The Antioxidant Activity of Prenylflavonoids

Prenylated flavonoids combine the flavonoid moiety and the lipophilic prenyl side-chain. A great number of derivatives belonging to the class of chalcones, flavones, flavanones, isoflavones and other complex structures possessing different prenylation patterns have been studied in the past two decades for their potential as antioxidant agents. In this review, current knowledge on the natural occurrence and structural characteristics of both natural and synthetic derivatives was compiled. An exhaustive survey on the methods used to evaluate the antioxidant potential of these prenylflavonoids and the main results obtained were also presented and discussed. Whenever possible, structure-activity relationships were explored.


Introduction
Flavonoids are oxygen heterocyclic compounds widespread throughout the plant kingdom. This class of secondary metabolites are responsible for the color and aroma of many flowers, fruits, medicinal plants and plant-derived beverages and play an important protective role in plants against different biotic and abiotic stresses. Flavonoids are also known for their nutritional value and positive therapeutic effects on humans and animals [1,2].
The basic structure of a flavonoid consists of a dibenzo-γ-pyrone framework and the degree of unsaturation and oxidation of the C ring defines the subclasses of this compounds such as chalcones, flavones, isoflavones and their dehydro derivatives. Different substitution patterns that includes hydroxyl, methyl, methoxyl, prenyl and glycosyl groups can be attached to the flavonoid unit, varying the number, type and position of the substituents [3].
Over the past two decades there have been an increasing number of reports on the isolation of prenylated flavonoids, belonging to the Leguminosae and Moraceae families, with some distribution among others such as Cannabaceae, Euphorbiaceae, Guttiferae, Rutaceae, Umbelliferae, etc. [4,5]. Barron and Ibrahim reviewed more than 700 prenylated flavonoids up to the end of 1994 [5] and Botta et al. compiled it from 1995 till 2004 [6]. Most of the prenylated flavonoids have been identified as chalcones, dihydrochalcones, flavones, flavanones, flavonols and isoflavones, being C-prenyl more common than O-prenyl derivatives. In addition, prenyl side-chains can include variations in the number of carbons, oxidation, dehydration, cyclization or reduction to give a huge array of compounds with an impressive antioxidant potential [4].
Along the manuscript, "prenyl" will be used a general term to identify prenyl/isopentenyl, geranyl and farnesyl side chains as well as furano or dimethylchromano derivatives.
The beneficial effects of flavonoids appear to be related to the various biological and pharmacological activities as anti-inflammatory, antimicrobial, antioxidant, antitumor, estrogenic, flavonoids can induce an increase in their bioactivities namely as antimicrobial and anticancer agents, however, a decrease in the bioavailability and plasma absorption is recorded, when compared to related non-prenylated derivatives [7][8][9][10][11].
The available information concerning the antioxidant activity of prenylated flavonoids is sparse, appearing some studies in a couple of review papers [7,8]. Taking into account our both interest (organic and medicinal chemistry) for the identification of prenylated flavonoids that has already been tested for their antioxidant activity, a systematic revision of the literature was made using PubMed® and Web of Knowledge ® databases. The research was limited to the 21st century, with publications from January 2000 to October 2019, using the terms "prenyl", "flavonoid", "prenylated flavonoid" in combination with "antioxidant", as keywords. After a careful analysis of nearly one hundred of papers, we excluded many of them due to the absence of the antioxidant effects for the target compounds, leaving 59 papers to be included and discussed in the present review.
Herein, it is our propose to organize and summarize the structural and chemical diversity of natural and synthetic flavonoids applied as antioxidants, providing the main in vitro and in vivo methodologies used in this field of research. This information is very useful for organic chemists to know the trends of structure-antioxidant activity relationship, in order to develop efficient routes for the total synthesis of natural derivatives, to develop improved strategies to maximize the synthesis of such natural and synthetic compounds or even in the design and synthesis of novel flavonoids with different prenyl substituents in different positions of the main skeleton. So, the studies here summarized and the promising results obtained highlights the importance of prenylated flavonoids as potential antioxidant agents.

Natural Occurrence and Structural Variation of Prenylflavonoids with Antioxidant Activity
From the natural prenylflavonoids with antioxidant activity most of them are from Moraceae and Fabaceae families with a limited number of derivatives from Apiaceae, Asteraceae, Cannabaceae, and Euphorbiaceae.
All the natural prenylflavones with antioxidant activity are C-substituted, being most of them mono-( Figure 4) and di-prenylated ( Figure 5). There is a single case of a triprenylated derivative, artelastoheterol (57), isolated from Artocarpus elasticus (Table 3 and Figure 6) [34].

