Chemical composition of dry extracts of <Emphasis Type="Italic">Gymnema sylvestre</Emphasis> leaves

Three certified preparations of dried extracts of Gymnema sylvestre with gravimetric concentrations of “25%,” “50%,” and “75%” were studied. After extraction with a water-alcohol mixture, the supernatants of all preparations were analyzed for the presence of phenolic acids and amino acids by ascending thin-layer chromatography. The samples were found to contain significant quantities of a diversity of organic compounds, including plant phenols. The presence of the latter was supported by the reaction with iron (III) chloride and the Pauli diazo reaction. The ninhydrin reaction showed that the samples contained some quantities of amino acids. These were greatest in the 50% preparation (0.15 mol/kg). Gymnema sylvestre was found to contain an interesting amino acid composition, in that there was no proline. According to the certification, the molar concentration of gymnemic acids in properties was 0.12 mol/kg, which was consistent with the concentrations of phenolic acids and amino acids identified in the present studies. Thus, purification of dry extracts of Gymnema sylvestre to obtain pure preparations of gymnemic acids requires procedures to reduce the contents of satellite compounds. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 42, No. 1, pp. 30–32, January, 2008.     

颜氏大疣蛛中挥发油、氨基酸及微量元素分析

目的 分析鉴定颜氏大疣蛛中的化学成分并测定含量。方法 采用水蒸气蒸馏法和索氏提取法提取挥发油,并用GC-MS进行分析鉴定;全自动氨基酸分析仪测定水解后氨基酸的含量;ICP-MS测定微量元素的含量。结果 水蒸气蒸馏法提取的挥发油共鉴定出22个化合物,索氏提取法18个化合物,共有化合物7个,其中油酸乙酯、亚油酸乙酯、十八酸乙酯含量较高;颜氏大疣蛛中含有16种氨基酸,包括7种人体必需氨基酸,氨基酸总量为10.53%,谷氨酸和精氨酸含量较高;富含11种人体必需微量元素,其中Zn、Fe、Mn含量较高,重金属元素As和Pb的含量符合限度要求,Cd和Cu的含量超出限度要求。结论 经鉴定2种方法提取的挥发油含量和成分具有一定的差异,其中一些成分具有一定的药用功效,此外,颜氏大疣蛛中还含有多种人体必需的氨基酸和微量元素。     

Analysis of sterols and fatty acids in natural and cultured Cordyceps by one-step derivatization followed with gas chromatography–mass spectrometry

Ten free fatty acids namely lauric acid, myristic acid, pentadecanoic acid, palmitoleic acid, palmitic acid, linoleic acid, oleic acid, stearic acid, docosanoic acid and lignoceric acid and four free sterols including ergosterol, cholesterol, campesterol and β-sitosterol in natural (wild) Cordyceps sinensis, Cordyceps liangshanensis and Cordyceps gunnii, as well as cultured C. sinensis and Cordyceps militaris were first determined using pressurized liquid extraction (PLE), trimethylsilyl (TMS) derivatization and GC–MS analysis. The conditions such as the amount of reagent, temperature and time for TMS derivatization of analytes were optimized. Under the optimum conditions, all calibration curves showed good linearity within the tested ranges. The intra- and inter-day variations for 14 investigated compounds were less than 3.4% and 5.2%, respectively. The results showed that palmitic acid, linoleic acid, oleic acid, stearic acid and ergosterol are main components in natural and cultured Cordyceps which could be discriminated by hierarchical clustering analysis based on the contents of 14 investigated compounds or the 4 fatty acids, where the contents of palmitic acid and oleic acid in natural Cordyceps are significantly higher than those in the cultured ones.     

Bioassay-guided isolation of a novel protein with antitumor activity from <Emphasis Type="Italic">Trachyrhamphus serratus</Emphasis> (Syngnathidae)

A novel protein with antitumor activity, Hailongin, was purified from the aqueous extract of the whole body of Trachyrhamphus serratus, which is commonly used in traditional Chinese medicine, by bioassay-guided fractionation. Hailongin exhibited strong inhibition of proliferation of the tested human cell lines, such as A549, HeLa, LoVo and CCRF-CEM. The IC₅₀ values of Hailongin ranged from 5.4 to 25.7 μ/mL. An in vivo study showed that the growth of implanted S-180 solid tumors in mice was significantly inhibited by Hailongin treatment, while the immunological function of the tumor-bearing mice was enhanced. The molecular weight and the isoelectric point of Hailongin were 57.074 kDa (by MALDI-TOF-MS) and 6.2 (by isoelectric focusing-polyacrylamide gel electrophoresis), respectively. Seventeen amino acids were identified in Hailongin. The acidic amino acids accounted for the majority of Hailongin’s amino acid composition. The N-terminal amino acid sequence of Hailongin was determined to be IVPYSHNAGNKGLTQMR and showed no significant homology with known proteins.     

