Effect of in vitro‐simulated gastrointestinal digestion on the stability and antioxidant activity of blueberry polyphenols and their cellular antioxidant activity towards HepG2 cells

In this study we investigated the influence of in vitro‐simulated gastrointestinal digestion on the bioaccessibility and antioxidant activity of polyphenols from blueberries (Vaccinium spp.). Total phenolic, anthocyanin, and flavonoid content was determined, and extract and digesta compositions were analysed by HPLC‐ESI‐MS/MS. The phenolic compounds were relatively stable under a gastric environment, whereas polyphenols and anthocyanins were unstable under an intestinal environment. The bioaccessibility of polyphenol, anthocyanin, and flavonoid was greatly decreased after the intestinal digestion, and the recoveries were only 13.93%, 1.95%, and 15.68% (the IN sample), respectively. Polyphenolic profile alteration occurred during in vitro‐simulated gastrointestinal digestion. Changes of phenolic compound antioxidant activity during digestion correlated with polyphenol, flavonoid, and caffeic acid concentrations. Digested extract cellular antioxidant activity was lower than non‐digested extract activity (P < 0.05). Polyphenol dose–response correlations with cellular antioxidant activity were observed. These results indicated that in vitro‐simulated gastrointestinal digestion significantly impact polyphenols and their antioxidant activity.     

南酸枣皮中酚类化合物体外模拟消化与溶剂提取的比较研究

对南酸枣皮进行体外模拟胃肠液消化与溶剂提取,研究不同提取方法对南酸枣皮中总多酚、总黄酮、原花青素释放的影响,并通过透析袋模拟肠道的吸收作用,考察其生物利用率。结果表明：体外消化过程中酚类化合物主要在胃部释放,经模拟胃液消化1 h后,总多酚、总黄酮、原花青素释放量分别为73.72、38.55、37.53 mg/g;肠液消化2 h后,总多酚、总黄酮释放量分别增加10.29 mg/g和8.78 mg/g,而部分原花青素发生分解,降幅为17.87%;透析袋内总多酚、总黄酮、原花青素释放量分别为5.94、5.48、3.21 mg/g,生物利用率分别为7.07%、11.59%和10.42%,因此黄酮类化合物最可能被人体吸收利用。不同处理方法的多酚类化合物的释放量为模拟胃肠液消化＜传统热水浸提＜超声波辅助乙醇提取（化学提取＞中药口服＞直接食用）。     

Solvent extraction and in vitro simulated gastrointestinal digestion of phenolic compounds from purple sweet potato

The total content of phenolic compounds in purple sweet potato (PSP) was determined and the release of such compounds from PSP in gastrointestinal digestion was studied in vitro. The extraction conditions for the maximum recovery of free phenol (FP) and bound phenol (BP) from PSP were determined by response surface methodology (RSM). The maximum recovery of FPPSP was 14.16 ± 0.87 mg GAE per g short for dry weight (DW), which was obtained using 60% (v/v) ethanol maceration with a liquid–solid ratio of 57.21:1 (mL g⁻¹) at 51.93 °C for 2.12 h. The maximum recovery for BPPSP was 7.54 mg GAE per g DW, which was obtained upon hydrolysis with 1.87 mol L⁻¹ NaOH at a liquid–solid ratio of 35.93:1 (mL g⁻¹) for 4.74 h. The maximum phenolic content was released after 1 and 2 h for the in vitro gastric and intestinal digestion respectively. The release of the phenolics was promoted by pepsin and gastric acid during gastric digestion, while it was further promoted by trypsin during intestinal digestion.     

