Study on Extraction and Antioxidant Activity of Flavonoids from Hemerocallis fulva (Daylily) Leaves

Hemerocallis fulva is a medical and edible plant. In this study, we optimized the ultrasound-assisted extraction (UAE) process of extracting flavonoids from Hemerocallis fulva leaves by single-factor experiments and response surface methodology (RSM). The optimum extraction conditions generating the maximal total flavonoids content was as follows: 70.6% ethanol concentration; 43.9:1 mL/g solvent to sample ratio; 61.7 °C extraction temperature. Under the optimized extraction conditions, the total flavonoid content (TFC) in eight Hemerocallis fulva varieties were determined, and H. fulva (L.) L. var. kwanso Regel had the highest TFC. The cytotoxicity of the extract was studied using the Cell Counting Kit-8 (CCK-8 assay). When the concentration was less than 1.25 mg/mL, the extract had no significant cytotoxicity to HaCaT cells. The antioxidant activity was measured via chemical antioxidant activity methods in vitro and via cellular antioxidant activity methods. The results indicated that the extract had a strong ABTS and •OH radical scavenging activity. Additionally, the extract had an excellent protective effect against H₂O₂-induced oxidative damage at a concentration of 1.25 mg/mL, which could effectively reduce the level of ROS to 106.681 ± 9.733% (p < 0.001), compared with the 163.995 ± 6.308% of the H₂O₂ group. We identified five flavonoids in the extracts using high-performance liquid chromatography (HPLC). Infrared spectroscopy indicated that the extract contained the structure of flavonoids. The results showed that the extract of Hemerocallis fulva leaves had excellent biocompatibility and antioxidant activity, and could be used as a cheap and potential source of antioxidants in the food, cosmetics, and medicine industries.     

Extraction of Phenolic and Flavonoid Compounds from Mung Bean (Vigna radiata L.) Seed Coat by Pressurized Liquid Extraction

Mung bean seed coat (MBC) is a by-product of the mung bean processing industry. It contains a large number of phenolic compounds with therapeutic anti-inflammatory, anti-diabetic and antioxidant properties. This research aimed to investigate the optimum conditions for phenolic and flavonoid extraction from MBC by pressurized liquid extraction (PLE). Response surface methodology (RSM) was used to study the effects of temperature (80–160 °C), pressure (1200–1800 psi) and ethanol concentration (5–95%) on total phenolic content (TPC), total flavonoid content (TFC) and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) scavenging activity (ABTS). Scale-up extraction was also performed. The optimum conditions for extraction were 160 °C, 1300 psi and 50% ethanol. Under optimum conditions, the TPC was 55.27 ± 1.14 mg gallic acid equivalent (GAE)/g MBC, TFC was 34.04 ± 0.72 mg catechin equivalent (CE)/g MBC and ABTS scavenging activity was 195.05 ± 2.29 mg trolox equivalent (TE)/g MBC. The TFC and ABTS scavenging activity of the extracts obtained at the pilot scale (10 L) was not significantly different from the laboratory scale, while TPC was significantly increased. The freeze-dried MBC extract contained vitexin and isovitexin 130.53 ± 17.89, 21.21 ± 3.22 mg/g extract, respectively. In conclusion, PLE was able to extract phenolics, flavonoids with ABTS scavenging activity from MBC with the prospect for future scale-up for food industry.     

双水相提取新疆圆柏果实中的总黄酮及抑制亚硝化反应的研究

本文采用超声集成丙醇-硫酸铵双水相体系提取新疆圆柏果实中总黄酮,并用分光光度法测定其含量。探讨了丙醇-硫酸铵双水相(醇-水)体系的形成及提取条件对总黄酮得率的影响,得到的最优条件为:醇-水比0.8∶1,硫酸铵的量8g,料液比1∶50;超声时间40min。结果表明,该方法可有效地从新疆圆柏实中提取总黄酮,在最优条件下提取率为11.33mg/g。在此条件下,圆柏果实总黄酮提取液对亚硝化反应的抑制能力接近抗坏血酸。     

Extraction and determination of bioactive flavonoids from Abelmoschus manihot (Linn.) Medicus flowers using deep eutectic solvents coupled with high‐performance liquid chromatography

