Ameliorating effects of ethyl acetate fraction from onion (Allium cepa L.) flesh and peel in mice following trimethyltin-induced learning and memory impairment

The anti-amnesic effects of onion (Allium cepa L.) flesh (OF)¹ and peel (OP)² on trimethyltin (TMT)³-induced learning and memory dysfunction were investigated to confirm learning and memory function. The inhibitory effect against cellular acetylcholinesterase (AChE)⁴ showed that the EtOAc fraction of OP (EOP⁵, IC₅₀ value = 37.11 μg/mL) was higher than the EtOAc fraction of OF (EOF⁶, IC₅₀ value = 433.34 μg/mL). The cognitive effects in ICR mice were also evaluated using Y-maze, passive avoidance, and Morris water maze tests. After the behavioral tests, AChE activity (control = 100%, TMT = 128%, EOF 20 = 108%, EOP 10 = 104%, and EOP 20 = 98%), superoxide dismutase (SOD)⁷ activity, oxidized glutathione (GSSG)⁸/total glutathione (GSH)⁹ and malondialdehyde (MDA)¹⁰ production were examined. These results indicate that both EOF and EOP improved learning and memory function. The main compounds of the EOF and EOP were analyzed by Q-TOF UPLC/MS, and the results were as follows: The EOF (quercetin and quercetin-4′-glucoside) and the EOP (quercetin-4′-glucoside and isorhamnetin-4′-glucoside). Consequently, our results suggest that both EOF and EOP could be efficacious in improving cognitive function through AChE inhibition and antioxidant activity in mice brains.     

Purification and characterization of an antioxidant protein from Ginkgo biloba seeds

A new antioxidation protein named G4b was purified from Ginkgo biloba seed albumin. The results of chemical colorimetry and chemiluminescence methods showed that the hemi-inhibitory concentration (IC₅₀) of G4b on the hydroxyl radical were 78.1 μg/mL and 115.1 μg/mL, and the IC₅₀ on DPPH was 100.7 μg/mL, while the IC₅₀ on the superoxide anion and DNA injury were 23 μg/mL and 27 μg/mL, respectively, and the abundance of sulfur amino acids and aromatic amino acids contained in G4b might contribute to its strong antioxidation properties. G4b is a kind of homogeneous and novel protein with a molecular mass of 29,247 Da, consisting of two peptides with similar molecular weights linked by a disulfide bond. Result of β-elimination reaction showed that G4b is connected with a small quantity of polysaccharide by an oxygen-glucosidic bond.     

Cyanidin-3-glucoside,nutritionally important constituents and in vitro antioxidant activities of Santalum album L. berries

Anthocyanins are ubiquitous plant pigments. They are hydrophilic, and have high tinctorial value hence, used as natural food colorant. The main aim of this study was characterization of Santalum album L. berries for its pigment content, nutritionally important phytoconstituents and functional attributes. Major pigment identified was cyanidin-3-glucoside (0.21% FW) as confirmed by spectral characteristics including LC–MS. Nutrients such as total carbohydrates (8.5), proteins (5.8), free amino acids (0.8 g), oil (1.5 g), ascorbic acid (1.5 μg), tocopherols (0.3 mg) and niacin (5.2 mg) per 100 g berries were determined along with total soluble solids (13.4°Brix) and phenols (3.1 mg GAE/g). Reducing power and DPPH· scavenging assays showed highest activity in methanol extract, which also efficiently protected bleaching of β-carotene (EC₅₀ 285 μg/mL) in β-carotene–linoleate model. Pigment rich crude extract was not toxic to HepG2 cells during 24 h exposure, whereas cyanidin-3-glucoside was cytotoxic (IC₅₀ 0.1 μg/mL).     

Antioxidant properties and polyphenolics composition of common hedge hyssop (Gratiola officinalis L.)

