Hindawi BioMed Research International Volume 2019, Article ID 7010467, 18 pages https://doi.org/10.1155/2019/7010467

Review Article A Review on Flavonoid Apigenin: Dietary Intake, ADME, Antimicrobial Effects, and Interactions with Human Gut Microbiota

Minqian Wang ,1,2 Jenni Firrman,2 LinShu Liu,2 and Kit Yam 1

 Food Science Department, Rutgers University, New Brunswick , USA Dairy and Functional Food Research Unit, Eastern Regional Research Center, ARS, USDA, Wyndmoor , USA

Correspondence should be addressed to Kit Yam; [email protected]

Received 19 April 2019; Revised 25 June 2019; Accepted 31 July 2019; Published 16 October 2019

Academic Editor: Jane Hanrahan

Copyright © 2019 Minqian Wang et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Apigenin is a favonoid of low toxicity and multiple benefcial bioactivities. Published reviews all focused on the fndings using eukaryotic cells, animal models, or epidemiological studies covering the pharmacokinetics, cancer chemoprevention, and drug interactions of apigenin; however, no review is available on the antimicrobial efects of apigenin. Research proves that dietary apigenin passes through the upper gastrointestinal tract and reaches the colon afer consumption. For that reason, it is worthwhile to study the potential interactions between apigenin and human gut microbiota. Tis review summarizes studies on antimicrobial efects of apigenin as well as what has been reported on apigenin and human gut microbiota. Various levels of efectiveness have been reported on apigenin’s antibacterial, antifungal, and antiparasitic capability. It has been shown that apigenin or its glycosides are degraded into smaller metabolites by certain gut bacteria which can regulate the human body afer absorption. How apigenin contributes to the structural and functional changes in human gut microbiota as well as the bioactivities of apigenin bacterial metabolites are worth further investigation.

1. Introduction Te largest number of published reviews on apigenin focus on its efects on various cancers [4, 6–14]. Tose Flavonoids are a type of phytochemicals called polyphenols, reviews include fndings relating to the pharmacokinetics, which are the secondary metabolites produced by plants [1]. cancer chemo-prevention, and drug interactions of apigenin By producing favonoids, plants possess a defense mechanism based on eukaryotic cells, animal models, or epidemiological to protect against UV-B and ward of microbial infection and studies. However, no review on the antimicrobial efects of herbivory [2]. “Te Handbook of Natural Flavonoids” pub- apigenin exists, although many research papers have reported lished in 1999 contains information on 6467 known favonoid on the bacterial inhibitory activities from natural favonoids. structures with formulae, references, and information on Another interesting aspect of the efects of apigenin on biological activities [3]. Among the over 6000 diferent bacteria is its interactions with gut microbes. Apigenin has favonoids, quercetin, kaempferol, myricetin, apigenin, and alowsolubility[15,16]andalowbioavailability[4],andthus luteolin are the fve most ubiquitous plant favonoids [4]. it may come into contact with the colon microbiota and be � Apigenin, 4 ,5,7-trihydroxy-favone (Figure 1), is one of the metabolizedintosmallerandmorebioavailablemolecules predominant monomeric favonoids found in a daily diet [5]. [17].Apigeninmayalsoafectthecompositionandfunction- Based on the chemical structure of its backbone, apigenin ality of gut microbiota. Terefore, there may exist interactions is a favone, one of the subclasses of favonoids. Apigenin between them. Te objective of this present review is to fll has gained attention among researchers partly due to its low the knowledge gap by providing an overview of reported toxicity and multiple benefcial bioactivities. results from antimicrobial tests using apigenin. To that end, 2 BioMed Research International

OH

HO O

OH O

� Figure 1: Apigenin (4 ,5,7-trihydroxy-favone).

the dietary sources of apigenin, certain relevant chemical only about 0.69 mg/day [12]. However, another study on the and biological properties, ADME (absorption, distribution, Dutch diet estimated that the average daily intake of apigenin metabolism, and excretion) of apigenin, the antimicrobial is about 1 mg [22]. A similar number, 1.5 ± 4.9 mg/day (range efects of apigenin and its interactions with gut microbiota 0-30.3), was reported in a group of female Flemish dietitians will be presented and discussed in this review. [23]. A more recent paper provided information on the mean intake of favonoid compounds in adults in the European 2. Apigenin: Dietary Source and Daily Union by country, region, and overall [26]. Te European average of apigenin intake is 3 ± 1mg/dayusingthefood Intake Levels consumption data from the European Food Safety Authority (EFSA) and the FLAVIOLA Food Composition Database. Before the discussion of the efects of dietary apigenin on gut Te daily intake level of apigenin is 4.23 mg/day in China [27], bacteria, it is necessary to look at the dietary intake level of while among middle-aged and older women in the US, it is apigenin. Te distribution of apigenin in the plant kingdom 0.13–1.35 mg/d [28]. Tis intake value among US women is is wide, as it has been found in many vegetables, herbs, similar to the reported 0.2-1.3 mg/day among 66,940 married and fruits [5]. Fresh parsley, vine spinach, celery seed, green womeninaUSnurses’healthstudyfrom1984through2002 celery heart, Chinese celery, and dried oregano are dietary [29].Bymeasuringtheintakeofapigeninfrommajordietary sourceswithhighapigenincontent[5].Otherplantsinwhich sourcesoffavonesinadultAustralians,themeandailyintake apigenin has been identifed include red and white sorghum, value was determined to be 0.45 mg/day [30]. rutabagas, oranges, kumquats, onions, wheat sprouts, tea, and Te above information indicates that, as a phytochemical cilantro[5,9,18].Driedparsleyhasaparticularlyhighlevel present in multiple food sources, quantifcation of dietary of apigenin that far exceeds any other vegetables or herbs [5]. intake for apigenin is difcult and has large variation [21]. Chamomile tea, high in apigenin content, is one of the most Diet varies with geographical location, culture, and spe- common sources of apigenin intake from a single ingredient cifc demographics; and it evolves with time. One primary [19]. In nature, apigenin is typically found in a glycosylated method for dietary intake level estimation relies on dietary form, with the tricyclic core structure linked to a sugar assessment, which includes a diet history interview or food moiety through hydroxyl groups (O-glycosides) or directly frequency questionnaires, and compares this against a food to carbon (C-glycosides). Te common apigenin glycosides composition database [21]. Te reliability of the information are apiin, apigenin-7-O-glucoside, apigenin-8-C-glucoside collected from subjects, as well as the inclusiveness, extensive- (vitexin), apigenin-6-C-glucoside (isovitexin), apigenin-7-O- ness [21], and demographic specifcity of the database can all neohesperidoside (rhoifolin), and apigenin-6-C-glucoside 8- impact the accuracy of intake levels obtained. Other than the C-arabinoside (schafoside) [4, 13, 20]. inaccuracy in the data, more information is available on the Sinceapigeniniswidelydistributedinfooditems,and daily amount of total or combined groups of favonoids than a diet high in favonoids has been reported with many a single favonoid of interest [21]. benefcial health efects, an estimation of the daily intake of apigenin could be useful in the correct interpretation of the relationship between health outcomes and apigenin. Tough 3. Chemical Properties: Solubility and