A highly efficient and ecofriendly extraction method using deep eutectic solvents was developed to extract bioactive flavonoids from Abelmoschus manihot (Linn.) Medicus flowers. First, a series of deep eutectic solvents using choline chloride as hydrogen bond acceptor with different hydrogen bond donors was successfully synthesized. Then, the types of deep eutectic solvents and the extraction conditions for bioactive flavonoids (hyperoside, isoquercitrin, and myricetin) were optimized based on the flavonoids extraction efficiencies. The optimized deep eutectic solvent for hyperoside and isoquercitrin extraction was composed of choline chloride and acetic acid with a molar ratio of 1:2. The optimized deep eutectic solvent for myricetin extraction was composed of one mole of choline chloride and two moles of methacrylic acid. The optimal extraction conditions were set as: solid to solvent ratio, 35:1 (mg/mL); extraction time, 30 min; extraction temperature, 30°C. Qualitative and quantitative analysis were performed using ultra high performance liquid chromatography with tandem mass spectrometry and high‐performance liquid chromatography. And the extraction efficiencies of hyperoside, isoquercitrin, and myricetin under optimal extraction conditions were calculated as 11.57, 5.64, and 1.11 mg/g, much higher than those extracted by traditional extraction solvents. Therefore, the prepared deep eutectic solvents can be selected as alternative solvent to extract bioactive flavonoids.     

Response surface optimized ultrasonic-assisted extraction of flavonoids from Sparganii rhizoma and evaluation of their in vitro antioxidant activities

An efficient ultrasound-assisted extraction technique was employed to extract total flavonoids from Sparganii rhizoma. The optimum extraction conditions for the highest yield of total flavonoids were ethanol concentration 53.62%, ultrasonication time 29.41 min and ultrasound power 300 W, which were determined using response surface methodology. The extraction yields of the optimal ultrasound-assisted extraction were higher than using conventional extraction. The crude extract was then purified on a polyamide resin, whereby the flavonoids content in the purified extract increased to 94.62%. The antioxidant activities of the purified flavonoids including DPPH radical scavenging activity, ABTS+ radical scavenging activity, reducing power, hydroxyl radical scavenging activity and superoxide anion scavenging activity, were evaluated in vitro, which suggested that the flavonoids showed significant antioxidant activities. Rutin, kaempferol and formononetin were identified in the extract by comparing relative retention times and UV-Vis spectra with those of reference standards.     

Combined Ultrahigh Pressure Extraction and High-Speed Counter-Current Chromatography for Separation and Purification of Three Glycoside Compounds from Dendrobium officinale Protocorm

As an alternative to Dendrobium candidum, protocorm-like bodies (PLBs) of Dendrobium candidum are of great value due to their high yield and low cost. In this work, three glycoside compounds, β-D-glucopyranose 1-[(E)-3-(4-hydroxyphenyl)-2-propenoat] (I), β-D-glucopyranose 1-[(E)-3-(3, 4-dihydroxyphenyl)-2-propenoat] (II), and 1-O-sinapoyl glucopyranoside (III), were extracted and isolated by ultrahigh pressure extraction (UPE) coupled with high-speed counter-current chromatography (HSCCC) from PLBs of D. officinale. First, the target compounds were optimized and prepared with 50% ethanol solution at a 1:30 (g/mL) solid/liquid ratio in 2 min under 300 MPa by UPE. Then, the crude extract was chromatographed with a silica gel column, and primary separation products were obtained. In addition, the products (150 mg) were separated by HSCCC under the solvent system of MTBE-n-butyl alcohol-acetonitrile-water (5:1:2:6, v/v/v/v), yielding 31.43 mg of compound I, 10.21 mg of compound II, and 24.75 mg of compound III. Their structures were further identified by ESI-MS, ¹H NMR, and ¹³C NMR. The antioxidant results showed that the three compounds expressed moderate effects on the DPPH· scavenging effect. Compound II had the best antioxidant capacity and its IC₅₀ value was 0.0497 mg/mL.     