Methods for the Evaluation of the Antioxidant Activity of Prenylflavonoids
Various methods have been applied to study the antioxidant properties of a wide variety natural and synthetic prenylated flavonoids. For the in vitro methods, the most common ones are those involving electron transfer mechanisms such as DPPH, FRAP and TEAC assays; hydrogen atom transfer mechanisms such as for the inhibition of ROS and RNS scavenging assays and metal chelation studies. In the former case, DPPH radical scavenging method is by far the most frequently used, probably due to its simplicity in terms of time effort, experimental procedure and cheap reagents. Considering the in vivo models, two methods were used to evaluate the antioxidant potential of several prenylated flavonoids that include lipid peroxidation assay and LDL oxidation assay.

Electron Transfer Mechanisms
DPPH Radical Scavenging Activity is a stable free radical characterized by an absorption band at about 517 nm. In the presence of an antioxidant molecule (AH), DPPH • trap a hydrogen atom to its reduced hydrazine form with consequent loss of the typical purple colour to a pale yellow one (Scheme 1).

Methods for the Evaluation of the Antioxidant Activity of Prenylflavonoids
Various methods have been applied to study the antioxidant properties of a wide variety natural and synthetic prenylated flavonoids. For the in vitro methods, the most common ones are those involving electron transfer mechanisms such as DPPH, FRAP and TEAC assays; hydrogen atom transfer mechanisms such as for the inhibition of ROS and RNS scavenging assays and metal chelation studies. In the former case, DPPH radical scavenging method is by far the most frequently used, probably due to its simplicity in terms of time effort, experimental procedure and cheap reagents. Considering the in vivo models, two methods were used to evaluate the antioxidant potential of several prenylated flavonoids that include lipid peroxidation assay and LDL oxidation assay.

Electron Transfer Mechanisms
DPPH Radical Scavenging Activity is a stable free radical characterized by an absorption band at about 517 nm. In the presence of an antioxidant molecule (AH), DPPH • trap a hydrogen atom to its reduced hydrazine form with consequent loss of the typical purple colour to a pale yellow one (Scheme 1). Scheme 1. Reaction scheme involved in DPPH radical scavenging activity assay.
The percentage of the DPPH • scavenging is calculated according to the following Equation (1): where Acontrol is the absorbance of the control (before the reaction take place) and Asample is the absorbance after the reaction occurred [56]. The percentage of the DPPH • scavenging is calculated according to the following Equation (1): where A control is the absorbance of the control (before the reaction take place) and A sample is the absorbance after the reaction occurred [56]. The activity is expressed as inhibitory concentration IC 50 , that is the amount of antioxidant necessary to decrease by 50% the initial DPPH • concentration. Some disadvantages of this method is the steric accessibility of the radical by large antioxidant molecules, spectrophotometric measurements can be affected by compounds that absorb at the same wavelength of the determination and cannot be applied for measuring the antioxidant capacity of plasma since precipitation of proteins may occur in alcoholic media.
ABTS Radical Cation Scavenging Activity ABTS •+ (2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) is a stable blue-green chromophore radical cation characterized by an absorption band at about 750 nm which losses its colour in the presence of an antioxidant molecule (Scheme 2). ABTS •+ is generated by reacting a strong oxidizing agent (e.g., potassium permanganate or potassium persulfate) with the ABTS salt. In this assay, Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) is usually used as antioxidant standard. The obtained results are expressed as Trolox equivalent antioxidant capacity (TEAC) values from the trolox standard curve [56]. This methodology can be applied to measure both hydrophilic and lipophilic antioxidant capacities since the ABTS •+ is soluble in both aqueous and organic solvents and is not affected by ionic strength of the medium.

