[Studies in Natural Products Chemistry] Volume 42 || Natural Antiviral Compounds
Transcript of [Studies in Natural Products Chemistry] Volume 42 || Natural Antiviral Compounds
Chapter 7
Natural Antiviral Compounds
A.E.D. Bekhit* and A.A. Bekhit{*Department of Food Science, University of Otago, Dunedin, New Zealand{Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University,
Alexandria, Egypt
Chapter OutlineIntroduction 195
Virus 198
Classes of Natural Antiviral
Compounds 199
Considerations for Antiviral
Activity of Compounds from
Natural Sources 217
Concluding Remarks 220
References 223
INTRODUCTION
Human relied on plants as medicine for centuries and their use for treatment
of ailments and diseases is practiced in many parts of the world despite the
widespread practice of modern medicine and the use of synthetic drugs world-
wide. Apart from their use in traditional medicine in many Asian, South
American, and African countries with an estimated 80% of the world’s popu-
lation using traditional medicine, plants contribute to about 25% of the pre-
scribed drugs worldwide [1]. Many natural products are used as adjuvant/
prophylactic or for therapeutic purposes. Several important synthetic drugs
have been isolated initially from plants (Table 1) and several other biologi-
cally active compounds are still obtained from cultivated or wild plants due
to limitations in synthesis techniques or economic viability [2].
The use of bioactive compounds from plants is attractive to many develop-
ing countries since the production cost is low compared to chemically synthe-
sized drugs rendering treatment to be affordable and accessible to all people.
Furthermore, the long history of medicinal plants in many societies has led to
improved understanding of the functions of certain plants and created a trust
and acceptability to herbal medicine. This knowledge is often treated as “heal-
ing art” and is often passed from one generation to another. The use of scien-
tific convention and systematic investigations to support the use of medicinal
Studies in Natural Products Chemistry, Vol. 42. http://dx.doi.org/10.1016/B978-0-444-63281-4.00007-0
© 2014 Elsevier B.V. All rights reserved. 195
TABLE 1 Examples of Common Drugs Originated from Plants
Drug (Trade Name) Plant Use Structure
Digoxin (Lanoxin) Digitalis spp. (e.g.,Digitalis lanata)
Heart conditions
OH
OH
O
H
O
O
O
O
OO
O
HHHO
HHOHO
HO
Quinine Cinchona spp. (e.g.,Cinchona officinalis)
Antipyretic
N
OH
NOCH3
Antimalarial
Quinidine Analgesic
Anti-inflammatory
Vincristine Catharanthus roseus Cancerchemotherapy
N
N
O
OHH O
O
O
H
OO
NH
N
H
OH
OO
Vinblastine
TABLE 1 Examples of Common Drugs Originated from Plants—Cont’d
Drug (Trade Name) Plant Use Structure
Atropine Solanaceae (Atropabelladonna)
Anticholinergic
HO
HOO
NH3C
Lowers theparasympatheticactivity
Morphine Papaver somniferum Potent opiateanalgesic drug
O H
H
HO
N
HO
CH3
Codeine
Paclitaxel (taxol) Taxus brevifolia Anticancer agent
OHO
O
O
O
HOH
O O
O O
O
NHO O
OH
Salicin (aspirin) Salix alba Analgesic
O
OH
O
O
Antipyretic
Anti-inflammatory
plants or to screen new plants has attracted much research over the past five
decades. The antiviral activity of plant extracts has been a very active research
area. A database search for antiviral and extract as keywords, excluding books
and references, retrieved 14,930 titles (Fig. 1). Exponential increase in the
number of investigations on antiviral activity of extracts from natural sources
in the period between 1994 and 2012 reflects the scientific community vision
for prospecting natural materials for antiviral compounds and supports the
potential benefits of using natural resources for that purpose.
VIRUS
The word virus originated from Latin which means poison or toxic. A virus is
a small infectious agent which can trigger an immune response which can
control the virus in some instances (e.g., common flu) but can lead to lethal
and pathological effects in many other cases (hepatitis and human immunode-
ficiency viruses), especially in individuals with compromised immune system.
Unlike bacterial, fungal, and parasitic organisms causing infection, virus is
not an autonomous organism and can only replicate inside the host cell envi-
ronment of another organism [3] and therefore they are obligate intracellular
pathogens.
There are millions of different types of viruses found in almost every eco-
system on earth. In fact, viruses are the most abundant type of biological enti-
ties [4–6]. They contain little more than bundles of gene strands of either
RNA or DNA and may be enclosed by a lipid-containing envelope [3].
Viruses have several invasion strategies. Each strain of virus has its own
unique configuration of surface molecules that precisely fit the membranes
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FIGURE 1 Number of publications between 1994 and 2012 for antiviral and extract as key-
words. The search was performed using Science Direct database on the November 15, 2012.
Studies in Natural Products Chemistry198
of target cells, which enable the entry of viruses into hosts [3]. These general
attributes lead to the success of viruses in evolution, genetic variation, variety
in means of transmission, efficient replication within host cells, and the ability
to persist in the host [4,5].
Viruses cause a range of structural and biochemical effects (cytopathic
effects) on the host cells. Most virus infections cause alterations to cell sur-
face membranes, cell lysis, and apoptosis (death to the host cell), though some
viruses result in no apparent changes to the infected cells [7]. Hundreds of
human-infecting viruses have been clinically investigated, but the majority
of natural antiviral extracts were tested against human immunodeficiency
virus (HIV), herpes simplex viruses (HSV), rhinoviruses, enteroviruses, hepa-
titis (Hep), and influenza viruses.
Classes of Natural Antiviral Compounds
Many crude and purified compounds have been reported to have antiviral
activity. The various classes of the majority of these compounds will be dis-
cussed below. It is worth noting that while the majority of these compounds
were examined using cell lines (i.e., in vitro), the beneficial effects reported
for these compounds should be used as indicator of the antiviral potential of
the examined compounds given that under metabolic in situ processes the
compounds may become inactive or higher concentrations will be required
for effective use in animal models which can be in some cases higher than
the toxic levels of compounds.
Photosensitizers
These are compounds which are expressed by plants and exhibit biological
activity under specific light wavelengths (Fig. 2). Based on their chemical
structure, the compounds can be alkaloids, furyl compounds or thiophenes,
and polyacetylenes, with the latter being the most potent against enveloped
viruses [7]. Photosensitizers are expressed in significant amounts (up to 1%
of the fresh weight) in plants belonging to Compositae (Asteraceae), Umbel-
liferae (Apiaceae), and Campanulaceae families. The compounds are also
found in several other organisms from marine ecosystems (e.g., algae, nudi-
branchs, sea hares, and sponges) as well as in fungi and insects [12]. These
compounds, as with most secondary metabolites, are suspected to be formed
as a defense mechanism against pests and are found in all plant parts. Several
reports documented the antiviral activity of plant photosensitizers, especially
thiophenes and polyacetylenes, which are activated by UV light [7,12] with
maximum activity observed at about 350–360 nm [7].
