Protective effects of flavonoids against microbes and toxins: The cases of hesperidin and hesperetin

    Protective effects of flavonoids against microbes and toxins: The cases of hesperidin and hesperetin Mehrdad Iranshahi, Ramin Rezaee, Hamideh Parhiz, Ali Roohbakhsh, Fatemeh Soltani PII: DOI: Reference:

S0024-3205(15)00366-5 doi: 10.1016/j.lfs.2015.07.014 LFS 14450

To appear in:

Life Sciences

Received date: Revised date: Accepted date:

7 April 2015 13 June 2015 11 July 2015

Please cite this article as: Iranshahi Mehrdad, Rezaee Ramin, Parhiz Hamideh, Roohbakhsh Ali, Soltani Fatemeh, Protective effects of flavonoids against microbes and toxins: The cases of hesperidin and hesperetin, Life Sciences (2015), doi: 10.1016/j.lfs.2015.07.014

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Full title: Protective effects of flavonoids against microbes and toxins:

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The cases of hesperidin and hesperetin

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Short title: Beneficial effects of hesperidin and hesperetin

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Mehrdad Iranshahi 1, Ramin Rezaee 2, Hamideh Parhiz 3, Ali Roohbakhsh 4,

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Fatemeh Soltani 1 * 1

Biotechnology Research Center and School of Pharmacy, Mashhad University of Medical

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Sciences, Mashhad, Iran

Department of Molecular Sciences, School of Medicine, North Khorasan University of Medical

Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences,

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Sciences, Bojnurd, Iran

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Mashhad, Iran; Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran. 4

Pharmaceutical Research Center, School of Pharmacy, Mashhad University of Medical Sciences,

Mashhad, Iran.

*To whom correspondence should be addressed: Fatemeh Soltani, Pharm.D., Ph.D. Assistant Professor of Pharmaceutical Biotechnology Biotechnology Research Center School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran E-mail: [email protected] Tel: +98-513-8823255 (337) Fax: +98-513-8823251

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Graphical Abstract: A schematic view of the protective effects of hesperidin and hesperetin against microbes and toxins.

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Abstract:

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Many plants produce flavonoids as secondary metabolites. These organic compounds may be involved in the defense against plant-threatening factors, such as microbes and toxins.

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Certain of these flavonoids protect their origin source against plant pathogens, but they also

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exhibit potential healthy properties in human organisms. Hesperidin (Hsd) and its aglycone,

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hesperetin (Hst), are two flavonoids from the Citrus species that exhibit various biological properties, including antioxidant, antiinflammatory and anticancer effects. Recent studies

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indicated that Hst and Hsd possess antimicrobial activity. Although the exact mechanisms behind their antimicrobial properties are not fully understood, several mechanisms such as

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the activation of the host immune system, bacterial membrane disruption, and interference

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with microbial enzymes, have been proposed.

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Hsd and Hst may also have protective effects against toxicity induced by various agents. These natural substances may contribute to the protection of cells and tissues through their antioxidant and radical scavenging activities. This review discusses the protective activities of Hsd and Hst against microbes and several toxicities induced by oxidants, chemicals, toxins, chemotherapy and radiotherapy agents, which were reported in vitro and in vivo. Furthermore, the probable mechanisms behind these activities are discussed.

Keywords: flavonoid; hesperetin; hesperidin; toxin; antimicrobial

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1. Introduction

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Polyphenols, a large class of biologically active substances, are distributed in plants as secondary metabolites. These substances provide color and flavor to different plant parts

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and they also play an important role in resistance against various microbial pathogens and

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protect against radiations and toxins. Many recent studies have mainly focused on

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polyphenols and their diverse biological effects (Quideau et al., 2011; Daglia, 2012).

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Flavonoids are one of the most common polyphenols, and they exhibit interesting and beneficial medicinal effects on human health. The various biological properties of

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flavonoids, such as antioxidant, antiinflammatory, anticancer, antibacterial, immune-

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2000).

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stimulating and antiviral activities, have been reported extensively (Harborne and Williams,

Hesperidin (Hsd) is the major flavonoid in citrus fruits, and it was isolated for the first time from citrus peel by Lebreton in 1827. The Hsd molecule is composed of an aglycone unit, namely hesperetin (Hst), and a disaccharide, rutinose (Figure 1).

Hesperetin: R=H; Hesperidin: R= Rutinose (glucose+rhamnose)

Figure 1: The chemical structures of hesperetin and hesperidin

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Hsd and Hst possess different activities, such as antioxidant, antiinflammatory, antimicrobial, anticarcinogenic and antiallergic effects. Hsd and Hst substances are also

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called bioflavonoids because of this wide range of effects. A descriptive review of the

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different biological activities and physicochemical properties of Hsd was published by

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Garg et al. in 2001 (Garg et al., 2001). In addition, neuropharmacological, antioxidant, antiinflammatory properties and the pharmacokinetics of Hsd and Hst were published in

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our recent review papers (Parhiz et al., 2014; Roohbakhsh et al., 2014). The protective

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effects of Hsd and Hst against toxicities induced by certain chemotherapy drugs have been widely investigated (Abdel-Raheem and Abdel-Ghany, 2009; Trivedi et al., 2011).