Stability dietary intake values have been obtained for some individual favonoids [21], when it comes to the favone, apigenin, Understanding of the solubility and stability of apigenin is relevant research is very limited [22, 23]. Higher estimation of the prerequisite of experiments that study the antimicrobial daily dietary intake of favonoids is 1 g as glycosides or 650 mg efects of apigenin in aqueous solutions. Flavonoids are ofen as aglycones [24], while another study estimated an average of found in nature as glycosides and phenolic acids, as esters only 23 mg/day in adults in the Netherlands [12]. In a study in the aqueous environment of the plant cell vacuole [7]. around 1990, the mean favanol and favone intake among Apigenin is the aglycone form and it is present in plants US health professionals was approximately 20-22 mg per day naturally as several apigenin glycosides as mentioned in the [25]. In the aforementioned study in the Netherlands in 1988, previous section. Tose glycoside conjugates, primarily as most of the average daily intake was attributed to quercetin, apigenin-7-O-glucoside, and acylated derivatives are more about 16 mg/day, and the actual daily intake of apigenin was water soluble than apigenin [10] and their structures have a BioMed Research International 3 majorimpactontheirabsorptionandbioavailability,withthe [45–50]. Terefore, the consumption of favonoids has been best bioavailability occurring when apigenin is bound to �- assumedtobesafe. glycosides [9]. Results of an Ames test, one of the oldest meth- Apigenin is practically insoluble in highly polar solvents ods employed for in vitro testing of carcinogenicity using such as water (0.00135 mg/mL), and nonpolar solvents such Salmonella strains [11], showed that apigenin was not muta- as silicon fuid (0.0728 mg/mL) and safower oil (0.0317 genic or toxic when tested alone [51]. Not only was apigenin mg/mL) [15, 16]. Other reports on apigenin’s solubility in not mutagenic [52], it protected against multiple genotoxic aqueous solutions show that it ranges from 0.001 to 1.63 agents, such as sodium azide, 9-amino acridine [11, 53]. Api- mg/mL in nonpolar solvents [6] and 2.16 �g/mL in phos- geninpreventedtherevertedmutationsasaresultofsodium phate bufer at pH 7.5 [31]. Apigenin is freely soluble in azide exposure using histidine auxotroph S. typhimurium dimethylsulfoxide (DMSO) [11]. One source estimated the TA100 strain and the hindrance percent of apigenin was solubility to be more than 100 mg/mL [16], while another 98.17% [54]. Apigenin-7-O-glucoside inhibited sodium azide showed that the approximate solubility of apigenin in ethanol, mutagenicity in S. typhimurium TA1535 at 6.67 nM in the top DMSO, and dimethylformamide (DMF) purged with inert agar when using the layered agar method with an inhibition gastobe0.3,15,and25mg/mL[32].Flavonoidsarealso rate of 27.2% and 9-aminoacridine in S. typhimurium TA1537 more soluble in methanol than in water [33]. As a result, at 3.33 nM with an inhibition rate of 91.1%. In the yeast organicsolventslikeDMSO[34]andTween80[31]areused deletion assay using mutagens ethyl methanesulfonate and to dissolve apigenin prior to their addition to an aqueous acridine, the inhibition rates were from 4% to 57.7% [53]. solution to increase solubility. Diferent carriers such as Apart from mutagenesis, hemolytic activity is another ethosomes [35], polymeric micelles of Pluronic P123 and measurement of safety. Hemolytic tests showed that the Solutol HS 15 [36], and carbon nanopowder [37], or self- percent hemolysis of apigenin was below the permissible microemulsifying delivery system [38] are also developed limit of 5% afer 30 min treatment. Being hemocompatible and tested to enhance the efcacy of apigenin. Taking into indicatesthatapigeninissafeforintravenousapplication, consideration its high permeability, apigenin is categorized as and suggests that apigenin is non-toxic in mammalian sys- a Class II drug according to Biopharmaceutics Classifcation tems [55]. Apigenin was able to signifcantly attenuate the System (BCS), whose characteristics are low solubility and hemolytic activity of the purifed Pneumolysin, the pore- high permeability [31, 39]. forming toxin secreted by S. pneumoniae,inaconcentration- Pure apigenin is generally regarded as unstable for long dependent manner [56]. termstorageatroomtemperature,andthusitrequiresstorage ∘ As can be concluded from the Ames tests and hemolytic at -20 C [9]. For use in experiments, it is recommended that tests, apigenin is not mutagenic nor hemolytic. Taking api- fresh solutions be made as needed [32]. Some researchers genin glucosides through a daily diet can hardly reach the have investigated the stability of apigenin under various con- therapeutic doses used in clinical trial [4] and has not been ditions. Afer heated refux in water for 30 min, maceration reported to be harmful. However, as consumers intentionally for 24 hours or microwave irradiation for 5 min under 500 increase apigenin intake from dietary supplements or phar- W, 93-95% of apigenin was recovered for each condition maceutical sources, safety concerns may arise with a higher [33]. However, ultrasonic extraction caused the product to be level of apigenin exposure. degraded with only an 86% recovery rate [33]. Another study showed that, in rat plasma in vitro, apigenin was stable under three conditions: 24 hours under room temperature, at least 5. Pharmacokinetics: Absorption, Distribution, ∘ 4 weeks when kept frozen at -20 C, and afer at least three Metabolism, and Excretion freeze-thaw cycles [40, 41]. Te decomposition for favonoids depends on the num- Te relevance of pharmacokinetics of apigenin in this review ber of substituents in the favonoid molecule. Hydroxyl is that it determines how much apigenin consumed orally is groups promote degradation of favonoids, whereas sugar available to the human gut microbiota. A summary of ADME moiety and methoxyl groups protect favonoids from degra- anddruginteractionsofapigenincanbefoundinthereview dation during microwave and ultrasonic-assisted extraction by Tang et al. [4]. In this section, information is provided [33]. Terefore, the glucosides, other than having a higher to show that oral bioavailability of apigenin is poor; it is solubility than the aglycone, are also chemically more stable, either excreted unabsorbed in the urine or feces or rapidly which may be one of the reasons why they have enhanced metabolized afer absorption [4]. biological activities [42]. .. Absorption. About 5-10% of total polyphenol intake, 4. Safety: Mutagenicity and Hemolysis Tests mostly monomers and dimers, may be absorbed in the small intestine [17]. Te gastrointestinal tract plays a signifcant role Safety is an important aspect of dietary components. In in the metabolism and conjugation of apigenin before the general, dietary plants containing favonoids have not been entry of apigenin into the systemic circulation and the liver associated with negative health impact, but they are rather [57]. In a perfused rat intestinal model, aglycone apigenin was considered to be benefcial. Apigenin is known for its low rapidly absorbed [57]. Apigenin can be transported by both toxicity [9, 11, 43, 44]. Evidence has also shown that a passive and active carrier-mediated saturable mechanism favonoid-rich diet is inversely associated with cancer risk in the duodenum and jejunum, and by passive transport 4 BioMed Research International mechanisms in the ileum and colon, primarily [31]. Oral more important than hepatic disposition in the frst-pass administration of apigenin resulted in a peak concentration metabolism of apigenin [63]. ± � (Cmax)1.33 0.24 g/mLandareaunderthecurve(AUC0–t) of 11.76 ± 1.52 �g ⋅ hour/mL in rats, which were very low . . Excretion. Excretion