Several alkaloids (Fig. 2A) exhibit antiviral activity and some have been
shown to be active upon exposure to long-wave ultraviolet (UVA,
300–400 nm). Several alkaloids such as b-carbolines (Rutaceae), atropine
Chapter 7 Natural Antiviral Compounds 199
NH
N
NH3C
CH3
Brevicollin
N
N
O
6- Canthione
NH
N
CH3
H3CO
Harmine
NH
N
CH3
Harmane
NH
N
CH3
HO
Harmol
NH
N
CH3
HO
Harmalol
NH
N
CH3
H3CO
Harmaline
NH
NH
COOH
Harmane-1,2,3,4-tetra-hydro-3-carboxylic acid
Alkaloids (A)
O
O
OO
HNO
O
Colchicine
N
N
O
OH
O
O
O
O
HN
N
H
O
O
OH
H
Vinblastine
FIGURE 2—CONT’D
Studies in Natural Products Chemistry200
Furyl compounds (B)
OO O
OCH3
8-Methoxypsoralen
OO
OCH3
O
Visnagin
O OO
Angelicin
OO
OCH3
OOCH3
Khellin
N O
OCH3
Dictamnine
Thiophenes and polyacetylenes (C)
S S S
A-TerthienylS S
C C C C CH
CH2CCH3C
Thiarubrine-A
ACBP-Thiophene
C C C C CH
CH2CCH3CS
Thiophene-A
2,5-Bis(2-thienylethynyl)thiophene
C C C C C C CH3
Phenylheptatriyne
3(1-Phenylethyneyl)-2,2 bithiophene
CC
CH
H2C
OCC
CS
CH3C Cl
CH3C
O
SC C
SC C
S
S SC C
FIGURE 2—CONT’D
Chapter 7 Natural Antiviral Compounds 201
Hypericin and its dervatives (D)OH
HO
OH
CH3
O
OH
HO
OH
CH3
O
Hypericin
OH
O
OCH3OH
O
OH
O
OCH3
OH
O
Cercosporin
OHOCH3
O
O
OH
OHOCH3
O
O
OH
Isocercosporin
OCH3
OH
H3CO
O
OCH3
OH
H3CO
O
OH
CH3
COCH3
Hypocrellin A
OCH3
OH
H3CO
O
OCH3
OH
H3CO
O
COCH3
CH3
Hypocrellin B
OHOCH3
OH
H3CO
O
OHOCH3
OH
H3CO
O
Phleichrome
OH
O
O
OH
O
O
O
O
OH
PhS
O
OCH3OH
O
OH
PhS
O
OCH3
OH
O
OH
H3CO
OCH3OCOPh
O
OH
H3CO
OCH3
OCO2(o-OH-Ph)
O
Calphostin C
OCH3
H3CO
OCH3
CO2Me
O
OCH3
H3CO
OCH3
CO2Me
O
FIGURE 2 Structure of well-known photosensitizers reported to have antiviral activity [7–11].
Studies in Natural Products Chemistry202
(Atropa belladonna), camptothecin (Camptotheca acuminate), castanosper-mine (Castanospermum australe), colchicines (Colchicum autumnale), indoli-zidines swainsonine (Swainsona canescens), and vinblastine (Catharanthusroseus) were found to have antiviral activity [13]. Several natural alkaloids
(yohimbine, vincamine, scopolamine, atropine, colchicine, allantoin, trigonel-
line, octopamine, synephrine, and capsaicin) were recently evaluated for their
anti-HSV-1 and anti-RNA virus parainfluenza (type-3) [14]. High inhibitory
effect against HSV-1 was reported with cytopathogenic effect is in the con-
centration range of 0.05–0.8 mg/mL. On the other hand, only atropine and
octopamine demonstrated an inhibitory effect against parainfluenza (type-3)
with a cytopathogenic effect at concentration of 0.05 mg/mL for both com-
pounds. This selective activity against viruses was previously reported for
some isoquinoline alkaloids (protopine, fumarophycine, chelidimerine, ophio-
carpine, and (þ)-bulbocapnine) which were potent inhibitors of parainfluenza
(type-3) while they had negligible effects on HSV-1 [15]. Harmine (Peganumharmala) and some harmine b-carbolines compounds are available in many
plants, marine, and mammalian cells. These compounds demonstrated broad
virucidal activity which requires UVA activation. Castanospermine is effec-
tive against viruses with membranes and target glycoprotein maturation step
in the virus development cycle. Thalimonine and indole alkaloid from Thalic-trum simplex L. and Uncaria rhynchophylla, respectively, demonstrated
potent anti-influenza A activity [16,17]. Several mechanisms have been sug-
gested for their biological activities, such as interaction with nucleic acid
and targeting macromolecules.
Furyl compounds (Fig. 2B) after exposure to long-wave ultraviolet (UVA,
300–400 nm) exhibit broad efficacy against viruses [7]. Several reports on the
antiviral activity of furocoumarins (psoralens) and furanochromones (visna-
gin) from Rutaceae and Umbelliferae (Apiaceae) are available [13]. The com-
pounds require UVA for their activity and appear to inhibit virus replication
by disrupting viral gene target expression by forming photo-adduct with the
virus DNA.
Thiophenes and polyacetylenes (Fig. 2C) compounds occur as polyines,
allenes, phenyl and thiophenyl derivatives, thioethers, and spiroketal enol
ethers in Asteraceae, Apiaceae, Campanulaceae Panax ginseng (Korean gin-
seng roots), Bidens sp., and Chrysanthemum sibiricum. The mechanism of
action of thiophenes and polyacetylenes against viruses is thought to be
mediated by membrane damage caused by singlet oxygen attack which is
released upon exposure to light [7,8], but other mechanisms may be possible
since the compounds show strong activity against virus with no membranes
[7]. The virus integrity is not compromised by the damage caused to its mem-
brane and remains able to occupy cells but lose its capability to replicate [7].
The presence of thiophene rings and the acetylenic substituent is important for
the antiviral activity. The activity was not affected by the presence of halide
groups but it was decreased by the presence of phenyl groups.
Chapter 7 Natural Antiviral Compounds 203
Hypericin (Fig. 2D) is another photosensitizer commonly found in plants
belonging to the genus Hypericurn. Upon exposure to visible light, hypericin
produces singlet oxygen [9], which is suggested as the main mechanism for
the antiviral effects of the compound. A series of related compounds
(Fig. 2D) showed that the antiviral activity against Sindbis virus was linked
to the ability to generate singlet oxygen in some compounds. However, some
compounds (1, 2, and 6) which have high singlet oxygen generation capability
did not possess antiviral activity and vice versa, suggesting the involvement of
other mechanisms in the inhibition of virus.
Photosensitizers exhibit different antiviral activities against different
viruses [7–9]. Therefore, it is recommended to screen the potential of antiviral
activity of extracted compounds against wide range of viruses.
Phenolics
Polyphenol compounds, such as epigallocatechin gallate (ECGC), epicatechin
gallate (ECG), epigallocatechin (EGC), and theaflavin digallate, are widely
found in plants. Polyphenol from tea, grape products, berries, and other plant
sources exhibits several mechanisms which promote the prevention of the
virus infectivity, such as by binding to the hemagglutinin of influenza
virus [18] or by altering the physical properties of the viral membrane [19].
