Food Research International 77 (2015) 221–235

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Food Research International

journal homepage: www.elsevier.com/locate/foodres

Review

Therapeutic potential of flavonoids and their mechanism of action

against microbial and viral infections—A review
Asif Ahmad a, Muhammad Kaleem a,⁎, Zaheer Ahmed b, Hammad Shafiq a
a
Department of Food Technology, PMAS-Arid Agriculture University Rawalpindi, Pakistan
b
Department of Home Health Sciences, Faculty of Sciences, Allama Iqbal Open University, H-8, Islamabad Pakistan

a r t i c l e i n f o a b s t r a c t

Article history: Flavonoids are polyphenolic phytochemicals generally found in vegetables, fruits, flowers, nuts, seeds, tea, honey
Received 7 April 2015 and propolis. In the past plants containing these compounds have been used to cure different diseases. Seeking
Received in revised form 11 June 2015 usage of plants for curing diseases many researchers carried out studies to analyze the antibacterial, antifungal,
Accepted 17 June 2015
antiviral, anti-inflammatory and antioxidant properties of extract of different plants containing flavonoids.
Available online 23 June 2015
Similarly, synergistic relationship was found between chemotherapeutics and flavonoids. Cancer is a serious
Keywords:
disease due to its high mortality rate. Anticancer mechanism of flavonoids has been described by elucidating
Antibacterial its point of intervention in cancer cell cycle. Flavonoids act as bactericidal and bacteriostatic by damaging
Antiviral cytoplasmic membrane, inhibiting energy metabolism and inhibiting synthesis of nucleic acids against different
Cancer microorganisms. Viral infections are more difficult to control as compared to bacterial. So antiviral drugs are least
HIV available, since flavonoids possess antiviral properties, so individual flavonoid compound having antiviral prop-
Toxins erties may be concentrated or their structure may be modified to enhance their antiviral activity. Phosphorylation
of protein by cytokines II is inhibited by flavonoids which help in the cell arrestation of HIV at integration traction
phage of virus. Structure activity relationship of flavonoids and human immune deficiency virus (HIV) has also
been discussed.
© 2015 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
2. Flavonoid chemistry and classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
3. Flavonoids: Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
4. Antibacterial properties of flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
4.1. Mechanism of flavonoid action . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
4.2. Neutralization of bacterial toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228
5. Antiviral properties of flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
5.1. Human immune deficiency virus inhibition by flavonoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
5.2. Structure activity relationship of flavonoids and human immune deficiency virus (HIV) . . . . . . . . . . . . . . . . . . . . . . . . 232
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232

1. Introduction

Phytochemicals are plant based substances that modulate metabo-

lism process to prevent humans from degenerative and chronic
⁎ Corresponding author at: PMAS Arid Agriculture University Rawalpindi, 43600,
Pakistan. diseases. Phenolic compounds are important class of plant based
E-mail address: kaleem.ft@gmail.com (M. Kaleem). secondary metabolites that are not essential for plant survival unlike