Ferric Reducing Antioxidant Power (FRAP) Method
This method is based on the reduction of the colourless complex of ferric iron and 2,3,5triphenyl-1,3,4-triaza-2-azoniacyclopenta-1,4-diene chloride (TPTZ) to the blue-coloured ferrous form at low pH (Scheme 3). FRAP reagent is produced by mixing acetate buffer (pH 3.6), TPTZ solution and FeCl3 6H2O. FRAP values are obtained by comparing the absorbance change at 593 nm in reaction mixtures with those containing ferrous ions in known concentration [17,53]. The redox potential of Fe(III) salt (−0.70 V) is comparable to that of ABTS •+ (0.68 V), therefore, the main difference between TEAC assay and the FRAP assay is that the first is carried out at neutral pH and latest under acidic (pH 3.6) conditions. Usually a calibration curve using antioxidant trolox is made and the results are expressed as trolox equivalents per kg (solid food) or per L (beverages) of sample [56]. Scheme 2. Reaction scheme involved in ABTS radical cation scavenging activity assay.

Ferric Reducing Antioxidant Power (FRAP) Method
This method is based on the reduction of the colourless complex of ferric iron and 2,3,5-triphenyl-1,3,4-triaza-2-azoniacyclopenta-1,4-diene chloride (TPTZ) to the blue-coloured ferrous form at low pH (Scheme 3). FRAP reagent is produced by mixing acetate buffer (pH 3.6), TPTZ solution and FeCl 3 6H 2 O. FRAP values are obtained by comparing the absorbance change at 593 nm in reaction mixtures with those containing ferrous ions in known concentration [17,53]. The redox potential of Fe(III) salt (−0.70 V) is comparable to that of ABTS •+ (0.68 V), therefore, the main difference between TEAC assay and the FRAP assay is that the first is carried out at neutral pH and latest under acidic (pH 3.6) conditions. Usually a calibration curve using antioxidant trolox is made and the results are expressed as trolox equivalents per kg (solid food) or per L (beverages) of sample [56]. solution and FeCl3 6H2O. FRAP values are obtained by comparing the absorbance change at 593 nm in reaction mixtures with those containing ferrous ions in known concentration [17,53]. The redox potential of Fe(III) salt (−0.70 V) is comparable to that of ABTS •+ (0.68 V), therefore, the main difference between TEAC assay and the FRAP assay is that the first is carried out at neutral pH and latest under acidic (pH 3.6) conditions. Usually a calibration curve using antioxidant trolox is made and the results are expressed as trolox equivalents per kg (solid food) or per L (beverages) of sample [56].

Hydrogen Atom Transfer Mechanisms
Superoxide Radical Anion Scavenging Activity Superoxide radical anion (O2 •− ) can be generated by two different approaches, using hypoxanthine or xanthine/xanthine oxidase (XOD) system at pH 7.4 [14,26,35] or using a nonenzymatic reaction of phenazine methosulphate (PMS) in the presence of nicotinamide adenine dinucleotide (NADH) [56] (Scheme 4). In both systems, superoxide anion radicals can reduce nitroblue tetrazolium (NBT) into formazan, and the effects are determined spectrophotometrically at 560 nm. Higher is the scavenging potential of the antioxidant molecule, lower is the formation of formazan and consequently, lower is the absorbance [56]. Other detectors can be used, being cytochrome c the second option, which reduction is followed spectrophotometrically at 550 nm [31,32]. The results are typically expressed as inhibitory concentration IC50 values. •− ) can be generated by two different approaches, using hypoxanthine or xanthine/xanthine oxidase (XOD) system at pH 7.4 [14,26,35] or using a non-enzymatic reaction of phenazine methosulphate (PMS) in the presence of nicotinamide adenine dinucleotide (NADH) [56] (Scheme 4). In both systems, superoxide anion radicals can reduce nitroblue tetrazolium (NBT) into formazan, and the effects are determined spectrophotometrically at 560 nm. Higher is the scavenging potential of the antioxidant molecule, lower is the formation of formazan and consequently, lower is the absorbance [56]. Other detectors can be used, being cytochrome c the second option, which reduction is followed spectrophotometrically at 550 nm [31,32]. The results are typically expressed as inhibitory concentration IC 50 values. The ORAC method can be applied to both hydrophilic and lipophilic environments and for the detection of both hydroxyl and peroxyl radicals, formed during lipid oxidation chain reactions (autoxidation) and involving hydrogen atom transfer reactions.
The system H2O2-CuSO4 is generally used as hydroxyl radical generator and β-phycoerythrin used as a redox-sensitive fluorescent indicator protein, which decay in the fluorescence is measured in the presence of free radical scavengers, using Trolox as standard (Scheme 5). The ORAC value is then calculated from the trolox equivalent and expressed as ORAC units or value by taking the difference of areas-under-the-decay curves between blank and sample and/or standard. Higher the ORAC value, higher the antioxidant potential of the tested compounds [56].

