Pretreatment of milk thistle seed to increase the silymarin yield: an alternative to petroleum ether defatting

Milk thistle (Silybum marianum L.) seed meal is extracted for the flavonolignans, silychristin, silydianin, silybinin A, silybinin B, isosilybinin A and isosilybinin B, which are collectively known as the silymarin complex. To obtain the flavonolignans, the meal is usually treated with successive washes of petroleum ether to remove the lipids, followed by extraction of the flavonolignans with ethanol. This work examines the possible replacement of petroleum ether and ethanol by water or other aqueous solutions in these processes. To replace petroleum ether, pretreatments with 1.2% NaOH (w/w), 1.5% H2SO4 (w/w), 2% NaHCO3 (w/w), 0.14% cellulase and water were investigated. Of these pretreatments, 1.5% H2SO4 and water produced similar flavonolignan yields as petroleum ether. Results established that pretreating the milk thistle seed meal with 1.5% H2SO4 (w/w) at 50 degrees C for 18 h could replace the petroleum ether pretreatment. In addition, it was shown that similar amounts of flavonolignan could be recovered with a 1.5% H2SO4/water (100 degrees C) extraction as with a petroleum ether/ethanol extraction. Although cellulase pretreatment was not examined extensively, significant advances in cellulase effectiveness and cost have occurred in the past few years by companies such as Genencor International and Novozymes. These advances should help to make enzyme use for cellulose conversion, as well as extraction pretreatment, technically and economically feasible.     

Silymarin content and antioxidant activity of seeds of wild Silybum marianum populations growing in Greece

Selection of milk thistle (Silybum marianum (L.) Gaertn.) genotypes for commercial cultivation depends on wild population resources, rather than on selection of varieties. The evaluation of these populations could contribute to the development of new milk thistle varieties with desirable traits such as high silymarin content. Therefore, we conducted a study to evaluate 30 wild populations occurring naturally in Greece, based on seed silymarin content. Seeds were collected from populations growing in localities presenting different environmental conditions. In addition, we also investigated the diversity in levels of silymarin constituents, as well as observed correlations among them. Significant differences in silymarin content were recorded among the populations studied, with values ranging from 2.31 to 7.71% (avg. 3.31%), as well as in flavonolignan and taxifolin content. The mean taxifolin content was 4.96 mg/g, while the highest silybin A + silybin B content (21.77–31.39 mg/g of dry weight (dw)) was recorded in seeds from the “Spata” population originating from central Greece. Our results showed noticeable levels of diversity in silymarin content and composition in native milk thistle populations, indicating that a valuable gene pool for exploitation in milk thistle breeding programmes exists in Greece.     

Green production of silybin and isosilybin by merging metabolic engineering approaches and enzymatic catalysis

Silymarin extracted from milk thistle seeds, is used for treating hepatic diseases. Silybin and isosilybin are its main components, and synthesized from coupling of taxifolin and coniferyl alcohol. Here, the biosynthetic pathways of taxifolin and coniferyl alcohol were reconstructed in Saccharomyces cerevisiae for the first time. To alleviate substantial burden caused by a great deal of genetic manipulation, expression of the enzymes (e.g. ZWF1, TYR1 and ARO8) playing multiple roles in the relevant biosynthetic pathways was selectively optimized. The strain YT1035 overexpressing seven heterologous enzymes and five native enzymes and the strain YC1053 overexpressing seven heterologous enzymes and four native enzymes, respectively produce 336.8 mg/L taxifolin and 201.1 mg/L coniferyl alcohol. Silybin and isosilybin are synthesized from taxifolin and coniferyl alcohol under catalysis of APX1t (the truncated milk thistle peroxidase), with a yield of 62.5%. This study demonstrates an approach for producing silybin and isosilybin from glucose for the first time.     

Extractability of polyphenols of sunflower seed in various solvents

The extractability of chlorogenic acid from defatted sunflower seed flour in water and salt solutions at different pH values and also in aqueous organic solvents was determined. It increased with increase in pH and at pH 8 in water nearly 70% chlorogenic acid was removed in a single extraction, while NaCl did not increase the extraction, and, MgCl₂ and CaCl₂ increased it, especially at higher concentrations. Methanol, ethanol, isopropanol and acetone, at 20% concentration in water, caused the maximum extraction of polyphenol. These organic solvents without added water were poor solvents for the extraction of polyphenol from the flour.     