体外模拟胃肠消化中山楂多酚及抗氧化活性的变化

本实验研究了山楂多酚、黄酮及抗氧化活性在体外模拟胃肠道环境下的变化规律,并利用高效液相色谱法测定了胃肠消化对山楂中5 种主要单体酚类化合物的影响。结果表明：山楂多酚在模拟胃液消化中稳定性较好,总多酚、黄酮含量及抗氧化能力均无显著性变化（P＞0.05）;而在模拟肠液消化中山楂多酚易发生降解,总多酚、黄酮含量在0.5 h内分别降低16.26%和7.46%,抗氧化能力也随之降低10.25%。高效液相色谱测定结果显示,5 种单体酚类化合物含量在胃液消化中无显著性变化（P＞0.05）;而在肠液消化中变化情况不相一致,其中金丝桃苷和异槲皮苷含量无显著性变化（P＞0.05）,表儿茶素在0.5 h内完全降解,绿原酸和原花青素B2在0.5 h内分别显著下降40.78%和28.99%（P＜0.05）。     

多酚化合物体外模拟消化的稳定性分析

选取7 种不同种类的酚类化合物,研究体外模拟胃肠道消化对其稳定性的影响。结果表明：不同酚类化合物在胃肠道消化过程中的稳定性不同。高效液相色谱分析结果显示,胃消化过程对酚酸类化合物的影响较小,其含量无显著变化,但类黄酮化合物在胃消化过程中稳定性较差,儿茶素含量下降了16.6%、表儿茶素含量下降了约6%。经模拟肠液处理后,除阿魏酸、对羟基苯甲酸外,其他酚类化合物含量均显著降低,此外,绿原酸在肠液消化过程中生成了新的物质。体外模拟胃肠液消化对混合标准品的作用结果有所不同,模拟胃液消化对混合标准品含量均无显著影响,模拟肠液消化后,仅表儿茶素、没食子酸含量分别降低为76.96%和50.30%,其他酚类化合物含量均无显著变化。     

Stability and antioxidant activity of phenolic compounds during in vitro digestion

The impact of phenolic compounds on the human body depended on the type, content, bioavailability, and antioxidant activity. After digestion, different phenolic compounds had different changes of bioavailability and antioxidant activity, which needed to be considered in the application. In this experiment, the structural stability and antioxidant activity of 27 phenolic compounds (phenolic acids, flavonols, flavonoids, and flavanones) were investigated during the in vitro simulated digestion. This experiment eliminated the influence of food matrix, provide a basis for regularity for the changes of phenolic substances in different materials. Results showed that the bioaccessibility of phenolic compounds with different structures varied, and there was a conformational relationship between the structure and stability. After oral digestion, most of the phenolic compounds underwent degradation and the cellular antioxidant activity (CAA) values decreased to a large extent (p < 0.05). After gastric digestion, the content (p > 0.05) and CAA values (p < 0.05) of most phenolic compounds increased. However, after intestinal digestion, the phenolic compounds were degraded to a greater extent, and different structures of phenolic compounds had different changes in CAA values (p < 0.05). In general, the CAA values of most phenolic compounds after in vitro digestion were lower than the initial value. The 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ehylbenzthiazoline-6-sulfonic acid) (ABTS), and ferric reducing antioxidant power (FRAP) values of phenolic acids and flavonols decreased after in vitro simulated digestion (p < 0.05), while the values of DPPH, ABTS, and FRAP of most flavonoids (p < 0.05) increased. The increased oxygen radical absorption capacity (ORAC) values were found in most phenolic acids, flavonols, and flavonoids (p < 0.05), and most flavanones showed unremarkable changes in ORAC values (p > 0.05). In general, the changing trend of chemical-based antioxidant activity was consistent with the content of phenolic compounds.     

Effect of addition of Agaricus blazei mushroom residue to milk enriched with Omega‐3 on the prevention of lipid oxidation and bioavailability of bioactive compounds after in vitro gastrointestinal digestion

The residue from a hydroalcoholic extract of the mushroom Agaricus blazei (MAR) was evaluated for phenolic compounds, flavonoids and antioxidant activity. The ability of MAR to slow the oxidation of Omega‐3 resulting from light exposure in milk matrix, and its bioavailability after in vitro digestion was investigated. MAR presented phenolic compounds and flavonoids and showed antioxidant activity. At each concentration, addition of MAR to Omega‐3‐supplemented milk inhibited the production of conjugated dienes and malonaldehyde compared with samples without MAR. The bioavailability assay showed that polyphenols were still present after in vitro digestion and had antioxidant activity.     