Oxygen Radical Absorbance Capacity (ORAC) Method
The ORAC method can be applied to both hydrophilic and lipophilic environments and for the detection of both hydroxyl and peroxyl radicals, formed during lipid oxidation chain reactions (autoxidation) and involving hydrogen atom transfer reactions.
The system H 2 O 2 -CuSO 4 is generally used as hydroxyl radical generator and β-phycoerythrin used as a redox-sensitive fluorescent indicator protein, which decay in the fluorescence is measured in the presence of free radical scavengers, using Trolox as standard (Scheme 5). The ORAC value is then calculated from the trolox equivalent and expressed as ORAC units or value by taking the difference of areas-under-the-decay curves between blank and sample and/or standard. Higher the ORAC value, higher the antioxidant potential of the tested compounds [56].

Oxygen Radical Absorbance Capacity (ORAC) Method
The ORAC method can be applied to both hydrophilic and lipophilic environments and for the detection of both hydroxyl and peroxyl radicals, formed during lipid oxidation chain reactions (autoxidation) and involving hydrogen atom transfer reactions.
The system H2O2-CuSO4 is generally used as hydroxyl radical generator and β-phycoerythrin used as a redox-sensitive fluorescent indicator protein, which decay in the fluorescence is measured in the presence of free radical scavengers, using Trolox as standard (Scheme 5). The ORAC value is then calculated from the trolox equivalent and expressed as ORAC units or value by taking the difference of areas-under-the-decay curves between blank and sample and/or standard. Higher the ORAC value, higher the antioxidant potential of the tested compounds [56]. Scheme 5. Reaction schemes involved in ORAC assay for the detection of hydroxyl and peroxyl radicals.

Scheme 5.
Reaction schemes involved in ORAC assay for the detection of hydroxyl and peroxyl radicals.
2,2 -Azobis-(2-amidinopropane)dihydrochloride (AAPH) is the most used peroxyl radical generator in hydrophilic systems [24] and as fluorescent probe can be used β-phycoerythrin or more recently fluorescein [16,25,28]. Thus, peroxyl radical are formed by thermodecomposition of AAPH, giving an alkyl radical that react with molecular oxygen to give peroxyl radical. The decay in fluorescence is recorded and the results of the scavenging activity is expressed as Trolox equivalents [56].

Other ROS/RNS Scavenging Activity
Other methodologies can be applied to evaluate the antioxidant potential of a series of natural and synthetic matrix against a series of reactive oxygen and nitrogen species (ROS and RNS). For prenylated flavonoids there are only a couple of papers referring the scavenging activity profile against hydrogen peroxide [31] and peroxynitrite anion [22], which led us to describe them in the next section, prior the discussion of the results obtained in such assays.

Metal Chelation
Copper and iron chelation by prenylated flavonoids were determined by the difference in thein the UV-vis spectra (190-900 nm) produced when this metal ions were incubated with the tested compounds. The results are expressed as the difference in the absorbance or spectral shift of sample in the presence and in the absence of the metal ions [31,44,56].

Lipid Peroxidation Assay
Lipid peroxidation is usually induced by metal ions such as iron and measured by the thiobarbituric acid method, being the levels of peroxides formed expressed as TBARS. Other systems used for prenylated flavonoids were Mb(IV)-induced arachidonic acid peroxidation [32] and metal-ion independent systems using tert-butyl hydroperoxide (TBHP)-induced lipid peroxidation in liver microsomes or oxidation of β-carotene/linoleic acid emulsion. One of the limitations of this technique is the time consuming that depends on the oxidation of a substrate which is influenced by temperature, pressure, matrix, etc., particularly important when a great numbers of samples are involved. [47].