http://dx.doi.org/10.1016/j.foodres.2015.06.021
0963-9969/© 2015 Elsevier Ltd. All rights reserved.
222 A. Ahmad et al. / Food Research International 77 (2015) 221–235
primary metabolites. These compound imparts organoleptic properties
to plant based foods importantly taste and color properties along and
improves the nutritional quality of food. Flavonoids are polyphenolic
phytochemicals which are biologically active (Messina & Redmond,
2006; Schneeman, 1987). Flavonoids are the most prevalent group
found in plants. About 9000 compounds are identified and there num-
ber is increasing day by day (Wood & Webster, 1986). Flavonoids act
as antioxidant, anti-depressant and anti-inflammatory in the human
body. Furthermore extract of plant containing flavonoids has been
shown to decrease the risk of cancer (Evers & Millar, 2002). Similarly
flavonoids protect the body from cardiovascular diseases because they
possess the ability to inhibit lipid per oxidation process (Manach,
Mazur, & Scalbert, 2005).
Initially drug resistant strains appear in hospitals where antibiotics
were mostly used (Levy, 1998). In military hospitals in 1930s sulfon-
amide resistant Streptococcus pyogenes appeared (Levy, 1982). London
civilian hospital confronted penicillin resistant Staphylococcus aureus
soon after the introduction of penicillin. In the same way Mycobacterium
tuberculosis which is resistant to streptomycin appeared in the patients
after the use of antibiotics (Barber & Rozwadowska-Dowzenko, 1948).
This shows that development of resistance against antimicrobial com-
pounds is not new but breadth of resistance in a single organism and
the number of resistant microorganisms is mounting (Levy, 2002). It is
a challenging problem for the whole world. In US hospitals out of 2 mil-
lion cases of bacterial infections 70% cases involve strains that are
resistant to antimicrobial agents. So the medicinal companies are
looking towards other classes of compounds with better antimicrobial
properties (Cushnie & Lamb, 2005). In antibacterial resistant infections
the standard method of treatment fails resulting in death two times
greater as compared to non-resistant antibacterial infections. As the
resistant microbial infections survive long time there are more chances
of diseases spread. Antimicrobial resistance increases the cost of health
care because infection cannot be treated with first line medicines lead-
ing to usage of expensive therapeutics (Wang, Ding, Liu, & Zheng, 2004).
Pharmaceutical companies are searching for new classes of compounds
with better antimicrobial properties. It has been suggested that such
formulations of drugs should be made that act on different target sites
as compared to that which are currently used and act on single site
(Levy & Marshall, 2004). Antimicrobial agents of tea were reviewed by
Bansal et al. (2013) which showed that tea is a rich source of polyphe-
nols which possess antimicrobial properties.
Mostly the drugs approved by the US food and drug administration
for treatment of viral infection are synthetic nucleoside analogues. In
vitro and in vivo studies show that viruses are developing resistance
to the synthetic nucleoside (Field, 2001). Similarly infection of adenovi-
rus occurs throughout the year and in all ages of individuals in many
countries. Pneumonia due to adenovirus is responsible for high
mortality rate specifically in children whose age is less than two years
(Avila et al., 1989). No chemotherapic medicine is available which is
effective in preventing infection. Therefore it is the need of time to
search new alternative natural antiviral compounds (Chiang, Chiang,
Liu, & Lin, 2003).
Enterovirus is a RNA virus belongs to the genus Enterovirus and fam-
ily Picornaviridae. EV71 is responsible for neurological diseases which
include brainstem encephalitis, aseptic meningitis and flaccid paralysis
but no vaccine or antiviral drug is available for the treatment of infec-
tions of EV71. 7 hydroxyisoflavone has been found effective in the inhi-
bition of EV71 replication and also inhibits viral protein synthesis which
is dose dependent. So there is need for more research on extract of plant
containing flavonoids to combat the problem of enterovirus 71 (Wang
et al., 2013). One of the most serious diseases in the world is cancer
due to its high mortality rate (Zini, Czerninski, & Sgan-Cohen, 2010).
The incidence of cancer has increased continuously not in developing
countries but also in developed countries of Europe and United States.
The incidence of cancer and mortality due to cancer is decreased by
chemotherapy, immunosuppressant and radiations but the number of
deaths due to cancer is greater than heart diseases in persons with age
less than 85 years (Doyle et al., 2006).
Cancer development takes place in three different phases which
include initiation, promotion and progression (Jones & Baylin, 2002).
Normal cell is converted into malignancy in many years which provide
opportunities to interfere in the development of malignancy which is
shown in Fig. 1 (Béliveau & Gingras, 2007). So it is the need of time to
search for compounds which possess cancer preventive properties
(Yoon & Baek, 2005). There is a strong correlation between phenolic
content and antioxidant activity of Camellia sinensis. Since free radicals
contribute to the cancer development so the foods like tea which are
rich source of polyphenolics and possess antioxidant activity might be
useful in decreasing the risk of cancer development (Oliveira, Calado,
Ares & Granato, 2014). Chemoprevention is a technique which utilizes
natural or synthetic or combination of both to control or reverse the
development of carcinogenesis (Atawodi et al., 2009).
Different studies have demonstrated that some flavonoids inhibit the
growth of cancer cell lines. The anti-proliferative effect in estrogen
depending tumor cells is related to the antiestrogenic properties of dif-
ferent flavonoids (Chiang et al., 2003). In some in vitro studies flavonoids
have been reported to affect cell signaling and in the progression of cell
cycle. Examples of such effects include inhibitory effect of tea flavonoids
on signal transduction pathways which are mediated by epidermal
growth factor and other factor that is platelet derived growth factor
that affects favorably downstream events like angiogenesis (Yazaki,
Sasaki, & Tsurumaru, 2009). Protein tyrosine kinase is inhibited by
quercetin and genistein which takes participation in cell proliferation
process (Gu et al., 2003). Apoptosis and cell cycle arrestation are caused
by quercetin, luteolin and apigenin by a p53-dependent mechanism
(Prior, Lazarus, Cao, Muccitelli, & Hammerstone, 2001).
2. Flavonoid chemistry and classification
Flavonoids are polyphenols that are biologically active (Hollman &
Katan, 1999). Research on flavonoids begin in 1936, when Albert
Szent-Gyorgyi was to establish synergistic effect between vitamin C
and lemon co-factor (Gil, Ferreres, & Tomas-Barberan, 1999) which
was known as citrin at that time and laterally vitamin P (Murray,
1998). Now flavonoids are not considered as vitamins because there de-
ficiency is not associated with deficiency symptoms. The basic structural
unit of flavonoid compounds consist of 2-phenyl-benzo α-pyrane or
flavane nucleus, which further composed of two benzene rings (A and
B) that are linked by a heterocyclic pyrane ring (C) as shown in Fig. 2
(Hollman & Katan, 1999; Kogawa, Kazuma, Kato, Noda, & Suzuki, 2007).
Mostly flavonoids are found as 3-o-glycosides with other sugar
conjugates except flavonols. Glucose is the most common glycosidic
unit however other sugar like arabinose, galactoserhamnose and
glucorhamnose conjugates are also present (Yazaki et al., 2009). Flavo-
noids polymerize by enzymatic oxidation or fermentation and results in
the formation of proanthocyanidins and tannins. Proanthocyanidins
which vary in size from dimmers to up to 17 unit flavonol polymers
(Gu et al., 2003; Prior et al., 2001). Proanthocyanidins are further classi-
fied into fifteen subclasses among which three are found commonly in
plants which are prodelephinidins, procyanidins and propelargonidins
(Gu et al., 2003). The structure of different classes of flavonoids has
been shown in Fig. 3.
The source of variance in flavonoids includes:
1. Structure of the aglycone ring vary and its state of oxidation or
reduction
2. The degree of hydroxylation of the aglycone and positioning of
hydroxyl group;
3. Difference in derivatisation. The derivative group may include alkyl
groups, isoprenoids or other carboxylic or alcoholic groups.
According to these sources of variation in theory more than 2 × 106
numbers of flavonoids are possible. Many of flavonoids have been
 A. Ahmad et al. / Food Research International 77 (2015) 221–235
Fig. 1. Carcinogenesis stages and potential effect of flavonoids in cancer progression.
identified till now and there number is increasing day by day (Havsteen,
1983).
Flavonoids may be classified on the base of biosynthetic origin. For
example chalcones, flavan-3-ols, flavanones, and flavan-3,4-diols, are
both intermediates in biosynthesis as well as end products that
Fig. 2. Flavonoid skeleton.
223
accumulate in plant tissues (Cushnie & Lamb, 2005). Other classes
are only end products of biosynthesis, for example anthocyanidins,
proanthocyanidins, flavones and flavonols (Sannomiya et al., 2005).
About 3000 flavones and 700 isoflavones exist in plants (de Rijke et al.,
2006). Classes of flavonoids and there representative compounds are pre-
sented in Table 1.
Based on molecular structure flavonoids are divided into eight groups
which include flavones, flavanone, flavonols, isoflavones, anthocyanidins,
catechin, dihydroflavanols and chalcone (Tsuchiya, 2010). Based on the
variance in heterocyclic ring C flavonoids are classified into six classes,
flavones, flavonols, flavanones, catechins, anthocyanidins, isoflavones
(Hollman & Katan, 1999). On the basis of oxidation state of central
pyran ring flavonoids are classified as flavonols, flavones, flavanonols, fla-
vanones, flavan-3-ol, anthocyanidins, isoflavon, and proanthocyanidins
(Hemingway, Karchesy, & Branham, 1989).
Since flavonoid polarity depends on the content of glycosides,
isoprenoids and either, aliphatic or aromatic substitution so for their
extraction a range of solvents from water to ethyl ether can be used
for extraction of them from a complex mixture such as propolis (bee
glue) bearing tissue, or plant extracts and they are often treacle
subfractionated on hydroxylapatite before the final separation is
performed by capillary electrophoresis (Aderogba, Ogundaini, & Eloff,
2006) or HPLC (Sultana & Anwar, 2008).
224 A. Ahmad et al. / Food Research International 77 (2015) 221–235

Fig. 3. Structure of major classes of flavonoids (Cushnie & Lamb, 2005).

 A. Ahmad et al. / Food Research International 77 (2015) 221–235 225

Table 1 containing 154.59, 146.57 and 104.46 mg PCY/L respectively. The

Flavonoid classes and representative compounds.
Flavonoid Examples of flavonoids
classes compounds
Flavones Apigenin, luteolin, chrysin,
diosmetin, vitexin, orientin,
isoorientin
Flavonol Quercetin, kaempferol,
myricetin, rutin
Catechins Catechin, epicatechin,
epigallocatechin gallate,
gallocatechin
Flavanones Hesperetin, naringenin,
eriodictyol, corylifolin,
kushenol, barachinin,
sophoraflavanone, kurarinone
Anthocyanidins Cyanidin, delphinidin,
petunidin
Peonidin, Pelargonidin,
Malvidin
Isoflavones Genistein, daidzein, glycitein,
formanantine, formonetin,
afromosin, medicarpin,
tectorigenin, calycosin,
daidzein
References
Hollman and Katan, (1999),
Xiao, Kai, Yamamoto, and Chen
(2013)
Cushnie and Lamb (2005)
Doublier and Wood (1995)
Hollman, Bijsman, van
Gameren, Cnossen, de Vries
and Katan (1999), Xiao et al.
(2013)
Bamforth (1985), Tanwar and
Modgil (2012)
Xiao et al. (2013)
major individual flavonoids constituent which were detected include
catechin, epicatechin and epigallocatechingallate (Granato, Grevink,
Zielinski, Nunes, & van Ruth, 2014). Similar compounds were also
detected in Brazilian tea which showed that Brazilian tea C. sinensis is
rich source of catechin, epicatechin, procyanidin B2, Mentha piperita is
rich source of quercetin and Peumus boldus contains significant amount
of catechin, epicatechin, procyanidin B1 and quercetin (Zielinski et al.,
2014). Hesperidin is the major flavonoid present in orange and manda-
rins while naringenin and narirutin are found in grape fruit while
fresh tomato skin contain naringeninchalcone which is changed to
naringenin during the processing of tomato to ketchup (Mälkki, Cho,
& Dreher, 2001; Wood, 2007). Cyclocarya paliurus is a plant used in
the folk medicine in China is source of quercetin, kaempferol,
kaempferol-7-O-a-L-rhamnoside, kaempferol-3-O-b-D-glucuronide
and quercetin-3-O-b-D-glucuronide (Xie et al., 2015). Phaseolus vulgaris
is a rich source of delphinidin glucoside, malvidin glucoside and
petunidin glucoside. Beside these compounds seventeen colorless
compounds were also detected in P. vulgaris among that the compound
which were detected in higher amount include myricetin 3-
Oarabinoside, catechin, vanillic acid and epicatechin (Mojica, Meyer,
Berhow, & de Mejía, 2015).