In vitro bio-accessibility and antioxidant activity of grape polyphenols

The bio-accessibility (the release of compounds from solid food matrices) of grape polyphenols using an in vitro model simulating gastro-intestinal conditions has been investigated. In vitro studies are needed to unravel factors affecting the release of antioxidants during digestion. The amount of bio-accessible polyphenols, flavonoids and anthocyanins increases during gastric digestion. The transition in the intestinal environment causes a decrease in all the analyzed classes of polyphenols followed by a renewal in the extraction of polyphenols and flavonoids but not of anthocyanins. The stability under gastro-intestinal conditions of pure phenolic acids, flavonoids and resveratrol has been analysed. Gastric digestion had no effect on any phenolic tested. Phenolic acids and resveratrol were degraded under pancreatic conditions whereas catechin and quercetin were not. Changes in antioxidant activity during digestion were correlated to the changes in polyphenols concentration as well as to the pH. Our results suggest that the gastro-intestinal tract may act as an extractor where polyphenols are progressively released from solid matrix and made available for the absorption or to exert their biological effects in the gastro-intestinal tract.     

Comparison of different methods for extracting polyphenols from Ipomoea batatas leaves,and identification of antioxidant constituents by HPLC-QTOF-MS2

This study aimed to compare the extraction efficiency of dynamic high pressure microfluidization-assisted extraction (DHPMAE) and other commonly used extraction methods on the antioxidant activities (AAs) of sweet potato leaves (Ipomoea batatas L., SPL), and to identify the antioxidant compounds by HPLC-QTOF-MS². Two highly consumed commercial varieties, orange-fleshed and cream-fleshed sweet potato leaves (OFL and CFL), were taken as materials. OFL was evidenced to be a better antioxidants resource than CFL due to higher polyphenols and AAs. DHPMAE had the best extraction efficiency on total phenolics and total flavonoids, with the values in OFL extract at 16.35 mg GAE/g DM and 16.51 mg RE/g DM, respectively. DHPMAE extract also exhibited the strongest DPPH· and ABTS·⁺ scavenging ability, chelating ability and reducing power. Multiple regression analysis revealed that polyphenols played an important contribution to the AAs of SPL extracts. After successive fractionation of DHPMAE extract by chloroform, ethyl acetate, and n-butanol, the ethyl acetate fraction showed the highest total phenolic (569.38 mg GAE/g Extract) and total flavonoid (43.15 mg RE/g Extract) content, as well as the highest AAs. HPLC-QTOF-MS² analysis of the ethyl acetate fraction resulted in identifying 37 compounds including 20 phenolic acids, 12 flavonoids, 3 organic acids, 1 nucleoside and 1 ester, and 20 of them were firstly detected in SPL. Caffeoylquinic acid derivatives and flavonoids were the most abundant antioxidant compounds. The presented results suggested that DHPM might be a promising assisted technology in extracting polyphenols from plants, and SPL could be utilized for the development of functional food.     

模拟胃肠道消化过程中迷迭香提取物总酚及抗氧化活性变化

本文研究了迷迭香提取物在模拟胃肠道消化过程中总酚含量及抗氧化活性的变化规律,并对其总酚含量变化与抗氧化活性进行相关性分析。结果表明,迷迭香提取物消化产物中总酚含量随模拟胃消化时间的延长而升高,而在模拟肠消化过程中显著降低(P<0.05)。经模拟胃消化180 min后,总酚含量显著升高了17.88%(P<0.05),而经模拟肠消化120 min后,总酚含量显著降低了19.64%(P<0.05)。经模拟胃消化180 min,消化产物对DPPH·和超氧阴离子自由基的清除能力分别显著提高了20.36%和22.07%(P<0.05),铁还原能力(Ferric ion reducing antioxidant power,FRAP)显著提高了11.76%(P<0.05)。而经模拟肠消化120 min后,消化产物对DPPH·和超氧阴离子自由基的清除能力分别显著降低了74.08%和50.00%(P<0.05),FRAP显著降低了52.13%(P<0.05)。此外,迷迭香提取物消化产物的抗氧化活性与其总酚含量之间存在良好的正相关性。以上结果表明,模拟胃肠道消化过程显著影响迷迭香多酚的含量和抗氧化活性。     

Fermented coconut jelly as a probiotic vehicle,physicochemical and microbiology characterisation during an in vitro digestion

