Analysis of solvent effects on polyphenols profile,antiproliferative and antioxidant activities of mulberry (Morus alba L.) extracts

A systematic study about the solvent effect on the mulberry polyphenol (MP) profile, antioxidant and antiproliferation capacities against human hepatoma HepG2 cells was conducted. Results indicated that MP profiles showed significant differences as a function of solvents including AA/W (acetic acid/water), MeOH/AA/W (methanol/ acetic acid/water), EtOH/AA/W (ethanol/ acetic acid/water) and ME2CO/AA/W (acetone/ acetic acid/water). Among the solvents, EtOH/AA/W took advantage in obtaining MP with the highest yield (49.81 mg GAE g^?1 dw), and cellular antioxidant capacity in the PBS no‐wash protocol (63.2 μmolQE/100 g), as well as low cell cytotoxicity (≥50.0 mg mL^?1). ME2CO/AA/W was the best choice for extracting MP with the most various compositions (two phenolic acids, four anthocyanins and four flavonols), greatest potential in inhibiting proliferation of HepG2 cells (EC₅₀ = 28.2 mg mL^?1) and highest intracellular antioxidant activity (38.0 μmolQE/100 g). Results provide baseline information for efficient solvent extraction to obtain MP that is promising as natural antioxidants and antitumor agents.     

小叶薜荔茎叶提取物的抗氧化活性研究

用95%乙醇对小叶薜荔茎叶进行浸提,浸膏分别用石油醚、乙酸乙酯、氯仿、正丁醇和水进行萃取,最后用大孔吸附树脂进行梯度洗脱,得到11种粗提物。采用Folin-Ciocalteu比色法、ORAC（氧自由基吸收能力）分析法和DPPH（1,1-二苯基-2-三硝基苯肼）法对粗提物进行总酚含量和抗氧化能力评价。同时测定了粗体物的羟基自由基的清除能力。结果表明,小叶薜荔茎叶粗提物的总酚含量在14.1%～68.6%之间,同时粗提物具有较强的抗氧化能力,它们的ORAC值在1.01×103～1.82×104μmolTE/g之间。粗提物的总酚含量与抗氧化能力之间具有明显的相关性。      

Antiproliferative and antioxidant activities of common vegetables: A comparative study

Epidemiological studies have consistently linked abundant consumption of fruits and vegetables to a reduction of the risk of developing several types of cancer. In most cases, however, the identification of specific fruits and vegetables that are responsible for these effects is still lacking, retarding the implementation of effective dietary-based chemopreventive approaches. As a first step towards the identification of foods endowed with the most potent chemopreventive activities, we evaluated the inhibitory effects of extracts isolated from 34 vegetables on the proliferation of 8 different tumour cell lines. The extracts from cruciferous vegetables as well as those from vegetables of the genus Allium inhibited the proliferation of all tested cancer cell lines whereas extracts from vegetables most commonly consumed in Western countries were much less effective. The antiproliferative effect of vegetables was specific to cells of cancerous origin and was found to be largely independent of their antioxidant properties. These results thus indicate that vegetables have very different inhibitory activities towards cancer cells and that the inclusion of cruciferous and Allium vegetables in the diet is essential for effective dietary-based chemopreventive strategies.     

Phenolic composition and antioxidant activity of white mulberry (Morus alba L.) fruits