Modulation of antibiotic activity by the hydroalcoholic extract from leaves of Caryocar coriaceum WITTM

The aim of this study was to identify the presence of tannins, phenols and flavonoids on the hydroalcoholic extract of Caryocar coriaceum leaves (HECCL) and to determine the antioxidant and antibacterial activity of this extract. The extract was tested alone (1024–1 μg/mL) or associated (MIC/8) with several antibiotics in order to identify any antibacterial activity against multiresistant bacterial strains (Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa). The existence of tannins, total phenols (901.31 mg/g) and flavonoids (89.68 mg/g) was confirmed in the HECCL. The presence of rutin and quercetin were confirmed by Thin-layer chromatography (TLC). Using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method, the antioxidant activity of the extract (9 μg/mL) was determined. Moreover, the Minimum Inhibitory Concentration (MIC) value found for HECCL was 1024 μg/mL and the association between HECCL (MIC/8) with benzylpenicillin significantly changed its minimum inhibitory concentration from 2500 to 625 μg/mL against E. coli.     

昆仑雪菊中黄酮类化合物提取与纯化

用超声法提取昆仑雪菊中黄酮类化合物,通过大孔树脂对提取液进行纯化,纯化物经聚酰胺层析柱梯度洗脱得到初步分离物,通过颜色反应和紫外光谱扫描进行定性分析。设计正交实验得到超声法提取的最佳工艺条件是65%乙醇、料液比为1∶40、超声时间60 min,此条件下提取3次,总黄酮得率可达132.6 mg/g。通过聚酰胺柱层析法分离出五个组分,初步判断它们属于不同的黄酮类物质。超声法提取总黄酮,提取率较高,聚酰胺树脂对黄酮类化合物的分离效果较好。     

超声提取-反相高效液相色谱法测定新疆大蓟中绿原酸和芦丁的含量

以黄酮提取量为指标,选用正交实验对新疆大蓟总黄酮的超声提取工艺进行优化,结果表明最佳提取条件为:超声电流250mA,料液比1∶30(g/mL),时间40min,总黄酮提取量为2.89mg/g。同时建立了高效液相色谱法测定新疆大蓟中绿原酸及芦丁含量的方法。采用SinoChrom ODS-BP色谱柱,甲醇-1%冰乙酸为流动相,检测波长为340nm,流速为0.9mL.min-1。方法测定绿原酸的线性范围为1.675～16.75μg/mL,相关系数R=0.99995;测定芦丁的线性范围为2～20μg/mL,相关系数R=0.9999,回收率分别为99.45%、99.65%。该法简单准确,适用于新疆大蓟中绿原酸及芦丁的定量分析。     

超声波提取含羞草种子中总黄酮工艺条件优化研究

采用超声提取法对含羞草种子中黄酮类物质进行提取,对提取工艺条件如溶剂、配料比、浸泡时间、提取温度、提取时间进行了优化。在单试验的基础上进行正交试验,得出黄酮类物质的最佳提取工艺条件:乙醇体积分数为60%,含羞草种子与60%乙醇配料比(g∶m L)为1∶8,超声温度范围40~44℃,超声时间为50 min。该方法操作简单,提取时间短,提取效率较高。     

Antimicrobial and Antioxidant Properties of Total Polyphenols of Anchusa italica Retz

Anchusa italica Retz has been used for a long time in phytotherapy. The aim of the present study was to determine the antioxidant and antibacterial activities of extracts from the leaves and roots of Anchusa italica Retz. We first determined the content of phenolic compounds and flavonoids using Folin–Ciocalteu reagents and aluminum chloride (AlCl₃). The antioxidant activity was determined using three methods: reducing power (FRAP), 2.2-diphenyl-1-picrylhydrazyl (DPPH), total antioxidant capacity (TAC). The antimicrobial activity was investigated against four strains of Escherichia coli, two strains of Klebsiella pneumoniae and coagulase-negative Staphylococcus, and one fungal strain of Candida albicans. The results showed that the root extract was rich in polyphenols (43.29 mg GAE/g extract), while the leave extract was rich in flavonoids (28.88 mg QE/g extract). The FRAP assay showed a strong iron reduction capacity for the root extract (IC₅₀ of 0.11 µg/mL) in comparison to ascorbic acid (IC₅₀ of 0.121 µg/mL). The DPPH test determined an IC₅₀ of 0.11 µg/mL for the root extract and an IC₅₀ of 0.14 µg/mL for the leaf extract. These values are low compared to those for ascorbic acid (IC₅₀ of 0.16 µg/mL) and BHT (IC₅₀ 0.20 µg/mL). The TAC values of the leaf and root extracts were 0.51 and 0.98 mg AAE/g extract, respectively. In vitro, the extract showed inhibitory activity against all strains studied, with diameters of zones of inhibition in the range of 11.00–16.00 mm for the root extract and 11.67–14.33 mm for the leaf extract. The minimum inhibitory concentration was recorded for the leaf extract against E. coli (ATB:57), corresponding to 5 mg/mL. Overall, this research indicates that the extracts of Anchusa italica Retz roots and leaves exert significant antioxidant and antibacterial activities, probably because of the high content of flavonoids and polyphenols.     