There is little information on the survival of probiotics in plant-based foods after simulated gastric and intestinal conditions, likewise the microstructure arrangement in the no-dairy fermented food. This work aimed to study if the agar–agar in a fermented coconut jelly confers protection to probiotics, phenolic and antioxidant compounds during in vitro digestion. Samples containing higher agar–agar amounts tend to retain (P < 0.05) antioxidant and phenolic compounds in their network better, even after the in vitro digestion. Also, a compact and homogeneous microstructure was observed by the Confocal Laser Scanning Microscopy. The texture profile analysis shows that 1% of agar samples presented the maximum hardness (P < 0.05) due to more bonding points and intermolecular interactions. Finally, the survival of probiotics remained above the recommended values (10⁶–10⁷ CFU g⁻¹) after the in vitro digestion of a product with probiotic potential.     

Effects of in vitro simulated digestion on the free and bound phenolic content and antioxidant activity of seven species of seaweeds

The effects of simulated gastrointestinal digestion on the content and antioxidant activity of phenolics of seven seaweeds were investigated: Two methods of digestion were used – simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) to mimic the stomach and upper intestinal environment. Results showed that SIF can significantly increase the free phenolic content of all tested seaweeds. The total phenolic content of the seaweeds increased in range from 4.16 to 17.24 mg GAE/g before simulated digestion to 4.08 to 40.37 mg GAE/g after digestion. The antioxidant activity of seaweed-bound phenolics was superior to that of free phenolics. Among the seven varieties of seaweeds, Sargassum thunbergii and Sargassum kjellmanianum contained the highest content of bound phenolics after SGF digestion. Bound phenolics of Undaria pinnatifida and Sargassum thunbergii showed the highest in vitro ABTS⁺ free radical scavenging ability, while Sargassum thunbergii and Sargassum fusiforme showed the highest in vitro FRAP antioxidant activity.     

Metabolic and microbial modulation of phenolic compounds from raspberry leaf extract under in vitro digestion and fermentation

Raspberry leaves, by-products in raspberry production, are also a rich source of bioactive phytochemicals. In this study, the changes of phenolic compounds in raspberries leaf extract (RLE) under in vitro digestion and colonic fermentation were further studied by HPLC-MS analysis and 16S rRNA. The results showed that the phenolic compounds in RLE were relatively stable during in vitro gastric digestion; however, in the subsequent intestinal digestion and colonic fermentation, their content decreased sharply. A large amount of hydroxyphenylpropionic acid, hydroxyphenylacetic acid and urolithins were produced under the action of gut microbiota. Moreover, compared with corresponding control, RLE significantly reduced the ratio of Firmicutes/Bacteroidetes in all volunteers, increased the relative abundances of some beneficial bacteria, Enterococcus, Prevotella, and decreased the relative abundances of potential pathogens, Clostridium and Faecalibacterium. These findings suggest that RLE during in vitro digestion and fermentation has positive effects on gut microbiota and potential value of maintaining intestinal health.     

Inhibition of phenolics on the in vitro digestion of noodles from the view of phenolics release

The inhibition mechanism of buckwheat phenolics on in vitro starch digestion was investigated using extruded noodles with buckwheat starch and phenolic extract (0.50%–2.00%). The cooking quality and reducing sugar released during in vitro digestion were studied, and the extractable phenolic content along digestion was also monitored to reveal a dose–effect relationship between reducing sugar released and the release of phenolics. Noodles containing increased phenolics released less reducing sugar (230–188 mg g⁻¹) during digestion. Cooking and digestion made phenolics more extractable, and most of the phenolics were released at the end of the gastric phase (85.6%–94.8%) compared with during the small intestinal digestion. The IC₅₀ of buckwheat phenolic extract (0.102 mg mL⁻¹) was four times that of acarbose (0.032 mg mL⁻¹). The inhibitory mechanism was further analysed using molecular docking, in which the activity of α-amylase was inhibited by phenolics that bind with active sites of α-amylase through hydrogen bonds and hydrophobic interaction. Phenolics interacting with starch and the released phenolics can both explain the reduction in starch digestion.     