Eight major mulberry cultivars [Nakhonratchasima 60 (NS 60), Buriram 60 (BR 60), Chumphon (CP), Wavee (WV), Chaingmai (CM), Pikultong (PT), Kamphaengsaen (KS) and Kamnanchul (KJ)] cultivated in Thailand were assessed for their flavonoid and phenolic acid composition using HPLC and tested for antioxidant potential using 2,2‐diphenyl‐1‐picrylhydrazyl hydrate (DPPH) assay. The total phenolic content (TPC) ranged from 104.78 to 213.53 mg GAE/100 g DW, and total flavonoid content (TFC) ranged from 69.58 to 211.01 mg CE/100 g DW. The major flavonoid compounds in mulberry fruit cultivars were (+)‐catechin (309.26–750.01 mg/100 g DW), procyanidin B1 (62.59–224.41 mg/100 g DW), quercetin (5.36–58.42 mg/100 g DW), rutin (18.73–26.90 mg/100 g DW) and (?)‐epicatechin (8.47–29.21 mg/100 g DW). Gallic acid, cinnamic acid and p‐hydroxybenzoic acid were found to be the major phenolic acids in mulberry fruit cultivars. The gallic acid and cinnamic acid contents ranged from 7.33 to 23.90 mg/100 g DW and from 11.64 to 15.05 mg/100 g DW, respectively. p‐Hydroxybenzoic acid content ranged from 1.77 mg/100 g DW (PT) to 7.13 mg/100 g DW (KJ). DPPH‐scavenging ability was excellent for ethanolic extract of NS 60, and EC₅₀ value of NS 60 (241.83 μg mL^?1) was significantly lower than those of the others (P < 0.05). TPC and TFC of the mulberry fruit were positively correlated with the DPPH‐scavenging ability.     

Microencapsulation of bioactive compounds from mulberry (Morus alba L.) leaf extracts by protein–polysaccharide interactions

Mulberry leaf extracts were generated using four concentrations of ethanol (50%, 60%, 70%, and 95% v/v). A 60% ethanolic mulberry leaf extract (60E) yielded a high total phenolic content (TPC) and antioxidant activity using 1, 1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging and a ferric reducing antioxidant power (FRAP) assay. Coating materials were derived using a combination of soy protein isolates (SPI) and low methoxyl (LM) pectin in a 1:1 ratio. The effect of various parameters on microencapsulation, such as pH (3.5, 4.0, and 4.5) and the concentration of coating materials (2.5, 5.0 and 7.5% w/v), was studied. Microcapsules produced using 60E as a core material at pH 4.0 with 7.5% of coating material showed a high encapsulation yield, encapsulation efficiency, TPC and antioxidant activity.     

Phytochemicals and antioxidant capacities in rice brans of different color

Rice bran, a byproduct of the rice milling process, contains most of the phytochemicals. This study aimed at determining the concentrations of lipophilic, solvent-extractable (free), and cell wall-bound (bound) phytochemicals and their antioxidant capacities from brans of white, light brown, brown, purple, and red colors, and broccoli and blueberry for comparison. The concentrations of lipophilic antioxidants of vitamin E (tocopherol and tocotrienols) and γ-oryzanols were 319.67 to 443.73 and 3861.93 to 5911.12 μg/g bran dry weight (DW), respectively, and were not associated with bran color. The total phenolic, total flavonoid, and antioxidant capacities of ORAC (oxygen radical absorbance capacity), DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging, and iron-chelating in the free fraction were correlated with the intensity of bran color, while variations of these in the bound fraction were less than those in the free fraction among brans. Compounds in the bound fraction had higher antioxidant capacity of ORAC than DPPH, relative to those in the free fraction. The bound fraction of light-color brans contributed as much to its total ORAC as the free fraction. Total proanthocyanidin concentration was the highest in red rice bran, while total anthocyanin was highest in purple brans. The predominant anthocyanin was cyanidin-3-glucoside. Red and purple brans had several fold higher total phenolics and flavonoids as well as ORAC and DPPH, from both free and bound fractions, than freeze-dried blueberry and broccoli. These results indicate that rice brans are natural sources of hydrophilic and lipophilic phytochemicals for use in quality control of various food systems as well as for nutraceutical and functional food application.     