of apigenin afer oral intake blood apigenin levels [58]. Conficting conclusions have been through feces is a good indication of the phenomenon drawn regarding the rate of absorption of apigenin. One that dietary apigenin is available for metabolism by the study on rats concluded that apigenin has a slow absorption gut microbiota. In one of the aforementioned experiments because afer a single oral administration of radiolabeled in Section 5.1 using rats, afer a single oral administration apigenin, radioactivity appeared in the blood 24 hours later of radiolabeled apigenin, 51.0% of the radioactivity was [59]. While another study reached the opposite conclusion recoveredinurineand12.0%infeceswithin10days.In that apigenin had a quick absorption rate in rats as the plasma thesameresearch,itwasdiscoveredthatsexandageofthe concentration of apigenin reached the peak level 3.9 hours rats afected apigenin conjugates eliminated via the urinary afer oral intake of apigenin in the form of Chrysanthemum route [59]. Tat research also concluded that apigenin has morifolium extract [60]. aslowmetabolismandasloweliminationphase.Tus,this favonoid possibly accumulates in the body [59]. .. Distribution. Multiple studies have been performed to .. Apigenin Glycosides. It is worth mentioning again that measure the distribution and excretion of apigenin in vivo. apigenin exists in plants as glycosides naturally; therefore, the Following an IV bolus injection of apigenin at 20 mg/kg, the absorption of the apigenin glycosides and the bioavailability mean value of systemic clearance was 6.12 ± 0.79 L/h/kg [61]. afer consumption of glycosides are useful information. Nev- In the previously mentioned study with rats, afer a single ertheless, such information on apigenin glycosides is rare, and oral administration of radiolabeled apigenin, the elimination most research done looked into other favonoids, in particular half-time was high with a value of 91.8 hours [59]. Te quercetin [66, 67]. In fact, in comparison to the other classes distribution volume was 259 mL, and the plasmatic clearance of favonoids, little is known about the bioavailability of was 1.95 mL/h [59]. Afer 10 days, 1.2% of the radioactivity favones [68]. was recovered in the blood, 0.4% in the kidneys, 9.4% in the Limited information on the absorption and distribution intestine, 1.2% in the liver, and 24.8% in the rest of the body of apigenin glycosides can only be inferred from a couple [59].Abouthalftheapigeninconsumedwentintourineand of studies available. In the frst one, purifed favonoid feces [59]. In another study, mice were fed a diet containing extract from parsley comprised a mixture of glycosides apigenin for 5, 6, or 7 days. It was found that with a dose of the favones: apigenin, luteolin and chrysoeriol, and of of 1.1 mmol/kg apigenin, plasma levels of apigenin reached the favonols: kaempferol, quercetin, and isorhamnetin were steady state afer 5 days and the steady-state concentrations administrated to rats by gavage [37]. Te dose corresponded of apigenin in plasma, liver, and the small intestinal mucosa to 6.9 mg aglycones per kg body mass. Glycosides were were 0.09 ± 0.08 nmol/mL, 1.5 ± 1.0 nmol/g, and 86 ± 47 detected by HPLC in the wall and the lumen of the stomach, nmol/g, respectively [62]. Tis is in the similar range to the the wall of the small intestine afer 1 hour and 4 hours as well other study. Apigenin distributes well into the tissues [57]. as the cecum wall afer 4 hours; however, no concentration values were provided and the glycosides were a mixture .. Metabolism. Te absorbed apigenin may go through of diferent favonoids. Te apigenin aglycone was detected extensive Phase I and Phase II metabolism [17]. In the in the lumen of the small intestine afer 1 and 4 hours rat liver, metabolism of apigenin was found to involve at the concentrations of 855.59 �g/gram dry matter and Phase I Enzymes in the presence of NADPH (nicotinamide 207.05 �g/gram dry matter, respectively; the concentration adenine dinucleotide phosphate), P450 (cytochrome P450 in cecum afer 4 hours was 353.38 �g/gram dry matter. enzymes), or FMO (favin-containing monooxygenase) [4]. Te concentrations of apigenin aglycone in the wall of the Phase II biotransformation of apigenin involves both enteric stomach, the small intestine, and the cecum were much and enterohepatic cycling [60]. Te conjugation reactions lower, 16.27 �g/gram dry matter in the stomach wall afer 4 glucuronidation and sulfation are the essential phase II hours and 12.58 �g/gram dry matter in the cecum wall. No metabolic pathways of apigenin [4]. In both rats and humans, apigenin was detected in the small intestine wall at either apigenin has been reported to produce major metabolites time points. As the treatment comprised of a mixture of of glucuronidated and sulfated conjugates [63]. Te major favonoids, the exact source of the apigenin detected was not hepaticmetaboliteofapigeninisluteolin[59].Teabsorbed able to be determined. Nevertheless, as can be seen from the apigenin in blood circulation and tissues is in the form results, the glycosides transported to the cecum were mostly of glucuronide, sulfate conjugates, or luteolin [4]. Tere absorbed or deglycosylated rapidly as the cecum luminal were three �-monoglucuronides that appeared afer a glu- contents favonoids contained only aglycones. Terefore, curonidation reaction, while only one product appeared afer when studying the impact of dietary apigenin in the colon by a sulfation reaction [4]. In human hepatic cell line, Hep in vitro simulation, such as when using bioreactors to culture G2,aswellasintestinalcellline,Caco-2,apigenin-induced gut microbiota, the treatment group should use apigenin, the phase II detoxifying enzyme UDP-glucuronosyltransferase aglycone form, rather than naturally present glycosides. In UGT1A1 [64, 65]. In addition, glucuronidation reactions also another study, three groups of rats received Apigenin-7-O-�- occur in the intestine, and intestinal disposition may be D-glucoside solution via tail vein injection at a single dose BioMed Research International 5 of 27.0, 18.0, or 9.0 mg/kg [69]. Plasma level of Apigenin-7- food in diferent cultures. Plants containing apigenin, along O-�-D-glucoside was measured over the course of 5 hours with other favonoids, have been used to battle diseases in afer drug administration. Estimated plasma concentrations many cultures. Apigenin has been identifed as an active of Apigenin-7-O-�-D-glucoside based on the fgure were ingredient in Scutellaria barbata D. Don (Lamiaceae) [75], about 1, 0.65, and 0.5 �g/gram right afer the injection, and Castanea sativa Mill. (Fagaceae) [76], Portulaca oleracea L. decreased to about 0.3, 0.25, and 0.38 �g/gram afer one hour [77], Marrubium globosum ssp. Libanoticum [78], Combretum for each group. erythrophyllum (Combretaceae) [79], Aquilegia oxysepala Depending on the sugar moiety, the absorption could take [80], and propolis [81], among which most are traditional place in the small intestine or in the colon afer deglycosi- herbal or alternative medicines. Chamomile tea, which is dation [67]. With some exceptions, glucosides are generally extremely rich in apigenin, has been used as a folk medicine the only glycosides that can be absorbed from the small for relieving indigestion or gastritis [9]. Chamomile is also intestine [66]. Te absorption of favonoid glycosides from usedinmouthrinse,skincareproducts,andvaporinhalantto the small intestine involves the glucose transport pathway reduce infammation [9]. Tere are two types of chamomile: [70]. Epithelial �-glucosidase-mediated deglycosylation is a theGermanchamomileandtheRomanchamomile,with critical step in the absorption and metabolism of dietary the former more common as a dietary