Viral inactivation in vitro is attributed to preferential binding of phenolics
to the protein coat of the virus thus arresting virus binding [20,21]. However,
the antiviral activity of polyphenol involves direct inactivation of the virus
and/or inhibition of the virus binding to the cells [22]. Several investigations
have drawn attention to possible antiviral activity attributable to other pheno-
lic compounds, such as proanthocyanidins, which are the oligomer or polymer
form of flavan-3-ol units, and resveratrol. Proanthocyanins (PACs) have been
shown to exhibit antiviral activity against poliomyelitis virus [23]. Three PAC
compounds existing in dimer, trimer, and tetramer form showed pronounced
antiviral properties against herpes simplex and coxsackieviruses [24,25]. Sev-
eral potential mechanisms have been reported for the antiviral activity of
PACs. For instance, PACs have been shown to inhibit enzymes involved in
the replication of rhinovirus and HIV virus [26]. Furthermore, PCAs A-1 pur-
ified from Vaccinium vitis-idaea had the ability to suppress HSV-2 infection
through the inhibition of viral attachment and penetration [27]. Several other
phenolics such as anthraquinone chrysophanic acid, caffeic acid, ellagitannin,
hypericin, tannins (condensed polymers); salicylates; and quinines (naphtho-
quinones, naphthoquinones and anthraquinones, in particular, aloe emodin)
have been reported to disrupt the synthesis of viral DNA [13]. Gallic acid,
chlorogenic acid, and quinic acid demonstrated good anti-HSV-1 and parain-
fluenza (type-3) inhibitory activity with cytopathogenic effect in the concen-
tration range of 0.05–0.4 mg/mL [14]. A selective effect was found for
caffeic acid which was effective against HSV-1 virus but had no effect on
Studies in Natural Products Chemistry204
parainfluenza (type-3). Grape extracts (skin and whole blue grapes), grape
juice, and wine were reported to inactivate various enteric viruses and HSV
type 1 [23]. More recently, wine residues were reported to have antiadenoviral
activity [28] and anti-influenza activity [29]. Pinot noir extracts exhibited pro-
tective effects of �50% against influenza A virus at concentrations
>1 mg/mL compared to virus alone. Pinot meunier pomace extracts had pro-
tective effects at concentration of 1 mg/mL, whereas both seed and skin
extracts were effective at 10 mg/mL.
Aqueous phenolic extracts from the Chinese plants Agrimonia pilosa, Pithe-cellobium clypearia, and Punica granatum showed anti-HSV-1 activity with
EC50 value ranging from 83.3 to 250 mg/mL, and selective indices (SI) ranging
from 3 to 12. In the same study, extracts from Blumea laciniata, Elephantopusscaber, Laggera pterodonta,Mussaenda pubescens, Schefflera octophylla, andScutellaria indica exhibited antihuman respiratory syncytial virus activity with
EC50 value ranging from 12.5 to 32 mg/mL and SI ranging from 11.2 to 40 [30].
The anti-HSV-1 activity (EC50) of A. pilosa aqueous extracts against standard,
acyclovir-resistant, and clinical strain was 125, 100, and 125 mg/mL, respec-
tively. Similarly, the EC50 of extracts from P. clypearia and P. granatumagainst the three strains varied (62.5, 125, and 100 mg/mL for P. clypeariaextract and 83.3, 62.5, and 50 mg/mL for P. granatum extract). This indicates
the need to examine the extracts on other strains in addition to standard virus
strains, especially those of clinical importance.
Resveratrol has been found to affect influenza virus replication both
in vitro and in vivo by several modes of action as follows: (1) by blockade
of the nuclear-cytoplasmic translocation of the viral ribonucleoprotein com-
plex, (2) by reducing the expression of late viral proteins, and (3) by the inhi-
bition of protein kinase C (PKC) activity and PKC-dependent pathways [31].
Resveratrol is able to inhibit the replication of HSV types-1 and -2 in a dose-
dependent and reversible manner [32]. Resveratrol also synergistically
enhances the anti-HIV activity of a number of nucleoside analogues for com-
bating infection in peripheral white blood cells [33]. In contrast to these
reports, Nakamura et al. [34] found resveratrol to increase the RNA replica-
tion in HepCV and the authors recommended that HepCV patients should
avoid supplements containing resveratrol. Resveratrol had anti-influenza
activity at low concentrations of 0.1–10 mg/mL and was the most effective
anti-influenza compound among several standards tested in our laboratory
(gallic acid, syringic acid, caffeic acid, b-coumaric acid, tannic acid, chloro-
genic acid, catechin, ECGC, keracyanin chloride, kuromanin chloride, delphini-
din chloride, cyanin chloride, cyanidin chloride, ideain chloride, pelargonidin
chloride, malvidin chloride, and quercetin) (unpublished data). It is worth
mentioning that resveratrol is very toxic at high concentration (only 11% viable
cells at 100 mg/mL).
Flavonoids demonstrated diverse antiviral activities against viruses includ-
ing HIV, respiratory and herpes viruses, and many others (adenovirus,
Chapter 7 Natural Antiviral Compounds 205
coxsackievirus, measles, pseudorabies virus, poliovirus, semliki forest virus,
and zoster virus). For example, amentoflavone, agathisflavone, robustafla-
vone, rhusflavanone, and succedaneflavanone from Rhus succedanea and
Garcinia multiflora [35], theaflavin from black tea [36], iridoids from Bar-leria prionitis [37], phenylpropanoid glycosides from Markhamia lutea [38],
chrysosplenol C from Dianella longifolia and Pterocaulon sphacelatum[39,40], morin from Maclura cochinchinensis [41], coumarins from Calophyl-lum cerasiferum [42], galangin from Helichrysum aureonitens [43], and baica-
lin from Scutellaria baicalensis [44] all have been shown to inactivate
different viruses at various levels depending on the virus type, concentration,
and the cell type used in the assay. Flavonoids exert their activity by blocking
RNA synthesis, protease inhibition, reverse transcriptase as well as direct
inhibition of viruses [13,45]. Some flavonoids exert their antiviral activity
through specific actions. For example, taxifolin (dihydroquercetin) from
Juglans mandshurica inhibited the cytopathic activity of HIV-1 virus [46], fla-
vonoid glucuronide from Chrysanthemum morifolium targeted integrase [47],
whereas Ginkgetin and tetrahydroxyflavone from Ginkgo biloba L. and
S. baicalensis, respectively, were targeting influenza virus sialidase [48,49].
The flavonoids glabranine and 7-O-methyl-glabranine were purified from the
Mexican plants Tephrosia madrensis, Tephrosia viridiflora, and Tephrosiacrassifolia and exhibited 70% inhibition of the dengue virus at a concentration
of 25 mM [50]. The efficacy of quercetin, apigenin, genistein, naringin,
silymarin, and silibinin was recently evaluated for their anti-HSV-1 and
anti-RNA virus parainfluenza (type-3) [14]. These compounds had high anti-
HSV-1 activity with cytopathogenic minimum inhibitory concentration
between 0.1 and 0.8 mg/mL with quercetin and silibinin being the most effec-
tive compounds. These compounds, however, were not effective against para-
influenza (type-3) except for genistein which had cytopathogenic minimum
inhibitory concentration of 0.2 mg/mL.
