Preparative isolation of bio-markers from the leaf exudate of Aloe ferox (“aloe bitters”) by high performance counter-current chromatography

One of the most crucial factors determining the safety and efficacy of any herbal medicine or natural product-based formulation is the quality of the raw material. The absence of readily available bio-markers (standards) is one of the hurdles which need to be overcome to develop robust and effective quality control protocols.Aloe ferox Mill. is a most coveted ethnomedicinally import plant indigenous to South Africa. A. ferox has been used since ancient times in folk medicine and recently it has gained popularity as an ingredient in cosmetic formulations and food supplements. This study aimed to develop a superior method for the isolation of bio-markers from “aloe bitters” (exudate) obtained from A. ferox.For separation by HPCCC the solvent system comprising of EtOAc/n-BuOH/H₂O (3.5:1.5:5, v/v/v) was used in reversed phase mode. By this method, and only in one run, eight bio-markers were separated and isolated on semi-preparative scale including aloesin, aloeresin C, aloeresin A, 5-hydroxyaloin, aloin B, aloinoside B, aloin A and aloinoside A. The isolation of bio-active molecules from A. ferox (Cape aloes) is presented to illustrate the efficiency and advantages of high performance counter-current chromatography (HPCCC).     

A reversed-phase high-performance liquid chromatographic method for the determination of aloesin, aloeresin A and anthraquinone in Aloe ferox

A reversed-phase HPLC method for the quantification of aloesin, aloeresin a and anthraquinone (as barbaloin) in Aloe ferox Miller and aloe-related products has been developed and validated. The method utilized a C18 column with a water-methanol gradient and UV detection at 297 nm. The method validation included linearity, accuracy, precision, limit of detection, limit of quantitation, specificity and standard solution stability. The method showed good linearity (r > 0.99 for all components) and recovery (>85% for all components). The detection and quantitation limits for barbaloin were determined to be 0.02 and 0.1 ppm at signal-to-noise ratios of approximately 3:1 and 10:1, respectively.     

Inhibitory effects of aloe carboxypeptidase fraction on streptozotocin-induced enhancement of vascular permeability in the pancreatic islets

The protective actions of components isolated from Aloe arborescens Miller var. natalensis Berger (Kidachi aloe in Japanese) on streptozotocin (Sz)-induced necrosis of B cells in the pancreatic islets of the mouse were investigated to clarify its action mechanism involved in anti-diabetic effects. In this experiment, phenol low molecular weight components of aloin and aloin A that were anti-oxidants and derived from the leaf skin or pulp extract, an aloe carboxypeptidase fraction that is a inhibitor of enhanced vascular permeability and a glycoprotein component that decreases blood glucose were tested with mice precedently administered with Sz which is known as a cytotoxin specific to B cells. The results showed that the treatment group receiving Sz followed by the aloe carboxypeptidase fraction increased the inhibition of dye leakage by 75.8% (p<0.001) in the extract of whole pancreas in comparison to the control group and the aloe carboxypeptidase fraction group also increased the inhibition effect by 68.4% (p<0.001) in the extract of pancreatic islets as compared to the control group. The carboxypeptidase is an aloe-derived protease known to inhibit the acetic acid-related enhancement of intraperitoneal vascular permeability in mice. Further, the elevation of blood glucose in Sz-induced diabetic mice intraperitoneally given the aloe carboxypeptitase fraction was significantly (p<0.01-0.001) restrained at 3, 7 and 14 days after the injection as compared to the control group given solvent only. The results of this experiment suggested that the inhibitory effect on the enhancement of vascular permeability related to the vascular acute inflammatory response at Sz-induced lesions of pancreatic islets was involved in the action mechanism of this enzyme.     

盐胁迫对库拉索芦荟叶片中NADP-苹果酸酶基因的诱导表达

为弄清景天酸代谢植物 库拉索芦荟中NADP 苹果酸酶 (NADP ME)基因的表达与其耐盐间的关系 ,根据已知NADP 苹果酸酶序列设计引物 ,从库拉索芦荟的 2个月幼苗中扩增克隆了NADP 苹果酸酶 4 96bp的cDNA片段 ,并对其进行了序列测定 ;选用敏感品种皂质芦荟和耐盐品种库拉索芦荟做材料 ,分别检测高盐胁迫条件下NADP ME的表达和NADP ME的活性。结果表明 ,两者在不同品种的芦荟中均被诱导 ,但诱导的强度与芦荟的耐盐程度相关。Northern杂交分析表明 ,高盐、干旱、外源ABA均能强烈诱导苹果酸酶的表达 ,但寒冷对其影响不大 ,这与库拉索芦荟的生物学特性相符合 ;此外 ,为了检测库拉索芦荟中NADP 苹果酸酶受盐诱导情况 ,利用Western印迹对样品进行了分析 ,结果显示高盐条件不仅明显诱导NADP ME的合成 ,而且随着处理时间的延长其合成量也在增多。     