Development of cyclodextrin-based extract of <Emphasis Type="Italic">Lotus corniculatus</Emphasis> as a potential substrate reduction therapy in mucopolysaccharidoses type III

A novel sustainable and eco-friendly procedure simultaneously combining the use of ultrasound irradiation and hydroxypropyl-β-cyclodextrin (HPβCD) complexation was used to extract phytochemicals from Lotus corniculatus and to prepare flavonoid rich extract having potential as substrate reduction therapy of mucopolysaccharidosis type III (MPS III), a rare autosomal recessive lysosomal storage disorder. For that instance, Box-Behnken design and response surface methodology was employed to statistically assess the influence of HPβCD concentration, ultrasonic power, and extraction time on the total phenolic compounds, total flavonoids, total phenolic acids and radical scavenging activity (RSA) of the extracts prepared, chosen as the responses indicative of the product quality and performance. The potential of such optimised extract to modulate accumulation of gylcosoaminoglycans (GAGs) was evaluated on the fibroblast obtained from patients suffering from MPS type III. Optimized extract prepared by 45 min extraction using HPβCD at concentration of 20 mM and ultrasonic irradiation of 648 W was rich in flavonoids (1.36 mg/mL) and showed notable RSA of 1.04 mg/mL. Freeze-dried extract, at concentration of 3 and 6 µg/mL, reduced GAG levels in skin fibroblasts by 33.97 and 50.08%, respectively, without any toxic effects at given doses, showing a potential to be considered as a part of the substrate reduction therapy of MPS III.     

Optimization of Enzyme-Assisted Extraction and Purification of Flavonoids from Pinus koraiensis Nut-Coated Film and Antioxidant Activity Evaluation

Pinus koraiensis nut-coated film is a kind of by-product of nut processing, which has been shown to contain flavonoids, polyphenols, and other substances that can be used to produce natural antioxidant extracts. In this study, response surface methodology (RSM) was used to optimize the extraction process of flavonoids of P. koraiensis nut-coated film (PNF), and macroporous resin HPD600 was used to purify PNF (P-PNF). Its antioxidant activity was examined by DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging capacity, oxygen free radical absorption capacity (ORAC), total oxygen radical capture (TRAP), and iron ion reduction capacity. Under the ideal extraction conditions comprising a cellulase dosage of 90 U/g, a material/liquid ratio of 1:20 (g/mL), and an extraction time of 2 h, the PNF yield was 3.37%. Purification conditions were sample concentration of 2.0 mg/mL, pH of 5, water washing volume of 3 bed volume (BV), eluent ethanol concentration of 50%, and volume of 2 BV. The P-PNF recovery was 84.32%, and purity increased from 33.80% to 61.70%. Additionally, P-PNF showed increased antioxidant activity compared to PNF. Cumulatively, this study obtained the optimal values for the process parameters in order to achieve the maximum rates of extraction of PNF for economically optimal production at an industrial scale.     