supplement [26]. favonoid glycosides. It is hypothesized that apigenin glu- Some preliminary studies suggest that a chamomile dietary cosides can be hydrolyzed into apigenin by cytosolic �- supplement might be helpful for generalized anxiety disorder glucosidase (CBG) and lactase-phlorizin hydrolase (LPH), (GAD). Researchers conducted a randomized, double-blind, which are enzymes produced by the liver or intestinal cells, placebo-controlled trial to test the efects of chamomile or the gut microbiota [4, 71, 72]. LPH has been shown to extract in patients diagnosed with mild to moderate GAD. hydrolyze favonoid glycosides and is proposed to be brush- Compared with placebo, chamomile was associated with border enzymes [57], and the resulting aglycone may then a clinically meaningful and statistically signifcant greater enter epithelial cells by passive difusion [68]. CBG functions reductioninmeanHamiltonAnxietyRating(HAM-A)scores inside cells [71]. Te sugar transporter SGLT1 may facilitate [82]. Although the researchers suggest that other chamomile the absorption of certain glycosides into epithelial cells [68]; species, preparations, and formulations might produce dif- however, apigenin and its various forms of glycosides (apige- ferent results [82]. It has been shown that apigenin and other nin-6-C-glucoside, apigenin-7-O-glucoside, apigenin-8-O- constituents of chamomile bind to BZ receptors and reduce glucoside) did not seem to be absorbed in intact form via the GABA-activated activity and may produce anxiolytic activity human sodium-coupled glucose transporter hSGLT1 [73]. [82]. Flavonoids that cannot be absorbed from the small As for the safety of chamomile, there have been reports of intestine, as well as the absorbed favonoids secreted with bile, allergic reactions as well as interactions between chamomile will be degraded in the colon by the microbiota [66]. and cyclosporine and warfarin, which can cause serious In one study, 28.6% and 16.6% of apigenin were recovered consequences [26]. in feces and in urine afer feeding Chrysanthemum morifo- lium extract to rats [60]. Te cumulative apigenin excreted in 7. Pharmacological Activities of Apigenin thebilewas6.34%ofthedose[60].Teaccumulationofapi- genin was also proposed in this study because of the quick ab- As one of the fve major favonoids in plants, apigenin has sorption rate and the slow elimination phase identifed [60]. been extensively studied for its biological functions. Several In one randomized cross-over human intervention study, reviews are available on the bioactivities of apigenin focusing a basic diet was supplemented with chopped parsley, pro- on diferent aspects including its health functionality [11] and viding 3.73-4.49 mg apigenin/ Megajoule (approximately 51 cancer chemoprevention potential [9, 10, 13]. Tese reviews mg of apigenin equivalent each day, mainly as apigenin-7- captured the large body of research on pharmacological apioside (apiin)) for one week, and the excretion of apigenin activities of apigenin and provided expert opinions on this was measured [74]. Te average urinary excretion of apigenin subject. was signifcantly higher during intervention with parsley Research on apigenin frst began in the 1960s, and it was (20.7-5727.3 �g/24 hours) than during the basic diet (0-1571.7 proposed to be chemo-preventative in the 1980s [9, 13, 51]. �g/24 hours). No diference between males and females was Recently, apigenin has received much attention because it has observed in the mean excretion of apigenin. Te amount of low intrinsic toxicity [4, 44] and it exerts diferential efects apigenin excreted ended abruptly afer parsley supplementa- on normal versus cancer cell growth, survival, or apoptosis in tion was halted. Terefore, urinary excretion and clearance of several diferent types of cells [9, 13, 52]. apigenin are quick, with the excretion half-life estimated to be Te reported biological functions of apigenin include approximately 12 hours. One missed opportunity in the study anti-oxidant, anti-mutagenic, anti-carcinogenic, anti- was that the plasma concentrations were not measured. infammatory, anti-proliferative, and anti-progression [9]. Apigenin is a moderate anti-oxidant compound because the 6. Usage as Herbal Medicine or double bond at the 2,3 carbon makes the structure more Functional Food reactive, despite the absence of the hydroxyl group at position 3 and a catechol structure in the B-ring [83, 84]. Apigenin Pharmacological potential of apigenin may be refected by the is a very efective anti-infammatory agent compared to use of plants containing it as herbal medicine or functional other favonoids. Apigenin was the most potent inhibitor of 6 BioMed Research International transcriptional activation of both inducible cyclooxygenase distribution of apigenin and hence concentration in diferent (COX-2) and inducible nitric oxide synthase (iNOS) parts of the body afer oral consumption of apigenin in in lipopolysaccharide (LPS)-activated RAW 264.7 cells humanisveryrare.Basedonavailableinformationthatthe among favonoids such as wogonin, luteolin, tectorigenin, dietary intake of apigenin ranged from 0-4.23 mg/Day, the kaempferol, and quercetin, etc., reducing the production of concentrations used in the above-mentioned researches tend nitric oxide [85, 86]. Apigenin also suppressed nitric oxide to be higher than what can be realistically achieved afer production in LPS/gamma-interferon (IFN-�)stimulated apigenin intake from diet. C6 astrocyte cells in a dose-dependent manner with an Apigenin showed synergistic efects with antitumor −3 IC50 less than 10 M [87]. When tested to see if favonoids drugs such as paclitaxel, 5-fuorouracil, PLX4032 and N- could inhibit LPS-induced TNF-� secretion in primary (4-hydroxyphenyl) retinamide by enhancing their bioavail- bone marrow-derived mouse macrophages, apigenin was ability or efcacy [4]. Apigenin may also serve as a dietary not as efective as quercetin, luteolin, or genistein, but was supplement along with small molecule inhibitors to improve similar to kaempferol, diosmetin, and hesperetin. It seems radioiodine therapeutic efcacy on invasive tumor margins that the double bond at C2-C3 and the position of the and thus minimizing future metastasis [15]. B-ring at 2 contribute to the high anti-infammatory efect [88]. Apigenin inhibits the production of proinfammatory cytokines IL-1�, IL-8, and TNF in lipopolysaccharide- 8. Antimicrobial Effects of stimulated human monocytes and mouse macrophages in Apigenin and Mechanism vitro (concentrations 0.1-25 �M or 0.027-6.756 �g/mL) [89]. It Te antimicrobial efects of dietary favonoids have been also inhibited TNF-induced NF-�B transcriptional activation studied extensively. Although there are many publications in NIH/3T3 cells (concentrations 10-30 �M or 2.702-8.107 reportingthefndingsonapigenin,thereisnoreviewthat �g/mL) [90]. Carrageenan can induce acute paw edema in summarizes the fndings. Te following section aims to mice, and the infammation could be alleviated by apigenin include available information in publications and to make [90]. Apigenin exhibited an anti-infammatory efect on meaningful conclusions. murine microglia cell line by reducing the production of nitric oxide and prostaglandin E2 and was found to be protective against ischemia in neuronal cells (concentrations .. Antibacterial Activities. Te antibacterial potential of 1-10 �M or 0.270-2.702 �g/mL) [91]. Apigenin exhibited apigenin has been tested against many bacteria species and the highest DNA protective efects against free radicals various strains within the same species. Broth microdilution 2+ generated by Fe among luteolin and quercetin at the and agar dilution methods are the most popular methods concentration of 1 �M in vitro, which indicated