桑叶降糖有效部位活性成分研究

目的 对桑叶中降糖活性部位进行化学成分研究.方法 利用体外α-糖苷酶活性测定和小鼠高血糖模型筛选降糖活性部位.氨基酸分析仪分析氨基酸组成及含量,利用纤维素、离子交换等色谱柱进行分离纯化,通过NMR、MS等波谱分析手段鉴定化合物结构.结果 桑叶中降糖活性成分主要集中在总碱部位,从中鉴定出15种氨基酸并测定了各种氨基酸含量;此外还分离得到8个化合物,除了以前报道过的DNJ (6)、fagomine (7)、1,4-dideoxy-1,4-imino-D-arabinitol (8)外,其余的分别鉴定为2-methyl-hydrazinecarboxylic acid (1)、zarzissine (2)、N-脒基脯氨酸(3)、去甲莨菪碱(1α,2β 3α,4β-tetrahydroxy-nor-tropane) (4)、grateloupinamide(5).结论 桑叶总碱部位为主要降糖活性成分,并且首次通过氨基酸分析鉴定桑叶总碱中氨基酸的种类和含量,分离出5个生物碱类成分,其中化合物1、2、3为首次从该属植物中分离得到,化合物3为新的天然产物.     

Isolation,cloning, and characterization of a partial novel aro A gene in common reed (Phragmites australis)

Abstract

Context: Among the essential amino acids, phenylalanine, tryptophan, and tyrosine are aromatic amino acids which are synthesized by the shikimate pathway in plants and bacteria. Herbicide glyphosate can inhibit the biosynthesis of these amino acids. So, identification of the gene tolerant to glyphosate is very important. It has been shown that the common reed or Phragmites australis Cav. (Poaceae) is relatively tolerant to glyphosate.

Objective: The aim of the current research is identification, cloning, sequencing, and registering of partial aro A gene of the common reed P. australis.

Materials and methods: The partial aro A gene of common reed (P. australis) was cloned in Escherichia coli and the amino acid sequence was identified/determined for the first time.

Results: This is the first report for isolation, cloning, and sequencing of a part of aro A gene from the common reed. A 670?bp fragment including two introns (86?bp and 289?bp) was obtained. The open reading frame (ORF) region in part of gene was encoded for 98 amino acids. Alignment showed high similarity among this region with Zea mays (L.) (Poaceae) (94.6%), Eleusine indica L. Gaertn (Poaceae) (94.2%), and Zoysia japonica Steud. (Poaceae) (94.2%). The alignment of amino acid sequence of the investigated part of the gene showed a homology with aro A from several other plants. This conserved region forms the enzyme active site.

Conclusion: The alignment results of nucleotide and amino acid residues with related sequences showed that there are some differences among them. The relative glyphosate tolerance in the common reed may be related to these differences.     

Facile synthesis of orthogonally protected amino acid building blocks for combinatorial N‐backbone cyclic peptide chemistry

Abstract: Protected N^α‐(aminoallyloxycarbonyl) and N^α‐(carboxyallyl) derivatives of all natural amino acids (except proline), and their chiral inverters, were synthesized using facile and efficient methods and were then used in the synthesis of N^α‐backbone cyclic peptides. Synthetic pathways for the preparation of the amino acid building units included alkylation, reductive amination and Michael addition using alkylhalides, aldehydes and α,β‐unsaturated carbonyl compounds, and the corresponding amino acids. The resulting amino acid prounits were then subjected to Fmoc protection affording optically pure amino acid building units. The appropriate synthetic pathway for each amino acid was chosen according to the nature of the side‐chain, resulting in fully orthogonal trifunctional building units for the solid‐phase peptide synthesis of small cyclic analogs of peptide loops (SCAPLs^?). N^α‐amino groups of building units were protected by Fmoc, functional side‐chains were protected by t‐Bu/Boc/Trt and N‐alkylamino or N‐alkylcarboxyl were protected by Alloc or Allyl, respectively. This facile method allows easy production of a large variety of amino acid building units in a short time, and is successfully employed in combinatorial chemistry as well as in large‐scale solid‐phase peptide synthesis. These building units have significant advantage in the synthesis of peptido‐related drugs.     