Reflux Extraction Optimization and Antioxidant Activity of Phenolic Compounds from Pleioblastus amarus (Keng) Shell

Phenols were extracted from the Pleioblastus amarus (Keng) shell (PAS) using ethanol. A Plackett–Burman assessment indicated that the factors affecting polyphenol extraction included the ethanol concentration, extraction temperature, liquid to solid ratio, extraction time, and reflux extraction times; the best extraction parameters were the ethanol concentration of 75%, a 20:1 liquid to solid ratio, and an extraction time of 2.1 h. The number of polyphenols was 7.216 mg/g. Furthermore, the phenol composition analysis showed the presence of p-Coumaric acid (196.88 mg /mL) and rutin (312.9 mg /mL), which were used for the in vitro extraction and determination of the antioxidant activity. According to the A, B, C, and D antioxidant activity assays, the ethyl acetate phase was the strongest with low IC₅₀ values of 0.169 ± 0.01 mg/mL, 0.289 ± 0.01 mg/mL, 0.372 ± 0.01 mg/mL, and 1.029 ± 0.03 mg/mL, respectively, confirming high antioxidant activity. For the n-butanol and petroleum ether phases, antioxidant activity was lower. This study showed that the polyphenol extract from Pleioblastus amarus (Keng) shell displayed excellent antioxidant activity, enhancing its practical application.     

Chemical Profile,Antimicrobial and Antioxidant Activity Assessment of the Crude Extract and Its Main Flavonoids from Tartary Buckwheat Sprouts

The purpose of this study was to investigate the major flavonoids content and bioactivities of Tartary buckwheat sprouts. The crude methanol extract (ME) of Tartary buckwheat sprouts was abundant in flavonoids, and six major flavonoids, including isoorientin, vitexin, isovitexin, rutin, quercetin, and kaemferol were successfully determined from the sprouts by the high-performance liquid chromatography (HPLC) method. Generally, the flavonoid content of buckwheat sprouts was in the order of rutin > quercetin > isovitexin > vitexin> isoorientin > kaemferol. The highest rutin content of the ME and sprout cultures was 89.81 mg/g and 31.50 mg/g, respectively. Antibacterial activity results indicated the ME displayed notable inhibitory activity against the five tested bacteria, and its minimum inhibitory concentration (MIC) values ranged from 0.8 mg/mL to 3.2 mg/mL. Among the six flavonoids, quercetin was the most active compound, which exhibited strong activity against all tested bacteria except for E. coli and S. epidermidis, with its MIC values ranging from 0.2 mg/mL to 0.4 mg/mL. For the antifungal activity assay, the ME of Tartary buckwheat sprouts and four flavonoids could significantly inhibit the spore germination of two pathogenic fungi, and their inhibitory efficiency was concentration dependent. Quercetin was the most active one, which significantly inhibited the spore germination of F. oxysporum f. sp. vasinfectum and F. oxysporum f. sp. cucumerinum, and its median effective inhibitory concentration (IC₅₀) value was 42.36 and 32.85 µg/mL, respectively. The antioxidant activity results showed that quercetin, kaemferol, and rutin displayed excellent antioxidant activity in the DPPH radical scavenging test, and their IC₅₀ value was calculated as 5.60, 16.23, and 27.95 µg/mL, respectively. This is the first report on the antimicrobial activity of the crude extract of Tartary buckwheat sprouts. These results indicated that the methanol extract of Tartary buckwheat sprouts could be used as a potential antimicrobial or antioxidant agent in the future.     

Ultra‐high‐pressure‐assisted extraction of wedelolactone and isodemethylwedelolactone from Ecliptae Herba and purification by high‐speed counter‐current chromatography

Ultra‐high‐pressure extraction combined with high‐speed counter‐current chromatography was employed to extract and purify wedelolactone and isodemethylwedelolactone from Ecliptae Herba. The operating conditions of ultra‐high‐pressure extraction were optimized using an orthogonal experimental design. The optimal conditions were 80% aqueous methanol solvent, 200 MPa pressure, 3 min extraction time and 1:20 (g/mL) solid–liquid ratio for extraction of wedelolactone and isodemethylwedelolactone. After extraction by ultra‐high pressure, the extraction solution was concentrated and subsequently extracted with ethyl acetate; a total of 2.1 g of crude sample was obtained from 100 g of Ecliptae Herba. A two‐phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (3:7:5:5, v/v) was used for high‐speed counter‐current chromatography separation, by which 23.5 mg wedelolactone, 6.8 mg isodemethylwedelolactone and 5.5 mg luteolin with purities >95% were purified from 300 mg crude sample in a one‐step separation. This research demonstrated that ultra‐high‐pressure extraction combined with high‐speed counter‐current chromatography was an efficient technique for the extraction and purification of coumestans from plant material.     