Water [51] and ethanolic [52] extracts of Brazilian propolis were effective
anti-influenza in several model systems. Shimizu et al. [52] investigated the
activity using A/PR/8/34 and A/WSN33 (WSN) strains in Madin–Darby
canine kidney (MDCK) cells and adapted influenza. The authors reported
wide range of effective and cytotoxic concentrations with one fraction at
10 mg/kg showing similar efficacy to oseltamivir (a standard anti-influenza
drug, 1 mg/kg). This antiviral activity is in agreement with strong documented
evidence for antiviral activity of propolis against HSV [53–56], poliovi-
rus [57], reovirus [58], HIV [59–61], and other viruses [62–65]. Several poly-
phenols, flavonoids, and phenylcarboxylic acids were identified from aqueous
and ethanolic extracts of propolis which exhibited very high antiviral activity
against HSV-1 with ethanolic extracts being about fivefold more effective
than aqueous extracts [55,56,63]. The extracts contained caffeic acid,
p-coumaric acid, benzoic acid, galangin, pinocembrin, and chrysin; however,
only galangin and chrysin demonstrated antiviral activity at concentrations
Studies in Natural Products Chemistry206
below their corresponding lethal levels and the extracts were far more effec-
tive as anti-HSV-1 compared to any of the individual compounds, suggesting
a synergistic effect among the various compounds. This is in contrast to the
findings reported for phenolic acids effects on HSV-1 and -2, and adeno-
viruses (3, 8, and 11) [66]. Aqueous extracts of Plantago major L. exhibitedweak inhibition on the viruses but pure compounds found in the extract of
the plant demonstrated potent activity. In particular, caffeic acid was the most
effective compound against HSV-1 and HSV-2 (EC50¼15.3 and 87.3 mg/mL,
SI¼671 and 118, respectively) and against adenovirus 3 (EC50¼14.2 mg/mL,
SI¼727). Chlorogenic acid exhibited the strongest antiadenovirus 11 activity
(EC50¼13.3 mg/mL, SI¼301). More, recently, Urushisaki et al. [51] reportedthe anti-influenza activity of aqueous extract of propolis and demonstrated
that the activity was mainly due to caffeoylquinic acids (Fig. 3). The authors
also found that the antiviral activity was due to a cytoprotective activity of the
cells since the extracts did not affect the viral RNA content per cell, suggest-
ing no direct effect on the influenza virus. The compound structure plays an
important role in determining the antiviral activity. For example, while 3,4-
dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-dicaffeoylquinic acid
have the exact molecular weight of 516.5 g/mol, their EC50 against influenza
A virus were 41.9, 107.3, and 144.9 mg/mL, respectively. The substitution of a
hydrogen group in chlorogenic acid by a caffeoyl group on R2 position
(Fig. 3) resulted in fourfold increase in the anti-influenza activity [51].
Terpenoids
Several sesquiterpene and triterpenoids (agastanol and agastaquinone from
Agastache rugosa, glycyrrhizin from Glycyrrhiza radix, moronic acid from
Rhus javanica, ursolic acid, maslinic acid, and saponin from Geum japoni-cum, Uvaol from Crataegus pinnatifida, Garciosaterpene A, C from Garciniaspeciosa, vaticinone from Vatica cinerea, and betulinic acid from many plants
spp.) demonstrated potent antiviral activities against HSV and HIV [67–75]
and improved the activity of synthetic drugs [76]. The antiviral activity was
attributed to: (1) a direct antiviral effect on the virus [76], (2) immunomodu-
latory effect through the production of interferon [21], (3) HIV-1 protease
R1O
OH OR2
OR3
HOOC
Quinic acid: R1 = R2 = R3 = H
O
(OH)HO
HO
Caffeoyl (caffeic acid)
Chlorogenic acid: R1 = caffeoyl, R2 = R3 = H4,5-Dicaffeoylquinic acid: R1 = H, R2 = R3 = caffeoyl3,5-Dicaffeoylquinic acid: R1 = R3 = caffeoyl, R2 = H3,4-Dicaffeoylquinic acid: R1 = R2 = caffeoyl, R3 = H3,4,5-Tricaffeoylquinic acid: R1 = R2 = R3 = caffeoyl
FIGURE 3 Chemical structures of several caffeoylquinic acids derived from propolis [51].
Chapter 7 Natural Antiviral Compounds 207
inhibition [77], and (4) interference with virus-cell binding [78]. Antiviral ter-
penoids can be found in marine sources. For example, Dolabelladienetriol
(Fig. 4) obtained from the marine brown alga Dictyota pfaffii inhibited
HIV-1 replication at EC50 of 8.4 mM through a noncompetitive inhibition of
reverse transcriptase [79].
Essential Oils
Essential oils (EOs) are mixtures of volatile compounds that can be isolated
from their original matrixes by distillation, solvent extraction, and expression
under pressure. These oils as mentioned above are complex mixture of various
compounds (Fig. 5), mostly terpenes (e.g., mono- and sesquiterpenoids) and
nonterpenes (e.g., benzoids and phenylpropanoids) and their composition
can vary depending on their source material. EOs with known biological
activities can be extracted from food plants (e.g., myristicin from nutmeg, cit-
ral from lemongrass oil, thymoquinone from black cumin, d-limonene from
orange, and b-myrcene from sweet fennel) but long list of food (dill seed, gar-
lic, basil, and so on) and nonfood materials (see below) has been reported as
sources of biologically active EOs extracts.
EOs have been used for therapeutic purposes and as cosmetics through
human history. Their antimicrobial effects and their use in skin formulations
have been known for centuries [82], which probably led to investigations of
their antiviral activities on viral skin diseases. Indeed, human herpes viruses,
for example, HSV-1 and HSV-2, are the most investigated viruses with EOs
[83–94]. Topical treatments containing extracts from lemon balm and sage
extracts are available for herpes labialis [80]. EOs obtained from Santolinainsularis [85], Melissa officinalis L. [82], Melaleuca species [87], Houttuyniacordata [88], Australian tea tree and eucalyptus [90], Mentha piperita [91],
and Salvia fruticosa [93] exhibited direct inactivation of HSV.
EOs from Artemisia arborescens L. (Asteraceae) exhibited potent inhibitory
activity against HSV-1 (EC50¼2.4 mg/mL) and HSV-2 (4.1 mg/mL) with a
CC50/EC50 ratio of 55 and 32.2, respectively, showing a good safety profile [95].
The antiviral activity was due to direct virucidal effects, which caused virus inac-
tivation and prevented cell-to-cell virus diffusion. EOs from Eugeniacaryophyllus and eugenol (the main EO in the obtained extract) inactivated
several herpes viruses at various levels directly and inhibited intracellular and
H
HO
OH
H
HO
FIGURE 4 Structure of Dolabelladienetriol [79].
Studies in Natural Products Chemistry208
Monoterpenes
α-Terpinene γ-Terpinene α-Pinene β-Pinene
OH
Terpinen-4-ol
OH
α-Terpineol
OH
OH
Thymol Carvacol
O
CHO
CHO
cis-Form trans-Form Citral p-Cymene
1,8-Cineole
H2C
OH
OH
Sabinene Geraniol Citronellol
Phenylpropanoids and sesquiterpenes
OCH3
trans-Anethole
OH
OCH3
Eugenol
OH
β-Eudesmol
HH
O
H
β-Caryophyllene β-Caryophyllene oxide
OH
Farnesol
CHOOH
OH
Cinnamaldehyde Cinnamyl alcohol Chavicol
OCH3 OCH3
O
O
Anethole Estragole Safrole
Isoprenoides
OO
OH
Ascaridole Menthol
FIGURE 5 Structure of various essential oils possesses antimicrobial and antiviral activities. Source: Refs. [80,81].
extracellular virus replication [96]. Direct virus inhibition of HSV-1 was reported
for several monoterpenes (a-terpinene, g-terpinene, a-pinene, p-cymene,
terpinen-4-ol, a-terpineol, thymol, citral, and 1,8-cineole), EOs from eucalyptus
(Eucalyptus sp., Myrtaceae), tea tree (Melaleuca alternifolia, Myrtaceae), and
thymol (Thymus sp., Lamiaceae) [81], and pure phenylpropanoids and sesquiter-
penes (trans-anethole, eugenol, b-eudesmol, farnesol, b-caryophyllene, and
b-caryophyllene oxide) and star anise oil [80]. Very high safety index of 160
and 140, for star anise oil and b-caryophyllene, respectively, was reported
(Table 2), suggesting a potential for practical application [80]. Furthermore, oils
from natural extracts (star anise oil and tea tree) had higher selectivity index and a
lower toxicity than their isolated pure compounds [80,81]. EOs from
A. arborescens demonstrated higher inhibition activity against HSV-1 compared
to HSV-2, EOs from E. caryophyllus were more effective against HSV-2 com-
pared with HSV-1 (EC50 was 42–74.4 mg/mL for HSV-2 compared with
62.0–153.0 mg/mL for HSV-1 depending on the virus strain) [96]. Therefore,
the source of EOs plays an important role in determining the efficacy of the
obtained extracts against various strains of the virus, which is related to the com-
position of EOs in the extracts and the sensitivity of the different strains to the
active compounds.