雅津蛋白桑多糖的分离纯化及生物活性研究

采用纤维素酶法提取桑叶多糖,通过单因素试验和正交试验,确定最优提取工艺参数;并采用Sephadex G-100型葡聚糖凝胶进行初级纯化,比较纯化前后多糖的抗氧化能力和α-葡萄糖苷酶抑制活性。结果表明,纤维素酶法提取的最优工艺为提取液pH 6.0、酶解温度50℃、酶浓度1.0%、酶反应时间50min;纯化后的桑叶多糖具有更高的总抗氧化能力、DPPH·和·OH清除能力;但纯化后的桑叶多糖对α-葡萄糖苷酶抑制效果低于纯化前的。优选得到的纤维素酶法提取桑叶多糖工艺具有较好的提取效果,且纯化后的桑叶多糖具有更强的抗氧化活性。     

Effect of flavonol glycoside in mulberry (Morus alba L.) leaf on glucose metabolism and oxidative stress in liver in diet‐induced obese mice

BACKGROUND: Mulberry therapies on type 2 diabetic patients or streptozotocin‐induced diabetic rats have been reported to improve fasting blood glucose levels. We investigated the effects of dietary consumption of mulberry‐leaf powder and purified quercetin 3‐(6‐malonylglucoside), the quantitatively major flavonol glycoside in mulberry leaves, on glucose and lipid metabolism in high‐fat diet‐induced obese mice. Male C57BL/6J mice aged 8 weeks were assigned to three groups (control, mulberry leaf powder (MLP), and quercetin 3‐(6‐malonylglucoside) (Q3MG)) and treated with their respective diets for 8 weeks. RESULTS: We found that dietary supplementation of 10 g MLP kg^?1 or 1 g Q3MG kg^?1 in high‐fat diet effectively suppressed blood glucose levels. We also noted increased expression of glycolysis‐related genes and suppression of thiobarbituric acid reactive substances concentrations in the liver of Q3MG group compared to control mice. CONCLUSION: Dietary consumption of Q3MG, the quantitatively major flavonol glycoside in mulberry leaves, improved hyperglycemia in obese mice and reduced oxidative stress in the liver. Copyright © 2010 Society of Chemical Industry     

基于HepG2细胞模型的褐赭色羊肚菌多酚抗氧化及抗增殖活性研究

本实验研究了褐赭色羊肚菌游离酚和结合酚体内抗氧化能力以及对HepG2细胞的毒性和抗增殖作用。采用HPLC对褐赭色羊肚菌多酚组分进行鉴定,测定多酚抗氧化能力指数(ORAC);以人肝癌细胞HepG2为模型,测定多酚细胞抗氧化活性(CAA值)及抗增殖活性(EC50值)。结果显示:褐赭色羊肚菌游离酚和结合酚含量分别为(4.27±0.07)、(0.14±0.01)mg GAE/g DW;其ORAC值分别为(4624.52±400)、(429.41±60)μmol TE/100 g DW;CAA值分别为(15.33±0.60)、(0.37±0.01)μmol QE/100 g DW(PBS清洗)和(51.84±0.83)、(1.27±0.04)μmol QE/100 g DW(不经PBS清洗)。游离酚和结合酚浓度为125μg/m L和70μg/m L时,对HepG2细胞增殖抑制率分别为77.28%、66.31%,且试验浓度范围内,羊肚菌多酚对HepG2基本无细胞毒性。褐赭色羊肚菌多酚具有一定的细胞抗氧化及抗增殖活性,可将褐赭色羊肚菌作为开发此类功能营养食品的原料,为羊肚菌的进一步综合利用提供理论依据。     