响应面法优化板栗花总黄酮微波提取工艺

应用响应面法优化了乙醇微波提取板栗花中总黄酮的工艺条件。用光度法在波长510 nm处测定提取液中总黄酮的含量(以芦丁为标准),从而判断提取条件的优劣。经试验确定最优提取条件为:①微波功率700 W;②提取时间80 s;③提取溶剂乙醇与水的体积比为40比60;④板栗花料与提取溶剂的比值为1比100。在此选定条件下板栗花总黄酮的提取率达到(3.47±0.14)g/100 g,与理论值(3.49 g/100 g)很接近。     

Antimicrobial activity and phenolic content of natural site and micropropagated Limonium avei (De Not.) Brullo & Erben plant extracts

This study reported the antimicrobial activity and phenolic content of natural site and micropropagated Limonium avei (De Not.) Brullo & Erben inflorescences. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of ethanolic extracts were determined according to the Clinical Laboratory Standards Institute guidelines. Individual phenolic acids and flavonoids were detected by a high performance liquid chromatography (HPLC-DAD) method. The samples showed a comparable antimicrobial activity, although the natural site extract possessed the lower MIC values. The best activity was detected against Gram-positive bacteria, such as Staphylococcus aureus including methicillin resistant strains (MIC and MBC values ranging from 7.81 to 62.50?μg?mL(-1) and from 500 to 2000?μg?mL(-1) respectively). In contrast, a low activity was found on Gram-negative bacteria and Candida albicans. The HPLC-DAD analysis revealed ten phenolic acids and four flavonoids with a major amount of m-coumaric acid, naringin and quercetin in the natural site extract.     

Analysis of flavonoids by capillary zone electrophoresis with electrokinetic supercharging

On-line concentration via Electrokinetic Supercharging (EKS) was used to enhance the sensitivity of the capillary electrophoretic separation of the four flavonoids naringenin, hesperetin, naringin and hesperidin. Separation conditions, including the background electrolyte pH and concentration, the length and choice of terminator and the electrokinetic injection time were optimized. The optimum conditions were: a background electrolyte of 30 mM sodium tetraborate (pH 9.5) containing 5% (v/v) of methanol, electrokinetic injection of the sample (130 s, -10 kV) followed by hydrodynamic injecting of 100 mM 2-(cyclohexylamino)ethanesulfonic acid (CHES) (17 s, 0.5 psi) as terminator, and separation with -20 kV. Under these conditions the four flavonoids could be separated with a sample-to-sample time of 15 min and detection limits from 2.0 to 6.8 ng mL(-1). When compared to a conventional hydrodynamic injection the sensitivity was enhanced between 824 and 1515 times which is 7.6-16 times higher than other CE methods for the on-line concentration of flavonoids. The applicability of the developed method was demonstrated by the detection of the four flavonoids in an aqueous extract of Clematis hexapetala pall.     

Optimized microwave‐assistant extraction combined ultrasonic pretreatment of flavonoids from Periploca forrestii Schltr. and evaluation of its anti‐allergic activity

Microwave extraction combined ultrasonic pretreatment of flavonoids from Periploca forrestii Schltr. was investigated in this study, extraction process was first performed in an ultrasonic cleaner, then treated by microwave irradiation. The optimum ultrasonic time of 25 min was selected by single‐factor experiments. A response surface methodology has been used to obtain a mathematical model that describes the process and analyzes the significant parameters ethanol concentration 59.92%, liquid to raw materials ratio 21.24 mL/g, microwave radiation time 209.53 s, and microwave power 274.14 w. In these optimum conditions, the yield of flavonoids from P. forrestii (TFPF) could be up to 9.11 ± 0.08%, which was increased by 14.30 and 19.86% compared microwave extraction and ultrasonic extraction, respectively. In vitro suppress hyaluronidase experimentation showed that TFPF purified using polyamide exhibited good anti‐hyaluronidase ability with IC₅₀ value of 1.033 mg/mL, possessing certain anti‐antiallergic and potential application prospect in pharmaceutical production of treating inflammation and other related fields.