Investigations against other viruses have been reported but to a lesser extent.
For example, the EO of Lippia junelliana and Lippia turbinate showed a potent
inhibition against Junin virus [100]. EOs from Pectis odorata, Gaillardia mega-potamica, Heterothalamus alienus, Buddleja cordobensis, and L. turbinate werevery effective against arenavirus Junin with a virucidal concentration 50%
(VC50, the concentration required to reduce virus titer by 50%) of <50 ppm
[101]. Also, EOs from P. odorata and Jungia polita were effective against
dengue virus at concentrations of <50 ppm, whereas EOs from P. odorata,G. megapotamica, and B. cordobensis had VC50 of 71.5, 99.1, and 54.1 ppm,
respectively. The viral envelope was suggested as the potential target of EOs
giving the lipophilic nature of the oil, which enables it to penetrate membranous
structures, and consequently would control the virus entry. The effects of EOs
from Lippia alba Mill. (Verbenaceae), Lippia origanoides Kunth. (Verbena-
ceae),Oreganum vulgare L. (Lamiaceae), and Artemisia vulgaris L. (Asteraceae)were investigated for their inhibitory actions on yellow fever virus [102]. The oil
from L. origanoides was the most active against the virus (11.1 mg/mL caused a
100% reduction of virus yield) and best safety profile with CC50/minimum inhi-
bition concentration ratio of 26. The antiviral activity of M. alternifolia EO and
the main EOs available in its oil (a-terpinene, g-terpinene, a-pinene, p-cymene,
terpinen-4-ol, and a-terpineol) were investigated against influenza A/PR/8 virus
subtype H1N1, polio type 1, Enteric Cytopathic Human Orphan 9, Coxsackie B1,
and adeno type 2 [103]. All the compounds did not have antiviral activities against
polio 1, adeno type 2, Enteric Cytopathic Human Orphan 9, and Coxsackie B1.
Tree tea oil and terpinen-4-ol, terpinolene, and a-terpineol inhibited influenza
A/PR/8 virus subtype H1N1 with the tea tree oil possessing much higher activity
than any of the individual compounds available in its composition.
Studies in Natural Products Chemistry210
TABLE 2 Selectivity Indices of Essential Oils Against HSV-1 and HSV-2
[80,81,97–99]
Essential Oil/
Compound
Max. Noncytotoxic
Concentration
(mg/mL)�SD (%)
TC50 (mg/mL)�SD
(%)a
IC50 (mg/mL)�SD
(%)bSelectivity
Index
HSV-1
Eucalyptus oil 200�3.5 290�5.8 55�8.2 5.3
Tea tree oil 75�8.3 120�9.6 2�4.2 60.0
Thyme oil 50�16.2 70�2.1% 11�13.3 6.4
Star anise oil 100�8.0 160�30.7 1�0.1 160
a-Terpinene 50�11.2 55�8.1% 8.5�16.3 6.5
g-Terpinene 35�8.1 38�12.7 7�2.8 5.4
a-Pinene 50�3.2 80�5.1 4.5�10.4 17.8
p-Cymene 30�12.7 65�13.0 16�16.2 4.1
Terpinen-4-ol 250�11.7 650�13.3 60�17.7 10.8
a-Terpineol 150�16.5 400�19.9 22�14.9 18.2
Thymol 35�8.1 85�12.8 30�6.5 2.8
Citral 20�1.7 45�8.9 3.5�10.1 12.9
1,8-Cineole 1250�9.6 2000�8.4 1200�8.9 1.7
trans-Anethole 70�3.0 100�6.4 20�1.1 5
Eugenol 60�11.1 85�8.1 35�6.2 2.4
b-Eudesmol 9�1.3 35�5.4 6�0.3 5.8
Farnesol 10�0.4 40�3.7 3.5�0.1 11.4
b-Caryophyllene 10�0.1 35�2.3 0.25�0.0 140
b-Caryophylleneoxide
9�1.1 18�1.2 0.7�0.1 25.7
Ginger oil 0.004 0.0002 20
Thyme oil 0.007 0.001 7
Hyssop oil 0.0075 0.0001 75
Sandalwood oil 0.0015 0.0002 7
Lemon balm oil 0.002 0.003 0.0004 7.5
Continued
Chapter 7 Natural Antiviral Compounds 211
Lignans
Lignans are widespread compounds in plants and many lignans exhibited
antiviral activities [13,45]. For example, peltatins from Justicia procumbensand Podophyllum peltatum, schizarin B and taiwanschirin D from Kadsuramatsudai, and rhinacanthin E and rhinacanthin F from Rhinacanthus nasu-tus were shown to inhibit HIV, hepatitis B virus (HepBV), and influenza
A by blocking the virus replication [13,45,104]. Honokiol is a very potent
anti-HepCV compound obtained from Magnolia officinalis. Honokiol inhib-ited HepCV (EC50¼1.2 mg/mL, SI¼29.1) through the disruption of the
HCV life cycle [105]. A less effective lignan compound, 3-hydroxy carui-
lignan, is obtained from the stems of Swietenia macrophylla (Meliaceae).
This compound decreased the RNA and inhibited HepCV at EC50 of
10.5 mg/mL [106].
Proteins and Peptides
Antiviral protein and peptide compounds are classified into five categories:
(1) single chain ribosome-inactivating proteins, (2) dimeric cytotoxins, (3)
lectins, (4) antiviral factor, and (5) meliacine [13]. The single chain
ribosome-inactivating proteins are found in extracts from Clerodendruminerme, Dianthus caryophyllus, Gelonium multiflorum, Momordica charantia,Phytolacca americana, Saponaria officinalis, Trichosanthes kirilowii, and
TABLE 2 Selectivity Indices of Essential Oils Against HSV-1 and HSV-2
[80,81,97–99]—Cont’d
Essential Oil/
Compound
Max. Noncytotoxic
Concentration
(mg/mL)�SD (%)
TC50 (mg/mL)�SD
(%)
IC50 (mg/mL)�SD
(%)
Selectivity
Index
HSV-2
Anise oil 0.016 0.003 5
Hyssop oil 0.0075 0.0006 13
Thyme oil 0.007 0.0007 10
Ginger oil 0.004 0.0001 40
Camomile oil 0.003 0.00015 20
Sandalwood oil 0.0015 0.0005 3
Lemon balm oil 0.002 0.003 0.00008 37.5
aTC50 = half-maximal toxic concentrationbIC50 = half-maximal inhibition concentration
Studies in Natural Products Chemistry212
Triticum aestivum. These proteinous fractions exhibit their antiviral effects
through inhibiting the synthesis of viral protein and interfering with the ribo-
some function in the infected cell through their effects on N-glycosidases andthe depurination of RNA. Recently, a protein-enriched fraction obtained from
larvae of housefly Musca domestica L. (Diptera: Muscidae) showed strong
antiviral activity against influenza virus H1N1 which was the result of direct
virucidal activity as well as interference with the virus interaction with the
cell [107]. A group of protein fractions isolated from P. americana,M. charantia, and G. multiflorum coined as Pokeweed antiviral proteins
(MRK29, MAP30, and GAP31) have been shown to be potent compounds
inactivating infective HIV and HIV-infected cells [108–112]. Panaxagin from
Panax ginseng [113] and a- and b-antifungal proteins from Vigna unguiculatainhibited the HIV-1 reverse transcriptase [114]. The dimeric cytotoxins are
found in Ricinus communis, Abrus precatorius, and Adenia digitata and they
have similar mechanism of action to single chain ribosome-inactivating pro-
teins. Lectins are sugar-binding proteins which possess the ability to specifi-
cally bind to carbohydrate moieties including cells. Several lectins from
Canavalia ensiformis, Lens culinaris, Phaseolus vulgaris, and Triticum vul-garis were found to have strong antiviral activities through viral membrane
interactions [13]. Recently, lectins from plant sources have been proposed
to act as anti-HIV drugs where they target the glycans present on the surface
of the external envelope protein of HIV.
