mitomycin biosynthetic pathway was generated in Streptomyces lavendulae to produce the DHS5373 mutant strain. Investigation of DHS5373 revealed continued production of mitomycin A and mitomycin C in addition to the accumulation of a new mitomycin analog, 9-epi-mitomycin C. The mitN gene was overexpressed in Escherichia coli, and the histidine-tagged protein (MitN) was purified to homogeneity. Reaction of 9-epi-mitomycin C with MitN in the presence of S-adenosylmethionine yielded mitomycin E showing that the enzyme functions as an aziridine N-methyltransferase. Likewise, MitN was also shown to convert mitomycin A to mitomycin F under the same reaction conditions. We conclude that MitN plays an important role in a parallel biosynthetic pathway leading to the subclass of mitomycins with 9alpha-stereochemistry but is not involved directly in the biosynthesis of mitomycins A and C.

Cinnamomum burmannii Blume (Lauraceae) is a source of Indonesian cinnamon and is widely used as a spice in Indonesia [1]. The chemical constituents of the leaves of this plant have not yet been reported. Recently, we reported a new amide, cinnabutamine, along with five known amides from the stems of C. burmannii [2]. In the course of screening for biologically and chemically novel agents from Formosan plants in the family Lauraceae [3–12], C. burmannii was chosen for further phytochemical investigation. In this study, the MeOH extract of its leaves was subjected to solvent partitioning and chromatographic separation to afford 11 pure substances. The chemical constituents of the leaves of C. burmannii were separated by column chromatography. Eleven compounds, including squalene (1) [13], ficaprenol-10 (2) [14], methyl vanillate (3) [15], coumarin (4) [16], -sitostenone (5) [17], -sitosterol (6) [17], vanillic acid (7) [15], syringic acid (8) [15], (+)-abscisic acid (9) [18], p-hydroxybenzoic acid (10) [15], and kaempferol-3-O-rhamnoside (11) [19], were isolated from the leaves of C. burmannii. Compounds 1–3, 5–8, 10, and 11 were found for the first time from this plant. The specimen of C. burmannii was collected from Chiayi County, Taiwan in January 2009. A voucher specimen (Cinnamo. 10) was identified by Dr. Fu-Yuan Lu (Department of Forestry and Natural Resources College of Agriculture, National Chiayi University) and deposited in the Department of Medical Laboratory Sciences and Biotechnology, School of Medical and Heath Science, Fooyin University, Kaohsiung, Taiwan. The air-dried leaves of C. burmannii (4.7 kg) were extracted with MeOH (80 L 4) at room temperature, and a MeOH extract (186.4 g) was obtained upon concentration under reduced pressure. The MeOH extract was placed on a silica gel column and eluted with CH2Cl2 gradually enriched with MeOH to afford five fractions. Part of fraction 2 (9.52 g) was subjected to silica gel chromatography by eluting with n-hexane–EtOAc (100:1) and enriched with EtOAc to furnish four further fractions (2-1–2-4). Fractions 2-1 (3.05 g) eluted with n-hexane– EtOAc (60:1) was further purified by silica gel column chromatography using the same solvent system to give squalene (1) (12 mg) and ficaprenol-10 (2) (15 mg). Fractions 2-2 (2.57 g) and 2-3 (1.62 g) eluting with n-hexane–EtOAc (30:1) were further separated by silica gel chromatography to give methyl vanillate (3) (5 mg), and purified by preparative TLC (n-hexane– acetone, 20:1) to give coumarin (4) (19 mg) and -sitostenone (5) (12 mg), respectively. A part of fraction 3 (21.07 g) was subjected to silica gel chromatography by eluting with CH2Cl2–MeOH (100:1) and enriched with MeOH to furnish four further fractions (3-1–3-4). Fraction 3-1 (6.52 g) eluted with CH2Cl2–MeOH (80:1) was further purified by silica gel column chromatography using n-hexane–acetone (10:1) to give -sitosterol (6) (10 mg). Fraction 3-2 (8.73 g) eluting with CH2Cl2– MeOH (60:1) was further purified by silica gel column chromatography using the same solvent system and preparative TLC (n-hexane–acetone, 4:1) to give vanillic acid (7) (6 mg) and syringic acid (8) (5 mg). Fraction 3-3 (0.43 g) was further purified by another silica gel column using n-hexane–acetone (3:1) to obtain (+)-abscisic acid (9) (9 mg). A part of fraction 4 (15.33 g) was subjected to silica gel chromatography eluting with CH2Cl2–MeOH (40:1) and enriched gradually with MeOH to give three fractions (4-1–4-3). Fraction 4-2 (8.21 g) was further separated by silica gel column chromatography using the same solvent system and purified by preparative TLC (CH2Cl2–MeOH, 20:1) to yield p-hydroxybenzoic acid (10) (6 mg). Kaempferol3-O-rhamnoside (11) (8 mg) was further purified on a silica gel column using CH2Cl2–MeOH (10:1) from fraction 4-3.