果皮中酚类物质含量、抗氧化活性及在体外消化过程中成分的变化

从21 种果皮中筛选出总酚、总黄酮含量较高的果皮,并选择1,1-二苯基-2-三硝基苯肼（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基、2,2’-联氨-双-3-乙基苯并噻唑啉-6-磺酸（2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid),ABTS）阳离子自由基清除力评价其抗氧化活性。对筛选出酚类物质含量高且抗氧化活性强的果皮进行体外模拟胃肠液消化,研究不同消化时间对总酚、总黄酮释放量的影响,并通过透析袋模拟肠道的吸收作用,考察其生物利用率,同时利用超高效液相色谱-二极管阵列检测器/电喷雾飞行时间质谱（ultra performance liquid chromatography-diode array detector/electrospray ionization-time of fight-mass spectrometry,UPLC-DAD/ESI-TOF-MS）研究模拟胃肠液消化前后酚类物质变化。结果表明：21 种果皮中石榴皮（总酚含量为（58.09±0.46）mg/g、总黄酮含量为（47.50±0.39）mg/g）和山竹皮（总酚含量为（52.09±1.52）mg/g、总黄酮含量为（36.07±0.46）mg/g）中酚类物质含量较高;石榴皮的抗氧化能力强于山竹皮;模拟胃液消化阶段,石榴皮中总酚和总黄酮释放量均增加,模拟肠液消化阶段总酚和总黄酮的释放量先增加后减少;与胃液消化阶段相比,肠液消化阶段总酚和总黄酮含量明显降低;生物利用率测定结果表明总酚的生物利用率在每个时间点均强于总黄酮;通过UPLC-DAD/ESI-TOF-MS从石榴皮提取物中共检测出的5 种酚类化合物（分别为α-安石榴苷、β-安石榴苷、鞣花酸己糖苷、鞣花酸脱氧己糖苷、鞣花酸）,其在胃液消化过程中损失率小于肠液消化,说明石榴皮中酚类物质在酸性环境中结构稳定。     

Stability of polyphenols in chokeberry (Aronia melanocarpa) subjected to in vitro gastric and pancreatic digestion

Berries and red fruits are dietary sources of polyphenols with reported health benefits. As part of the diet, polyphenols are ingested as complex mixtures immersed in a food matrix which is digested in the gut. Epithelial cells lining the gut are regularly exposed to these digested mixtures. To understand the effects of dietary polyphenols on human gut health it is essential to determine their stability and fate in the lumen. In this work, we investigated the effects of an in vitro gastric and pancreatic digestion on the stability and composition of the major polyphenols in chokeberry juice. Digestion was carried out with a mixture of pepsin-HCl for 2 h, followed by a 2 h incubation with pancreatin and bile salts at 37 °C with shaking, in the absence of light and under N₂. After digestion, the chokeberry samples were acidified, filtered and HPLC-DAD/HPLC–MS–MS analysed to determine the content of total soluble recovered phenolics. Gastric digestion had no substantial effect on any of the major phenolic compounds in chokeberry, namely anthocyanins, flavan-3-ols, flavonols and caffeic acid derivatives. However, these compounds were significantly altered during the pancreatic digestion and this effect was more marked for anthocyanins (approximately 43% was lost during the 2 h treatment with pancreatin). Flavonols and flavan-3-ols decreased by 26% and 19%, respectively. Neochlorogenic acid decreased by 28% whereas chlorogenic acid was increased by 24%. In vitro digestion of standard phenolic compounds, representing each of the groups of phenolics in chokeberry, confirmed some of the observed changes. Interactions with the digestive enzymes were not responsible for the observed losses which were mostly due to the chemical conditions during pancreatic digestion. Our results, in accordance with previously published results, show that dietary polyphenols are highly sensitive to the mild alkaline conditions in the small intestine and that a good proportion of these compounds can be transformed into other unknown and/or undetected structural forms with different chemical properties and, consequently, different bioaccessibility, bioavailability and biological activity.     