Lectins have the potential to inhibit HIV infection and prevent HIV trans-
mission from virus-infected cells to uninfected CD4T lymphocytes [115,116].
Meliacine, a protein isolated from Melia azedarach, demonstrated strong
activities against HSV-1 strain in mice, inhibited Junin hemorrhagic fever
virus in Vero cells, and foot and mouth disease virus in baby hamster kidney
cell [13]. Meliacine inhibits the virus replication cycle and interfere with the
virus penetration step into cells. The peptides mirabamides A, C, and D (Fig. 6)
were isolated from the sponge Siliquariaspongia mirabilis inhibited HIV-1
cell fusion (EC50¼0.21–6.1 mg/mL) through interaction with the virus enve-
lope glycoproteins [117].
A purified peptide from the seeds of Sorghum bicolor L. (MW¼2000 Da)
effectively inhibited the replication of HSV type 1 (HSV-1) in a dose-
dependent manner, at 40–90% of the control level, after incubation with
20–10 mg/mL of the peptide, with EC50 of 12.5 mg/mL and had an CC50 value
of 500 mg/mL against Vero cells [118]. The peptide was not only able to
inhibit the initiation and the spread of infection but also had an in vitro pro-
phylactic effect against HSV-1 infection. The virucidal activity was suggested
to be caused by the disintegration of the entire HSV particles; the solubiliza-
tion of the virus envelope; or the chemical modification, degradation, or
masking of some of the essential envelope proteins. The peptide had weak
activity against poliovirus type 1, a nonenveloped virus.
Chapter 7 Natural Antiviral Compounds 213
Polysaccharides
Extracts containing polysaccharides from Achyrocline flaccida, Aloe barba-densis, Bostrychia montagnei, Cedrela tubiflora, Prunella vulgaris, Sclero-tium glucanicum, Stevia rebaudiana, Rhizophora apiculata, and Rhizophoramucronata showed protective effects against several viruses [13]. The
mechanisms suggested for their effects were immunostimulation of antibody
production against capsid protein epitopes of nonenveloped picornavirus,
and prevention of the virus binding to the cell and the formation of syncytia.
Polysaccharides possessing antiviral activity from marine sources have been
reported. Talarico et al. [119] isolated an L-galactan hybrid C2S-3 (Fig. 7A)
from Cryptonemia crenulata, which exhibited a potent antiviral activity
(EC50¼0.8–16 mg/mL) against three clinical strains of dengue virus serotype 2.
NO
NH
NHOR1
OMe
NH
OO
O
O
O
R2
MeO
HN
NMe
OO
OH
HN
HN
NH2
O
O
O
HN
H2N
OHHN
NH
OO
O
HO
OH
Mirabamide A R1 = O
OH
HOHO
MeR2 = Cl
R2 = Cl
R2 = H
Mirabamide B R1 = H
Mirabamide C R1 =O
OH
HOHO
Me
FIGURE 6 Structure of the peptides mirabamides A, C, and D which were isolated from the
sponge Siliquariaspongia mirabilis.
Studies in Natural Products Chemistry214
A sulfated polymannuroguluronate (Fig. 7B) isolated from Laminaria japon-ica [120] and a sulfated xylomannan isolated from Scinaia hatei [121]
inhibited HIV and HSV, respectively. The compounds interfered with the
virus multiplication cycle.
Galactofucan, a sulfated polysaccharide obtained by aqueous extraction of
the seaweed Undaria pinnatifida, was evaluated for antiviral activity against
32 clinical strains of HSV in which 14 strains of HSV-1 and 18 strains of
HSV-2 were examined [122]. Among the investigated strains, 12 strains
(4 HSV-1 and 8 HSV-2) were resistant to acyclovir and 20 strains (10
HSV-1 and 10 HSV-2) were susceptible to acyclovir. The median EC50 of
galactofucan for the 14 strains of HSV-1 was 32 mg/mL, whereas the EC50
for the 18 strains of HSV-2 was 0.5 mg/mL and exhibited significantly higher
efficacy against clinical strains of HSV-2 than HSV-1. The mode of action of
the galactofucan was shown to be the inhibition of viral binding and entry into
the host cell. The cytotoxicity of galactofucan was >4.0 mg/mL, suggesting a
potentially high safety margin. Synthetic sugar analogues demonstrated that
the sugars interfere with glycosylation of influenza virus glycoprotein leading
to virus inhibition [123]. Sugars containing a benzyl group were able to
inhibit the virus and higher concentrations of 2-deoxy-D-glucose and
D-glucosamine promoted the disruption of the glycosylation step.
An extract obtained from the red marine alga Ceramium rubrum (Huds.) Ag.,
from the Bulgarian Black Sea, inhibited the replication of 11 strains of influenza
viruses’ type A and B in vitro and in vivo [124]. The extract induced a
O
HO OH
O3SO
O
O
O
O OSO3
HOHO
HO
O
OH
O
OSO3
O
OSO3
HOHO
n
D,L-Galactan hybrid C2S-3
OO
HO
HOOC
O
OSO3
OO
O3SO
HOOCOH
OO
O3SO
HOOCOH
OO
HO
HOOCOSO3
n
Sulfated polymannuroguluronate
A
B
FIGURE 7 Structure of polysaccharides obtained from seaweeds with high antiviral activity.
Chapter 7 Natural Antiviral Compounds 215
cytopathogenic effect at concentration range (0.12–1.1 mg/mL, depending on the
virus strain) and exhibited a selective virus-inhibitory effect in a dose-dependent
manner. The selectivity indices ranged 9.5–68.3 for the influenza viruses and the
activity was attributed to inhibition of virus adsorption as well as to inhibition of
the intracellular stages of viral replication. The extract also inhibited the
replication of HSV-1 and -2 in cell cultures with SI ranged from 4.9 to 10.8.
Chitosan is the deacetylated form of chitin, a polymer that is widely abundant
in nature (Fig. 8). Chitosan and its derivatives have been extracted from several
fungal, insect, and marine sources and their antiviral efficacies were evaluated
using various viruses [125–128]. Most of the available literature documents the
antiviral activity of chitosan against many plant viral infections [125–127] with
blocking the replication of the virus being described as the major mechanism
of action. Chitosan oligosaccharide lactate (MW¼5 kDa) and water-soluble
chitosan (MW¼53 kDa) were investigated at concentrations of 1.4%, 0.7%,
and 0.35% against murine norovirus 1, feline calicivirus F-9, and bacteriophage
MS2 [129]. Higher MW chitosan was more effective than oligosaccharide lactate
derivative and the highest antiviral activity was found at the concentration of
0.7%. The compounds were more effective against feline calicivirus F-9 than
bacteriophageMS2 but had no effect on murine norovirus 1. These results under-
pin the importance of elucidating the specificity of the tested antiviral compounds
toward certain viruses. The effects of chitosan appear to be linked to nonspecific
binding between the positively charged chitosan and the negatively charged virus
surface which causes weakening and disruption to the capsid structure [129].