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However, there has not been a comprehensive report on the protective effects of Hsd and

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Hst against invading pathogens and various toxicities induced by environmental toxins,

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occupational hazards, radiotherapy and chemotherapeutic agents. Accordingly, this review provides a detailed overview of the current state of knowledge of the protective effects of the bioflavonoids, Hsd and Hst, against the aforementioned toxicities. The probable mechanisms governing these particular activities are also discussed.

2. Protective effects of Hsd and Hst against invading pathogens Plant flavonoids play an important role in the protection against pathogenic microorganisms, such as bacteria, fungi and viruses. However, the high rate of microbial resistance to conventional antibiotics suggested flavonoids as suitable alternatives to antibiotics. Furthermore, flavonoids can be considered natural food preservatives because of their antimicrobial activities (Rodriguez Vaquero et al., 2010). Generally, flavonoids are

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found in glycosylated forms in plants, and the presence of a sugar moiety is an important factor that determines their bioavailability. However, the antimicrobial efficacy of

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flavonoids varies depending on their chemical structure and the strain of microorganism.

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Flavonoids, such as Hsd and Hst, exhibit anti-infective and anti-replicative effects against

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several microorganisms.

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2.1. Antibacterial activities

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The antibacterial activity and bioavailability of flavonoids are affected by various parameters, such as molecular conformation, hydrophobicity, solubility, presence or

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absence of sugar moiety and the type of sugar in the chemical backbone (Manach et al.,

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2005). However, the exact mechanisms of the antibacterial effects of flavonoids are not

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clear, but several mechanisms, such as interference with bacterial DNA synthesis, bacterial movement, cytoplasmic membrane permeability and the inhibition of bacterial metalloenzymes, have been proposed (Mirzoeva et al., 1997; Havsteen, 2002; Cushnie and Lamb, 2005). Different studies evaluated the inhibitory effects of plant flavonoid-rich extracts and pure flavonoids, including Hsd derivatives, against some pathogenic microorganisms (Table 1). For example, an investigation in 2004 demonstrated that the ethanolic extract of grapefruit seed and pulp containing flavanones, mainly naringin and Hsd, significantly inhibited only Gram-positive bacteria using an agar diffusion method, however, this extract was effective against Gram-positive and Gram-negative bacteria using a broth dilution susceptibility test (Cvetnic and Vladimir-Knezevic, 2004). In 2008, Liang evaluated the antimicrobial effects of a flavonoid extract of Pericarpium citri reticulatae (FEPCR) and its major constituents, including Hsd, nobiletin and 6

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tangeretin, against Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Salmonella typhimurium and Enterobacter cloacae using the agar

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dilution method. This study demonstrated that Hsd and FEPCR exhibited a wide range of

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antibacterial activity, but the two other FEPCR flavonoids, tangeretin and nobiletin, were

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nearly inactive against the tested microorganisms. In general, Hsd and FEPCR exerted higher inhibitory activity against Gram-positive bacteria than Gram-negative bacteria. The

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lowest and highest minimum inhibitory concentrations (MICs) were observed for S. aureus

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(MIC: l00 µg/ml) and E. cloacae (MIC: 1600 µg/ml), respectively. The polymethoxylated structure of tangeretin and nobiletin likely produced their low antibacterial activity (Yi et

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al., 2008). The antibacterial activity of Hsd against Proteus mirabilis and S. aureus was

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also shown in another study, in which its MIC90 was 12 times lower than that of

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chloramphenicol (de Gregorio Alapont et al., 2000). Although Hsd was active against S. aureus is these studies, it was almost ineffective in an earlier study against some tested pathogenic strains, including S. aureus, B. subtilis, Streptococcus beta-haemolyticus, Enterotoxigenic E. coli, Klebsiella species, Pseudomonas aeruginosa, S. typhimurium, Shigella dysenteriae, Shigella flexneri and Vibrio cholera. (Nazrul Islam and Ahsan, 1997). Hsd pretreatment before infection with S. typhimurium aroA in a mouse model reduced bacterial numbers in the spleen and liver, and it also reduced plasma lipopolysaccharide (LPS) levels. The decrease in LPS levels also down-regulated early and late endotoxin shock mediators in plasma, namely tumor necrosis factor and high-mobility group protein B1, respectively. Although Hsd reduced the number of bacteria in this study, Hsd did not exhibit direct antibacterial activity against S. typhimurium aroA. Hsd increased the influx of immune cells, such as neutrophils, into the peritoneal cavity, which suggests that the 7

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antibacterial activity may be due to the activation of host defense systems rather than LPS binding (Kawaguchi et al., 2004).