Acylation/deacylation reactions represent a basic requirement of triglyceride as well as phospholipid metabolism, and maintenance of membrane lipid composition. In order to examine enzymes participating in these pathways, we synthesized 18-(4'-azido-2'-hydroxybenzoylamino)-oleic acid, an iodinable photoaffinity analogue of oleic acid as a new tool for analyzing enzymes, especially those binding unsaturated fatty acids or acyl-CoAs. For the synthesis of omega-amino-oleic acid, coupling two bifunctional Cg-components was used. The described synthesis scheme is also suited for the specific generation of other fatty acid analogues with distinct positions of the double bond. The functionality of 18-(4'-azido-2'-hydroxybenzoylamino)-oleic acid was investigated with the enzyme lysophosphatidylcholine:acyl-CoA-O-acyltransferase (LAT) [EC 2.3.1.23], an enzyme that shows high specificity towards (poly)unsaturated fatty acyl-CoAs. It could be shown that the photolabel, esterified with coenzyme A, acts in the dark as a reversible inhibitor of the enzyme activity, but photolysis of the label results in irreversible inactivation of LAT. This inactivation could be prevented by addition of the native substrate arachidonyl-CoA during photolysis. Several proteins could be specifically visualized using the iodinated analogue. The data indicate that this new photoaffinity label may have application to identify and characterize lipid biosynthetic enzymes using unsaturated fatty acids as well as acyl-CoA binding proteins and the active site of these proteins.

Increased intake of fish oil rich in the omega-3 fatty acids eicosapentaenoic acid (EPA, C20:5 omega-3) and docosahexaenoic acid (DHA, C22:6 omega-3) reduces the incidence of human disorders such as atherosclerotic cardiovascular disease. However, mechanisms that contribute to the beneficial effects of fish oil consumption are poorly understood. Mounting evidence suggests that oxidation products of EPA and DHA may be responsible, at least in part, for these benefits. Previously, we have defined the free radical-induced oxidation of arachidonic acid in vitro and in vivo and have proposed a unified mechanism for its peroxidation. We hypothesize that the oxidation of EPA can be rationally defined but would be predicted to be significantly more complex than arachidonate because of the fact that EPA contains an addition carbon-carbon double bond. Herein, we present, for the first time, a unified mechanism for the peroxidation of EPA. Novel oxidation products were identified employing state-of-the-art mass spectrometric techniques including Ag(+) coordination ionspray and atmospheric pressure chemical ionization mass spectrometry. Predicted compounds detected both in vitro and in vivo included monocylic peroxides, serial cyclic peroxides, bicyclic endoperoxides, and dioxolane-endoperoxides. Systematic study of the peroxidation of EPA provides the basis to examine the role of specific oxidation products as mediators of the biological effects of fish oil.