Chitosan obtained from the larvae of houseflyM. domestica L. (Diptera: Musci-
dae) demonstrated effective antiviral activities against Autographa californicamulticapsid nucleopolyhedrovirus and Bombyx mori nuclear polyhedrosis virusin Spodoptera frugiperda 9 cell line and Silkworm larvae, respectively [128].
A 1 log reduction in virus titer and 30% reduction in larvaemortality after 20 days
were achieved at chitosan concentration of 1 mg/mL. The availability of several
functional groups and the cationic nature of chitosan render the compound as an
important template for the generation of several derivatives, whichmay have bet-
ter antiviral activity. Kulikov et al. [130] and Davydova et al. [126] using chemi-
cal and enzymatic hydrolysis techniques showed that the antiviral of chitosan
against tobacco mosaic virus was not affected by the compound deacetylation
OOO
OH
OH
CH2OH
NHCOCH3
CH2OH
OH
NHCOCH3
CH2OH
OH
NHCOCH3
OHOO
n
OOO
OH
OH
CH2OH
NH2
CH2OH
OH
NH2
CH2OH
OH
NH2
OHOO
n
Chitin Chitosan
FIGURE 8 Structure of chitin and its derivative chitosan.
Studies in Natural Products Chemistry216
degree but it was increased with the decrease in the degree of polymerization and
the molecular weight of the chitosan derivatives. Similar effects on human
viruses are yet to be investigated.
Considerations for Antiviral Activity of Compounds fromNatural Sources
Extraction Conditions
The successful evaluation of medicinal plants and the screen of novel natural
sources for antiviral activity are dependent on applying systematic procedures
to obtain the targeted compounds. Despite the availability of standard meth-
ods for the extraction of natural products [131], the reporting of extraction
procedures in the literature is normally not sufficient. The extraction temper-
ature is an important factor in successful extraction of bioactive compounds.
Aqueous extracts Agrimonia eupatoria, A. pilosa, and A. coreana pilosella
have been reported to possess antiviral activity against HepBV. Aqueous
extracts of A. eupatoria were extracted at 37, 45, 55, and 60 �C and investi-
gated for inhibition of HBsAg release against HepBV [132]. The extracts
obtained at 60 �C were found to have the greatest inhibition (104%, 41%,
and 32% decrease in production of HBsAg compared with 35, 45, and
55 �C, respectively). This is probably due to better extraction of active com-
pounds at 60 �C without compromising their activity. The study also high-
lighted the seasonal effects on the examined activity and the inhibitory
activity of A. eupatoria was highest at mid-July.
Furthermore, efficient use of extraction system to obtain the active
compound(s) is crucial for appropriate screening of materials. Methanol, hex-
ane, and chloroform extracts from Jatropha curcas were found to produce
moderate cytoprotective effect against HIV in cultured human lymphoblastoid
cells but not ethyl acetate or water extracts [133]. In the same study, methanol
extracts of Alchornea cordifolia, Maprounea africana, and Mangifera indicaexhibited weak cytoprotective effect against HIV but this activity was abol-
ished when other solvents were used, with the exception of chloroform when
was used to extract M. Africana. Similarly, Ben Sassi et al. [134] screened15 species of Tunisian traditional medicinal plants against HSV-1 using petro-
leum ether, acetone, methanol, or water as the extraction solvent and signifi-
cant effects from the solvent were evident. Generally, methanol produced
higher yields and in some instances better selectivity index which was depen-
dent on the material. Twenty-one Ethiopian medicinal plants were extracted
using various solvents and were screened for activity against HIV-type 1 and
type 2 [135]. Methanol extracts of Combretum paniculatum (Combretaceae)
and B. abyssinica (Melianthaceae) had SI of 4.7 and 3.8 against HIV-type 1
only, whereas the acetone extract of C. paniculatum only exhibited activity
against HIV-type 1 and type 2 with SI of 6.4 and 32, respectively. The antiviral
activity of seven Panamanian plant extracts (Hybanthus prunifolius, Ouratea
Chapter 7 Natural Antiviral Compounds 217
lucens, Trichilia cipo, Tetragastis panamensis, Piper cordulaturn, Alseis black-iana, and Aspidosperma megalocarpon) using dichloromethane, petroleum
ether, ethanol, or water as the extraction solvent indicated the poor efficacy of
dichloromethane and petroleum ether in extracting the active compounds from
the plants [136]. Generally, the extracts exhibited selective effects, with ethanol
extracts were more effective against HSV-1 than HSV-2 while the aqueous
extracts exhibited the opposite effect. Moreover, the extracts were more effec-
tive against HSV-1 compared with HSV-2, with the exception of O. lucensand T. panamensis extracts. The antiviral activity of the extracts were in the fol-lowing order: poliovirus>parainfluenza>vesicular stomatitis virus>HSV-1,
but this order was slightly affected by the cell line used in the assays.
Giving the various compounds that can be expressed differently in the
plant, the relationship between the best solvent to be used with a plant can
be complicated by the part of plant (leave, stem, root, or bark of these parts)
to be extracted from and this was reported in several studies [133–136]. Sev-
eral other factors need to be considered during the extraction and evaluation
of the compounds such as the distribution of the compounds in the plant,
extraction system (solvent, time, temperature, and solid:liquid ratio), the
effects of storage, chemicals used, dialysis, use of enzymes, pH, and
centrifugation.
Several reports compared the efficacy of plant crude extracts with their
active pure compounds [137,138]. These studies were generally reported a
higher antiviral activity of pure compounds compared with the crude extracts.
For example, the anti-HSV-1 activity of an ethyl acetate extract of Tanacetumvulgare was 0.008% the activity of its active compound, parthenolide [137].
Similarly, higher anti-HSV-1 was found in isolated ursolic acid and an active
fraction obtained from Mallotus peltatus compared to crude methanolic
extract of the plant [138]. While the purification of active compounds seems
to be a logical way to obtain active compounds with higher efficacy, several
disadvantages may be posed by carrying the purification step. For example,
active compounds are generally having low CC50 which can pose risk of
higher toxic effects at lower doses [138]. Also, the cost and technology asso-
ciated with the purification of these compounds can be prohibitive for certain
communities and reduce access and affordability of the treatment. Further-
more, some plant extracts may contain several active compounds which can
synergistically lead to higher antiviral activity or other component that can
lead to better bioavailability. For example, the anti-influenza activity of crude
grape extracts was equivalent (at the same concentration) to that found with
the most active antiviral compounds (catechin, ECGC, delphinidin chloride,
cyanidin chloride, or pelargonidin chloride) found in grapes [unpublished
data] despite the fact that they are available in much smaller concentrations
in the crude extract. The bioavailability of polyphenols is greatly enhanced
with natural compounds such as pectin [139] which will be lost during further
purification. Synergistic effects of active compounds in crude extracts have
Studies in Natural Products Chemistry218
been reported for J. curcas [140] and other medicinal plants [141] in other
medical conditions. Therefore, more research on the bioavailability of anti-
viral active compounds, the measurements of the compounds and their meta-
bolites, and their synergistic effects is needed.