4. Antibacterial properties of flavonoids

3. Flavonoids: Sources
A most commonly found group of plant phenolic compounds is fla-
vonoid and they are found in almost all parts of the plant (Cushnie &
Lamb, 2005; Toda, Okubo, Ikigai, Suzuki, Suzuki, Hara and Shimamura,
1991a, 1991b). They are present in plants as secondary metabolites
meaning that they do not affect growth of the plant but they impart
color to flowers and fruits and may protect the plant from insect pests
and ultraviolet radiations. As flavonoids are phytochemicals they cannot
be synthesized by human or animal body and they are taken from plant
source (Cook & Samman, 1996). Good sources of flavonoids include
fruits, vegetables, wine, tea, seeds, flowers, nuts, blue berries, black
berries, can berries, red beans, propalis and honey (Atawodi et al.,
2009; Cook & Samman, 1996; Hollman et al., 1999; Tsuchiya, 2010).
Black rice (Oryza sativa) is rich source of flavonoids and anthocyanins
containing 51.26 mg/100 g and 116.58 mg/100 g respectively (Pedro,
Granato, & Rosso, 2015). Extract or raw fermented cocoa beans from
Cameroom has been shown to contain significant amount of flavan-3-
ols, sulfated flavan-3-ols and dimmers of proantocyanidin (Patras,
Milev, Vrancken, & Kuhnert, 2014). Flavonoid compounds their
structure and sources are summarized in Table 2.
Quercetin is the most commonly found flavonol in the diet. Quercetin
is present in vegetables and fruits but the highest concentration is
present in onion (Campbell & Bedford, 1992). The importance of foods
as a source of quercetin varies within different countries. According to
Hertog, Hollman, Katan, and Kromhout (1993) tea is the major source
of quercetin in Netherlands and Japan. While in Italy, wine is the major
source of flavonoids (Jenkins, Jenkins, Zdravkovic, Würsch, & Vuksan,
2002). Mauritian fresh tea leaves and black tea contains significant
amount of polyphenolics and flavonoids which contribute to its antioxi-
dant potential of Mauritian black tea. Among flavonoid compounds it is
good source of epicatechin, catechin, epigallocatechin 3-gallate, epigallo-
catechin and dimmers of procyanidin B1 and B2 (Luximon-Ramma et al.,
2005). Similarly, 16 flavonoids were detected in the tea made from water
lily flowers which include the derivatives of quercetin, kaempferol,
isorhamnetin and chrysoeriol (Yin et al., 2015). In the United States the
daily intake of flavonols and flavones varies between 20 and 22 mg/d
of which 73 to 76% was quercetin (Würsch & Pi-Sunyer, 1997).
Flavanones are mostly found in citrus fruits. Maximum concentra-
tion is found in solid tissue but their amount in juice is several hundred
milligrams per litre (Lazaridou, Biliaderis, Micha-Screttas, & Steele,
2004). Green, red, and yellow teas are rich source of flavan-3-ols
Extract obtained from plants that were used in folk medicine have
been screened by many researcher for their antibacterial activity. It
has been reported that extract obtained from Hypericum and
Chromolaena possesses antibacterial activities (Satoh, 2005; Satoh,
Ishii, Shimizu, Sawamura & Nishimura, 2002a, 2002b). Similarly propo-
lis contains high amount of flavonoids and it is analyzed by researcher
several times and results are positive for antibacterial properties
(Nichenametla, Taruscio, Barney, & Exon, 2006; Sivakesava &
Irudayaraj, 2001b; Xiao et al., 2013). Table 3 describes the antibacterial
activity of different flavonoid compounds.
Researcher moved one step ahead by isolating and identifying differ-
ent flavonoids that possess antibacterial activity. These flavonoids
include luteolin and luteolin 7-glucoside (Ruiz-Matute, Soria,
Martínez-Castro, & Sanz, 2007; Zhu et al., 2010), naringin and
naringenin (Garcia-Alvarez, Huidobro, Hermida, & Rodriguez-Otero,
2000; Lichtenberg-Kraag, Hedtke, & Bienefeld, 2002), apigenin
(Horvath & Molnár-Perl, 1997), pinocembrin (Sivakesava & Irudayaraj,
2001a), galangin (Kelly, Downey, & Fouratier, 2004), quercetin, 3-O-
methylquercetin and various quercetin glycosides (Batsoulis et al.,
2005; Won, Lee, Ko, Kim, & Rhee, 2008), ponciretin (Baroni,
Chiabrando, Costa, & Wunderlin, 2002), and kaempferol along with its
derivatives (Verzera, Campisi, Zappalà, & Bonaccorsi, 2001). Other fla-
vones, flavone glycosides, flavonols, flavanones, isoflavones, isoflavans,
isoflavanones having antibacterial activities have been identified
(Alcaraz, Blanco, Puig, Tomas, & Ferretti, 2000; Basile, Giordano,
López-Sáez, & Cobianchi, 1999; Cushnie, Hamilton, & Lamb, 2003;
Özçelik, Orhan, Özgen, & Ergun, 2008; Paradkar & Irudayaraj, 2002).
Many flavonoids show antibacterial activity. Out of one hundred and
eighty two flavonoid twenty five have been shown active against num-
ber of bacteria. Flavanones without sugar moiety showed antimicrobial
activity while none of the flavanolignans and flavonols tested showed
antimicrobial activity against test microorganisms. A number of dis-
eases in humans and animals are caused by S. aureus (Satoh, Ishii,
Shimizu, Sawamura & Nishimura, 2002a, 2002b). There is considerable
mortality and morbidity due to S. aureus infections (Sakane,
Sawamura, Ohno, Nishimura, & Umehara, 2002). Flavonoid chalcone,
2′(OH)-chalcone, 2′,4′(OH)2-chalcone and 2′,4(OH)2-chalcone can be
used as therapeutic agents against infections of methicillin-resistant
S. aureus strains (Alcaraz et al., 2000). Extract of green tea (C. sinensis)
possesses antistaphylococcal activity. In vitro gastric and duodenal di-
gestion did not affect the antistaphylococcal activity of green tea extract
226 A. Ahmad et al. / Food Research International 77 (2015) 221–235

Table 2
Flavonoid compound, their structure and sources.