LDL Oxidation Assay
Cu(II)-induced low-density lipoprotein (LDL) oxidation is determined by measuring, in an initial stage, the formation of conjugated dienes through the increase of absorbance at 234-250 nm and at the end, the generated amount of lipid peroxides by the TBARS assay at 532 nm, using MDA for the standard curve [12]. 3-Morpholinosydnomine (SIN-1), a peroxynitrite generator, can also be used to induce LDL oxidation [15]. Two flavanones, propolin A (103) and propolin B (104), were isolated and characterized from Taiwanese propolis glue collected from hives located in the area of Bagwa Shan, Taiwan. Both compounds were tested in concentrations ranging from 3.125 to 25 µg/mL and exhibited strong scavenging effects against DPPH • with IC 50 values of 5.0 and 9.0 µg/mL, respectively [48].
Pyranocycloartobiloxanthone A (139) was isolated from the stem bark of the endemic and rare A. obtusus collected from Sarawak, Malaysia and showed strong DPPH • scavenging activity with an IC 50 value of 2.0 µg/mL. No information was given for the positive control [55].
Two prenylated pterocarpans-phaseollin (154) and shinpterocarpin (155) (31). All the compounds were tested for their ability to scavenge DPPH • and the results expressed as Trolox equivalents, defined as the concentration of Trolox (µM) having the same activity as 1 g of the tested compound. Thus, the most active compound was 25 (15 µM TEAC/g), a two-fold stronger scavenger than 31 (37 µM TEAC/g) and 1 (38 µM TEAC/g). The values for the remaining compounds 25-30 ranged from 44 to 70 µM TEAC/g [27]. Two positive controls were used in this assay being BHA a stronger scavenger (IC 50 49.87 µM), better than the analyzed compounds, and BHT considerably weaker, with an IC 50 value of 231.6 µM [19].
From the leaves of Macaranga pruinosa collected in Samarinda, Indonesia the flavone glyasperin A (71) was isolated and its antioxidant potential tested using a DPPH • scavenging system. The IC 50 value of 71 (443.0 ± 8.0 µg/mL) was almost twice higher than that of the positive control, kaempferol (IC 50 238.0 ± 3.3 µg/mL) [41].
From Macaranga gigantea (Euphorbiaceae) leaves collected in Indonesia the flavones glyasperin A (71) and broussoflavonol F (72) were isolated. The antioxidant activity was evaluated by their ability to scavenge DPPH • , showing IC 50 values of 125.10 and 708.54 µM, respectively. In addition, the results pointed out that 71 was twice as active as the positive control ascorbic acid (329.01 µM) [42].

ABTS Radical Cation Scavenging Activity (TEAC Method)
Rajendran et al. verified the suppression of the absorbance of ABTS •+ in a concentration-dependent manner for artocarpin (47) and cycloartocarpin (132). The results demonstrate that the reaction with these compounds show small inhibitory effects even up to 4 min of reaction, when compared with the reaction of the positive control quercetin which is completed within 1 min. Artocarpin (47) has two hydroxyl groups in the B ring at 4 and 6 and registered a TEAC value of 910 µM while cycloartocarpin (132) has a fused partially saturated six member heterocyclic ring between rings C and B and had a TEAC value of 690 µM. Meanwhile, the control quercetin possesses a catechol structure in the B ring, a C2=C3 double bond in conjunction a 3-OH and 4-carbonyl groups, allowing resonance stabilization for electron delocalization and therefore, an higher TEAC value (1230 µM) when compared with 47 and 132. These results demonstrate the importance of electron delocalization across the molecule for stabilization of the aryloxyl radical [32].
The two prenylated flavones cycloartocarpesin B (56) and cudraflavone B (65) and three flavanones-euchrestaflavanone B (94), euchrestaflavanone C (95) and a novel flavanone A (96)-had similar ABTS •+ scavenging activity (IC 50 4.2-8.3 µM) to that of quercetin (IC 50 4.0 µM). In addition, the most active compound 65 had the same TEAC value (expresses the numbers of µmols of Trolox having an antioxidant capacity corresponding to 1.0 µmol of the test substance) than quercetin (TEAC value of 5.0). These results point out the importance of the prenyl group for the antioxidant effect against ABTS system [38].
Xanthohumol (1) was a stronger scavenger in the ABTS •+ scavenging system than in the FRAP system, with 0.27 ± 0.04 µM Trolox equivalents in the FRAP assay system, and was completely inactive in the scavenging of DPPH • [17].
The FRAP value of chalcone dimer 44 was calculated from the calibration curve derived from dilutions of a vitamin C standard, measuring the decrease in absorption of the complex at 660 nm. Compound 44 exhibited better activity in the FRAP assay than the positive control, gallic acid (648.44 and 531.02 mg/mM, equivalent amounts of vitamin C) [29].
It was only possible to determine the FRAP potential of three of the seven flavonoids isolated from A. scortechinii King. Artocarpin (47), artonin E (55) and cycloartobiloxanthone (140) exhibited FRAP values of 0.19 ± 0.19, 1.32 ± 1.17 and 2.79 ± 0.19 Trolox equivalents, respectively. A closer look into the FRAP values of artocarpin (47) and artonin E (55) we may question the reliability of such results since the error associated is too high. The two positive controls used in this assay, BHA and HBT, provided values of 0.60 ± 0.06 and 1.89 ± 0.02 Trolox equivalents, respectively [19].