Croton campestris A. St.-Hil. (Euphorbiaceae) has been used in folk medicine as anti-inflammatory and antitumor treatment [1]. Our aim was evaluate anticancer and anti-inflammatory properties of the fraction CFqb14 obtained from C. campestris on in vitro and in vivo experimental models respectively. C. campestris leaves were extracted with ethyl acetate and crude extract was fractionated by column chromatograph (silica gel 1:10) with gradients of polarity solvents. The antiproliferative activity was evaluated on 8 human cancer cell lines. After 48h of treatment, cell viability was determined by sulforhodamine B assay. Fraction CFqb14 was evaluated in paw edema model by inoculation of carrageenan in the right hind paw of balb/C female mice. Five groups were treated intraperitoneally with vehicle (NaCl 0.9%), indomethacin 20 mg/kg (positive control) and CFqb14 1.25, 2.5 and 5 mg/kg 30 min before carrageenan inoculation. The edema was measured 1.5, 3, 4.5, 6, 24, 48, 72 and 96h after edema induction. Early inflammation phase inhibition was observed and, considering the high expression of COX-2 of NCI-H460 (lung) and relating anti-proliferative activity with selectivity mainly for that cell line (GI50= 0.21 µg/mL), the in vivo anti-inflammatory activity may be related to COX-2 inhibition and consequently reduction of prostaglandin production, a fundamental mediator of early phase of inflammation process. Fraction's composition was screened by Thin Layer Chromatography (TLC) via exposure to chemical reagents as cerium sulfate and Liebermann-Burchard specific for terpenes. The phytochemical reagents analysis identified terpenes as major compounds in the CFqb14, suggesting that these compounds are related to anticancer and anti-inflammatory activity observed. Further studies to identify the mechanism of action and the active principles are in progress. All protocols were approved by Committee for Ethics in Animal Research at the State University of Campinas protocol 3026 – 1.

Breadfruit is one of the Indonesian plants traditionally used in medication. The main active compound in breadfruit leaves is a geranylated flavonoid namely 2-geranyl-2',3,4,4'-tetrahydroxy dihydrochalcone (GTD). Previous study showed that the separation and isolation of GTD from sukun leaves is time consuming and laborious as it requires a long procedure (extraction, liquid-liquid partition, Vacuum Liquid Chromatography (VCC), Sephadex Column Chromatography (SCC), and preparative Thin Layer Chromatography (TLC). This process is ineffective and inefficient. Thus, the more effective and shorter method of isolation is needed. This study aimed to isolate GTD from breadfruit leaves utilizing flash column chromatography (FCC). The breadfruit leaves were extracted using ethanol and the extract was partitioned with the solvent n-hexane: ethyl acetate: methanol: water (3:1:3:1). The lower phase containing GTD was subjected to VCC and the fraction containing GTD was purified with FCC (using n-hexane, ethyl acetate, and methanol in a gradient polarity as mobile phases; and silica gel as a solid phase) to isolate GTD. The isolated GTD was analyzed by thin-layer chromatography (TLC) and purity was determined using high-performance liquid chromatography. This method was able to produce 138 mg of GTD (purity of 88.49 %) from 15 g of breadfruit leaf extract (0.92% yield). This study demonstrated that GTD, a main bioactive compound of breadfruit leaves, could be effectively isolated by using FCC instead of SCC and preparative TLC.

Experiments were carried out in order to elucidate the conditions for the quantitative analysis of cholesterol by thin-layer chromatography (TLC) and to compare the quantitative values of TLC with those of digitonin-method.The pure cholesterol refined by column chromatography was used as the standard sample for the calibration curve of TLC.Silica gel G and silica gel G-10% AgNO3 for the stationary phase were suited to the determination of cholesterol by TLC. Activation temperatures, activation times, developing solvents, detection agents, charring times and standing times after charring were examined with the both stationary phases.In the determination of cholesterols by TLC with silica gel G· and silica gel G-10% AgNO3·plate, the purities of cholesterol in sample 2 was 99.2% 99.1% and that in sample 3 was 97.3% respectively. Their purities described before agreed nearly in both methods. The data of standard deviation showed in the range of 0.670.70% and 0.830.90% respectively.It was found that values in the determination of these cholesterols (sample 2 and 3) by digitonin-method were slighty higher than those obtained by TLC.Solubilities of cholesterol-digitonide for 95% ethanol in range of 15.025.0°C were measured. The value obtained was in good agreement with the data of Windaus at 18.0°C. The solubility described before was used in order to correct the purities of the cholesterols.