Stability and antioxidant activity of anthocyanins from purple sweet potato (Ipomoea batatas L. cultivar Eshu No. 8) subjected to simulated in vitro gastrointestinal digestion

The study was aimed to investigate the stability and antioxidant activities of anthocyanins obtained from purple sweet potato via an in vitro digestion system. Three fractions of anthocyanin-rich extracts were obtained via ODS packing column. Anthocyanins and copigment (primary phenolic acids) of fraction 2 were investigated during simulated an in vitro gastrointestinal digestion. Anthocyanins and copigment were recognised by UPLC-LTQ-MS/MS. Besides, anthocyanins were found effectively stable under the acidic gastric digestion conditions. However, the anthocyanins recovery was greatly decreased at around 10% after intestinal digestion. An association between the type, number of acylated group and stability to intestinal digestion was found. Di-acylated anthocyanins possessed higher stability compared with mono-acylated anthocyanins and the stability of acylated group in digestion process followed the order of p-hydroxybenzoyl > feruloyl > caffeoyl. However, there was no much difference in copigment content which was found during digestion process. Moreover, the radical scavenging ability and xanthine oxidase (XO) inhibition showed that the digestion products possessed good biological activities mainly due to its anthocyanin composition.     

Polyphenols bioaccessibility and bioavailability assessment in ipecac infusion using a combined assay of simulated in vitro digestion and Caco-2 cell model

In this report, we investigated for the first time the total polyphenols content (TPC) and antioxidant activity before and after digestion of Carapichea ipecacuanha root infusion, better known as ipecac, prepared at different concentrations. An in vitro digestion system coupled to a Caco-2 cell model was applied to study the bioavailability of antioxidant compounds. The ability of ipecac bioaccessible fractions to inhibit reactive oxygen species (ROS) generation at cellular level was also evaluated. The findings revealed that water volume of 50 mL g⁻¹ of sample provided the maximum yield of extraction of TPC and antioxidant activity. Polyphenols increased in content and activity after digestion and they were highly bioavailable (75% of intestinal absorption). Polyphenols were also present in the residual parts which indicate a possible local activity. Results also suggest that ipecac infusion could represent a promising source of effective bioavailable antioxidants to be exploited in functional foods field.     

In vitro bioaccessibility,bioavailability and plasma protein interaction of polyphenols from Annurca apple (M. pumila Miller cv Annurca)

The in vitro bioaccessibility, bioavailability and plasma protein interaction of polyphenols from Annurca apple and other conventional cultivars were evaluated. Salivary digestion concentrated into the medium 27–35% of native apple polyphenols, suggesting the potential bioavailability through the oral mucosal epithelium of significant amounts of bioactive compounds that could be gastric sensitive and/or poorly absorbed in the intestine. Annurca flesh revealed the highest content and provided the best intestinal bioaccessibility and bioavailability of oligomeric procyanidins among all of the apple peel and flesh tested. Since 49.4% of native procyanidins were not absorbed, they are expected to accumulate in the intestinal lumen where a potential inhibition capacity of cellular cholesterol uptake could be assumed. The permeated procyanidins (6.7% of their native pattern, 12.0% of intestinal procyanidins) significantly bound (58.7%) to plasma HDLs, suggesting a major role in cholesterol metabolism. Our results would indicate Annurca apple and its potential nutraceuticals as effective in the regulation of plasma cholesterol levels.     

Simulated gastrointestinal digestion and in vitro colonic fermentation of date (Phoenix dactylifera L.) seed polyphenols

Date seeds are a by‐product of date fruit industry and a rich source of polyphenols. In this study, in vitro bioaccessibility and colonic fermentation of major polyphenols from date seed powder (DSP) and DSP‐fortified yoghurt (DSPY) were investigated using HPLC. Catechin, epicatechin and procyanidin A2, B1 and B2 were stable during simulated gastric and sequential intestinal digestion. Bioaccessibility was significantly (P < 0.05) higher for all compounds from DSPY compared with DSP. After in vitro colonic fermentation of insoluble digestion materials, most of the target compounds were metabolised by faecal bacteria to ferulic acid, 3‐hydroxyphenylacetic acid, 3‐phenylpropionic acid and 3‐(4‐hydroxyphenyl) propionic acid. DSPY contained significantly (P < 0.05) higher level of free polyphenols as indicated by higher bioaccessibility; however, the stability of the polyphenols and their fermentation products from DSPY were similar to that of DSP alone. These data would be useful in product developments incorporating DSP as a source of polyphenols in food products.