薏米中酚类提取物测定及抗氧化能力指数分析

选取3种薏米样品,经提取获得游离型和结合型多酚类化合物,采用改进的Folin-Ciocalteu比色法及硼氢化钠/氯醌(SBC)比色法,用酶标仪测定其多酚和黄酮含量;采用抗氧化能力指数(ORAC)方法,于荧光酶标仪中测定提取物中的总抗氧化活性。研究表明,游离型多酚含量中龙薏1号薏米显著性高于贵州黑谷薏米和辽宁5号薏米,结合型多酚含量中龙薏1号薏米显著性高于贵州黑米薏米和辽宁5号薏米,所有样品中其平均结合型多酚含量占总多酚含量的45.28%;游离型黄酮含量中辽宁5号薏米显著高于贵州黑谷薏米和辽宁5号薏米,结合型黄酮含量中龙薏1号薏米高于辽宁5号薏米和贵州黑谷薏米,总黄酮含量最高的是辽宁5号薏米。总抗氧化能力指数(ORAC)最高的是龙薏1号薏米,其值是(668.0 ± 32.7)mg Trolox /100g样品(以干质量计),其次是贵州黑谷薏米和辽宁5号薏米,所有样品中平均结合型ORAC值占总ORAC值的48.08%;3个品种的薏米其总多酚含量与总抗氧化能力指数之间相关系数为0.933,有显著相关性(P＜0.05)。     

不同品种荔枝果肉细胞抗氧化及抗增殖活性评价

为明确荔枝果肉的生物抗氧化和抗肿瘤保健功能,采用HepG2人肝癌细胞模型对我国南方地区主要种植的10种代表性荔枝品种果肉的细胞抗氧化活性(CAA值)和抗肿瘤细胞增殖活性进行了评价。结果表明,10种荔枝品种中,妃子笑荔枝果肉具有最大的CAA值,淮枝则最小;妃子笑抗HepG2肿瘤细胞增殖活性最强,糯米糍则最弱。荔枝果肉的CAA值与总多酚含量、总黄酮含量、多酚化学抗氧化能力指数(ORAC值)之间都有极显著的正相关性(p0.01),且与黄酮含量之间正相关性最大,说明荔枝果肉中起生物抗氧化作用的主要成分是黄酮类化合物;但荔枝果肉对HepG2肿瘤细胞的抗增殖活性与总多酚含量、总黄酮含量、ORAC和CAA值之间均呈现较弱的负相关性(p0.05),提示某些特殊酚类单体或其协同作用在抗肿瘤细胞增殖过程中发挥主要作用。     

干燥方式对桑叶降糖活性成分含量的影响

本实验探讨了冷冻干燥、真空干燥、微波干燥和热风干燥4 种不同干燥方式对桑叶降糖活性成分的影响。采用高效液相色谱法测定桑叶1-脱氧野尻霉素（1-deoxynojirimycin,DNJ）含量、苯酚-硫酸法测桑叶多糖含量、亚硝酸钠法测定桑叶总黄酮含量。结果表明：冷冻干燥法制备的桑叶色泽最好,与新鲜桑叶最为接近,但DNJ和总黄酮的含量都较低;真空干燥法制备的桑叶DNJ和多糖含量最低,且桑叶的色泽也最差;微波干燥对桑叶的降糖成分和色泽影响较小,当功率为140 W处理10 min时,其DNJ和多糖的含量最高,分别为（297.15±6.35） mg/100 g和（11.71±0.01）%;热风干燥的时间较短,对桑叶的颜色影响也较小,当干燥温度为75 ℃,干燥时间为60 min,所得桑叶的DNJ、总黄酮、多糖的含量都较高,分别为（227.47±5.79）mg/100 g、（49.51±1.18）mg/g和（11.29±0.01）%。因此热风干燥更适合于工业化生产中大批量桑叶的干燥。同时测定对α-葡萄糖苷酶和α-淀粉酶的抑制作用可知干燥后桑叶降糖物质结构未发生改变,降糖效果依然存在。     

HPLC-DAD-ESI-MS/MS Analysis and Antioxidant Activities of Nonanthocyanin Phenolics in Mulberry (Morus alba L.)