The antioxidant potential of Gratiola officinalis was evaluated by the off-line and on-line HPLC/UV/DPPH radical scavenging assays, phytochemical composition was analyzed by LC/MS. On-line method was validated by using reference antioxidants and linear dependence was found between their concentration and radical scavenging peak area. Radical scavenging capacity (RSC) of methanol and acetone extracts expressed in their concentration required to scavenge 50% of DPPH^? was 0.10% and 0.13%, respectively; the RSC in ABTS^?+ assay was 1093 ± 104 and 746 ± 18 μM of trolox equivalents in 1 g, respectively. Good correlation was observed between total amount of phenolic compounds and RSC. Preliminary HPLC/UV/MS analysis revealed that the main compounds possessing antioxidant activity in the extracts might be phenylpropanoid glycosides; UPLC/UV/ESI-QTOF-MS analysis suggested 15 structures: 2,5-dihydroxy-p-benzenediacetic and caffeic acids, apigenin 6,8-di-C-β-d-glucopyranoside (vicenin-2), apigenin 8-C-α-l-arabinoside 6-C-β-d-glucoside, (shaftoside), forsythoside B, arenarioside, verbascoside (acteoside), amioside, quercetin-6-O-(2-O-acetyl-glucopyranosyl)-glucopyranoside, isoverbascoside, quercetin glucuronide, linariifolioside, methoxy luteolin-7-O-(6-O-acetyl-glucopyranosyl)-glucopyranoside, methoxy luteolin-glucuronide and luteolin glucuronide.     

Production of nutraceutics from chestnut burs by hydrolytic treatment

Chestnut (Castanea sativa) burs (CB) were processed by solvent extraction (with ethanol, toluene–ethanol or n-hexane) and/or isothermal autohydrolysis, and the potential as nutraceutics of the solubilized fractions of dietary fiber and antioxidants was evaluated. Among the studied alternatives, the most suited consisted on ethanol extraction, which yielded 21.0 g gallic acid equivalents (GAE)/100 g ethanolic CB extract, with EC_50,DPPH = 0.38 g/L. Ethanol-extracted CB were treated with water at 120 °C, and the soluble fraction was mixed with ethanol to precipitate 8.8 g soluble fiber/100 g CB, with a Water Retention Capacity of 2.4 g/g and an oil absorption capacity of 23.7 g/g. The mother liquors from the above precipitation stage were concentrated and extracted with ethyl acetate to yield an organic phase containing soluble phenolics with high antioxidant activity (EC_50,DPPH = 0.103 g/L) at a yield of 7 g/100 g CB. When the aqueous phase before the ethyl acetate extraction was treated with sulfuric acid, an additional antioxidant fraction (EC_50,DPPH = 0.395 g/L) was obtained. The properties and characteristics of these extracts produced make them good candidates to be used as active ingredients.     

High molecular weight persimmon tannin is a potent antioxidant both ex vivo and in vivo

The antioxidant activities of high molecular weight persimmon condensed tannin (HMWPT) were evaluated in an ex vivo tissue system and in vivo. Addition of HMWPT to mouse liver homogenate protected the samples against auto-peroxidation or H₂O₂- or Fe²⁺/ascorbic acid-induced lipid peroxidation. The IC₅₀ values for HMWPT were 4.32 ± 0.20 μg/mL (auto-oxidation), 1.36 ± 0.40 μg/mL (H₂O₂-induced peroxidation) or 0.20 ± 0.09 mg/mL (Fe²⁺/ascorbic acid-induced peroxidation). Mice were oxidatively stressed with bromobenzene to test the antioxidant activity of HMWPT in vivo. An oral dose of HMWPT at 200 or 400 mg/kg HMWPT significantly (P < 0.01) prevented the bromobenzene-induced decrease in serum and liver superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, and decreased liver malondialdehyde levels in bromobenzene-treated mice (P < 0.01). The results suggest that dietary HMWPT may provide protection from oxidative damage both ex vivo and in vivo.     

Purification and characterization of angiotensin I-converting enzyme inhibitory peptides of small red bean (Phaseolus vulgaris) hydrolysates

Angiotensin I-converting enzyme (ACE) inhibitory activity was investigated for small red bean (Phaseolus vulgaris) protein hydrolysate produced by sequential digestion of Alcalase, papain followed by in vitro gastrointestinal simulation. The hydrolysate had ACE inhibitory activity with IC₅₀ of 67.2 ± 1.8 μg protein/mL. Peptides responsible for potent ACE inhibitory activity were isolated by a three-step purification process, including ultrafiltration, gel filtration and preparative reverse phase high performance chromatography (RP-HPLC). The fraction obtained after RP-HPLC fractionation with the highest activity yielded an IC₅₀ of 19.3 ± 1.4 μg protein/mL. Enzymatic kinetic studies using this fraction demonstrated competitive inhibition with K_i of 11.6 ± 1.7 μg protein/mL. Mass spectrometric characterization identified for the first time the octapeptide PVNNPQIH which demonstrated an IC₅₀ value of 206.7 ± 3.9 μM. The results expand the knowledge base of ACE inhibitory properties of small red bean protein hydrolysate and should be useful in further identification of specific ACE inhibitory peptides in beans.     