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Narbad’s group investigated the antimicrobial properties of flavonoid-rich fractions of

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bergamot peel against Gram-negative bacteria (E. coli, P. putida, S. enterica), Gram-

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positive bacteria (Listeria innocua, B. subtilis, S. aureus, Lactococcus lactis) and the yeast Saccharomyces cerevisiae and demonstrated that the analyzed fractions were active only

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against Gram-negative bacteria. These studies found that treatment of these fractions with

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Pectinase 62L, which converts flavonoid glycosides into their aglycones, increased the antimicrobial activity of the flavonoids. Additionally, they studied the antimicrobial effects

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and possible interactions between some pure bergamot flavonoids (neohesperidin, Hst,

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neoeriocitrin, eriodictyol, naringin and naringenin). The aglycones exhibited MIC values

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ranging from 250-1000 µg/ml, and a synergistic antimicrobial effect was observed between eriodictyol and Hst against E.coli and S. enterica and between eriodictyol and naringenin against S. enterica and P. putida. A slight antagonistic interaction was observed for naringenin and Hst against E. coli and S. enterica and eriodictyol and Hst against P. putida. These synergistic and antagonistic effects may occur through various mechanisms, such as a similarity of targets (Mandalari et al., 2007). Lee’s group sought to improve the bioavailability and the solubility of Hsd, and they converted Hsd into hesperetin-7-O-glucoside (Hst-7-G) using naringinase from Aspergillus sojae. This bioconversion increased the bioavailability of Hsd as well as its solubility in 10% ethanol. Hst-7-G exhibited a higher inhibitory effect against Helicobacter pylori than Hst, but its efficacy was similar to Hsd. The relatively lower inhibitory effect of Hst compared Hsd and Hst-7-G is likely related to its poor water solubility (Lee et al., 2012). 8

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Plant-derived antimicrobial substances are suitable templates for designing more potent antimicrobial drugs. For example, Duganath’s group synthesized and evaluated the

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antibacterial activity of some novel Hsd analogues against B. subtillis, S. aureus,

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Micrococcus luteus, E. coli, P. aeruginosa and P. fluorescens. The analyzed structure-

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activity relationships (SAR) revealed that the replacement of a carbonyl moiety with hydrazone enhanced the inhibition zone, and the presence of electron-donating groups

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further improved the inhibitory effects (Duganath et al., 2014). One of the proposed

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mechanisms behind the antimicrobial effects of flavonoids is the inhibition of hyaluronidase, which plays an important role in the penetration of sperm and microbes into

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a substrate. It was shown that flavonoids interact with hyaluronidase, mainly through

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electrostatic and hydrophobic forces, and form enzyme-flavonoid complexes. This binding

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affects the microenvironment of enzyme active site, and results in reduction or inhibition of the enzyme activity (Zeng et al., 2015). Garg et al. (2005) introduced a new sulfonated derivative of Hsd (S-Hsd) as an anti-hyaluronidase agent and a novel contraceptive microbicide. They discovered that S-Hsd had a potent antimicrobial effect against sexually transmitted infection (STI)–causing bacteria, including Chlamydia trachomatis and Neisseria gonorrhoeae, but it did not inhibit the growth of normal vaginal flora, such as Lactobacillus gasseri. These findings suggested that S-Hsd lacked toxicity in normal and healthy cells (e.g., sperm, vaginal tissue), and the microbial flora of the vagina are a potential candidate to prevent the spread of STIs and conception (Garg et al., 2005). Polyphenols possess antibacterial activity against human pathogenic microorganisms, but they also inhibit physiological microbiota that are present in the human gut and other tissues (Parkar et al., 2008). This commensal microbiota plays a protective role against 9

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invasive pathogens through the release of antimicrobial substances. They metabolize the glycoside form of polyphenols into the corresponding aglycones with higher

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bioavailability. The released aglycones inhibit the growth and activity of intestinal bacteria.

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Therefore, excessive consumption of polyphenols in the diet may negatively impact human

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health. An example for this influence was presented by Duda-Chodak in 2012, who assessed the effects of some polyphenols, including Hsd and Hst, on human intestinal (Bacteroides

galacturonicus,

Lactobacillus

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bacteria

sp.,

Enterococcus

caccae,

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Bifidobacterium catenulatum, Ruminococcus gauvreauii and E. coli). The results demonstrated that Hsd did not impact the tested bacteria, but its aglycone Hst inhibited

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2.2. Antiviral activities

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almost all of the tested bacteria (MIC>250 µg/ml) (Duda-Chodak, 2012) .