ABSTRACT:  Qualitative analysis of the nonanthocyanin phenolics from 2 mulberry cultivars, da-10 and hongguo, was performed by the HPLC-DAD-ESI-MS/MS method. Six nonanthocyanin phenolics compounds were identified (procatechuic acid, chlorogenic acid, 4-caffeolyquinic acid, taxifolin, rutin, quercetin) and 3 others (3,5-diCQA, taxifolin-hexoside, kamepferol-hexoside) were tentatively identified. Quantification of nonanthocyanin phenolic was performed by HPLC-DAD, which revealed that rutin (111.38 and 90.79 μg/g FW) was the major nonanthocyanin phenolics in the 2 mulberry cultivars. Phenolic acids in cultivar da-10 (54.68 μg/g FW) were far more than in cultivar hongguo (14.93 μg/g FW). CQA and its isomers (40.02 μg/g FW) were the major phenolic acid in cultivar da-10. The nonanthocyanin phenolics in the 2 cultivars of mulberry had higher antiradical activities in superoxide anion and DPPH radical and antioxidant activity in β-CLAMS assay than the Trolox. These results were relevant to not only the control of color stability and organoleptic characteristics of mulberry juice and wine but also the exploitation of the functional foods made from mulberry.     

A simple preparative method for isolation and purification of polysaccharides from mulberry (Morus alba L.) leaves

An effective method for preparative extraction and purification of the polysaccharides from mulberry leaves was studied. Orthogonal experiment was carried out to get the optimal conditions (T = 70 °C, pH = 6, t = 60 min, D = 0.4 g g^?1 material) of activated carbon decolorisation. The staged, isocratic and multiple ethanol precipitation procedures were also investigated. Through the activated carbon decolorisation and 20% ethanol precipitation procedure, the polysaccharides yield reached 2.91%, and the content of neutral sugar and uronic acid were improved to 21.87% and 57.43% respectively. This process was superior to the macroporous resin adsorption and gel permeation chromatography (GPC) methods. The structure analysis indicated that the purified mulberry leaves polysaccharide (MLP) was composed of Man, GalA and Rha in the ratio of 1.0:7.2:20.0. The molecular weight measured using GPC was approximately 557 062 D. The IR and ¹H NMR spectra indicated that the MLP was mainly pectin, which was classified as high‐methoxyl pectin.     

Comparative antioxidant capacities of phenolic compounds measured by various tests

The purpose of this study was to compare the antioxidant capacities of standard compounds (phenolic compounds, ascorbic acid, and glutathione) as measured by various assays. Five methods were selected so as to span a diversity of technical approaches: TEAC (radical 2,2′-azino-bis(3-ethylbenzothiazoline)-6 sulphonic acid), DPPH (radical 2,2-diphenyl-1-picryhydrazyl used to measure reducing capacity), ORAC (oxygen radical scavenging capacity), red blood cell haemolysis (protection of biological sample), and ESR (electron spin resonance for direct free radical evaluation). Most compounds showed significant differences in free radical scavenging activity according to the method used. Of the 25 tested compounds, only a few, such as myricetin and gallocatechin, gave comparable activities in the various tests. To standardise reporting on antioxidant capacity, it is proposed to use a weighted mean of the values obtained using the DPPH, ORAC, resistance to haemolysis, and ESR assays.     

桑叶水提物预防小鼠高脂血症的作用研究

目的探讨桑叶水提取物(mulberry leaf water extract,MWE)的降血脂功效,为桑叶保健品降血脂的开发利用提供依据。方法在饲喂高脂饲料的同时,给予小鼠不同剂量的桑叶水提取物,28 d后摘眼球取血,测定不同试验组小鼠血清血脂水平、载脂蛋白A1(Apo A1)和载脂蛋白B(Apo B)含量、小鼠肝脏中3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)、胆固醇-7α-羟化酶(CYP7A1)和卵磷脂胆固醇酰基转移酶(LCAT)活性和总胆汁酸(total bile acid,TBA)含量以及粪便含水率和TBA含量。结果与高脂组小鼠相比,MWE可降低高脂饮食小鼠肝脏系数及脂肪系数,并降低高脂饮食小鼠血脂水平,使肝脏HMG-COA和LCAT活性降低,提高CYP7A1活性,并抑制肝脏胆汁酸的积累,促进胆汁酸的排出。结论 MWE对高脂小鼠具有显著的降血脂功效,可有效减少脂肪积累,促进胆固醇代谢从而预防动脉硬化的发生。     