Evaluation and improvement of spectrophotometric assays of TTC reduction: maize (Zea mays) embryo as an example

The triphenyl tetrazolium chloride (TTC) reduction assay was evaluated and improved with maize seed (Zea mays cv. Zhengdan958). The reduced TTC in embryo was extracted with three kinds of organic solvents: trichloroacetic acid (TCA)/acetone, ethanol, and acetone. The absorbance spectra of the three extracts were similar, with a maximum at 485 nm. The efficiency of TCA/acetone in extracting the reduced TTC was higher than that of acetone and ethanol. A negative correlation between TTC reduction and malondialdehyde content in embryo was demonstrated. The TCA/acetone extraction may be used as a routine protocol for TTC reduction assay of seed vigor in cereal (e.g. maize, rice, wheat and barley) seeds.     

狭叶柴胡种子萌发与内源抑制物质

分别测定了柴胡种子的水浸提液、水浸泡液与乙醚浸提液对柴胡种子萌发以及对白菜和小麦种子萌发和幼苗生长的抑制活性;用水、乙醚、乙醇和丙酮浸泡柴胡种子,比较其对种子中抑制物质的去除效果;运用原位实验方法探讨柴胡种子萌发与内源抑制物质之间的关系。结果表明：柴胡种子中含有一定活性的内源抑制物质,其对白菜幼根生长的抑制活性高于对白菜种子萌发的抑制活性,对小麦叶绿素含量、鲜质量和α-淀粉酶的活性高于对小麦种子萌发的抑制活性,并且对柴胡自身种子萌发也有明显的抑制作用;室温用乙醚浸种24 h可以提高种子发芽率。据此,建立了柴胡种子的催芽技术。     

Effective recovery of poly‐β‐hydroxybutyrate (PHB) biopolymer from Cupriavidus necator using a novel and environmentally friendly solvent system

This work demonstrates a significant advance in bioprocessing for a high‐melting lipid polymer. A novel and environmental friendly solvent mixture, acetone/ethanol/propylene carbonate (A/E/P, 1:1:1 v/v/v) was identified for extracting poly‐hydroxybutyrate (PHB), a high‐value biopolymer, from Cupriavidus necator. A set of solubility curves of PHB in various solvents was established. PHB recovery of 85% and purity of 92% were obtained from defatted dry biomass (DDB) using A/E/P. This solvent mixture is compatible with water, and from non‐defatted wet biomass, PHB recovery of 83% and purity of 90% were achieved. Water and hexane were evaluated as anti‐solvents to assist PHB precipitation, and hexane improved recovery of PHB from biomass to 92% and the purity to 93%. A scale‐up extraction and separation reactor was designed, built and successfully tested. Properties of PHB recovered were not significantly affected by the extraction solvent and conditions, as shown by average molecular weight (1.4 × 10⁶) and melting point (175.2°C) not being different from PHB extracted using chloroform. Therefore, this biorenewable solvent system was effective and versatile for extracting PHB biopolymers. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:678–685, 2016     

Unconventional Protein Sources: I. Lupinus albus

Three different treatments were tested for their effectiveness in removing alkaloids, and producing protein products suitable for human consumption from Lupinus albus seeds. The treatments included: a) water leaching of the seeds, b) ammonium salt treatment of the seeds, and c) multistage extraction of the defatted meal.

Results revealed that treatments a) and c) were highly effective in the removal of alkaloids. Protein contents of the resulting three products were about 62, 47 and 58%, respectively, compared to 39% in the seed. Amino acid analysis showed that the protein preparations lack sulphur amino acids, lysine, tryptophan and valine.     

葡萄籽中原花青素提取方法优化处理

溶剂提取法是提取葡萄籽原花青素的常用方法 ,然而 ,在不同提取条件下 ,提取效果并不一致。利用水、甲醇、乙醇、丙酮及它们的水溶液提取葡萄籽中的原花青素 ,而后用铁盐催化比色法测原花青素含量 ,考察了提取剂的浓度、粉碎度对提取的影响。在优化处理的基础上 ,获得了有效的提取条件 :葡萄籽粉碎度 10 0目 ,提取剂 70 %甲醇水溶液。     

Antioxidant content of oil and defatted meal obtained from borage seeds by an enzymatic-aided cold pressing process

Polyphenols content (as catechin equivalents) and tocopherol content were determined in borage defatted meal and borage oil, respectively. In addition, antioxidant activity of extracts obtained from borage defatted meal was evaluated. A cold pressing process was used for the extraction of Borago officinalis oil, resulting in a defatted meal (by-product). Polyphenols from this defatted borage meal were extracted using several solvents. An extract containing highly soluble solids and phenolic compounds with antioxidant activity (as free radical-scavenging, DPPH) was obtained when methanol was used. The tocopherol content was higher in oil extracted by cold pressing than in oil extracted with petroleum ether as organic solvent. An enzymatic treatment was applied (45 °C, 20% moisture, 0.25% E/S ratio, 1:1 Olivex:Celluclast enzymatic mixture) previously to borage oil extraction, which improved the antioxidant content in the borage defatted meal by three-folds, as compared to the values obtained by a nonenzyme-aided process.     