Viruses are a large group of human pathogens that cause severe infectious diseases. Public health concern on the prevalence of chronic viral infectious diseases, such as human immunodeficiency virus (HIV), hepatitis C virus (HCV), and influenza virus, and their resistance to conventional antiviral therapies have increased the demand for new antiviral drugs (Lou et al., 2014) . Several recent investigations of developed antiviral substances with high efficacy and low toxicity were conducted. The antiviral effects of Hsd, Hst and several flavonoids were reported many years ago (Table 1). For example, an early study using means of dye uptake measurements

(Finter)

evaluated

the

effects

of

Hsd,

Hsd-methylchalcone,

trihydroxyethylrutin, quercitrin, rutin and aurantiin against vesicular stromatitis virus (VSV) in mouse fibroblasts. Additionally, the inhibitory activity of Hsd against influenza 10

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virus in HeLa cells was also determined using the plaque reduction test. Hsd protected HeLa cells against VSV and influenza virus. The protection of HeLa cells disappeared after

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the addition of hyaluronidase, which assists microbe penetration into target tissues.

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Therefore, the antiviral activity of Hsd was due to its anti-hyaluronidase activity, which

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was shown in previous reports (Wacker and Eilmes, 1978; Garg, Anderson et al., 2005). Another early study evaluated the inhibitory effect of flavonoids, including quercetin,

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quercitrin, Hsd and rutin, on the growth of Human (alpha) herpesvirus 1 (HSV-1) and Suid

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(alpha) herpesvirus 1 (SHV-1) (pseudorabies virus). Quercetin and quercitrin suppressed viral growth in a concentration-dependent manner, but Hsd and rutin did not show antiviral

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activity. In addition, quercetin and quercitrin enhanced intracellular cAMP levels, but the

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Hsd and rutin did not affect cAMP. This finding suggests a relationship between cAMP

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levels and the antiviral effect against some of viruses (Mucsi and Pragai, 1985). The inhibitory effects of Hst, naringenin and its glycosides against the replication of a neuro-adapted strain of Sindbis (NSV) was studied in 2003 Hst and naringenin suppressed viral replication with an ID50 of 15.9 and 14.5 µg/ml, respectively, in the MTT assay and an ID50 of 20.5 and 14.9 µg/ml, respectively, using the plaque reduction assay. In contrast, the corresponding glycosides bearing the rutinoside sugar did not block NSV replication (Paredes et al., 2003). Hsd and diosmin showed potent inhibitory activity against rotaviruses in another study, but their aglycones were inactive. The rutinoside moiety in Hsd and diosmin appears important to protection against the invasion of rotavirus into cells. However, previous reports showed that this moiety negatively impacts antiviral activity against NSV (Bae et al., 2000; Paredes, Alzuru et al., 2003). 11

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Furthermore, sulfonated hesperidin (S-Hsd) completely inhibited Herpes simplex virus type 2 (HSV-2) and HIV at a concentration of 100 µg/ml, but Hsd did not exhibit an inhibitor

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effect on these viruses (Garg, Anderson et al., 2005). Sialidase is an important enzyme in

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the initial and late stages of influenza virus infection processes, and it is a drug target for

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the prevention of the spread of influenza infection. Glucosyl hesperidin (G-Hsd), which has higher water solubility than Hsd, decreased the replication of three influenza virus strains

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(H1N1, H3N2 and H5N3) when it was added to the cell culture after or at the time of

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infection. However, pretreatment caused no reduction. This observation might be attributed to viral sialidase inhibition activity rather than receptor binding inhibition (Saha et al.,

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2009).

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Lin et al. found that Hst, Hsd and some other flavonoids exhibited significant antiviral

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activity against enterovirus 71 (EV-71) without toxic effects. The probable antiviral mechanism was evaluated using the anti-oxidant and inhibition of viral internal ribosomal entry site (IRES) activities. None of these substances had significant anti-oxidant activity in the trolox equivalent antioxidant capacity assay, but 7, 8-dihydroxyflavone, kaempferol and Hst exhibited the most effective inhibition of viral IRES activity. The internal ribosomal entry site is required for viral protein translation, which enables it as a suitable drug target against EV-71. Therefore, the antiviral mechanism of these flavonoids might occur through the elimination of viral protein translation (Tsai et al., 2011). One recent study demonstrated that Hsd inhibits canine distemper virus in vitro, especially at the times of adsorption, penetration and intracellularly during the infectious cycle. Therefore, Hsd may demonstrate anti-distemper activity through the direct inactivation of

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virus particles and other internal mechanisms, such as the inhibition of nucleic acid

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synthesis (Carvalho et al., 2013). 2.3. Antifungal activities

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Fungal infections and resistance to current antifungal drugs have been topics of research

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over the past decade. This focus can be associated with, at least partially, with increasing

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incidence of cancer and AIDS. Therefore, it is necessary to develop newer antifungal substances. A wide range of biological activities of Hsd and Hst was studied, but their

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antifungal activities are limited to only a few reports in the literature (Table 1). Therefore, a clear structure-activity relationship for these substances and their analogues cannot be

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discerned, and more investigations in this area are needed.