Effect of germination and elicitation on phenolic composition and bioactivity of kidney beans

Ready-to-eat sprouts are becoming popular healthy fresh foods. Germination and elicitation may be used to improve phytochemical and functional quality of sprouts. The aim of this study was to unveil the efficacy of germination and the use of elicitors (500 μM ascorbic, 50 μM folic and 5 mM glutamic acid) to enhance the phenolic composition and antioxidant and angiotensin I converting enzyme (ACE) inhibitory activities of 8 day-old Phaseolus vulgaris L. var. Pinto sprouts. Sprouting produced a general decrease in flavan-3-ols and anthocyanins of sprouts that was compensated with a higher content of flavanones and flavonols. Although elicitor treatments reduced total phenolic content of sprouts, they promoted the synthesis of specific flavanones and flavonol O-glycosides, effect that was dependent on the type of elicitor. Antioxidant activity was not affected by ascorbic and folic acid treatments whereas it was slightly reduced in glutamic acid-treated sprouts. Folic acid was the only treatment that caused an outstanding increase in the ACE inhibitory activity of sprouts. In conclusion, elicitation may produce positive changes in the phenolic profile and improve health-promoting potential of sprouts, although selection of elicitor is crucial to deliver marketplace ready to eat sprouts enriched in specific bioactive phytochemicals.     

Magnoflorine from Tinospora cordifolia stem inhibits α-glucosidase and is antiglycemic in rats

Antidiabetic potential of Tinospora cordifolia stem is well proven. In the course of screening of useful α-glucosidase inhibitors, we prepared alkaloid fraction (AFTC) and isolated three isoquinoline alkaloids, namely, jatrorrhizine, palmatine and magnoflorine as active candidates for α-glucosidase inhibition. The enzyme kinetics was studied using sucrose and maltose as substrates. Michaelis–Menten constant (K_m) and maximal velocity (V_max) values were estimated. A significant decrease in V_max and unaltered K_m was observed in case of jatrorrhizine and palmatine (non-competitive inhibition). Magnoflorine was found to increase apparent K_m and shown to be reversible, competitive inhibition. The IC₅₀ value as sucrase inhibitor was 36.25, 23.46 and 9.8 μg/mL for jatrorrhizine, palmatine and magnoflorine, respectively, and as maltase inhibitor was 22.05, 38.42 and 7.6 μg/mL for jatrorrhizine, palmatine and magnoflorine, respectively. In vivo studies were conducted on rats to determine oral glucose tolerance test (OGTT), using different substrates: glucose, sucrose and maltose. The increase in plasma glucose level was significantly suppressed (P < 0.01) by all the three alkaloids at 20 mg/kg b.w. Magnoflorine possessed the most potential activity as α-glucosidase inhibitor in vitro and in vivo.     

NMR-based quantification of rosmarinic and carnosic acids,GC–MS profile and bioactivity relevant to neurodegenerative disorders of Rosmarinus officinalis L. extracts

A comparative study of phytochemicals content and biological properties of eight Rosmarinus officinalis (rosemary) populations (RO1–RO8) collected in different areas of Tunisia was carried out. Two of the main rosemary constituents, rosmarinic and carnosic acids, were quantified by an NMR technique. Carnosic acid content was higher than that of rosmarinic acid. The non-polar constituents were examined by GC and GC–MS. Total phenols and flavonoids content were also determined in order to discuss the possible correlation between these phytochemicals and bioactivity. Antioxidant activity was investigated through different in vitro assays. Sample RO3 from a sub-humid area showed the highest potency in 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay (IC₅₀ of 6.74 μg/mL) while RO5 from arid area exhibited the highest radical 2,2′-azino-bis-3-ethylbenzthiazoline-6-sulphonic acid (ABTS) scavenging activity (IC₅₀ of 5.92 μg/mL). The most active sample in Ferric Reducing Antioxidant Power (FRAP) assay was RO4 (62.21 μM Fe(II)/g), followed by RO1–RO3 and RO8 harvested in sub-humid areas with values in the range 49.61–58.72 μM Fe(II)/g. Extracts were able to inhibit acethylcholinesterase (AChE) and butyrylcholinesterase (BChE). Sample RO5 exhibited a promising AChE inhibitory activity (IC₅₀ of 4.47 μg/mL). Thus, we suggest rosemary extracts as a functional food ingredient for preventing AD.     