Hydroxyl Radical Detected by ESR
The ability of the prenylated flavonoids to scavenge HO • in a hydrophilic environment can also be measured by ESR spectroscopy, where HO • radicals generated by a Fenton-type reaction are trapped as DMPO spin adducts, giving rise to the corresponding ESR signals. The intensity of the DMPO-OH spin adduct signal is reduced in the presence of radical scavengers and the results are given as the difference of the respective ESR signal intensities of the sample with and without the flavonoid. Lee [31].
The ONOO − scavenging activity reported by Jung et al. was measured by monitoring the ONOO − induced oxidation of non-fluorescent DHR to fluorescent rhodamine 123. The assay was performed at 37 • C, reacting the tested compounds dissolved in 10% DMSO, ONOO − and DHR 123 and the fluorimetric signal detected after a 5 min incubation period. The fluorescence intensity of the oxidized DHR 123 was measured at the excitation and emission wavelengths of 485 nm and 530 nm, respectively, and the results were expressed the percent inhibition of oxidation of DHR 123. L-Penicillamine was used as the positive control. From the eight flavonoids analyzed (the chalcones kuraridin (18) and kuraridinol (19), the flavonol kushenol C (70) and the flavanones leachianone (89), kushenol E (90), sophoraflavanone G (91), kurarinone (92) and kurarinol (93)), the flavonol 70 (IC 50 0.62 ± 0.01 µM) was the most active scavenger, even better than the positive control, L-penicillamine (IC 50 (3), isoxanthohumol (102) and 8-prenylnaringenin (113). Spectra (200-600 nm) were recorded after preparation of the mixtures and 10 min later, in the absence and in presence of copper ions. Small variations were observed in the spectra of 1, 102 and 113. The chalcones 2 and 3 developed new maxima around 290 nm over a 10-min period, which was attributed to conversion of the chalcones to their isomeric flavanones rather than chelation of copper ions. The importance of 3 ,4 -dihydroxy substituents (catechol moieties) on the B-ring for copper or iron chelate formation is known and none of the flavonoids is this study had such a profile [12]. In continuation of their studies, 1, 2 and 3 were also tested for their chelation of iron ions, recording the absorbance between 190 and 600 nm, and no changes were induced by this metal in the UV-vis spectra [13]. Unlike Miranda's work, Dufall et al. tested three prenylated flavonoids bearing a catechol unit in the B-ring: 6,8-diprenyleriodictyol (97), dorsmanin C (67) and dorsmanin F (98). Methanolic solutions of the compounds were mixed with copper solution and their interaction was measured between 200 and 800 nm after 10 s and compared with the flavonoid alone. Interestingly, only 67 showed any interaction with Cu 2+ ions indicated by significant bathochromic shift in major absorbance bands upon addition of equimolar concentrations of Cu 2+ ions. These results led us to infer t the importance of the catechol unit for the chelating properties but also of the presence of C2=C3 double bond in combination with a 3-OH, the structural characteristics of flavonol 67 absent in flavanones 97 and 98 [37]. Meanwhile, 3,5,7,2 -tetrahydroxy-6-methoxy-8-prenylflavanone, known as floranol (76) exhibits two absorption bands at 297 and 340 nm related to the π-π* transitions of chromophores A and B, respectively, which were significantly changed by both coordination to Cu 2+ and Fe 3+ ions. The authors pointed out that these metals probably bind to a site on A-ring through a bidentate coordination involving the 5-OH and 4-carbonyl groups [44].