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Islam and Ahsan showed that Hsd did not exhibit any inhibitory effects against Aspergillus

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fumigatus, A. niger, Candida albicans, Trichoderma sp., and S. cerevisiae. However, Krolicki and co-workers demonstrated that Hsd and naringin possessed antifungal activity against Botrytis cinerea, T. glaucum and A. fumigatus (Krolicki and Lamer-Zarawska, 1984; Nazrul Islam and Ahsan, 1997). Some investigations demonstrated that these substances are natural additives in food industries. For example, Hsd and ten other flavonoids isolated from citrus species showed a range of inhibitory effects on food fungal contaminants, including A. parasiticus, A. flavus, Fusarium semitectum and Penicillium expansum (Salas et al., 2011). Salas et al. (Salas et al., 2012) demonstrated that Hsd reduced patulin accumulation, which is a type of mycotoxin produced by different fungi, such as P. expansum, A. terreus and Byssochlamys

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fulva. The results demonstrated that there was no significant difference between the

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antifungal activities of flavanones.

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2.4. Antiparasitic effects

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Recently, the development of natural compounds with anti-parasitic activity has been highly encouraged. The antifilarial effects of Hst against the human lymphatic filarial

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parasite, Brugia malayi, have been studied (Table 1). Hst killed the adult worm and

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microfilariae in vitro, but it was ineffective in in vivo models. This difference between in vitro and in vivo results may be explained by the host metabolism, which may transform Hst into its inactive metabolite (Lakshmi et al., 2010).

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Allam’s group investigated the in vitro and in vivo schistosomicidal activities of Hsd. Hsd demonstrated effective schistosomicidal activity at 200 µg/ml, but it was almost inactive at

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lower concentrations. The results from the in vivo study indicated that Hsd reduced both male and female worm burdens. Hsd likely exerted its schistosomicidal activity first through a direct effect on Schistosoma mansoni by inhibiting vital worm enzymes and second by boosting the humoral immune response and increasing the IgG against this parasite (Allam and Abuelsaad, 2014).

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Table 1: Protective effects of Hsd and related compounds against invading pathogens Types

Compounds

Proposed Mechanisms

Gram positive bacteria

S. aureus, S. epidermidis, E. faecalis, S. betahaemolyticus, L. innocua, B. subtilis, L. lactis, M. luteus E. coli, S. typhimurium, E. cloacae, P. mirabilis, Klebsiella , S. dysenteriae, S. flexneri, V. cholera, P. putida,S. enterica, H. pylori, P. aeruginosa, P. fluorescens, N. gonorrhoeae VSV e, influenza virus, NSVf, Rotavirus, HSV1g, HSV-2, SHV-1h, HIVi, j EV-71 , Canin distamper

e.g. Hsda, Hstb, Hst-7-Gc, SHsdd, nobiletin, tangeritin, eohesperidin, neoeriocitrin, riodictyol, naringin, naringenin

Interfering with DNA synthesis, bacterial movement, membrane permeability, bacterial enzymes, activation of host defense and etc.

(Cushnie and Lamb, 2005; Cvetnic and VladimirKnezevic, 2004; Daglia, 2012; Yi et al., 2008; Kawaguchi et al., 2004; Lee et al., 2012; Mandalari et al., 2007; Garg et al., 2005)

e.g. Hsd, Hst, Hsdmethylchalcon, SHsd, quercitrin, quercetin, rutin, aurantiin, naringenin, diosmin, kaempferol, 7,8dihydroxyflavone

Direct virus inactivation, inhibition of Hyaluronidase, sialidase, viral replication, IRESk, DNA synthesis, affecting cAMP and etc.

e.g. Hsd, Hst

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(Garg, Anderson et al., 2005; Mucsi and Pragai, 1985; Paredes et al., 2003; Bae et al., 2000; Paredes, Alzuru et al., 2003; Garg, Anderson et al., 2005; Tsai et al., 2011; Carvalho et al., 2013) (Krolicki and LamerZarawska, 1984; Nazrul Islam and Ahsan, 1997; Salas et al., 2011 and 2012)

e.g. Hsd, Hst

Inhibition of worm enzymes, boosting host immune

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Viruses

Fungi

B. cinerea, glaucum, fumigatus, parasiticus, flavus, semitectum, expansum

Parasites

B. malayi, S. mansoni

T. A. A. A. F. P.

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References

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Gram negative bacteria

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Pathogens

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(Lakshmi et al., 2010; Allam and Abuelsaad, 2014)

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response a

hesperidin.bhesperetin, chesperetin-7-O-glucoside, dsulphonated hesperidin, evesicular stromatitis virus, neuro-adapted strain of Sindbis, gHerpes simplex virus, hSuid (alpha) herpesvirus 1, ihuman immunodeficiency virus, jenterovirus 71, kinternal ribosome entry site, lNP: not proposed

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3. Protective effects of Hsd and Hst against toxicities induced by various factors

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Human health is threatened by pathogenic microorganisms, parasites and exposure to toxic materials, primarily occupational and environmental contaminations, such as heavy metals.