Structure–Activity Relationships
A series of thyrsiflorin and scopadulane compounds (methyl thyrsiflorin A,
methyl thyrsiflorin B acetate, thyrsiflorin C, thyrsiflorin C diacetate,
13-scopadulanone, 13a-scopadulanol, 7b-hydroxy-13-scopadulanone, 7b-acet-oxy-13b-scopadulanol, 8(14)-scopadulen-13-one, 8(14)-scopadulen-13,15-
dione, 7a-hydroxy-8(14)-scopadulen-13-one, cyclopropane intermediate, 7a-hydroxy-8a-scopadulan-13-one, and a rearranged scopadulane-type diterpene
ketone) derived from the natural scopadulane-type diterpenes were investigated
for their in vitro anti-HSV-2 activity [142]. The activity of the compounds
indicated that a polar substituent at C-13 had a hydroxyl group in the case of
13a-scopadulanol, which is essential to improve the antiviral activity since the
presence of a carbonyl group at C-13 in 13-scopadulanone exhibited a lower
antiviral activity and a complete loss of activity when the hydroxyl group was
esterified in methyl thyrsiflorin A. The location of the substitution is important
since the substitution at C-7 with a polar group and the presence of a carbonyl or
ester group at C-13 in thyrsiflorin C resulted in a complete loss of the activity.
The antiviral activity of scopadulciol against HSV-1 increased when the substi-
tution at C-13 was with a hydroxyl group compared with acetyl group [142,143].
Indeed, the substitution with hydroxyl groups appears to improve the anti-
viral activity of some phenolic acids (Table 3) and improve their selectivity
toward the virus strain; however, the availability of hydroxyl groups do not
necessarily mean a better antiviral activity. The antiviral activity of flavonols
against HSV-1 was higher than flavones [144] and their activity was found to
decrease with the increase in the number of their hydroxyl substitution, that is,
galangin>kaempferol>quercetin (Table 4). The glycosides compounds of
kaempferol and quercetin (kaempferol 3-O-rutinoside, kaempferol 3-O-robi-nobioside, quercetin 3-O-rutinoside) had much higher antiherpes activity than
their aglycon (Table 4). This form has very high safety profile and exhibited a
much higher SI value compared to their aglycon [144]. It is worth mentioning
that this form is the natural form that can be found in many plants. Therefore,
particular attention should be given to the purification system to avoid the
activities of endogenous glucosidases which can catalyze the removal of the
sugar group.
The antiviral activities of three photosensitizer compounds (hypericin, tet-
rabromohypericin, and gymnochrome B) were evaluated against dengue
viruses [146,147]. Gymnochrome B exhibited higher anti-dengue 4 activity
while tetrabromohypericin had lower activity compared to hypericin
(EC50¼0.035, 0.91, and 2.31 mg/mL for gymnochrome B, hypericin, and
Chapter 7 Natural Antiviral Compounds 219
tetrabromohypericin, respectively). The antiviral activity of hypericin and
related analogues against herpes viruses was negatively correlated with the
level of substitution of chlorine in the hypericin structure in position 7 (7,70-dichlorohypericin) [148]. The substitution of functional groups on hypericin
can modify its nature and the antiviral mechanism becomes independent of
photoactivation [149].
CONCLUDING REMARKS
Antiviral compounds can be successfully obtained from various plants,
marine, insects, and animal sources. The compounds vary in their selective
nature and efficacy toward different viruses. The future of natural antiviral
compound is very promising since many of these compounds have multibio-
logical functions. Several health-promoting properties (anticancer, antioxi-
dant, immunomodulation, antibacterial, antiparasitic activities) have been
reported for many of the compounds discussed in this chapter (e.g., phenolic
compounds). The multifunctionality of these compounds makes them a very
appealing alternative to synthetic drugs. The economical advantage of obtain-
ing antiviral compounds from plants is obvious. Many of the reported
promising plants are cultivated in developing countries and can be beneficial
to their economies. Many plants have been reported to possess antiviral activ-
ities against plants viruses [150]. Some of those already shown to be effective
against human viruses such as Acacia arabica [151], Chenopodium
TABLE 3 The Structures of the Pure Compounds from Plantago major,
Their Anti-HSV Activities [66]
R3
O
R1
R2
Compound
Functional Group
Anti-HSV Activities
(EC50, mg/mL)
R1 R2 R3 HSV-1 SI HSV-2 SI
Caffeic acid dOH dOH dOH 15.29 671 87.25 118
Chlorogenic acid dOH dOH X* 47.6 83.9 86.44 46.2
Ferulic acid dOH dOCH3 dOH >100 – >100 –
p-Coumaric acid dOH dH dOH >200 – 32.78 14.9
X*: 1,3,4,5-tetrahydroxycyclohexane carboxylic acid.
Studies in Natural Products Chemistry220
TABLE 4 The Structure of Quercetin, Kaempferol, and Their Glycosides
Found in the Leaves of Ficus benjamina [145]
O
O
HO
OH
OH
O
O
HO
OH
OH
OHOH
OH
Galangin
OHO
OH
OH
OH
OO
H
OHOH
HO
O
O
O
H
OH
OH
HO
Quercetin 3-O-rutinoside
OHO
OH
OH
OO
H
OHOH
HO
O
O
O
H
OH
OH
HO
Kaempferol 3-O-rutinoside
OHO
OH
OH
OO
OH
HOH
HO
O
O
O
H
OH
OH
HO
HO
Kaempferol 3-O-robinobioside
O
O
HO
OH
OH
Kaempferol Quercetin
Continued
Chapter 7 Natural Antiviral Compounds 221
ambrosiodes L. [152], and Zingiber officinale [153] and the potential of other
plants is promising. The screening of pure compounds, such as in the case of
caffeic acid, resulted in different outcomes [29,56,66], which may be partially
explained by the differences in the antiviral screening system (i.e., the type of
virus and cells used in the assay). However, these compounds in their natural
sources will be complexed with other molecules and available in different
forms. The majority of available studies report the screening and evaluation
of natural antiviral compounds in in vitro systems. The inclusion of animal
models is encouraged where possible to determine any practical use. Any
extrapolation of antiviral activity obtained from pure compounds cannot be
extended to the composition of the extracts since the compounds will be in
different forms. The pure compounds, however, provide the template to syn-
thesize novel compounds and improve our understanding about structure–
activity relationship of antiviral drugs.
ABBREVIATIONS
CC50 the concentration causes the reduction of cell viability by 50%
EC50 half-maximal effective concentration
EOs essential oils
HIV human immunodeficiency virus
HSV herpes simplex viruses
Hep hepatitis
ppm part per million
SI selectivity index
VC50 the concentration required to reduce virus titer by 50%
TABLE 4 The Structure of Quercetin, Kaempferol, and Their Glycosides
Found in the Leaves of Ficus benjamina [145] —Cont’d
Compound
Anti-HSV-1
Activity (EC50,
mg/mL)
Plaques Number (% of
Control) During and After
Infection
HSV-1 SI HSV-1 HSV-2varicellazoster virus
Quercetin 3-O-rutinoside 0.92 266.7 18 28 100
Kaempferol 3-O-rutinoside 1.78 100 28 39 101
Kaempferol 3-O-robinobioside 1.38 666.7 6.1 10 102
Quercetin 18.12 7.1 – – –
Kaempferol 7.15 3.2 – – –
Studies in Natural Products Chemistry222
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