Flavonoid Structure Source Reference

Apigenin Mentha longifolia Kogawa et al. (2007)

Baicalin Oroxylum indicum Sannomiya et al. (2005)

Genistein Butea monospermea Evans (2009)

Kaempferol Acalypha indica, Momordica charantia Ghoulami, Il Idrissi, and Fkih-Tetouani (2001), Nahrstedt, Hungeling,
and Petereit (2006)

Luteolin Aloe vera, Bacopa monnieri, Cannabis sativa, Momordica Aderogba et al. (2006), Argolo et al. (2004), Ghoulami et al. (2001),
Lopez-Lazaro (2009)

Myricetin Betula pendula, Azadirachta indica Gupta, Taneja, Dhar, and Atal (1983), Nahrstedt et al. (2006)

Orientin Cannabis sativa, Mimosa pudica, Passiflora incarnate Lopez-Lazaro (2009), Sannomiya et al. (2005)

Quercetin Azadirachta indica, Bauhinia monandra, Calendula officinalis, Ghoulami et al. (2001), Gupta et al. (1983), Nahrstedt et al. (2006)
Momordica charantia

Quercetrin Betula pendula, Tilia cordata Gupta et al. (1983)

Rutin Tilia cordata Gupta et al. (1983)

Vitexin Passiflora incarnate Aderogba et al. (2006)

but gastroduodenal digestion of green tea extract minimized the
antistaphylococcal activity. Phytochemical analysis showed that poly-
phenols are stable to gastric conditions, but they are degraded to a
great extent by gastroduodenal conditions specifically gallocatechins
which are responsible for antibacterial activity (Marchese et al., 2014).
The utilization of flavonoids against bacterial, protozoan, and fungal
infections has two aims:
(1) To kill the microbial cells and
(2) To act against the spread and the effects of the bacterial toxins
(Hollman et al., 1997).
Most of the strains that are encountered by humans might be con-
trolled by flavonoids. Mechanism by which they control microbial
growth is complex. Since eicosanoids are not formed by bacteria
apparently, so the target of the flavonoids may include the PG COX
and other related lipoxygenase enzyme. Bacteria similar to other pro-
karyotic cells do not have cAMP PDE enzyme which can be targeted
by the flavonoids but they rather contain metalloenzymes, heavy
metal atom present in them forms strong ligand complexes with flavo-
noid compounds example include phosphatases (Wiseman, 1999).
Therefore, the bacteriocidal activity of flavonoids is due to its metabolic
perturbation. Ion channels that are sensitive components of animal and
bacterial cells are specific inhibition points of flavonoids. In animals the
ion channels are regulated by phosphorylation/dephosphorylation
reactions (Kelly et al., 2004).
Beside from the active role that the flavonoids have in the destruc-
tion of infectants, they strengthen loose connective tissues by control-
ling some enzymes that can hydrolyze their proteoglycan and protein
network. This network sterically hinders the diffusion of infectants
 A. Ahmad et al. / Food Research International 77 (2015) 221–235 227

Table 3
Flavonoid compounds and their antibacterial activity.

Flavonoid Antibacterial and antiviral activity against References

bacteria

Quercetin Staphylococcus aureus, Bacillus subtilis, Bacillus cereus, Akroum, Bendjeddou, Satta, and Lalaoui (2009),
Escherichia coli, Helicobacter pylori, Pseudomonas Doublier and Wood (1995), Narayana, Reddy, Chaluvadi,
aeruginosa, P. fluorescens, Enterobacter aerogenes and Krishna (2001), Ramos et al. (2006),
Rattanachaikunsopon and Phumkhachorn (2010)
Apigenin Streptococcus pyogens, Streptococcus viridans, (Akroum et al. (2009), Basile et al. (1999), Doublier and
Streptococcus jaccalis, Enterobacter cloacae, Vibrio cholera, Wood (1995), Narayana et al. (2001), Srinivas and
Enterococcus faecalis, Escherichia coli, Staphylococcus Reddy (2012)
aureus, Pseudomonas aeruginosa, Bacillus cereus, Bacillus
subtilis, Klebsiella pneumoniae, Bacillus subtilis Salmonella
typhimurium
Rutin Bacillus anthracis, Pseudomonas aeruginosa, E. coli, Narayana et al. (2001), Novy, Urban, Leuner, Vadlejch,
Klebsiella pneumoniae, Salmonella typhimurium, Bacillus and Kokoska (2011), Srinivas and Reddy (2012), Sun,
cereus Kuang, and Li (2011), Tapas et al. (2008)
Baicalin Staphylococcus aureus, Pseudomonas aeruginosa
Chrysin Streptococcus jaccalis, Streptococcus baris, Streptococcus Darwish, Ra'ed, Zarga, and Nazer (2013), Hollman et al.
pneumonia, E. coli (1999), Speciale et al. (2013), Tereschuk, Riera, Castro,
and Abdala (1997)
Hydroxyethylrutoside Clostridium perfringens Bukvički, Veljić, Soković, Grujić, and Marin (2012),
Datisetin Proteus vulgaris Kujumgiev et al. (1999), Narayana et al. (2001)
Hydroxyethylrutoside Pseudomonas aeruginosa
iso-liquiritigenin Staphylococcus aureus
Saponarine Enterobacter cloacae, E. aerogenes, Pseudomonas Basile et al. (1999), Thomas (2013)
aeruginosa
Lucenin Enterobacter cloacae, E. aerogenes, Pseudomonas
aervuginosa
Lucenin Enterobacter cloacae, E. aerogenes, Pseudomonas
aeruginosa
Bartramia flavone Enterobacter cloacae,, E. aerogenes, Pseudomonas
aeruginosa
5,7-dimethoxyflavanone-4′-O-B-D-glucopyranoside Klebsiella pneumonia Özçelik et al. (2008)
5,7-dimethoxyflavanone-4′-O-[2″-O-(5‴-Otranscinnamoyl)- Klebsiella pneumonia
B-D-apiofuranosyl]-B Dglucopyranoside
5,7,3′-trihydroxyflavanone- 4′-O-B Dglucopyranoside Klebsiella pneumonia
Naringenin-7-O-B-Dglucopyranoside Klebsiella pneumonia
Quercetin-3-Orutinoside (Rutin) Klebsiella pneumonia
Kaempferol-3-Orutinoside (nicotiflorin) Klebsiella pneumonia
Kaempfero-3-o-glucoside Escherichia coli, Staphylococcus aureus, Pseudomonas Akroum et al. (2009)
aeruginosa, Bacillus cereus, Bacillus subtilis
Morin-3-O-arabinoside P. fluorescens, V. cholera, L. monocytogenes Balangcod et al. (2012), Rattanachaikunsopon and
Phumkhachorn (2010)
Genkwanin Vibrio cholera, Enterococcus faecalis, E. coli Cushnie, Hamilton and Lamb (2003), Lee, Moon, Kim,
Mendonca, and Paik (2010)
Rhamnocitrin Vibrio cholera, Enterococcus faecalis, Micrococcus luteus, (Liu, Mou, Zhao, Wang, Zhou, Wang, et al. (2010),
Shigella sonnei Martini, Katerere, and Eloff (2004), Teffo et al. (2010)
Quercetin-5,3′dimethylether Vibrio cholera, Enterococcus faecalis, Micrococcus luteus,
Shigella sonnei
Rhamnazin Vibrio cholera, Enterococcus faecalis
Kaempferol Vibrio cholera, Enterococcus faecalis