Structure determination of neoefrapeptins A to N: peptides with insecticidal activity produced by the fungus Geotrichum candidum

The structures of neoefrapeptins A to N, peptides with insecticidal activity, were elucidated. They showed a close similarity to efrapeptin. However, all neoefrapeptins contained the very rare amino acid 1-amino-cyclopropane-carboxylic acid and some of them also contained (2S,3S)-3-methylproline. The neoefrapeptins are the first case, in which these amino acids are found as building blocks for linear peptides. They were identified by comparison of the silylated hydrolyzate to reference material by GC/MS (EI-mode). The sequence was elucidated using mass spectrometry (ESI+ mode). Full scan spectra showed two fragments in high yield, even under mild ionization conditions. MS/MS spectra of these two fragments yielded fragment rich spectra from which the sequence of the compounds was determined almost completely. The proteolytic cleavage with the proteinase papain yielded products that allowed to prove the rest of the sequence and the identity of the C-terminus to efrapeptin. The proteolytic cleavage products allowed furthermore to determine the position of the isobaric amino acids, pipecolic acid and 3-methylproline in neoefrapeptin F, as well as the location of R-isovaline and S-isovaline. Papain digestion was such established as a tool for structure elucidation of peptides rich in alpha,alpha-dialkylated amino acids. CD spectra suggested a 3(10) helical structure for neoefrapeptins A and F.     

Amino acid and mineral compositions of the vegetative part of <Emphasis Type="Italic">Chamerion angustifolium</Emphasis>

The amino acid and mineral compositions of the above-ground part of narrow-leaved fireweed (Chamerion angustifolium (L.) Holub) collected in the Krasnoyarsk region have been studied. A total of 16 free amino acids was identified by HPLC, and 24 elements were detected by atomic-emission spectrometry. This plant may be recommended as a valuable source of amino acids and trace elements. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 41, No. 7, pp. 27–29, July, 2007.     

β-Naphthoflavone induced CYP1A1 and 1A3 proteins in the liver of rainbow trout (Oncorhynchus mykiss)

1. Two CYP1A proteins, designated HAP 1 andHAP 2, were isolated from the liver of the β-naphthoflavone(BNF)-treated rainbow trout. The proteins were initially resolved by chromatography on a DEAE sepharose column and were further purified by hydroxylapatite chromatography. 2. Both HAP 1 and HAP 2 proteins exhibited high 7-ethoxyresorufin, methoxy resorufin and phenacetin O-dealkylase activities and were good catalysts for the oxidation of 7,12-dimethylbenz[a]anthracene (DMBA). No qualitative difference was observed between the two proteins in their ability to catalyse the formation of the individual metabolites of DMBA. 3. The two purified proteins showed identical amino acid sequence for the first 13 amino acids. However, the 14th amino acid was valine for HAP 1 protein and alanine for HAP 2 protein. 4. Alignment ofthe amino acid sequences showed that HAP 1 protein was identical to the deduced protein ofthe previously reported troutCYP1A2 (renamed CYP1A1) gene for the first 24 amino acids at the N-terminal region. HAP 2 protein corresponded to the deduced protein sequence of CYP1A3 gene for the first 14 amino acids. However, unlike the deduced sequences of CYP1A1 and 1A3 the N -terminal methionine was absent in the purified proteins. 5. We conclude thatHAP 1 and HAP 2 are the products ofthe CYP1A1 and CYP1A3 genes respectively, and are found in the liver of the BNF-treated rainbow trout.     

Amino acids and cyclic dipeptides in stinging nettle (<Emphasis Type="Italic">Urtica dioica</Emphasis> and <Emphasis Type="Italic">U. urens</Emphasis>) homeopathic matrix tinctures

Qualitative and quantitative contents of amino acids and N-containing substances isolated from lipophilic fractions of tinctures derived from both freshly collected plants and dried material of Urtica dioica L. and U. urens L. in accordance with homeopathic technology were studied using amino-acid analysis and GC-MS techniques. The amino-acid composition includes 18 compounds. The content of free amino acids amounted to 0.02 – 0.21%; that of bound amino acids, 0.03 – 0.29%, both being higher in tinctures obtained from freshly collected raw material. A comparison of the amino-acid contents before and after hydrolysis shows that amino acids are present in tinctures mostly in the free form. The analyses of tinctures showed the presence of pyrazine and pyrazole derivatives, including 4-ethyl-4,5-dihydro-5-propyl-1H-pyrazol-1-carboxyaldehyde isomers and hexahydropyrrolo[1, 2-a]pyrazin-1,4-dione derivatives with 3-alkyl and 3-phenylmethyl substituents in addition to 5,10-diethoxy-2,3,7,8-tetrahydro-1H,6H-dipyrrolo[1,2-a; 1′,2′-d]pyrazine. These biologically active compounds may contribute to the pharmacological effect of urtica-based phytopreparations. Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 42, No. 11, pp. 49–52, November, 2008.     