α-Glucosidase and α-amylase inhibitory constituent of Carex baccans: Bio-assay guided isolation and quantification by validated RP-HPLC–DAD

Carex baccans is used extensively as food additive for its medicinal and nutritional properties. Its extract demonstrated significant inhibition of α-glucosidase and α-amylase with IC₅₀ 43.32 ± 1.22 and 562.18 ± 5.98 μg/mL, respectively. A bio-assay guided approach was employed to identify the active constituent(s). Fractionation and purification of the extract led to the isolation of a potent inhibitor, (+)-α-viniferin, and of weak inhibitors smiglasides A and B. (+)-α-Viniferin was quantified in the extract and fractions using HPLC and the method was validated for linearity, limit of detection, limit of quantification, precision, and accuracy. The calibration curve showed good linearity (r² > 0.999) in the range of 7.813–250 μg/mL. The identification of α-glucosidase and α-amylase inhibitory activity in C. baccans supports the possible use of the plant as functional food for the management of diabetes. The validated HPLC method for the study of plant extracts will be useful in future research.     

Licochalcone A: A lipase inhibitor from the roots of Glycyrrhiza uralensis

In order to develop a functional food and anti-obesity drug through the inhibition of absorption of dietary lipids, we investigated the inhibitory effects on pancreatic lipase of extracts from more than 800 species of herbs in Korea. In this study, licochalcone A was isolated from the ethyl acetate/n-hexane fraction of ethyl acetate extract of the roots of Glycyrrhiza uralensis. Its structure was elucidated by ¹H NMR, ¹³C NMR and HR-EI mass spectroscopy. Licochalcone A substantially inhibited activity with IC₅₀ value of 35 μg/mL (103.4 μM). In an investigation of reversibility, licochalcone A was shown to be reversible to pancreatic lipase. The inhibition mode of licochalcone A was a non-competitive inhibitor, determined by Lineweaver–Burk plot analysis, and K_i value was 11.2 μg/mL (32.8 μM). Furthermore, licochalcone A significantly reduced the production of oleic acid with not only artificial substrate DNPB but natural substrate triolein by pancreatic lipase.     

The impact of high pressure on glucosinolate profile and myrosinase activity in seedlings from Brussels sprouts

The potential of high pressure (HP) to control bioactive components using seedlings of Brussels sprouts as a simple non-chopped vegetable system was examined. Enzyme activity in situ compared to purified enzyme and residual enzyme substrate in situ are used as three complementary measures for the HP effect. Purified myrosinase and seedlings of Brussels sprouts were submitted to HP 200–800 MPa at 5 °C for 3 min. The myrosinase activity decreased for both myrosinase systems upon increasing pressure to 800 MPa. Applying first-order kinetic to determine activation volumes revealed a linear relationship from 400 to 600 (ΔV^# = − 19.04 mL/mol) and 450–600 MPa (ΔV^# = − 37.79 mL/mol) for seedlings and purified myrosinase, respectively, indicating a protective effect of the plant matrix against enzyme inactivation. Purified myrosinase was activated at 200 MPa but at 800 MPa the glucosinolate degradation due to pressure induced disruption of the plant matrix seems to be partly counter-acted by myrosinase inactivation.Industrial relevanceHigh Pressure (HP) processing is an effective non-thermal preservation treatment for liquid and solid food. Moreover, over the last years, the potential of this technology to improve health and safety attributes of foods has been demonstrated. In particular, the ability of HP to preserve bioactive compounds has been established. There are only few studies evaluating the impact of HP on the complex bioactive glucosinolates-myrosinase. Therefore, this study opens the doors through the application of HP to preserve the bioactive glucosinolates in cruciferous vegetables by creating new processing solutions through controlled enzyme inactivation. Thus, HP could be an effective tool to achieve more effective solutions to obtain the new generation of convenient food and meet the need for new bioactive food products.     