Isolation and purification of silychristin,silydianin and taxifolin in the co-products of the silybin refined process from the silymarin by high-speed counter-current chromatography

A preparative high-speed counter-current chromatography (HSCCC) method for isolation and purification of silychristin, silydianin and taxifolin in the co-products of the silybin refined process from the silymarin was successfully established by n-hexane–chloroform–methanol–water (0.5:11:10:6 (0.5 acetic acid), v/v/v/v) as the two-phase solvent system. 146 mg silydianin, 280 mg silychristin and 63 mg taxifolin from 1.463 g co-products sample in one separation were obtained with the purities of 95.1%, 99.3% and 98.2%, respectively, determined by HPLC. The structures of the compounds were identified by means of ESI-MS-MS, TOF-MS, ¹H NMR, ¹³C NMR and 2DNMR-HMBC. Silychristin, silydianin and taxifolin had been separated as standards by HSCCC for the first time. A comparative study between HSCCC and RPLC for separation and isolation of taxifolin, silychristin and silydianin was investigated. The differences between the two preparative chromatographic methods were all discussed. The results demonstrated that HSCCC was a powerful separation tool and could contribute to identifying and quantifying plant ingredients.     

Downregulated Lipid Metabolism in Differentiated Murine Adipocytes by Procyanidins from Defatted Grape Seed Meal

Recent reports have proposed possible anti-obesity mechanisms for antioxidants involving increased energy expenditure, pre-adipocyte differentiation and proliferation, decreased lipogenesis and increased lipolysis, and fat oxidation. The aim of this study was to examine and to confirm the anti-obesity effect of the oligomeric and polymeric procyanidin fractions from defatted grape seeds. The lipid metabolism-related mRNA level in the mouse preadipocytes, 3T3-L1 cells, was determined to evaluate the anti-obesity effect of the phenolic fractions from a grape seed meal. Lipid accumulation was reduced by 19% of the control level by the procyanidin fraction originating from the grape seed meal. Emerging from the effect of the treatment on HSL and LPL mRNA expression, lipolytic enzyme activity was not involved in the anti-obesity effects of CPE and FPP from the defatted grape seed meal. We tested and confirmed in this study the effect of the biological activities of oligomeric and polymeric procyanidins from the defatted grape seed meal. It is suggested from the results of this brief study that further studies would be desirable to focus on the anti-obesity effect of the purified extracts of a defatted grape seed meal.     

'Callose' in the Impermeable Seed Coat of Sesbania punicea

Dry legumes of Sesbania punicea (Cav.) Benth. contain impermeableseeds which are either beige-greenish or reddish. When keptin water for over one year they do not swell. Quick imbibitionwas induced by a deep cut, abrasion with coarse abrasive paper,immersion in concentrated sulphuric acid, in boiling water andin boiling KOH solutions. Neither lipid solvents (acetone, chloroform,cyclohexane, diethyl ether, ethanol, xylene), nor, at room temperature,KOH solutions up to 50% per cent, permeabilized the seeds. Astrong barrier to water entry appeared to be located in thepalisade layer which showed ‘light points’ in places,and which seemed to be transversal thickenings. In the testa,‘ callose’, which may play a role in the impermeabilityof the Sesbania punicea seed, was detected. Sesbania punicea, germination, seed dormancy, seed coat impermeability, ‘callose’     

一种提取高质量油菜种子和种皮RNA的方法

提取高质量的RNA是从基因表达水平上研究油菜种子和种皮发育的必要条件。现有方法因为油菜种子脂肪、多酚和多糖,难以快速获得完整、高纯度的油菜种子总RNA。本试验针对油菜种子和种皮特点,利用苯酚-氯仿抽提后用无水乙醇沉淀RNA,建立了在油菜种子和种皮中快速提取高质量总RNA的提取方法,电泳分析表明28S rRNA亮度约为18S rRNA的2倍;紫外分光光度计检测A260/A280介于1.8～2.0之间。用该法分离的RNA,已成功用于RT-PCR、Northern blot分析和基因全长的克隆等分子生物学研究。     

红花种子氨基酸分析

本文用日立８３５—５０型氨基酸自动测定仪进行去油后不同地区红花种子粉末氨基酸的测定,实验结果表明,红花种子含１６—１９种氨基酸,其中有７—８种人体必需氨基酸。     

Structural features of an l-arabinan derived from mustard seed meal

Aqueous extraction of defatted mustard seed meal yielded an arabinan. Methylation analysis revealed a main chain of 1,5-linked l-arabinofuranosyl residues substituted at O-2 and/or O-3 with additional arabinose, both in furanoside and pyranoside forms.     