Lipid Peroxidation
Ko et al. studied lipid peroxidation promoted by iron ions in rat brain homogenate and measured by the decrease of absorbance at 532 nm. Tetramethoxypropane was used as a standard, and the results were expressed as nanomoles of MDA equivalents per milligram of protein of rat brain homogenates. Cycloheterophyllin (143), artonin A (144) and artonin B (145) inhibited Fe(II)-induced lipid peroxidation in a concentration-dependent manner with IC 50 values calculated to be 0.96 ± 0.21, 0.47 ± 0.24 and 0.71 ± 0.13 µM, respectively. BHT also inhibited this Fe(II)-induced lipid peroxidation with an IC 50 1.33 ± 0.05 µM. In contrast, no effect on Fe(II)-induced lipid peroxidation in rat brain homogenate was observed for artocarpin (47) and artocarpetin A (58) at 100 µM concentration [31].
Studies on artocarpin (47) and cycloartocarpin (132) as inhibitors of lipid peroxidation was studied by following Mb(IV) reduction, induced by fatty acid, arachidonic acid. Decrease in the absorbance at 532 nm was recorded and the results were expressed as % inhibition of TBARS. At 100 µM concentration, cycloartocarpin (132, 34% inhibition) inhibited the formation of MDA more efficiently than ascorbic acid (27% inhibition) and even better than artocarpin (47, 24% inhibition) [32].
LDL oxidation was induced by the peroxynitrite generator, 3-morpholinosydnonimine (SIN-1), and measured by the formation of conjugated dienes and TBARS. Conjugated diene formation was monitored by recording the absorbance at 250 nm every 30 min for 8 h and then at 18 and 24 h. After a 24 h incubation at 25 • C, TBARS were measured. The results pointed out that xanthohumol (1) inhibited SIN-1-induced oxidation of LDL in a dose-dependent manner in the assay for conjugated diene formation and in the TBARS assay. High inhibitions were also recorded in the TBARS formation for a series of other prenylated chalcones at 5 µM, (xanthogalenol (2), desmethylxanthohumol (3), 5 -prenylxanthohumol (5), dehydrocycloxanthohumol (6) and dehydro-cycloxanthohumol hydrate (7)), while the highly lipophilic prenylchalcones 3 -geranyl-chalconaringenin (11) and 4 -O-5 -C-diprenylxanthohumol (23) as well as prenylated flavanones 102 and 112-116 were considerably less active. Generally, chalcones are more potent inhibitors of LDL oxidation than flavanones due to their α,β -unsaturated keto group that can act as a Michael acceptor system for peroxynitrite. The introduction of additional prenyl groups further enhances the nucleophilicity of the 2 -OH group but also increases the compound's lipophilicity and reduces its water solubility, making their oxidation products less effective ROS/RNS scavengers in aqueous medium [15].
Five derivatives (cycloartocarpesin B (56), cudraflavone B (65), euchrestaflavanone B (94), euchrestaflavanone C (95) and novel flavanone A (96)) isolated from C. tricuspidata were assessed for their potential as inhibitors of Cu(II)-induced oxidation of LDL. All compounds exhibited modest antioxidant activity against LDL oxidation in TBARS assay with IC 50 values ranging from 27.2 to 65.6 µM, in comparison with an IC 50 of 3.6 µM obtained for the positive control probucol [39].
The antioxidant activity of floranol (76) isolated from the roots of Dioclea grandiflora, was evaluated by the inhibition of Cu(II)-induced oxidation of human LDL by measuring the formation of conjugated dienes. Floranol (76) inhibited the LDL oxidation, in a dose-dependent manner. Thus, in the absence of 76 a lag phase of 33 ± 1 min was measured; while in the presence of 76, lag-phases of 68 ± 1 and 178 ± 2 min were obtained for 3 and 10 µM concentration, respectively. Concentrations above 30 µM practically prevent LDL oxidation [44].

Conclusions
A good number of prenylated flavonoids (more than 150 derivatives) have been studied over the past two decades focusing on their antioxidant properties. Most of them were isolated from different parts of plants belonging to the Moraceae and Fabaceae families, with a limited number of derivatives from the Apiaceae, Asteraceae, Cannabaceae, and Euphorbiaceae. Few Oand C-derivatives obtained by synthesis belong to the chalcone, flavone and flavanone class of compounds. A detailed description of all in vitro and in vivo methodologies used to study the antioxidant effects of natural and synthetic derivatives were made, including reactants, temperature, time and type of detection. DPPH radical scavenging assay is undoubtedly the most frequently used technique, probably due to its simplicity and low cost. From the published results, most of the analyzed prenylated flavonoids exhibited high inhibitory effects and some structure-activity relationships were also described. However, it is not easy to make a comparison between compounds since different methods and positive controls were applied. In conclusion, the importance of prenyl groups for the antioxidant properties of flavonoids in several in vitro and in vivo models is highlighted in this review.