Release of multifunctional peptides by gastrointestinal digestion of sea cucumber (Isostichopus badionotus)

Sea cucumber is a benthic marine organism distributed worldwide and used as food in several Asian countries. The species Isostichopus badionotus is captured intensively off the Yucatan Peninsula, Mexico. Boiled I. badionotus was subjected to in vitro simulated gastrointestinal digestion using pepsin and a pepsin–Corolase PP^® mixture. ACE-inhibitory and radical scavenging activities, iron reducing capacity and cytotoxic effects against colorectal cancer cells were evaluated in the hydrolysates and their ultrafiltered fractions. ACE-inhibitory activity was potent in fractions containing peptides <3000 Da, an effect augmented with combined action of gastric (pepsin) and intestinal (Corolase PP^®) enzymes (IC₅₀ = 0.038 ± 0.004 mg/mL). Antioxidant activity was exerted by peptides with low and high molecular weights, depending on hydrolysis method. This is the first report of cytotoxic capacity against colorectal HT-29 cells in peptides from sea cucumber. Sea cucumber hydrolysates and ultrafiltered fractions are potential ingredients for development of functional foods.     

Radical scavenging activity of black currant (Ribes nigrum L.) extract and its inhibitory effect on gastric cancer cell proliferation via induction of apoptosis

A black currant extract (BCE) was prepared and its antiproliferative activity against gastric cancer SGC-7901 cells was investigated. Strong 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) radical scavenging activities and a high reducing power were confirmed with BCE. BCE inhibited the proliferation of SGC-7901 cells in a dose- and time-dependent manner, and the IC₅₀ were 12.7, 10.2 and 9.0 mg/mL for 12, 24 and 48 h, respectively. Morphologic observations with inverted and fluorescence microscopes yielded vivid evidence of cell shrinkage, formation of cytoplasmic filaments, condensation of nuclear chromatin, and cell apoptosis in the presence of BCE. Flow cytometric analysis also showed that BCE treatment at concentrations of 10–20 mg/mL resulted in marked reductions of viable cells. The high concentration of phenolic compounds present in the BCE (12.2 mg/mL), including six prominent anthocyanins identified by HPLC–ESI-MS², appeared to be responsible for BCE’s antiradical activity and anticancer effects. These findings of inhibition of SGC-7901 cells and induction of apoptosis suggest that black currant may contribute to the reduction in gastric cancer risk.     

Phenolic profile and antioxidant activity of the Algerian ripe date palm fruit (Phoenix dactylifera)

The aim of this study was to determine the phenolic profile of seven different varieties of ripe date palm fruit (Phoenix dactylifera) from Algeria by LC–DAD–MS (ESI+), to investigate their respective antioxidant activities by the DPPH^· method and to estimate their phenolic content using the Folin–Ciocalteu method. The total phenolic content was in the range of 2.49 ± 0.01 to 8.36 ± 0.60 mg gallic acid equivalents (GAE) per 100 g fresh fruit. This fruit was shown to possess an antioxidant activity, giving values of antiradical efficient (AE) from 0.08 ± 0.00 to 0.22 ± 0.00. The phenolic contents and the antiradical efficiencies of the different varieties were highly correlated (R²=0.975). All the varieties were found to contain mainly p-coumaric, ferulic and sinapic acids and some cinnamic acid derivatives. Three different isomers of 5-o-caffeoylshikimic acid were detected. Different types of flavonoids were identified, mainly flavones, flavanones and flavonol glycosides.     

Nondestructive estimation of fatty acid composition in soybean [Glycine max (L.) Merrill] seeds using Near-Infrared Transmittance Spectroscopy

The potential of Near-Infrared Transmittance (NIT) Spectroscopy for estimation of fatty acid composition in soybean seed samples was studied. Total 612 whole seed samples with wide range of variability for major fatty acids were used to develop calibration equations by applying SNV de-trend and first derivative mathematical treatment in the range of 850–1048 nm. Useful chemometric models for most important fatty acids present in soybean seed oil were developed using Modified Partial Least Squares (MPLS) regression method. In external validation oleic (r² = 0.89, SEP = 1.61), linoleic (r² = 0.86, SEP = 1.50) and palmitic (r² = 0.89, SEP = 0.17) acids were predicted with good accuracy, while the predictions for linolenic acid (r² = 0.78, SEP = 0.36) and stearic acid (r² = 0.63, SEP = 0.11) had relatively poor accuracy. The whole-seed NIT spectroscopy equations for fatty acid estimation would be useful for improving efficiency of breeding programs aimed at altering fatty acid composition in soybean.     