Dietary aloin, aloesin, or aloe-gel exerts anti-inflammatory activity in a rat colitis model

AimsAloe has been a very popular folk remedy for inflammation-related pathological conditions despite the lack of studies reporting its efficacy in vivo. The present study evaluated the anti-inflammatory effects of aloe components (aloin, aloesin and aloe-gel) known to be biologically active in the rat model of colitis.Main methodsMale Sprague Dawley rats were fed experimental diets for 2 weeks before and during the induction of colitis. Drinking water containing 3% dextran sulfate sodium (DSS) was provided for 1 week to induce colitis. At the end of the experimental period, clinical and biochemical markers were compared.Key findingsPlasma leukotriene B₄ (LTB₄) and tumor necrosis factor-α (TNF-α) concentrations were significantly decreased in all groups supplemented with aloe components compared to the colitis control group (p < 0.05). Animals fed both a 0.1% and 0.5% aloesin supplemented diet showed colonic myeloperoxidase (MPO) activities which were decreased by 32.2% and 40.1%, respectively (p < 0.05). Colonic mucosa TNF-α and interleukin-1ß (IL-1β) mRNA expressions were significantly reduced in all animals fed aloin, aloesin, or aloe-gel (p < 0.05).SignificanceDietary supplementation of aloe components ameliorates intestinal inflammatory responses in a DSS-induced ulcerative colitis rat model. In particular, aloesin was the most potent inhibitor. Further studies are required for a more complete understanding of the specific mechanism of the action of these supplements.     

Modulation of melanogenesis by aloesin: a competitive inhibitor of tyrosinase

Aloesin, [2-acetonyl-8-beta-d-glucopyranosyl-7-hydroxy-5-methylchromone], a compound isolated from the Aloe plant, is shown in these studies to modulate melanogenesis via competitive inhibition of tyrosinase. Aloesin inhibits purified tyrosinase enzyme and specifically inhibits melanin production in vitro. Enzyme kinetics studies using normal human melanocyte cell lysates and cell-based melanin production demonstrated that aloesin is a competitive inhibitor of tyrosinase from mushroom, human and murine sources. Tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (DOPA) oxidase activities of tyrosinase from normal human melanocyte cell lysates were inhibited by aloesin in a dose dependent manner. In a percutaneous absorption study a finite dose of aloesin penetrated the skin slowly and was recovered primarily in the surface wash. Aloesin shows promise as a pigmentation-altering agent for cosmetic or therapeutic applications.     

Cape aloes—A review of the phytochemistry,pharmacology and commercialisation of Aloe ferox

Aloe ferox Mill. (= A. candelabrum A. Berger), commonly known as the bitter aloe or Cape aloe, is a polymorphic species indigenous to South Africa. The plant has been used since ancient times as a generic chemopreventive and anti-tumour remedy in folk medicine and it has a well-documented history of use as a laxative. In addition to the plethora of traditional medicinal uses, A. ferox has recently gained popularity as an ingredient in cosmetic formulations and food supplements. Anti-oxidant, antimicrobial, anti-inflammatory, anticancer and antimalarial activities, etc. have been reported. In addition, the ability of Cape aloes to enhance the transport of poorly permeable drugs has enjoyed recent research interest. Due to its medicinal and commercial importance it has been a popular research topic for natural product scientists who have isolated several chromones and anthrones from the leaf exudate and finished product (bitters). A summary of the historical and modern day uses, commercialisation, chemical composition and biological properties of this coveted ethnomedicinally and commercially important species is presented.     