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Additionally, chemotherapy and radiotherapy have side effects and toxicities on the human body. These factors damage tissues and cells through different mechanisms, such as the

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induction of reactive oxygen species (ROS) production and oxidative stress (Figure 2).

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Therefore, natural compounds that possess antioxidant properties may contribute to the

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protection of cells and tissues against the deleterious effects of free radicals. Hsd and Hst are the most abundant flavonoids with strong radical scavenger activity.

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Figure 2: Schematic of oxidative stress pathways and the protective role of Hsd and Hst

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3.1. Heavy metals

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One of the major environmental and human health concerns is related to toxic heavy

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metals, particularly arsenic, lead, cadmium and mercury. The generation of reactive oxygen and nitrogen species are primary inducers of toxicity and carcinogenicity of these metals.

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These species bind to structural proteins, enzymes, nucleic acids and other vital cellular

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components and cause oxidative deterioration of biological macromolecules. Regimens that contain antioxidants and a chelating agent provide more efficient treatments of metal

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poisoning (Flora et al., 2008).

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Hsd, Hst and other derivative flavonoids have antioxidant and metal chelating properties.

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Several studies investigated the protective effect of these flavonoids against toxicities induced by heavy metals.

Cadmium (Cd) has many industrial uses, but it has serious pathological consequences on several tissues because of its long retention time and the induction of early oxidative stress. Cd-induced injury is mediated by different mechanisms, including ROS creation and the induction of oxidative stress (Bernhoft, 2013). Pari et al. (Pari and Shagirtha, 2012) demonstrated that Hst treatment in male Wistar rats at 40 mg/kg body weight/day for 21 days efficiently maintained liver functions against Cdinduced oxidative hepatotoxicity and returned the status of liver marker enzymes, oxidative stress markers, and enzymatic and non-enzymatic antioxidants to their normal levels. Consistent with this study, Hst exhibited nephron protective effects and attenuated Cdinduced nephrotoxicity markers in another study. The protective role of Hst likely occurred 17

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through its potent antioxidant, metal-chelating and membrane-stabilizing properties

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(Shagirtha and Pari, 2011).

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Additionally, Hsd revealed a potent protective activity against lesions induced by acute exposure to arsenic, a major environmental toxicant and a human carcinogen, in mouse

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liver and kidney. Various mechanisms, including antioxidant, anti-inflammatory, vascular

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protection and enzyme modulation activities, are likely responsible for the multiply protective effects of Hsd (das Neves et al., 2004).

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The protective activity of alpha-glucosyl Hsd (G-Hsd) against testicular toxicity and sperm nuclear DNA damage mediated by vanadium, a metal mainly utilized as ferrovanadium or

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as a steel additive, was studied in male Sprague-Dawley rats. Vanadium at 1 µg/kg for a period of 90 days induced a notable toxicity in rat testis. The co-administration of G-Hsd

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(25 and 50 µg/kg) with vanadium significantly restored the sperm and histological parameters, and it also overcame the oxidative stress situation induced by this metal (Vijaya Bharathi et al., 2014).

3.2. Chemotherapeutic agents

Hst and Hsd also exhibit a potential to attenuate the toxic side effects of several chemotherapeutic drugs. Doxorubicin (DOX) is a potent anthracycline chemotherapeutic agent that possesses different toxicities and side effects to non-target tissues. Several hypotheses were proposed to explain the mechanisms of DOX-induced side effects. An increase in ROS leading to apoptosis has been implicated in several toxicities of DOX. Therefore, the consumption of

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potent antioxidants or antioxidant-rich plants might play an important role in maintaining the ROS balance and consequently protect against adverse effects. For example, an in vitro

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study showed that Pummelo (Citrus maxima) fruit juice, which has antioxidants including

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ascorbic acid (AA), Hsd, naringin, and gallic acid, protected rat cardiac H9C2 cells against

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DOX- induced cytotoxicity (Chularojmontri et al., 2013). Another in vivo investigation demonstrated that the co-administration of Hsd with DOX protected this tissue against

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DOX-induced cardiotoxicity by maintaining serum cardiac enzyme levels (Abdel-Raheem

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and Abdel-Ghany, 2009). Moreover, oral Hst administration significantly reduced the testicular toxic effects of DOX and suppressed the expression of some apoptosis-associated

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genes, such as NF-kB, p38 and caspase-3, in rat testicular cells (Trivedi et al., 2011).