through the tissue. One example is the reticence of hyaluronidase by dif-
ferent flavonoids. Thus, the latter contribute to the control and encapsu-
lation of the infectants. In this state, the infectants will steadily be
decomposed by scavenging and repair processes (Özçelik et al., 2008).
Many pathogenic bacterial strains including Escherichia coli, S. aureus
and Pseudomonas aeruginosa are called as multi drug resistant (MDR)
organisms and are a great challenge for medical practices. No or limited
antibodies are available for treatment of infections from such organ-
isms. This necessitates the need for formulations of new antibacterial
drugs a critical need (Flint, Enquist, Racaniello, & Skalka, 2009). To
search these compounds a study was carried out on quercetin, morin
and their corresponding derivatives of sulfonic acid against bacterial
species of P. aeruginosa, S. aureus and E. coli along with their antibiotic
resistant strains isolated from the infected humans. Against standard
gram negative microorganisms morin was found effective in limiting
the growth at lowest concentrations. In comparison to this morin is
less effective against S. aureus. All the compounds were found effective
against drug resistant microorganisms and in this case also morin was
found most effective (Wang et al., 2013). Growth of Lactobacillus and
Bifidobacterium spp. is promoted by epigallocatechingallate (EGCG),
gallocatechingallate (GCG) and epigallocatechin 3-O-(3-O-methyl)
gallate (EGCG3) extracted from Chinese Oolong tea, while they possess
inhibitory effect against Clostridium histolyticum and bacteroides group
of bacteria (Zhang et al., 2013).
Many bacteria have been reported to possess enzymes that have
possessed extended spectrum β lactamases (ESBLs) that are resistant
to lactam possessing antibiotics. Enterobacteriaceae family possesses
plasmid mediated enzymes A. Mostly these microorganism gram nega-
tive rods cause pneumonia in humans (Landolfo, Gariglio, Gribaudo, &
Lembo, 2003). For controlling such infections it is necessary to look for
new antimicrobial agents that are effective against such resistant micro-
organisms. Since plant secondary metabolites are produced in response
to infections. So these secondary metabolites can be effective source for
controlling such infections. Since flavonoids are secondary metabolites
and there antimicrobial properties have been reported in many studies
against gram negative bacterias (Basile et al., 1999). In vitro study has
shown that flavonoids are effective against the isolated strains of Klebsi-
ella pneumoniae, which are similar to that produced by the control of
loxacin at the concentration of 32–64 μg ml/l; in comparison, another
control, ampicillin, showed no activity. On the basis of different studies
228 A. Ahmad et al. / Food Research International 77 (2015) 221–235
flavonoids are considered as potential therapeutic compounds for infec-
tions that may be caused by ESβL-producing bacteria in the future
(Evers, Wang, & Huang, 2004).
It is interesting to know that whether these compounds kill the bacte-
ria or they inhibit the growth of bacteria. Bactericidal activity is measured
by minimum bactericidal concentration (BMC) assay or time kill method
(Gu et al., 2003) and this activity is defined as 99.9% reduction in bacterial
number. These methods are mostly applied to check flavonoids and in
many studies this activity has been seen that flavonoids act by aggregat-
ing or clumping cells (Barber & Rozwadowska-Dowzenko, 1948; Levy,
1998). In 2007, it was proved that the flavonols galangin possess bacteri-
cidal activity. A significant implication of this aggregatory effect is that
traditional procedures are not suitable now for representing flavonoid
bactericidal activity because decrease in colony forming unit (CFU)
might be associated with clumping of cells and cells may not be dead in
this state. Instantaneously no apparent solutions to this methodological
problem, but an approach might be to come to an end MBC testing in
favor of time–kill studies, with microscopic testing of treated bacteria
(Levy, 2002).
4.1. Mechanism of flavonoid action
Earlier research on flavonoids shows their direct bactericidal activity
in three different ways
• Damage of cytoplasmic membrane by perforation mechanism (Plaper
et al., 2003) and decrease in membrane fluidity (Wu et al., 2008)
• Inhibitory effect on energy metabolism (Ávila, Smânia,, Monache, &
Smânia, 2008)
• Inhibition of synthesis of nucleic acids (Mirzoeva, Grishanin, & Calder,
1997).
Evidences in the support of abovementioned mechanism have been
provided laterally. Mechanism of action of flavonoids against bacteria
has been presented in Fig. 4. Flavolan and flavonol classes of compounds
destroy the cytoplasmic membrane (Alvarez, Zarelli, Pappano, &
Debattista, 2004). Flavonol, flavan-3-ol and flavone classes of
compounds inhibit the energy metabolism process (Nielsen, Boesen,
Larsen, Schønning, & Kromann, 2004) while isoflavones have been
shown to inhibit the process of nucleic acid synthesis (Ávila et al., 2008).
Evidences about another two mechanism of flavonoid action have
been provided. Firstly inhibitory effect on cell membrane synthesis
process (Suresh Babu et al., 2005) and secondly inhibition of cell wall
synthesis (Šmejkal et al., 2008).
There are three hypotheses about the mechanism of action of
flavonoids (i) flavonoids having different structure have different
mechanism of action while flavonoids having similar structure have
one or similar mechanism of action. (b) All flavonoids have different
mechanism of action (c) all flavonoids have same mechanism of action.
The possibility that different flavonoids have different mechanism of ac-
tion was thought to be least probable because all flavonoids show great
structural similarity by sharing flavan nucleus. Secondly many studies
are performed on same flavonoid for example quercetin is widely
studied and different mechanisms are described on the basis of same
compound (Mirzoeva et al., 1997; Plaper et al., 2003; Wu et al., 2008).
Structure activity relationship showed that different structural configu-
rations have different bactericidal activities like in chalcone hydroxyl-
ation at carbon 2 is necessary for antibacterial activity (Ávila et al.,
2008). Similarly hydroxylation at C4 in ring A improves the antibacterial
activity (Alvarez et al., 2004). Methoxylation or acetylation at C2 and
fluorination at C3 decreases antibacterial activity (Ávila et al., 2008;
Nielsen et al., 2004). Lipophilicity of ring A is important for antibacterial
activity but geranyl or prenyl group at C3 improves this activity (Ávila
et al., 2008). Beside from chalcone in flavone alkyl amino chain or
o-acyl at C3 in A ring enhances antibacterial activity (Suresh Babu
et al., 2005). In flavanone hydroxylation at C3 in ring C improves
bactericidal activity (Šmejkal et al., 2008). This shows that difference
in structure has difference in bactericidal activity (Teffo, Aderogba, &
Eloff, 2010) which may be due to different mechanism of action but con-
troversially carboxylation at C4 in ring A increases the solubility of
chalcone 60 times with negligible change in antibacterial activity
(Nielsen et al., 2004). Similarly in another study the compounds of
apigenin were modified to synthesize different lipophilic derivatives
and then their antibacterial activity was measured. Result showed that
the different derivatives of apigenin have significant higher antibacterial
activity which is in accordance with the theory that difference in the
structure has different mechanism of action (Liu et al., 2013).
4.2. Neutralization of bacterial toxins
Toxins are very imperative in bacterial pathogenesis and sometimes
these toxins cause fatal disease even the bacteria have been killed (Xu,
Wan, Dong, But, & Foo, 2000). Some studies show that these flavonoids
can neutralize these toxic factors. In vitro and in vivo S. aureus toxins are
neutralized by catechins. Similarly isoflavone genistein inhibits exotox-
in. Genistein had a protecting effect against Vibrio vulnificus infection