its function in which the minimal inhibitory concentrations (MICs) are as an antioxidant (concentration at 10 �Mor2.702�g/mL) determined as the lowest concentration of treatment that [92]. Te anti-infammatory activity of apigenin has also showednogrowthaferincubation[100,103,105,108]. been reported in lipopolysaccharide-induced infammation However, results obtained from the two methods may not in acute lung injury in situ (concentrations 10 and 20 mg/kg necessarily coincide [106]. It has been suggested this might body weight) [93]. result from the diferent solubilities of the tested compounds Te anti-mutagenic efect has been reported in in vitro cell in liquid and the agar gel culture media, and the variability models, in vivo experiments, and AMES test using bacterial in MIC judgment criteria [110]. Results of the antibacterial models showing that apigenin could prevent, inhibit, or and antifungal tests of apigenin measured by its MICs are reverse chemically induced genotoxicity [9, 94–96]. Te anti- summarized in Table 1. Due to the limited amount of apigenin that could dissolve in the media, apigenin is considered not carcinogenic efect of apigenin has been widely reported. Api- active if the MIC is above 128 �g/mL in this review. MIC geninhasbeenfoundtobeprotectiveagainstmultipletypes values are strain-specifc, making it difcult to summarize the of cancer including breast cancer [18], cervical cancer, colon efects of apigenin in a shorter and more general way. cancer, leukemia (concentrations 0-200 �M or 0.0-54.048 � Apigenin could not inhibit the growth of Staphylo- g/mL) [97], lung cancer, prostate cancer (concentrations coccus aureus (8325-4, ATCC 29213, wood 46, and BAA- � � 0.0-80 M or 0.0-21.619 g/mL) [98], skin cancer, thyroid 1717) [108]. Despite the reported lack of activity against S. cancer, endometrial cancer, neuroblastoma, and adrenocorti- aureus, apigenin was found to remarkably decrease the level cal cancer [9, 10]. Derivatized compounds based on apigenin of �-hemolysin at low concentrations in a concentration- displayed higher antiproliferative activity in human lung, dependent manner in S. aureus culture supernatants [108]. cervical, hepatocellular liver and breast cancer cell lines than Alpha-hemolysin is a pore-forming cytotoxin that is secreted apigenin itself (concentrations 62.5-2000 �g/mL) [99]. by most S. aureus strains, essential for the pathogenesis of Concentrations used in research on the pharmacological S. aureus pneumonia [108]. Apigenin protected the adeno- activities of apigenin should be based on the concentrations carcinomic human alveolar basal epithelial cells (A549 cells) systemically reached following dietary intake of the favonoid. from �-hemolysin-mediated injury in the A549 cells and While researchers can use concentrations much higher in the S. aureus co-culture system [108]. Terefore, the protective experiments, results obtained from the higher concentrations efect apigenin did not come from a reduction in bacterial may not accurately describe the efects in real life when quantity, but more likely the altered cell physiology. What is the source of apigenin is from the diet. Information on the more promising is that apigenin alleviated injury of the lung BioMed Research International 7 [76] [76] [101] [103] [103] [103] [103] [103] [102] [100] [100] [100] [100] [104] [100] Reference lection; RSKK, Refk Saydam Central � L+, extended-spectrum beta-lactamases; NCTC, National ;ES Method of determining MIC Broth microdilution with no growth Broth microdilution with no growth Broth dilution with no visible growth Broth dilution with no visible growth Staphylococcus aureus Broth microdilution with no growth under the microscope Broth microdilution with no growth under the microscope Broth microdilution with no growth under the microscope Broth microdilution with no growth under the microscope Broth microdilution with no growth under the microscope Broth microdilution with no growth under the microscope Agar dilution with strong, moderate,Agar dilution weak, with or strong, absent moderate, growth weak, or absent growth Agar dilution with strong, moderate, weak, or absent growth Agar dilution with strong, moderate, weak, or absent growth Agar dilution with strong, moderate, weak, or absent growth 512 2 8 � g/mL 400 16 64 64 64 Antibacterial activity 128 > > Not active Not active Not active Not active Not active Not active MIC ; MSSA, methicillin-susceptible Staphylococcus aureus strain NCTC 7171 Not active Agar dilution with strong, moderate, weak, or absent growth [103] ATCC 6633 8 Broth microdilution with no growth under the microscope [100] NCIMB 775 ATCC 19433 NCTC 12201 ATCC 29212 RSKK 02026 ATCC 13048 ATCC 10699 NCTC 12203 NCTC 12202 NCTC 12204 clinical isolate clinical isolate clinical isolate clinical isolate Strain information cefazidime-resistant Species Acinetobacter baumannii Bacillus cereus Bacillus subtilis Enterobacter aerogenes Enterobacter cloacae Enterococcus faecium Enterococcus faecalis Collection of Type Cultures; NCIMB, National Collection of Industrial, Food and Marine Bacteria; MYA, Meyer et Yarrow; OD, Optical Density. Hygiene Institute. MRSA, methicillin-resistant Table 1: Te antibacterial and antifungal efects of apigenin measured by its minimum inhibitory concentration (MIC). ATCC, American type culture col 8 BioMed Research International [76] [76] [101] [101] [101] [101] [101] [103] [103] [103] [105] [106] [106] [100] [100] [100] [100] [100] [100] [104] [106] Reference agar afer incubation Method of determining MIC Broth microdilution with no growth Broth dilution with no visible growth Broth dilution with no visible growth Agar dilution with no visible bacterial growth Agar dilution with no visible bacterial growth Agar dilution with no visible bacterial growth Broth microdilution with no growth under the microscope Broth microdilution with no growth under the microscope Broth microdilution with no growth under the microscope Broth microdilution with no growth underBroth the microdilution microscope with no growth under the microscope Broth microdilution with no growth under the microscope Broth microdilution with no growth under the microscope Broth microdilution with no growth under the microscope Broth microdilution with no growth under the microscope Broth microdilution with no growth under the microscope Broth microdilution with no growth under the microscope Agar dilution with strong, moderate, weak, or absent growth Agar dilution with strong, moderate, weak, or absent growth Agar dilution with strong, moderate, weak, or absent growth Broth microdilution with no visual turbidity and no growth on Table 1: Continued. 