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Melzig et al. evaluated the influence of several selected flavonoids on daunomycin

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cytotoxicity, an anthracycline chemotherapeutic agent, and obtained various results. Some of these compounds, including flavonols such as quercetin and rutin, decreased the toxic effects of daunomycin, but others, including myricetin, flavone and apigenin, inhibited and improved the cytotoxicity. In this study, Hsd, naringin and catechin did not change the proliferation inhibition induced by daunomycin in endothelial cells (Melzig et al., 1997). Cisplatin is another effective chemotherapeutic agent, but its utility is limited because of its nephrotoxicity. Several mechanisms, such as apoptosis, necrosis, inflammation and oxidative stress, are involved in the pathogenesis of cisplatin-induced renal injury. Hsd treatment in rat minimized cisplatin-mediated nephrotoxicity, and oral administration of Hsd for several days prior to cisplatin injection decreased oxidative stress, apoptosis, necrosis and inflammation induced by cisplatin (Sahu et al., 2013). Hsd also ameliorated gentamycin-induced acute nephrotoxicity, which is characterized by an increase in serum 19

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parameters such as urea, uric acid, creatinine and NO, a decrease in the content and activities of anti-oxidant enzymes and tubular necrosis, in rat model (Anandan and

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Subramanian, 2012).

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Gastrointestinal toxicity primarily occurs during methotrexate (MTX) chemotherapy, which

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is a dihydrofolate reductase antagonist that is used in the treatment of malignancies. The efficacy of Hsd in preventing intestinal damage during MTX treatment was reported by

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Acipayam’s group using histopathological and immunohistochemical methods (Acipayam

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et al., 2014).

Hsd reduced blood toxicity of cyclophosphamide (CTX). Pretreatment of tumor-containing

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mice with a dose of 200mg/kg of Hsd prior to CTX administration resulted in a notable

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protection against hematotoxicity induced by CTX. However, Hsd administration with

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CTX in colon carcinoma (CT-26)-bearing mice suppressed the antitumor activity of CTX. These results suggest that these natural compounds may be beneficial in reducing some of the side effects of chemotherapeutic agents, but changes in the efficacy of these chemotherapies by flavonoids in patients should be considered (Hosseinimehr et al., 2012).

3.3. Radiations 3.3.1. Radiation therapy Radiation therapy is one of the major methods for the treatment of patients with cancerous tumors. Ionizing radiation induces oxidative stress, DNA damage and apoptosis. The high radiation doses can affect normal cells and result in deleterious side effects, such as genomic instability.

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Kalpana group (Kalpana et al., 2011), demonstrated that orally administered Hsd, at an optimum dose of 25 mg/kg body weight, protected the liver of mice against 4Gy X-ray

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radiation hepatocellular damage as evidenced by the restoration of the activity of

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antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT) and

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glutathione peroxidase (GPx), and a decline in the lipid peroxidative index (thiobarbituric acid reactive substances) and genetic damage parameters in blood and liver. Furthermore,

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this group revealed that the pretreatment of human peripheral lymphocytes with 16.38 µM

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of Hsd in vitro 30 minutes prior to 4 Gy gamma radiation exposure protected the cells against genetic damage (micronuclei frequencies, dicentric aberrations, comet parameters

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and DNA fragmentation) and alterations in biochemical factors (SOD, CAT, GPx and

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thiobarbituric acid reactive substances) induced by γ-irradiation. Interestingly, the optimum

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concentration of Hsd (16.38 µM) was lower than the radioprotectors, cimetidine (1mM) and vanillin (0.5mM) (Kalpana et al., 2009). These in vitro results are consistent with Park’s in vivo findings in which they evaluated the radioprotective activity of Hsd (50 mg/kg and 100 mg/kg orally administered for 7 days) against 1 Gy, 3 Gy and 5 Gy of γ radiation in Sprague–Dawley rats (Pradeep et al., 2008). Hsd also protected rat hearts and kidneys against the toxic damages of irradiation and improved changes in the corresponding parameters (Pradeep et al., 2012). These results were further supported by another study from Hosseinimehr and co-workers who designed an in vitro/in vivo method to evaluate the radioprotective effect of Hsd. They demonstrated that a single oral ingestion of Hsd (250 mg) by human volunteers one hour before in vitro exposure of isolated lymphocytes to 150 cGy of 60Co γ-irradiation protected the cells against induced genotoxicity (Hosseinimehr et al., 2009). Moreover, their earlier 21

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study demonstrated that the pre-administration of Hsd (80mg/kg) before 2 Gy radiation protected mouse bone marrow cells and reduced the frequency of micronucleated

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polychromatic erythrocytes (Hosseinimehr and Nemati, 2006).