芦荟多糖含量测定及芦荟膏体外透皮吸收的实验研究

目的:研究芦荟膏中各功能成分体外透皮吸收的能力。方法:以Wistar大鼠的背部皮肤为透皮实验原料,每隔一定时间通过分光光度法和高效液相色谱法测定透皮后接收池内芦荟多糖及蒽醌类含量。结果:随着芦荟膏剂量的增加,渗透量逐渐增加,芦荟膏中芦荟多糖、芦荟大黄素、芦荟苷的渗透量随时间延长逐渐增加,但是渗透速率逐渐降低。结论:芦荟膏有较强的体外透皮吸收能力,芦荟膏经皮给药能充分发挥其作用。     

Inhibition of collagenase and metalloproteinases by aloins and aloe gel

The effects of Aloe barbadensis gel and aloe gel constituents on the activity of microbial and human metalloproteinases have been investigated. Clostridium histolyticum collagenase (ChC) results dose-dependently inhibited by aloe gel and the activity-guided fractionation led to an active fraction enriched in phenolics and aloins. Aloins have been shown to be able to bind and to inhibit ChC reversibly and non-competitively. Aloe gel and aloins are also effective inhibitors of stimulated granulocyte matrix metalloproteinases (MMPs). The remarkable structural resemblances between aloins and the pharmacophore structure of inhibitory tetracyclines, suggest that the inhibitory effects of aloins are via an interaction between the carbonyl group at C(9) and an adjacent hydroxyl group of anthrone (C(1) or C(8)) at the secondary binding site of enzyme, destabilizing the structure of granulocyte MMPs.     

A C-glucosylated 5-methylchromone from Kenya aloe

A new bitter C-glucoside, aloeresin D, was isolated from a commercial sample of Kenya aloe. Its structure was assigned as 8-C-β-d-[2-O-(E)-p-coumaroyl]glucopyranosyl-2-[(R)-2-hydroxy]propyl-7-methoxy-5-methyl-chromone by spectral data and chemical transformations.     

Aloeresin c,a bitter c,o-diglucoside from cape aloe

A new bitter C,O-diglucoside, aloeresin C, was isolated from commercial Cape aloe. Its structure, 2-acetonyl-7-O-β-D-glucopyranosyl-8-C-β-D-[2′-O-(E)-p-coumaroyl]glucopyranosyl-5-methylchromone, was established by spectral and chemical methods.     

芦荟属植物叶内蒽醌类物质的分布与其化学防御的关系

芦荟属植物是一类生长在干旱或半干旱沙漠环境的肉质植物,木立芦荟和海藻芦荟的肉质叶的植物研究结果表明,叶内含高浓度的芦荟素,高那特芦荟素,芦荟苦素和芦荟宁等蒽醌类物质,其中,海莱芦荟整叶的蒽醌类物质总含量占其泌出物干重的44．89％。两种芦荟叶内蒽醌类物质的分布有共同的规律,即幼叶的含量高于老叶;叶上部的含量高于中,基部,叶缘则高于叶的中央。但由于木立芦荟和海莱芦荟的个体形态不同,蒽醌类物质的分布在种间还存在各自的特点,根据实验结果推测,蒽醌类物质在芦荟属植物体内的累积和分布与其化学防御机制有关。     

Specificity of acid proteinase A from Aspergillus niger var. macrosporus towards B-chain of performic acid oxidized bovine insulin.

1. A comparative study on the mode of action of two highly purified acid endopeptidases (EC 3.4.-) from Aspergillus niger var. macrosporus, acid proteinase A and B, on the B-chain of performic acid oxidized insulin was performed, putting emphasis on the quantitative analysis of the effects of enzyme A. Acid proteinase A behaved very specifically towards the substrate and hydrolyzed four peptide bonds exclusively: three major sites, where hydrolysis proceeded rapidly and almost completely, Asn3-Gln4, Glu13-Ala14, and Tyr26-Thr27; and a minor one, Gly20-Glu21, at which hydrolysis was much slower. 2. The effects of four protease inhibitors, pepstatin, diazoacetyl-D,L-norleucine methyl ester/Cu(II), di-isopropyl phosphorofluoridate, and 1,2-epoxy-3-(p-nitrophenozy) propane on acid proteinases A and B were studied. Acid proteinase A preparations, treated with the former two inhibitors, were used to establish that the major sites of attack were really affected by enzyme A and not by contaminating proteinase B.     