Investigating glucosyltransferase inhibitory activities of polyphenols from kapur (<Emphasis Type="Italic">Dryobalanops</Emphasis> sp.) heartwood extracts

The inhibitory effects of 50% aqueous ethanol extracts obtained from 36 tropical woody plants species on glucosyltransferase (GTase) activity were studied. Out of the 36 species examined, those obtained from kapur (Dryobalanops sp.), a species growing in Kalimantan (Indonesia), showed the highest level of GTase inhibition. Kapur extracts were further subjected to fractionation using column chromatography (LH-20 gel, cellulose and C-18 silica gel column). LH-20 gel provided the most successful method of fractionation. The separated fractions showed positive with Folin-Ciocalteau’s reagent and negative with vanillin-HCl reagent, indicating that the main constituents of the active fractions were polyphenols but not proanthocyanidin (condensed tannins). Results of the assay for protein precipitating ability with bovine serum albumin (BSA) solution suggested these polyphenols have strong protein-precipitating ability. The predominant compound produced after acid hydrolysis was ellagic acid, indicating that the GTase-inhibitory components were mainly ellagitannins. Two polyphenolic compounds referred to as compounds 1 and 2 were isolated from the water eluate fraction with LH-20 gel column, and these compounds showed comparatively strong GTase-inhibitory activities and relatively low molecular weight. Using a combination of two-dimensional, ¹H and ¹³C nuclear magnetic resonance analysis, compound 1 was identified as 4-methoxy-2-[tetrahydro-3,4,5-trihydroxy-6-(hydroxymethyl)pyran-2-yl]-α-resorcylic acid δ–lactone (bergenin), and 2 was identified as 4-O-(α-rhamnopyranosyl) ellagic acid (eschweilenol C). Bergenin has been previously isolated from the roots of Bergenia crassifolia, and eschweilenol C has been isolated from the bark of Eschweilera coriacea. Both compounds were found in kapur for the first time.     

Design,Synthesis and Biological Evaluation of Peptidyl Epoxyketone Proteasome Inhibitors Composed of β‐amino Acids

A series of novel di‐ and tripeptidyl epoxyketone derivatives composed of β‐amino acids were designed, synthesized and evaluated for their proteasome inhibitory activities and anti‐proliferation activities against two multiple myeloma cell lines RPMI 8226 and NCI‐H929 and normal cells (peripheral blood mononucleated cells). Among these tested compounds, tripeptidyl analogues showed much more potent activities than dipeptides, and four tripeptidyl compounds exhibited proteasome inhibitory activities with IC₅₀ values ranging from 0.97 ± 0.05 to 1.85 ± 0.11 μm . In addition, all the four compounds showed anti‐proliferation activities with IC₅₀ values at low micromolar levels against two multiple myeloma cell lines and weak activities against normal cells. Furthermore, Western blot analysis was performed to verify the proteasome inhibition induced by compounds 21d and 21e . All the experimental results validated that the β‐amino acid building block has the potential for the development of proteasome inhibitors.     

裸花紫珠叶中的酚酸类化学成分

目的研究裸花紫珠(Callicarpa nudiflora Hook.Et Arn.)叶的化学成分。方法采用硅胶柱色谱分离化合物,运用理化性质和波谱技术确定所得化合物的结构。结果从裸花紫珠叶的水提取物中分离得到6个酚酸类化合物,分别鉴定为:香草酸(1)、对-羟基桂皮酸(2)、咖啡酸(3)、阿魏酸(4)、原儿茶酸(5)、原儿茶醛(6)。结论化合物2-6为首次从紫珠属植物中分得,6个化合物均为首次从该植物中分离得到。     

A convenient general method for synthesis of Nα- or Nω-dithiasuccinoyl (Dts) amino acids and dipeptides: application of polyethylene glycol as a carrier for functional purification*

N ^α-Dithiasuccinoyl (Dts) amino acids ( 1 ) needed for solid-phase peptide synthesis have been prepared in good yields and excellent purities by a new method that exploits the solubility properties of polyethylene glycol (PEG; bifunctional with average molecular weight 2000 was found to be optimal). Suitably side-chain protected amino acid derivatives are first reacted with a polymeric xanthate ( 11 ), following which the free α-carboxyl is blocked by silylation and the Dts heterocycle is elaborated in the same pot by reaction with chlorocarbonylsulfenyl chloride ( 4 ). Upon aqueous workup, the polymeric carrier removes any urethane blocked amino acids which arise during the process. Exaggerated conditions were explored to prove the power of this functional purification approach, and mechanisms of formation of polymer-bound urethanes are proposed and supported by solution model studies. The preparation and characterization of the companion N-(iso-propyldithio)carbonyl derivative of proline is also presented.     