The recent finding that p-nitrobenzofurazan (NBD)-FA is incorporated into and released from the acylglycerols of isolated rat adipocytes in an insulin-sensitive manner [G. Muller, H. Jordan, C. Jung, H. Kleine, and S. Petry. 2003. Biochimie. 85: 1245-1246] suggests that NBD-FA-labeled acylglycerols are cleaved by rat adipocyte hormone-sensitive lipase (HSL) in vivo. In the present study, we developed a continuous, sensitive in vitro lipase assay using a monoacylglycerol (MAG) containing NBD (NBD-MAG). NBD-MAG was found to provide an efficient substrate for rat adipocyte and human recombinant HSL. Ultrasonic treatment applied in the presence of phospholipids leads to the incorporation of NBD-MAG into the phospholipid liposomes and to a concomitant change of its spectrophotometric properties. The enzymatic release of NBD-FA and its dissociation from the carrier liposomes is accompanied by the recovery of the original spectrophotometric characteristics. The rate of lipolysis was monitored by measuring the increase in optical density at 481 nm, which was found to be linear with time and linearly proportional to the amount of lipase added. To assess the specific activity of recombinant HSL, we determined the molar extinction coefficient of NBD-FA under the assay conditions. This convenient assay procedure based on NBD-MAG should facilitate the search for small molecule HSL inhibitors.

Fractionation and purification of membrane lipids from the archaeon Thermoplasma acidophilum DSM 1728/10217

As a series of studies to clarify clonorchicidal substances in body surface mucus of some fresh-water fishes, the substance in the epidermal mucus of Cyprinus carpio was isolated by silica gel column and thin layer chromatography and analyzed for its chemical nature. Wormicidal trial was done in vitro, and the results obtained are summarized as follows: The mucus was extracted by ethyl ether and separated into 4 fractions by column chromatography using benzene as solvent. The second fraction with yellowish red colour among them showed the strongest clonorchicidal effect. The yellowish red fraction obtained by column chromatography was then fractionated into 6 spots by thin layer chromatography with petrol. ether/chloroform(30/70, v/v), and the Rf. 0.714 spot among the 6 spots showed the strongest effect. The Rf. 0.714 spot was further fractionated into 6 spots by thin layer chromatography with benzene/acetone (90/10, v/v), and the Rf. 0.800 spot among the later 6 spots revealed the strongest effect. The Rf. 0.800 spot was chromatographed on column with benzene and 2 fractions were obtained. The second fraction of light brown colour represented the final purified fraction. By these purification procedures, clonorchicidal substance was purified 15-fold with 0.03 percent yield from the mucus of C. carpio, and 10mg of the final fraction killed the cercaria in 26 min, the metacercaria in 115 min, and the adult in 443 min. Infra red and nuclear magnetic resonance spectrometric analysis of the purified substance revealed that the substance belongs to an ethyl ester of unsaturated fatty acid with 2 double bonds, 15 methylene groups and l methyl group.

n- Hexane defatted leaf of Mitracarpushirtus was extracted with Methanol. The extracts were screened for the active components present. The N-hexane extract showed the presence of Flavonoids, Alkaloids, Saponin, Tannin, Glycoside, Anthraquinone, Resins and Steroids while the Methanolic extract showed the presence of Flavonoids, Saponin and Tannin. Methanolic extract (6g) was chromatographed. The flavonoid fraction was isolated using Column Chromatography over Silica gel Column (230-400 mesh) and eluted with the solvent mixture of CH3C1/CH3OH/H2O in the ratio (70:30:1 V/V). The Flavonoid fraction collected was purified using re-crystallization method and a yield of 17.90% was obtained. Key Word: Isolation, Flavonoids, Mitracarpushirtus