in vivo, as shown by using CD-1 mice. Similarly catechin polymers and
epicatechin neutralize endotoxin (Lopez-Lazaro, 2009). This toxin neu-
tralizing effect was observed by incubating LPS with the flavonoids that
resulted in decrease in the amount of LPS that is attached to beads that is
coated with binding agent (Vlietinck, De Bruyne, Apers, & Pieters, 1998).
Flavonoids block the interaction between LPS (lipopolysaccharides) and
its receptors TLR4/MD2 (toll like receptor 4/myeloid differentiation fac-
tor 2) and CD14, a stage in the development of septic shock. Therefore,
these phenolic compounds can also be used for decreasing the risk of
gram-negative bacterial diseases (Lopez-Lazaro, 2009).
There are two types of bacterial toxins which include exotoxins and
endotoxins. The latter are poisonous glycolipids (gram-positive bacte-
ria) or Teichoic acids (gram-negative bacteria), which are parts of the
outside surface of the bacterial membrane which is surrounded by a
network of proteoglycans and peptidoglycans (Harwood et al., 2007).
When enzyme attacks on these aglycans in the blood of the patient, en-
dotoxins are exposed. They are occupied by the immune cells and the
antibodies, which results in complement activation, anaphylaxis,
fibrogenesis, and eicosanoid formation. The concluding process starts
with the start of PLA2 (phospholipases A2), which liberates arachidonic
acid. The toxic effect of lipid A moiety of the lipopolysaccharide A of the
most important endotoxin, is owing to an interface with the CD14 re-
ceptor of macrophages. The latter starts the production of the TNF-a
(tumor necrosis factor), as well as of IL-1 and IL-6 (Harwood et al.,
2007). Myricetin and other flavonoid compounds suppress the TNF-a-
mediated NF-kB activity by low-regulating the activity of the kinase
IkB kinase (Erdman et al., 2007).
Most of the bacterial toxins are protein in nature and there virulence
is determined by 3-D structural conformations. Thus change in the
structure of these proteins inactivates them by inhibiting their interac-
tion with the cell membrane receptor cite of host cell and other biomol-
ecules that bacteria require for their survival. So it is the need of time to
develop such foods that change bacterial toxins to non-toxic material.
Bacillus anthracis is a spore forming, toxin producing bacteria that
causes skin infection in humans. Anthrax lethal factor (LF) which is
produced by B. anthracis is strongly inhibited by ECGC (epigallocate-
chin-3-gallate) (Dell'Aica et al., 2004). The toxin induced deaths of mac-
rophages are prevented by catechin and it also resulted in the survival of
Fischer 344 rats. Anthrax toxin inactivation mechanism may include the
binding of Zn atom with the –OH of phenyl ring of epicatechin-3-gallate
because of metalloproteinase nature of toxin or it may be due to antiox-
idant properties of catechins (Benelli, Venè, Bisacchi, Garbisa, & Albini,
2002; Cabrera, Giménez, & López, 2003).
Clostridium botulinum is a spore forming bacteria that causes botu-
lism disease, which results in paralysis as a result of blocking of motor
nerve terminals at myoneural junction. The neuromuscular action of
 A. Ahmad et al. / Food Research International 77 (2015) 221–235
Fig. 4. Different ways of flavonoid action on bacterial cells.
neurotoxin of botulinum is blocked by thearubigin fraction of black tea
(Satoh, Ishii, Shimizu, Sawamura & Nishimura, 2002a, 2002b). This anti-
toxin activity may be due to chelating activity of catechin to metallopro-
teinase portion of neurotoxin. The antitoxin activity against botulinum
neurotoxin is also observed in kaempferol, kaempfenol and quercetin
glycosided (Sakane et al., 2002).
Helicobacter pylori produces vacuolating cytotoxin VacA results in
gastritis and ulceration. VacA induced gastritis in rats was effectively
inhibited by high molecular weight polyphenols that are extracted
from hop bracts. This antitoxin activity may be due to complex forma-
tion between toxin and polyphenol or by inhibiting the binding of
toxin to gastrointestinal tract (Yahiro et al., 2005).
Vibrio cholerae causes the disease cholera and this microbe is found
in drinking water or sea foods. Cholera is characterized by profuse diar-
rhea. This disease result from the interaction of adenylatecyclase of the
mucosa of gastrointestinal tract with the cholera enterotoxin resulting
in water flow from open ion channels through osmosis. Toda, Okubo,
Ikigai, Suzuki, Suzuki, et al. (1991a, 1991b) showed that tea catechins
cosseted against experimental infection by V. cholera.
When spores of Clostridium tetani infest a wound release a neurotox-
in named as tetanospasmin after germination. This toxin acts on the
central nervous system and results in muscle contraction that lead to
death unless the host has been vaccinated previously. The thearubigin
constituent of black tea has been shown to possess such properties
which protect the mouse from paralysis (Satoh, Ishii, Shimizu,
Sawamura and Nishimura, 2002a, 2002b). This antitoxin effect may be
due to covalent binding of thearubigin with the toxin as mentioned
above (Satoh, 2005).
5. Antiviral properties of flavonoids
Many traditional plants have been identified to possess antiviral ac-
tivity and few of them had been used to treat viral infections in humans
and animals (Nielsen et al., 2004). Study in the development of antiviral
agents started in 1952 after World War II in Europe, 288 plants was ex-
amined against influenza virus A in embryonated eggs. Results showed
that 12 plants were found effective in suppressing virus amplification
229
(Spencer, Abd El Mohsen, Minihane, & Mathers, 2008). In the past
many screening program has been started in different regions of the
world to study the antiviral activity of medicinal plants in vivo and
in vitro assays. Canadian scientists showed antiviral activities of apple,
grape, strawberry and other fruit juices against poliovirus I, herpes sim-
plex virus (HSV), coxsackievirus B5 and echovirus 7 (Spencer et al.,
2008). Some flavonoid compound and their activity against various
viruses have been presented in Table 4.
Naturally occurring flavonoids with antiviral activity are recognized
since the 1940s but now efforts are made to modify natural compounds
to improve their antiviral activity (Tapas, Sakarkar, & Kakde, 2008). Fla-
vonoids like rutin, morin, quercetin, apigenin, hesperidin and catechin
have been found effective against 11 different types of viruses. This
antiviral activity might be associated with nonglycosidic compounds.
Similarly hydroxylation at 3-position is found to be apparently prereq-
uisite for their antiviral activity. Flavonols are found to be more active
in comparison to flavones against herpes simplex type I virus and the
order of importance was galangin N kaempferol N quercetin (Dillard &
German, 2000).
Viral infection is the most difficult disease to be controlled, because
viruses consist of only a few building units, which they have common
with all living organisms. Yet, viruses are equipped with powerful
tools to help their invasion into cells and perceptive plans for the de-
struction of the cell metabolism. The similarity between the building
units of viruses and that of biological cells renders the development of
antiviral drugs difficult because uninfected cells will get these drugs
and die (Wiseman, 1999). Most of the antiviral drugs are more danger-
ous to the patient than to the infectant. An extra difficulty in controlling
viral infections is the great mutability in viral genomes. Which results in
the detailed structure of the proteins in the protective capsid or mem-
brane surrounding the viral genome persistently changes. Therefore, at-
tempts to control viruses with specific antibodies against surface
epitopes are mostly futile.
The infectious matter of a virus is its nucleic acid which may be RNA
or a DNA. Genes are present in the viral nucleic acids, which in the case
of RNA viruses are overlapping. This saves space and disturbs the
enemy. The viral nucleic acids may consist of one or two copies. In the
230 A. Ahmad et al. / Food Research International 77 (2015) 221–235