Phytonutrients affecting hydrophilic and lipophilic antioxidant activities in fruits,vegetables and legumes

The antioxidant activities of fruits (n = 21), vegetables (n = 67) and legumes (n = 7) commonly consumed in Korea were determined by both the lipophilic antioxidant performance assay (LAP) and the hydrophilic oxygen radical absorbance capacity assay (ORAC). The LAP assay used the lipophilic radical initiator MeO‐AMVN [2,2′‐azobis(4‐methoxy‐2,4‐dimethylvaleronitrile)] and the lipophilic probe BODIPY 581/591 [4,4‐difluoro‐5‐(4‐phenyl‐1,3‐butadienyl)‐4‐bora‐3a, 4a‐diaza‐s‐indacene‐3‐undecanoic acid]. The ORAC assay used the hydrophilic radical initiator AAPH [2,2′‐azobis(2‐amidinopropane) dihydrochloride] and the hydrophilic probe fluorescein. In addition, the lipid‐soluble phytonutrients, carotenoids and tocopherols were determined by a reverse‐phase HPLC system using a C30 column with a UV detector. The water‐soluble phytonutrient, ascorbic acid, was analyzed using an HPLC system with an electrochemical detector. Total phenols were determined by UV spectrophotometry. Tocopherols (r = 0.774, p < 0.0001) and carotenoids (r = 0.569, p < 0.0001) were significantly correlated with LAP in total samples (n = 95). ORAC was significantly correlated with total phenols (r = 0.893, p < 0.0001), but not with ascorbic acid (r = 0.009, p = 0.929) in total samples (n = 95). These data indicate that carotenoids and tocopherols and total phenols are the major contributors to the lipophilic and hydrophilic antioxidant capacities, respectively. Therefore, the contribution of both the hydrophilic and lipophilic components of fruits and vegetables should be considered when determining the actual ‘total’ antioxidant activity of fruits and vegetables. Copyright © 2007 Society of Chemical Industry     

Impact of extraction parameters and their optimization on the nutraceuticals and antioxidant properties of aqueous extract mulberry leaf

An investigation into the efficient use of water as a solvent and the influence of extraction temperature, extraction time, water to leaf powder ratio, particle size, and extraction cycle on the nutraceutical and antioxidant profile of aqueous mulberry leaf extract were conducted using a single-factor experiment approach. All the assessed extracting parameters showed a significant effect on the nutraceutical compounds and antioxidant properties. The optimum extraction conditions were as follows: extraction temperature of 70°C, extraction time of 40 min, water to leaf powder ratio of 40:1 ml/g, particle size of 25 µm, and two extraction cycles. Based on these optimal conditions, chlorogenic acid (62.10 mg/g), caffeic acid (32.21 mg/g), kaempferol-7-O-glucoside (19.30 mg/g), quercetin-3-rutinose (15.69 mg/g), quercetin-3-O-glucoside (32.38 mg/g), kaempferol-3-(6-rhamnosylglucoside) (42.52 mg/g), quercetin-3-(6-malonylglucoside) (65.19 mg/g), kaempferol-3-glucoside (66.27 mg/g), kaempferol-3-(6-malonylglucoside) (50.18 mg/g), 1-deoxynojirimycin (15.58 mg/g), and gamma-aminobutyric acid (5.05 mg/g) were obtained. The optimal aqueous extract had high antioxidant properties of 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (39.98 mM/g), cupric ion reducing capacity (58.93 mM/g), 1,1-diphenyl-2-picrylhydrazyl (101.33 mM/g), and ferric reducing antioxidant power capacity (233.77 mM/g) of dried mulberry leaf extract.