8 4 4 � g/mL 200 25 128 128 128 128 128 200 > 400 135.12 > Not active Not active Not active Not active Not active Not active Not active Not active MIC SS1 JM109 RSKK 574 ATCC 7002 ATCC 10031 ATCC 11229 ATCC 35218 ATCC 35218 NCTC 12241 NCTC 11954 NCTC 11560 ATCC 10240 ATCC 14948 ATCC 25922 ATCC 25922 ATCC 43504ATCC 43504 25 Agar dilution with no visible bacterial growth [106] clinical isolate clinical isolates Strain information ES � L+ clinical isolate ES � L+ clinical isolate ES � L+ clinical isolate Species Escherichia coli Helicobacter pylori Klebsiella pneumoniae Mariniluteicoccus flavus Proteus mirabilis BioMed Research International 9 [75] [76] [76] [76] [101] [101] [103] [103] [103] [103] [103] [103] [103] [103] [103] [105] [105] [107] [107] [108] [108] [108] [108] [108] [100] [109] [100] [100] [104] [100] [104] [104] Reference 600 nm 600 nm 600 nm 600 nm 600 nm Disc-difusion test agar afer incubation agar afer incubation Method of determining MIC Agar dilution with no growth Broth microdilution with no growth Broth microdilution with no growth Broth microdilution with no growth Broth dilution with no visible growth Broth dilution with no visible growth Broth dilution with no visible growth Broth microdilution with no visible growth Broth microdilution with no visible growth Broth microdilution with no bacterial growth by OD Broth microdilution with no bacterial growth by OD Broth microdilution with no bacterial growth by OD Broth microdilution with no bacterial growth by OD Broth microdilution with no bacterial growth by OD Broth microdilution with no growth under the microscope Broth microdilution with no growth under the microscope Broth microdilution with no growth under the microscope Broth microdilution with no growth under the microscope Broth microdilution with no growth under the microscope Broth microdilution with no growth under the microscope Agar dilution with strong, moderate, weak, or absent growth Agar dilution with strong, moderate,Agar weak, dilution or with absent strong, growth moderate, weak, or absent growth Agar dilution with strong, moderate, weak, or absent growth Agar dilution with strong, moderate, weak, or absent growth Agar dilution with strong, moderate, weak, or absent growth Agar dilution with strong, moderate, weak, or absent growth Agar dilution with strong, moderate, weak, or absent growth Agar dilution with strong, moderate, weak, or absent growth Broth microdilution with no visual turbidity and no growth on Broth microdilution with no visual turbidity and no growth on Table 1: Continued. 2 128 128 4 � g/mL 16 32 1024 1024 1024 1024 1024 64 4000 400 400 > 400 > > > > > > > > 3.9-15.6 Not active Not active Not active Not active Not active Not active Not active Not active MIC Low activity Low activity Low activity Low activity Low activity Low activity no inhibition 10708 strains 6538P) isolates Serotype DU 1090 BAA 1717 NCTC 6571 ATCC 6538 NCTC 8203 NCTC 8506 strain Mu50 NCTC 11561 ATCC 10145 ATCC 33591 ATCC 10832 ATCC 29213 ATCC 13709 ATCC 12454 NCTC 11940 ATCC 27853 ATCC 27853 ATCC 27853 ATCC 27853 ATCC 25923 ATCC 25923 NCTC 10788 NCTC 10662 NCIMB 9968 NCTC 8325-4 clinical isolate 34 MRSA clinical Strain information MRSA ATCC 10442 Choleraesuis ATCC 20 MRSA and MSSA MRSA clinical isolate NCTC 7447 or ATCC FDA209P(thesameas Species Proteus vulgaris Pseudomonas aeruginosa Salmonella enterica Staphylococcus aureus 10 BioMed Research International [105] [105] [105] [104] Reference agar afer incubation agar afer incubation agar afer incubation Method of determining MIC Broth microdilution with no growth Broth microdilution with no visual turbidity and no growth on Broth microdilution with no visual turbidity and no growth on Broth microdilution with no visual turbidity and no growth on Table 1: Continued. Antifungal activity � g/mL 50 67.56 135.12 Not active MIC ATCC 10231 8 Broth microdilution with no growth under the microscope [100] ATCC 19615 ATCC 22019 16 Broth microdilution with no growth under the microscope [100] ATCC 12344 ATCC 90028 ATCC MYA2950 Strain information Species Streptococcus pyogenes Candida albicans Candida glabrata Candida parapsilosis BioMed Research International 11 tissueanddecreasedcytokinelevelsinthebronchoalveolar lipophilicity and consequently diminishes the ability to pene- lavage fuid in the mouse model of S. aureus pneumonia trate bacterial membrane [116]. Some derivatives of apigenin [108]. When apigenin was applied with LysGH15, the lysin showed increased antibacterial activity against S. aureus, derived from phage GH15 with high efciency and a broad Bacillus subtilis, Escherichia coli,andPseudomonas aerugi- lytic spectrum against MRSA, synergism was observed using nosa, especially 7-[3-(Morpholin-4-yl)propoxy]-5-hydroxy- amouseS. aureus pneumonia model [111]. 2-(4-hydroxyphenyl)-4H-chromen-4-one [99]. Given the Reverse antibiotics (RA) are chemicals that are inefective prevalence of antibiotic resistance, apigenin or its derivatives against antibiotic-susceptible bacteria but active against the couldbeacandidateasanewantibioticorasadietary relevant antibiotic-resistant bacteria [112]. RAs can help supplement to enhance the performance of antibiotics. put a stop to the accumulation of antibiotic resistance by Tere are many other studies that tested apigenin- bacteria, because treatment with RA kills the bacteria that containing plant extracts’ antimicrobial efects. Plant extracts have acquired the antibiotic resistance and leaves only the could have several other bioactive components, thus fur- bacteria that possess the original phenotype resistant to ther research is needed to accurately attribute the cause RA and susceptible to that antibiotic [112]. Apigenin has of antimicrobial efect. Tose studies that did not test the RA activities against quinolone-resistant S. aureus [107]. efects of apigenin independent of other potential bioactive Te minimum inhibitory concentrations (MICs) of api- compounds are not included in this review. genin against quinolone-resistant S. aureus strain Mu50 and Some insight is available on the mechanism of the quinolone-susceptible S. aureus FDA 209P are 4 mg/L and antibacterial activity of apigenin. It has been previously morethan128mg/L[107].Apigeninwasalsofoundtoreverse indicatedthatthemaintargetsofapigeninonbacteriamaybe bacterial resistance to cephalosporin cefazidime in Enter- the nucleic acid processing enzymes and cell wall/membrane obacter cloacae [102]. Te MIC of apigenin in cefazidime- [34, 106]. In a study where apigenin inhibited S. aureus,mode resistant E. cloacae (CREC) was higher than 512 �g/mL, which of action was compared to other antibiotics with known indicated no inhibitive efects [102]. Cefazidime applied in mechanism by clustering the treatments based on intracellu- combination with apigenin showed a synergistic efect with lar metabolites. Apigenin was clustered with rifampicin and a fractional inhibitory concentration index smaller than 0.01 norfoxacin which target RNA polymerase, and gyrase and � [102]. Te 5,7- OH group of A ring and one 4 -OHgroup topoisomerase IV, respectively [80], which indicates that the of the B ring in apigenin were found important in reversing target of apigenin could be nucleic acid processing enzymes antimicrobial resistance [102]. Te signifcantly enhanced [75].TetargetDNAgyrasewasalsoreportedinoneother activities of cefazidime by apigenin may have been the result study [117]. Apigenin afects the d-Alanine: d-Alanine ligase of peptidoglycan synthesis inhibition, certain �-lactamase and the type II fatty acid synthetic pathway, both of which are enzymes inhibition, and alteration of outer membrane and involved in cell membrane/wall synthesis [34]. In apigenin- cytoplasmic membrane permeabilization [102]. treated E. caccae, the expression of stress response genes Contrasting results have been observed on apigenin’s and protein chaperone genes were found to be up-regulated, efect on Helicobacter pylori in vitro. While one study saw no which indicated the overall adverse efects of apigenin on the inhibitory efect against thirteen randomly selected clinical bacterium [34]. strains of H. pylori from antral biopsies and a reference Although the methods employed in determining the strain H. pylori ATCC 43504 [101], apigenin showed mod- inhibitory efects are diferent, nearly all studies focused on erate antibacterial activity in another, with a MIC of 25 pathogenic bacteria and cultured the bacteria under aerobic �g/mL against both H. pylori SS1 and H. pylori ATCC conditions. Since dietary apigenin will enter the colon, it is 43504 [106]. An in vivo experiment using Mongolian ger- worthwhile to study the efects of apigenin on commensal gut bils, apigenin treatments (30–60 mg/kg body weight/day) bacteria under anaerobic culture conditions. efectively decreased H. pylori-induced atrophic gastri- � tis and N -methyl-N’-nitro-N-nitroso-guanidine (MNNG)- .. Antiviral Activity. Apigenin has been reported to be able induced dysplasia/gastric cancer rates [113]. Te dose of to inhibit multiple viruses, including enterovirus 71 (EV71), 60 mg apigenin/kg bodyweight/day signifcantly decreased herpes simplex virus HSV-1 and HSV-2, hepatitis C virus, H. pylori colonization and H. pylori-induced histologi- infuenza virus, hand, foot, and mouth disease virus, and cal changes of neutrophil and monocyte infltrations and African swine fever virus (ASFV), but not coxsackievirus A16 atrophic gastritis [113]. (CAV16). Te details are described as follows. Streptococcus mutans isthemainpathogenresponsiblefor Oneofthemajorcausativeagentsforhand,foot,and the development of dental caries in humans. Te organism mouth disease (HFMD), EV71, is a member of genus synthesizes glucans during adhesive interactions with the Enterovirus in Picornaviridae family [118]. Research found tooth surface and other oral bacteria. Apigenin was found that apigenin inhibited EV71-mediated cytopathogenic efect capable of inhibiting water-insoluble glucans synthesis and and EV71 replication in vitro [118]. Viral polyprotein expres- reducing the incidence of dental caries with minimal efects sion, EV71-induced cell apoptosis, intracellular reactive oxy- on the viability of oral microbiota populations in vivo when gen species (ROS) generation and cytokines up-regulation applied topically [114, 115]. were inhibited [118]. Apigenin could interfere with viral Te antibacterial activity of apigenin-C-8 glucoside was internal ribosome entry site (IRES) activity and inhibit EV71- reported to be weak compared to the apigenin aglycone [116]. induced c-Jun N-terminal kinase (JNK) activation which It was hypothesized that glycosylation causes a reduction in is critical for viral replication [118]. Another study tested 12 BioMed Research International methanol extract of Paulownia tomentosa fower for antiviral Trichophyton mentagrophytes and lesions were treated with activity against enterovirus 71 (EV71) and CAV16 in vitro two concentrations of apigenin ointment, 2.5 mg/g, and [119]. CAV16 is another one of the predominant etiologic 5mg/g. Apigenin exhibited a similar efect as the reference agents of hand, foot, and mouth disease [119]. Te extract drug Terbinafne [125]. Applied at 5 mg/g for both apigenin had no efect against CAV16 infection; however, apigenin and Terbinafne, complete recovery from the infection was was again identifed as an active component to inhibit EV71 recorded on the 12th day in both groups. With 2.5 mg/g [119]. Te EC50 value, inhibitory concentration of compound ointment, the infection was cured on the 16th day [125]. A that produces 50% inhibition of virus-induced cytopathic recentpaperreportedthatapigenininducedcellshrinkagein efects, for apigenin to block EV71 infection was 11.0 �M, C. albicans, altering the cell membrane potential and causing with a selectivity index (SI) of approximately 9.3 [119]. Similar leakage of intracellular components [126]. compounds like naringenin and quercetin were not active against EV71 infection [119]. . . Antiparasitic Activity. Leishmaniasis is a disease that In another study, apigenin, as a bioactive compound in afects more than 12 million people worldwide with around Ocimum basilicum, also known as sweet basil, showed a broad 2millionnewcaseseachyear.Itiscausedbytheprotozoa spectrum of antiviral activity [120]. Te highest activities were parasite L. amazonesis [127]. Apigenin treatment for 24 hours against Herpes simplex virus HSV-2 (EC50 =9.7mg/L;SI= resulted in concentration-dependent inhibition of cellular 6.2), adenovirus ADV-3 (EC50 = 11.1 mg/L; selectivity index proliferation with IC50 equals 23.7 �M and increased reactive SI = 5.4), hepatitis B surface antigen (EC50 =7.1mg/L;SI= oxygen species (ROS) generation. Other mechanisms of the 2.3) and hepatitis B e antigen (EC50 =12.8mg/L;SI=1.3). negative efects of apigenin on L. amazonesis include exten- Apigenin inhibited HSV-1 in Madin-Darby canine kidney sive swelling in parasite mitochondria, altered mitochondrial (MDBK) cells within the concentrations of 0.4–1.6 �g/ml membrane potential, rupture of the trans-Golgi network, and [100]. cytoplasmic vacuolization [128]. Apigenin was found to be able to inhibit Hepatitis C virus (HCV) replication in vitro [121]. Apigenin decreased 9. Apigenin and Human Gut Microbiota the expression levels of mature microRNA miR122, a liver- specifc miRNA that positively regulates HCV replica- As shown earlier in the ADME section of the review, part tion, through inhibition of TRBP (transactivating response of the apigenin intake is excreted in the feces and apigenin RNA-binding protein) phosphorylation without signifcantly aglycone was detected in cecum luminal content, thus the afecting cell growth [121]. Terefore, apigenin intake, either bacterial community in the colon is exposed to dietary through regular diet or supplements, may be benefcial for apigenin. Once apigenin enters the colon, it becomes the chronically infected patients [121]. substrate of the pool of various enzymes produced by the gut Apigenin showed signifcant anti-infuenza virus activi- microbiota. Human gut microbiota has been found to harbor ties with IC50 of 1.34 �g/mL in MDBK cells [122]. enzymes that could degrade apigenin. Animal disease related viruses were also found to be Some, not all, commensal gut bacteria are capable of inhibited by apigenin. Foot-and-mouth disease (FMD) is a degrading apigenin on their own. Bacteroides distasonis was highly contagious and clinically acute viral disease of domes- found to be capable of converting apigenin-7-O-glucoside tic and wild cloven-hoofed animals worldwide caused by to apigenin; however, it was not the case with E. coli [129]. FMD virus (FMDV) [123]. FMDV belongs to the Aphthovirus Eubacterium ramulus, a strictly anaerobic human gut bac- genus of the Picornaviridae family [123]. In vitro experiment terium, is able to metabolize apigenin, as well as quercetin, shows that apigenin inhibited FMDV infection at the viral naringenin, daidzein and genistein [130]. It possesses a post-entry stage with no extracellular virucidal activity [123]. phloretin-hydrolase able to break the phloretine C-C bond Similar to EV71, apigenin interfered with the translational [131]. Another anaerobic bacterium isolated from human activity of FMDV by internal ribosome entry site [123]. feces, Clostridium orbiscindens, was found to degrade api- ASFV causes serious diseases in domestic pigs. A dose- genin to 3-(4-hydroxyphenyl)propionic acid with phloretin dependent anti-ASFV efect of apigenin was reported in vitro and naringenin as the two intermediates [132]. Tis degrada- [124]. Apigenin was highly efective at the early stages of tion product 3-(4-hydroxyphenyl)propionic acid, also known infection and was able to achieve a more than 3-log reduction as desaminotyrosine (DAT) is benefcial during infuenza by in ASFV yield when it was added to Vero cells at 1-hour post- triggering type I interferon signaling and in turn augmenting infection [124]. Apigenin inhibited ASFV-specifc protein antiviral responses by phagocytic cells; therefore prevent- synthesis and viral factory formation. Continuous apigenin ing infammation and severe disease [133]. It is possible treatment prevented cytopathic efect in ASFV-infected cells that the anti-infammatory activity and protective efects [124]. on lung tissues mentioned in the previous sections are mediated by microbial metabolites from apigenin as well .. Antifungal Activity. Candida albicans ATCC 10231 and [93, 108]. Degradation of apigenin and its glycosides most C. parapsilosis ATCC 22019 were reported to be inhibited likely involves multiple bacteria, with complementary and by apigenin with