Table 4
Antiviral activities of flavonoid compounds.

Flavonoid Source Activity against virus References

Orientin Trollius chinensis Para influenza type3 virus Pang et al. (2013), Peterson (1991), Pilon, Carneiro,
Carnevale Neto, da S. Bolzani, and Castro-Gamboa (2013)
Vitexin Trollius chinensis Para influenza type3 virus Peterson (1991)
Quercetin Kalanchoe pinnata, Genus crataegus HCV, polio, herpes simplex Ashfaq and Idrees (2014), Chwil et al. (2014), Kardonob,
Shimizu, Sudarmono, and Hotta (2014)
Glabranine Tephrosia madrensis, Dengue virus Abd Kadir, Yaakob and Zulkifli (2013), Wood and Webster
7-O-methyl-glabranine Tephrosia viridiflora and Tephrosia crassifolia (1986)
Ladanein Marrubium peregrinum HCV Haid et al. (2012)
Naringenin Citrus paradisi HCV Ashfaq and Idrees (2014), Nahmias et al. (2008)
Chrysosplenol C Pterocaulon sphacelatum Polio virus Bhatty (1999), Rocha Martins et al. (2011)
Tanacetum parthenium
5-hydroxy-7,8-Dimethoxyflavone Mosla scabra Anti-influenza viruses Wu, Yu, Yan, Chen, Zhang and Wen (2010)
Apigenin Mosla scabra Anti-influenza viruses, HCV, Ashfaq and Idrees (2014), Grienke et al. (2012),
Enteovirus-71 Wu, Yu et al. (2010), Zhang et al. (2014)
Moralbanone Morus alba Herpes simplex type 1 virus Du et al. (2003), Farmer, Hunter, and Organ (2012)
Acacetin Mosla scabra Anti-influenza viruses Wu, Yu et al. (2010)
Eudraflavone Morus alba Herpes simplex type 1 virus Du et al. (2003)
B hydroperoxide
Leachianone G Morus alba Herpes simplex type 1 Du et al. (2003), Zafar et al. (2013)
Liquiritigenin Glycyrrhiza uralensis HCV Adianti et al. (2014)
G. glabra
Quercetin and 3-O-glycosides Bauhinia longifolia Mayaro virus dos Santos et al. (2014)
7-hydroxyisoflavone Hedysarum theinum Enterovirus71 Wang et al. (2013)