Characteristics of Protease Production by Cephalosporium sp

We investigated protease formation by Cephalosporium sp. strain KM388, which produced trypsin inhibitor in the same cultures, in medium containing polypeptone, meat extract, and glucose (natural medium) and in medium containing NaNO₃, glucose, and yeast extract (semisynthetic medium). In natural medium, protease was secreted into the culture broth after cessation of growth caused by consumption of the polypeptone, the growth-limiting substrate. Enzyme formation in the stationary growth phase was due to de novo and so-called preferential synthesis, because cycloheximide immediately inhibited enzyme formation. In semisynthetic medium, protease was produced in parallel with mycelial growth, but production was repressed by the addition of polypeptone to the medium; protease production began after the added polypeptone was consumed. On the other hand, if glucose was eliminated from natural medium, the lag period of initiation of enzyme production was reduced until the late exponential phase. The addition of phosphate up to a concentration of 1.0% to natural medium also shortened the lag period and damped the pH change of the broth during cultivation.     

不同生境中国芦荟凝胶活性成分的分析

中国芦荟产于云南元江地区,是一种极具发展潜力的芦荟品种,在全国均有引种,特别在东北地区的温室中已开始大面积种植。本项研究对不同生境的中国芦荟凝胶中活性成分的含量进行了分析。生境不同的中国芦荟凝胶中活性成分含量有明显差异。从测定结果上看,云南元江产的中国芦荟凝胶中所含的活性成分要高于黑龙江温室内产的中国芦荟;积温高、土壤呈弱酸性的条件下,中国芦荟凝胶中的蛋白质、有机酸含量较高。     

Membrane-bound enzymes. III. Protease activity in leucocytes in relation to erythrocyte membranes.

Protease activity was detected in membranes of human bovine erythrocytes prepared by the conventional procedures which include washing and removal of the "buffy layer". The enzyme was extracted by 0.75 M KCNS or (NH4)2SO4 and was activated by 0.4 to 0.5 M of the same salts. Colored, particulate hide powder-azure, membrane fractions and soluble proteins such as hemoglobin, casein or albumin were susceptible to hydrolysis by the membraneous protease. Partial purification of the enzyme was accomplished through disc-gel electrophoresis on polyacrylamide in the presence of 0.25% positively charged detergents like cetyltrimethylammonium bromide. An alkaline protease (pH 7.4) with properties similar to those of the erythrocyte enzyme was found in leucocytes. The similarity between the properties of the leucocytic and erythrocytic proteases and the correlation of the activity in erythrocyte membranes with content of white cells in these preparations, suggest that enzymatic activities in the contaminating leucocytes are responsible for the activity of membraneous proteases in erythrocytes.     

A Purified Zinc Protease of Pea Chloroplasts,EP1, Degrades the Large Subunit of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase

A previously reported endopeptidase (EP1) from pea chloroplasts was purified over 11,000-fold using a four-step protocol involving ultrafiltration, sucrose gradient centrifugation, isoelectric focusing, and high performance liquid chromatography gel filtration. The enzyme was determined to be a metalloprotease requiring bound Zn2+ and added Mg2+ or Ca2+ for proper activity. Its localization in the stroma of pea chloroplasts was confirmed by demonstrating its insensitivity to thermolysin when the envelope was intact. A contaminating serine protease that attacks EP1 was found. The contaminating protease was inhibited by 4-(2-aminoethyl)-benzenesulfonyl fluoride, but not by o-phenanthroline, whereas EP1 sensitivities were the reverse. EP1 is able to hydrolyze the large subunit of native ribulose-1,5-bisphosphate carboxylase/oxygenase under physiological conditions.     

遮荫对库拉索芦荟蒽醌类物质的影响

采用高效液相色谱技术,研究了遮荫65%处理6个月对库拉索芦荟叶片中主要有效成分蒽醌类物质的影响,以探讨遮荫导致芦荟有效成分变化的原因.结果显示:(1)遮荫使芦荟叶中总蒽醌、芦荟素和芦荟大黄素含量分别比自然光下显著减少了39.22%、18.65%和40.96%.(2)遮荫处理下芦荟叶片中大部分物质含量较对照减少,且化学成分种类也明显减少,但在24、46 min两个时间遮荫处理出现的物质含量明显高于对照.(3)遮荫使芦荟叶中可溶性蛋白和可溶性糖含量较自然光下分别显著减少33.33%和73.05%,MDA含量显著减少48.78%.研究表明,遮荫使芦荟叶片中可溶性糖和可溶性蛋白含量下降,致使同化产物合成效率降低,次生代谢物合成前体减少,最终影响总蒽醌、芦荟素和芦荟大黄素的合成和积累.