Cocoa brew inhibits in vitro α-glucosidase activity: The role of polyphenols and high molecular weight compounds

Cocoa brew showed a dose-dependent inhibitory activity against α-glucosidase (IC₅₀ 7.87 mg/mL). The cocoa brew was fractionated by ultrafiltration in a low molecular weight fraction (LMW < 10 kDa) and two melanoidin-rich fractions called high molecular weight (HMW > 30 kDa) and intermediate molecular weight (IMW 10–30 kDa) fractions. All fractions tested caused some inhibition with IMW that was the most active (IC₅₀ 2.37 mg/mL). LMW fraction was separated with Sephadex LH-20 in an unbound (containing monomeric and dimeric catechins) and a bound fraction. All the inhibitory activity was recovered in the unbound fraction. All the phenolic compounds identified with HPLC showed α-glucosidase inhibitory activity. IMW and HMW fractions were fractionated by ethanol precipitation. The fractions from IMW precipitated with 75 and 25% ethanol were found to contain power inhibitors of α-glucosidase activity (IC₅₀ 0.87 and 1.01 mg/mL, respectively). In the HMW sample, the fractions precipitated with 50 and 75% ethanol were found to be active against α-glucosidase activity. Most of the inhibitory activity against α-glucosidase of cocoa brew was due to the LMW fraction (56%) whereas IMW and HMW contributed for about 47% of the inhibitory activity. This study suggests that different components of cocoa may influence α-glucosidase activity.     

Air-dried capsicum annuum var. acuminatum medium and big: Determination of bioactive constituents,antioxidant activity and carbohydrate-hydrolyzing enzymes inhibition

In the present study the total phenols, flavonoids, carotenoids and capsaicinoids content, the in vitro antioxidant and hypoglycemic activities of extracts of air-dried fruits from two Capsicum annuum cultivars were investigated. A different composition between the two cultivars was evidenced. C. annuum var. acuminatum medium extract presented a major content of phenols, carotenoids, capsaicin and dihydrocapsaicin while C. annuum var. acuminatum big extract is characterized by the highest quercetin, luteolin and kaempferol content with 68.0, 87.6 and 29.7 μg/g dried weight, respectively, analyzed by HPLC. Medium pepper showed the highest radical scavenging activity in DPPH assay (IC₅₀ of 85.3 μg/ml) while big pepper have an interesting activity in ABTS assay (IC₅₀ of 16.4 μg/ml) and the highest inhibition of linoleic acid oxidation with an IC₅₀ value of 1.2 μg/ml after 30 min of incubation. A selective inhibitory activity against α-amylase was demonstrated for C. annuum var. acuminatum big lipophilic fraction (IC₅₀ values of 8.7 μg/ml). The obtained results suggest that C. annuum cultivars could be used as valuable flavor with functional properties for foods.     

Podocarpane,isopimarane, and abietane diterpenoids from Isodon lophanthoides var. graciliflorus

Four new diterpenoids including two podocarpanes, graciliflorins A (1) and B (2), an isopimarane acetal, graciliflorin C (3), and a rearranged abietane, graciliflorin D (4) were isolated from the aerial parts of Isodon lophanthoides var. graciliflorus (Lamiaceae) along with podocarpa-8,11,13-triene-3α,13-diol (5) and micranthin B (6). Their structures were elucidated based on the spectroscopic data. The in vitro cytotoxicity of compounds 1–4 and 6 against human carcinoma A549, MCF-7, and HeLa cell lines were evaluated using the MTT colourimetric assay. Micranthin B (6) showed moderate activity against all the cells with IC₅₀ values of 16.29, 18.20, and 22.25 μM, while compounds 1–4 were inactive (IC₅₀ > 50 μg/ml).