latter case, the strands are complementary to each other (Hooper et al.,
2008). Thus nucleic acids, which are packed into a spherical bundle, are
covered by a protective protein capsid that consists of nucleic acids and
total of small, simple proteins. While some other viruses have in addi-
tion an outer lipid membrane that have glycoproteins imbedded in
them. These glycoproteins checks weak points on the host cells, at
which the virus particle enters. In the case of HIV infection, this weak
point is the K+ channel of T4-lymphocytes (helper T-Ly), which is asso-
ciated with a chemokine receptor (Hooper et al., 2008). Moreover, some
viruses carry enzymes, e.g., neuraminidase or lysozyme, on their surface
with which they can hydrolyse protective structures in the plasma
membrane of the victim's cells (Wiseman, 1999). Antiviral activities of
different flavonoids have been summarized in Table 4.
The viral genome must possess at least three different genes named
pol (polymerase), gag (a gene-regulating protein), and envelope. In the
RNA viruses, which are the most dangerous because they are potentially
oncogenic, the polymerase is a reverse transcriptase that can form a
DNA chain complementary to an RNA, thus breaking the formerly
central dogma of molecular biology that DNA makes RNA, which in
turn, makes protein.
Once the virus particle has passed the plasma membrane of the tar-
get cell, it fuses its lipid membrane with a lysosome. Then, the proteases
in the latter hydrolyze the protein capsid around the viral genome and
the nucleic acids somehow escape into the cytoplasm. They reach the
chromosomes through a nuclear pore. If it is an RNA virus, the poll
gene delivers the reverse transcriptase that another virus may have pro-
vided during a previous infection of the host cell, and synthesizes a
DNA-strand complementary to the viral RNA. The viral DNA is then in-
corporated into the cellular genome. The DNA viruses do not need the
reverse transcription, but insert their genomes directly into the chromo-
somes of the cell with the aid of enzymes from the host (Moon, Wang, &
Morris, 2006).
Now the integrated viral genome can enter into one of two states, ly-
sogenic or lytic. In the former state, the viral genes may remain silent for
a prolonged period, but upon adequate provocation, perhaps a second
infection or an irradiation, it becomes active, i.e., it enters the lytic
state. In the latter, the viral genome takes command over the metabo-
lism of the cell (Egert,& Rimbach, 2011). It uses the enzymes and the
substrates in the cell to provide energy and building blocks for new
viral particles. These processes deprive the cell of its essential sub-
strates. Therefore, the infected cell starves and suffers an early death,
while scores of new virus particles leave the dying cell. Alternatively,
the infected cell becomes “immortalized” and provides a source of
new viral particles during its extended life time.
Evidence shows that substances close related to flavonoids inhibit
the fusion of the viral membrane with that of the lysosome (Lambert,
Sang, & Yang, 2007). Therefore, many claims from lay medical practi-
tioners of the prophylactic effect of flavonoids against viral attack have
substantial support (Jerome‐Morais, Diamond, & Wright, 2011). The
mechanism by which flavonoids inhibit viral infection remains unclear,
but it looks that PGs take part in the fusion of cell membranes.
Flavonoids inhibit formation of PGs, a justification can be made for the
protective effect of flavonoids against viral infections (Prasain, Carlson,
& Wyss, 2010). Besides, the lysosomes can only be acidified and activat-
ed by their proton pump, and that can be inhibited by flavonoids
(Corcoran, McKay, & Blumberg, 2012).
However, if the viral nucleic acids have reached the cytoplasm, then
it is very difficult to prevent their integration in the cellular genome. In
contrast, viral genes, which are imprisoned in their protein capsid, are
harmless. Some flavonoids, e.g., quercetin, have been shown to be inhib-
itors of the reverse transcriptase of RNA viruses (Finke, Tomaso, &
Frangoulidis, 2012). This property is very desirable since the currently
used inhibitors, e.g., arabinosides and acyclovir, of this enzyme are ex-
tremely toxic, not only to virally infected cells, but also to normal cells.
An additional advantage of the flavonoids is that they induce the pro-
duction of IFNs. These substances have several antiviral effects, includ-
ing fortification of the cellular membrane; induction of nucleases,
which attack the viral genome; and modification of the phosphorylation
pattern of the protein translation eIFs (eukaryotic initiation factors),
which stop the biosynthesis of all proteins, including those of the virus
(Cliver, 2009).
Synergistic effect has been established between different combina-
tions of flavonols and flavones like luteolin and kaempferol shows syn-
ergistic effect against HSV. This is the reason that propolis is more active
than its isolated constituents (Clarke et al., 2002). Synergistic effect has
been found between antiviral agents and flavonoids. For example quer-
cetin enhances the effects of acyclovir (Clarke et al., 2002) and 5-ethyl-
2_-dioxyuridine (Hemingway et al., 1989) against pseudorabies HSV in-
fection. Similarly antiviral activity of acyclovir is improved by apigenin
against different viruses (Clarke et al., 2002). Flavonoids also displayed
antiviral, including anti-HIV activity. Flavonols are found to be more ac-
tive than flavones against herpes simplex virus type 1 and the order of
importance was galangin N kaempferol N quercetin (Argolo, Sant'Ana,
Pletsch, & Coelho, 2004).
 A. Ahmad et al. / Food Research International 77 (2015) 221–235
5.1. Human immune deficiency virus inhibition by flavonoids
Anti HIV drugs have been developed now and they have improved
the quality of lives of AIDS patients but the emergence of drug resistant
virus necessitates the need to search for new compounds that have been
screened from natural sources having antiHIV properties include cou-
marins, alkaloids, polyphenolics, flavonoids, sulfated polysaccharides,
triterterpenes, tannins, iridoids, lactones, lignans, saponins, ribosomes
inactivating proteins, quinines and phospholipids (Lopez-Lazaro,
2009; Vlietinck et al., 1998).
An important and interesting area of research is the apparent inhib-
itory effect of flavonoids against human immune deficiency virus (HIV).
In vitro studies show that HIV I infection and replication is inhibited by
baicalin that was purified from the plant Scutellaria baicalensis (Dell'Aica
et al., 2004). Similarly baicalin extracted from Chinese herbal medicine
has properties that have antagonistic properties against HIV I reverse
transcriptase. Baicalin forms complexes with chemokines and progres-
sively reduces their ability to bind receptors on the surface of cell.
Baicalin below the cytotoxic concentration inhibited monocyte tropic
(R5) and T cell tropic (X4) HIV Env protein mediated union with cells
expressing CD4/CXCR4 or CD4/CCR5 (Benelli et al., 2002).
As HIV spreads more rapidly during the 1980's the research focus
was on determining antiviral activities of flavonoids against HIV
(Lopez-Lazaro, 2009). As a result it has been shown that HIV I proteases
are inhibited by various flavonoids including demethylated gardenin A
Fig. 5. HIV life cycle and intervention of flavonoids.
231
and 3,2-dihydroxyflavone (Cabrera et al., 2003). Among 28 flavonoids
tested flavans showed better antiviral activity as compared to flava-
nones in the selective inhibition of HIV-1 and HIV-2 and other related
immunodeficiency virus infections (Evans, 2009). The HIV life
cycle with point of intervention of flavonoids has been demonstrated
in Fig. 5. Out of twenty-eight flavonoids tested, flavan-3-o1 was more
effective than flavones and flavanones in selective inhibition of HIV-1,
HIV-2 and similar immunodeficiency virus infections (Wei et al., 2004).
In another study, effect of different flavonoids was studied on
human immune deficiency virus (HIV)-1 proteases. Among the differ-
ent classes of flavonoids, flavanones, flavones, catechols, flavonols and
chalcones, flavonols were found most effective while flavanones and
chalcones possess less antiviral activity. Among different flavonoid
compounds quercetin was found to be most potent inhibitor of HIV pro-
tease while luteolin and butein possess moderate activity (Xu et al.,
2000).
Xanthohumol is a prenylated flavonoid that is constituent of beer
and can be purified from Humulus lupulus. At noncytotoxic concentra-
tion xanthohumol inhibited the cytopathic effect induced due to
HIV-1. Similarly the C8166b lymphocytes reverse transcriptase and pro-
duction of viral p24 antigen is inhibited by xanthohumol. Its therapeutic
index was found to be 10.8. These results suggest that xanthohumol is
effective in controlling HIV-1 infection and it can be developed into
novel chemotherapeutic agent for prevention of HIV-1 infection
(Wang et al., 2004).
232 A. Ahmad et al. / Food Research International 77 (2015) 221–235
Fig. 6. A. 2-methoxy-3-methyl-4,6-dihydroxy-5-(30-hydroxy) cinnamoylbenzaldehyde.
B. 3-methyl-4,6-dihydroxy-5-(30-hydroxy)cinnamoylbenzaldehyde.
5.2. Structure activity relationship of flavonoids and human immune
deficiency virus (HIV)
Acacetin-7-o-β-D-galactopyranoside which is extracted from chry-
santhemum morifolium flower has been shown to possess antiviral ac-
tivity against HIV-1 virus with a therapeutic index of 5 at relatively low
toxic level i.e. IC50 = 37 μM. This compound has flavone skeleton with
galactose at C7 and –OH group at C5 and in the B ring CH3 at C4.
When this methoxyl group is replaced by hydroxyl group that is
apigenin-7-o-β-D-galactopyranoside its anti HIV activity is greatly de-
creased that is therapeutic index of 2. By adding another sugar group
that is Acacetin-7-o-(6″-rhamnopyranosyl)-β-D-galactopyranoside
completely removes its antiviral activity. Luteolin, luteolin-7-o-β-D-
glucopyranoside and acetate of luteolin-7-o-β-D-glucopyranoside have
antiviral activity similar to acacetin-7-o-β-D-galactopyranoside but at
toxic level. A nonglycosylated flavone luteolin having hydroxyl group
at C5, 7, 3′ and 4′ have antiviral activity EC50 = 10 pM but nearly at
toxic level (Hu et al., 1994).
2-methoxy-3-methyl-4,6-dihydroxy-5-(30-hydroxy)
cinnamoylbenzaldehyde is a chalcone that has a therapeutic index of
489 against HIV is shown in Fig. 6a while 3-methyl-4,6-dihydroxy-5-
(30-hydroxy)cinnamoylbenzaldehyde lacking –CH3 at C2 as shown in
Fig. 6b does not show any antiviral activity so the presence of methyl
group at C2 may be necessary for antiviral properties of chalcone (Wu,
Wang, Yi, & Lee, 2003).
Lawinal showed that anti-HIV activity with EC50 2.3 μg/Ml with
adequate therapeutic index of 45.2 while desmethoxyatteucinol was
comparatively less active having Ec50 4.97 μg/ml and therapeutic
index of 4.18. This shows that aldehyde group is more active in antiviral
activity against HIV then a methyl group at C6 in flavanones while in
comparison to this, aldehyde group is more desirable than aldehyde at
C8 in flavanones (Wu et al., 2003).
6. Conclusion
Antibiotic resistance development in bacteria and viruses is a global
problem which necessitates the need to search for new therapeutic
compounds of natural origin to overcome this problem. Flavonoids are
phytochemicals which possess antimicrobial, anti-inflammatory and
antioxidant properties. So flavonoids could be extracted from plants
and optimized through structural modification to develop new pharma-
cologically active antimicrobial group of agents. Beside from pharmaco-
logically active agents, flavonoids may be extracted and fortified into
staple foods for developing nutraceutical products.
Acknowledgment
The project was funded by the Higher Education Commission of
Pakistan (112-34055-2AV1-048) as a MS leading to PhD Fellowship
Program.
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