MIC of 8 and 16 �g/mL, respectively overlapping functionalities. [100]. Apigenin could be used as an antifungal agent in Some information is available on how the human gut the clinical treatment of dermatophytosis [125]. Mice were microbiota metabolizes apigenin glycosides using in vitro experimentally induced to develop dermatophytosis with experiments or animal models. Hanske et al. performed a BioMed Research International 13 study demonstrating that the bioavailability of apigenin-7-O- fatty acid levels as products of carbohydrate fermentation glucoside is infuenced by human intestinal microbiota using were detected between incubations with diferent phenolic rats and in vitro culture of human gut microbiota in test compounds [134]. In another short-term in vitro fermenta- tubes [129]. Te in vitro culture experiment was carried out tion experiment that tested the efects of apigenin on the by incubating 10 mL culture of human gut microbiota and humangutmicrobiota,apigeninslightlyenhancedtheoverall apigenin-7-O-glucoside mixture in airtight tubes for 24 hours microbial density and the microbial diversity [34]. Some ∘ at 37 C [129]. Results showed that human fecal suspensions microbial community composition changes were observed at converted apigenin-7-O-glucoside completely within 5 hours the phylum level between the apigenin-treated group and the of incubation [129]. Apigenin-7-O-glucoside concentration control group [34]. However, those diferences may not be big remained largely stable without the presence of bacteria enough to make a practical signifcance. [129]. Apigenin and naringenin were transiently formed as intermediate metabolites from apigenin-7-O-glucoside 10. Conclusion [129]. Te end products of apigenin-7-O-glucoside microbial degradation were 3-(4-hydroxyphenyl)propionic acid (4- Apigenin has been shown to possess antibacterial, antiviral, HPPA) and trace amounts of 3-(3-hydroxyphenyl)propionic antifungal, and antiparasitic activities. Te inhibitory efects acid (3-HPPA) [129]. In rat models, germ-free rats excreted on bacteria are strain-specifc. In some cases, although via urine apigenin-7-O-glucoside, apigenin, and luteolin apigenin was not able to inhibit the growth of pathogenic uniformly within 48 hours afer application of apigenin- bacteria, it decreased the production of toxin and alleviated 7-O-glucoside, both in free and conjugated forms [129]. injurycausedbythepathogen[108].Synergyhasbeen Additional metabolites were excreted by human-microbiota observed between apigenin and other antibiotics [105, 110]. associated (HMA) rats in urine: naringenin, phloretin, 3-(3,4- Broth microdilution and agar dilution are the most popular dihydroxyphenyl)propionic acid (3,4-DHPPA), 4-HPPA, 4- methods to determine the minimal inhibitory concentra- hydroxycinnamic acid (4-HCA), and 3-HPPA, with apigenin- tions; however, due to system diferences, there are discrep- 7-O-glucoside, apigenin, luteolin, naringenin, phloretin, 4- ancies between the values obtained from the two methods. HPPA, and 4-HCA in both their free and conjugated forms Tere seems to be no consensus on which method is better and only free forms of 3,4-DHPPA and 3-HPPA [129]. in general. Te antimicrobial efects of apigenin are strain- Apigenin-7-O-glucoside, apigenin, and luteolin, mainly as specifc and limited by its solubility. Still, it is a good starting conjugates, were observed in germ-free rats’ fecal excretion, point to investigate the antimicrobial efects and mechanisms while they were excreted at a considerably lower level in of apigenin, or how to use it to complement or enhance the HMA rats [129]. Tis study strongly supports that the gut efectiveness of antibiotics. microbiota plays a major role in the metabolism of dietary Nearly all studies looking at the antibacterial efects of apigenin. apigenin used pathogenic bacterial and cultured the bacteria Another study using an in vitro system to study the under aerobic conditions. Some published research looked fermentation of apigenin showed that the number of at other similar dietary phytochemicals [135–137]. Te efect metabolites formed is donor-dependent with 3-(4-hydrox- of apigenin on commensal gut bacteria under anaerobic yphenyl)propionic acid, 3-hydroxyphenyl-acetic acid, and conditions is a new territory to explore. 3-phenylpropionic acid being the common metabolites Initial information on the interactions between apigenin detected with microbiota samples from all three diferent andthehumangutmicrobiotaisavailable.Itisnowknown donors [134]. Tis study also found that fermentation rates that a few gut bacteria are able to degrade apigenin into of C-glycosides are slower compared to the rates of O- smallermolecules,someofwhicharebetterabsorbedthan glycosides. apigenin and are more biologically active. Inconclusive results While microbiota facilitates degradation of apigenin, have been reported on how apigenin afects the structure apigenin and its metabolites may also modify the structure of the gut microbiota; in the real-life scenario, it is most and function of gut microbiota considering its efects on likely that the efects would be mild due to the limited bacteria. Te efect on the modulation of gut ecology is still amount of apigenin from dietary sources and the ability poorly understood [17]. Troughout the study by Hanske et of the microbiota to degrade apigenin. We also know that al., the similarity of the intestinal microbiota composition the changes in the microbial community and the rate of of rats to the human fecal sample used to associate the rats degradation of apigenin depend on the form of apigenin, was determined by PCR-coupled denaturing gradient gel whether it is glycosylated and which type of glycoside, and electrophoresis and was reported to be ranged from 63.3 the gut microbiota composition [134]. Also because of the to 75.8%. However, no detailed information was available complexity of human gut microbiota, there is still much to on compositional changes of the community although there investigate. seemed to be a change. Te in vitro experiment was carried Our knowledge of how apigenin afects human gut in test tubes without pH control and a freshly inoculated microbiota and how it in turn modulates human health microbiota. It would be more ideal to perform the in vitro isstillverylimited.Apowerfultooltostudythestruc- incubation of apigenin and gut bacteria in a chemostat and tural changes in the gut microbiota is Next-Generation establish a stable community. In the aforementioned research Sequencing. High-throughput sequencing platforms have that used samples from three diferent donors, no signifcant enabled rapid determination of microbiota composition by changes in the microbiota composition and short-chain sequencing and analyzing either targeted regions of certain 14 BioMed Research International

genes (e.g., 16S gene for bacteria, Internal transcribed spacer [12]M.G.L.Hertog,P.C.H.Hollman,M.B.Katan,andD. gene for fungi) or the whole genome of the community Kromhout, “Intake of potentially anticarcinogenic favonoids (shot-gun metagenomics). It is yet to be discovered what and their determinants in adults in Te Netherlands,” Nutrition other gut bacteria can metabolize apigenin and what are the and Cancer,vol.20,no.1,pp.21–29,1993. metabolic products. Tere might be a potential cross-feeding [13] E.´ C. Lefort and J. Blay, “Apigenin and its impact on gastroin- phenomenon in apigenin degradation, as the metabolites testinal cancers,” Molecular Nutrition & Food Research,vol.57, from one bacterium could be the substrate for another. Te no. 1, pp. 126–144, 2013. impact of dietary apigenin on human health through gut [14] J. Madunic,´ I. V. Madunic,´ G. Gajski, J. Popic,´ and V. 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