Identification of major aroma compounds in the leaf of <Emphasis Type="Italic">Morinda citrifolia</Emphasis> Linn.

Morinda citrifolia, commonly named noni, has been used as food and as a folk medicine throughout the tropics. The use of the leaves to make hot water beverages is increasing in popularity, especially in Japan and the United States. To better understand the effects of processing on the content of the major aroma compounds, volatile oils were collected from samples of frozen, dried and roasted leaves by steam distillation and then analyzed by GC-MS. Drying of the leaves reduces the quantity of aroma compounds by more than half. Palmitic acid and E-phytol were identified as the major components of the volatile oil. With the exception of E-phytol, all of the known volatile compounds identified in the leaf samples were done so for the first time.     

TOXINS ASSOCIATED WITH MEDICINAL AND EDIBLE SEAWEEDS

Toxins associated with medicinal and edible seaweeds are reviewed with an emphasis on chemistry. The red alga Digenea simplex has been used for the treatment of roundworm disease for centuries. Its active principle is kainic acid . The related domoic acid is a constituent of another red alga, Chondria armata, used for the same purpose. These compounds known as kainoids are potent neurotoxins and excitatory amino acids. Kainoids are important tools in neurophysiological research. Domoic acids are also produced by diatoms and were responsible for the shellfish poisonings known as amnesic shellfish poisonings which occurred in Canada in 1987. Caulerpin and Caulerpicin have been described as toxic constituents of edible species of the green algal genus Caulerpa, but evidences in later studies indicate that they have no acute toxicity. Caulerpin, which has a structure related to auxin, promotes plant growth. Caulerpenyne, a toxic constituent of Caulerpa taxifolia and other inedible species, has been evaluated for its ecotoxicological effect in the Mediterranean where C. taxifolia bloomed explosively. Three different classes of compounds have been identified in the poisonings with species in the genus Gracilaria. They are prostaglandin E₂ from G. verrucosa in Japan, aplysiatoxins and related compounds from G. coronopifolia in Hawaii, and polycavernosides from G. tsudai in Guam.     

Solid-phase synthesis of human osteocalcin by using a γ-carboxyglutamic acid derivative

Human osteocalcin, also called bone Gla protein (BGP), consisting of 49 amino acids with two to three γ-carboxyglutamate residues, was chemically synthesized for the first time by a novel solid-phase peptide synthesis. An l -enantiomer of N-tert-butyloxycarbonyl-γ,γ′-dicyclohexyl-γ-carboxyglutaniic acid was designed, prepared and utilized as a monomeric compound and proven to be useful for the solid-phase peptide synthesis of human osteocalcin. The synthesis and optical resolution of the γ-carboxyglutamic acid (Gla) derivative are first described, followed by the synthesis and characterization of Gla¹⁷-human osteocalcin.     

大戟根化学成分的研究

从中药大戟(Euphorbia pekinensis)根中分得9个化合物,经理化常数和波谱数据分析,分别鉴定为羊毛甾醇(I),3-甲氧基-4-羟基反式苯丙烯酸正十八醇酯(II),β-谷甾醇(III),伞形花内酯(IV),2,2′-二甲氧基-3,3′二羟基-5,5′-氧-6,6′-联苯二甲酸酐(V),d-松脂素(VI),槲皮素(VII),3,4-二甲氧基苯甲酸(VII)和3,4-二羟基苯甲酸(IX)。其中II和V为两个未见文献报道的新化合物,其余化合物均为首次从该植物中分离得到,VI为首次从该属植物中分得的木脂素类化合物。     

豹皮菌中氨基酸类成分的研究

从豹皮菌Amanita panthorina新鲜子实体的乙醇提取物中分离得到了4个氨基酸类化合物(I～IV)。根据光谱分析及理化性质,鉴定Ⅰ的结构为3-amino-2-hydroxypentandioicacid,Ⅱ为2-amino-3-(1,2-dicarboxyethylthio)propanoicacid。其中I为一新化合物,Ⅱ是首次从该菌中提取得到的。药理实验证明Ⅰ及Ⅱ分别是谷氨酸NMDA受体兴奋剂及阻断剂。