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3.3.2. Ultraviolet irradiation

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Solar UV irradiation, particularly ultraviolet B (UVB), has hazardous effects, including sunburn, inflammation, skin aging and cancer. Numerous studies showed that some natural

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compounds reduced the photodamage induced by short-term exposure to UVB radiation. For example, Marnewick’s group indicated that polyphenolic extracts of honeybush, Hsd

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and mangiferin-rich, topically applied on the dorsal skin of SKH-1 mice for a period of 10

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days, 30 minutes before exposure to UVB (180 mJ/cm2), reduced sunburn effects and the

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expression of inflammatory and cell proliferation markers. Moreover, this treatment decreased oxidative stress and modulated the induced oxidative damage. However, the pure forms of the two most abundant flavonoids in honeybush, Hsd and mangiferin, were less effective. One of the reasons that these substances showed lower activity than the plant extract might be because of the synergistic effects between herbal phenolic compounds. Moreover, the conversion of Hsd into Hst might enhance its photoprotection through improving its absorption, as shown in another study by Lee et al. (Petrova et al., 2011). Lee et al. showed that the conversion of the glycoside contents (Hsd and narirutin) of aqueous extracts of Citrus unshiu peel into their corresponding aglycones resulted in lower levels of matrix metalloproteinase expression in human dermal fibroblasts exposed to UVA. These enzymes are one of the major enzymes that mediate UV-induced skin aging. The protective

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activity of Hsd was lower than the plant source, but it protected Balb/C mice epidermis against UVB-induced cyclobutane pyrimidine dimers via activation of DNA repair

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mechanisms accompanied by enhanced p53 expression (Jin et al., 2011).

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3.4. Miscellaneous properties

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In addition to aforementioned toxicities, several substances, such as ethanol, nicotine, insecticides, and food additives, also have toxic effects on human health.

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Alcoholic liver disease is a major health concern in modern dietary life, and it occurs after prolonged or excessive alcohol intake. Dangerous byproducts, namely acetaldehydes, are

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produced during the hepatic metabolism of alcohol via several enzymes, such as alcohol

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dehydrogenase, CYP2E1 and CAT. Acetaldehydes react with macromolecules and generate

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adducts that consequently induce hepatic inflammation, fibrosis, cirrhosis and other injuries. The administration of Hsd with ethanol to male ICR mice for 8 weeks significantly maintained the prognostic parameters of hepatocellular damage. Hsd was enzymatically modified to hespretin-7-O-glucoside to improve bioavailability. The resulting product

showed higher hepatic protection, in terms of the recovery of hepatic antioxidant systems and cytokine release, compared to Hsd (Park et al., 2013). In a recent study, Hsd (100 or 200 mg/kg) in rat exhibited antifibrotic effects against liver fibrosis induced by 10 mg/kg/day, i.p., dimethylnitrosamine, which is used as a preservative in the meat industry. Hsd prevented the increase in serum and hepatic parameters and gene expression of caspase-3, inducible nitric oxide synthase and α-smooth muscle actin (Elshazly and Mahmoud, 2014).

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Hsd at a dose of 25 mg/kg demonstrated a significant protective effect against nicotineinduced lung toxicity in male Wistar rats and returned the levels of lipid peroxidative and

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biochemical indices to a normal state (Balakrishnan and Menon, 2007).

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In addition, Hsd inhibited DNA damage induced by diazonin. Treatment of lymphocytes

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with Hsd 3 hours before exposure to 750  µM diazonin for 24 hours significantly inhibited micronucleus formation and DNA damage induced by this nonsystemic organophosphate

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insecticide (Shokrzadeh et al., 2014). Glucosyl-Hsd inhibited DNA double-strand breakage

4. Conclusion

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procedures (Yoshikawa et al., 2004).

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induced by YOYO, which is a green fluorescent dye that is widely used in DNA staining

Hst and Hsd are citrus flavonoids that exhibit various biological activities. Numerous papers have been published on certain beneficial effects of Hsd, Hst and their derivatives. These substances play an important role in plant defense systems to combat different pathogens. Therefore, they may be useful polyphenolic substances that possess antibacterial, antiviral and antifungal activities in humans. The exact mechanisms of antimicrobial activity are not fully understood. However, different mechanisms, including bacterial membrane perturbation, the inactivation of metalloenzymes, interference with bacterial DNA synthesis machinery, and the activation

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of the host immune system, have been proposed. Although the antimicrobial effects of some plant-derived substances might be lower than those of standard antimicrobial agents,

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in order to understand their possible mechanisms of actions, further investigations on these

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compounds are still needed. On the other hand, they can be considered as suitable templates

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for designing more potent and less toxic natural based-antimicrobial drugs.

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In addition, these flavonoids may play an important role in the protection of populations exposed to occupational, environmental and therapeutic agents. These substances exert

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their protective activities primarily through antioxidant effects.

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Although polyphenols are natural compounds with low toxicity, but the presence of sugar

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moiety in their backbone might affect beneficial microbiota by altering the micro-ecology in the gut. However, commensally intestinal microbiota may increase the bioavailability

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and biological effects of flavonoids and thereby more investigations are required to determine the suitable doses and structures to use in humans. In conclusion, Hsd and Hst can be considered as potential protective agents against various harmful factors, however further studies are necessary to elucidate the beneficial and protective effects of these compounds in human populations. Acknowledgments This study was partially supported by the Mashhad University of Medical Sciences. Conflict of Interest statement The authors declare that there are no conflicts of interest.

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