Control of storage rot of cassava tuber caused by Rhizopus oryzae using some plant extracts

Abstract

The potency of Piper nigrum seed and leaf, Aframomum meleguata seed and Ageratum conyzoides leaf extracts in the control of cassava tuber rot caused by Rhizopus oryzae was investigated. Water, ethanol and petroleum ether were used as extracting solvents. These extracts were fungitoxic both in vitro and in vivo against the test pathogen. P. nigrum seed extracts were the best followed by those of A. meleguata seed, A. conyzoides leaf and then P. nigrum leaf. The extracts were more effective in controlling rot development in unwounded than wounded tubers especially when they were applied before inoculation with the test pathogen. Ethanol extracts gave the highest growth inhibition in vivo followed by water and the petroleum ether extract whereas water extracts showed marked superiority over the extracting solvents in checking rot development in cassava tuber. Water and ethanol extracts of P. nigrum seed and leaf, A. meleguata seed and A. conyzoides leaf could be used as pesticide of plant origin in the control of R. oryzae causing cassava tuber rot in storage.     

Study of different Trichoderma strains on growth characteristics and silymarin accumulation of milk thistle plant

Abstract

Biological control involves the use of beneficial organisms such as Trichoderma that promote positive responses by the plant. Trichoderma species produce and/or release a variety of compounds, including cell wall degrading enzymes and secondary metabolites, which enhance root development, crop productivity and resistance to biotic and abiotic stresses. This study examines the effects of the five Trichoderma strains (M7, KHB, G124-1, G46-3 and G46-7) on milk thistle including morphological characteristics and silymarin accumulation. This research indicated that treating milk thistle by M7 strain, significantly increased the highest branch length of plants, while strain KHB induced the production of silychristin, isosilybin and silymarin. Finally, strain G46-7 dramatically increased silydianin content in milk thistle plant. The findings suggested that certain Trichoderma strains are positive elicitors for promoting growth characteristics and silymarin accumulation in Silybum marianum (L.) Gaertn.     

沙蛤中牛磺酸的制备研究

以沙蛤提取液中牛磺酸含量为指标,考察乙醇回流法、水煮法和预处理方法,并采用单因素和正交实验法优化工艺条件,确定了沙蛤中提取牛磺酸的最佳工艺。结果:提取牛磺酸的最佳工艺条件为:超声波破碎15 m in,60℃下80%乙醇回流40 m in,该条件下所得牛磺酸质量分数达6.41 mg.g-1原料。结论:超声波破碎与乙醇回流方法相结合,利于牛磺酸溶出,其含量提高26.9%。     

Optimisation of enzyme assisted extraction of silybin from the seeds of Silybum marianum by Box–Behnken experimental design

Introduction – Silybin, a standardised extract of flavanolignans from the seeds of Silybum marianum, has been used for centuries as a natural remedy in the treatment of hepatitis and cirrhosis. The higher yield of silybin by using more efficient extraction technique is of particular interest in the herbal products manufacture. Objective – To systematically investigate the important factors of enzyme‐assisted extraction of flavanolignans from the seeds of Silybum marianum to enhance the extraction yield of silybin. Methodology – The important factors of enzyme‐assisted extraction were optimised by employing Box–Behnken design with the aid of the orthogonal array design (OAD) OA₈ (2⁷). The effects of enzyme incubation temperature (EIT), the pH of enzyme solution (PES) and the size of seeds (SS) on the yield of silybin were visualised as three‐dimensional response surface and contour plots. Results – The predictive yield was 24.6 mg/g defatted seeds under the optimum enzymolysis conditions (EIT 40°C, PES 4.5 and SS 7003 μm). The coefficient of the model was r² > 0.97 (n = 15). The actual yield of silybin was 24.81 ± 1.93 mg/g defatted seeds, higher by 138 and 123.6% than that from ethanol extraction in this study and in the previous literature, respectively. IR spectra and HPLC of the extracts by EAE were in agreement with those from ethanol extraction. SEM and TEM pictures of defatted seeds by variant extractions demonstrate that the extraction of silybin depends on the destruction of cell walls. Conclusion – The results suggest that EAE is a promising alternative for the extraction of silybin by the use of traditional ethanol extraction. Copyright © 2009 John Wiley & Sons, Ltd.