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TABLE 13.1 (Continued)
Plant species Family Active form Activity
Leucaena leucocephala (Lam.) de
Wit
Fabaceae Seeds extract Demonstrated activity against DENV-1 in vitro and in vivo level (doi:
10.1016/
S0166-3542(03)00175-X
)
Meristiella gelidium (J. Agardh)
D.P. Cheney & P.W.
Gabrielson
Solieriaceae Extract and carrageenan
derivatives
Antiviral activity against DENV-2 (doi:
10.1016/j.carbpol.2005.09.020)
Mimosa scabrella Benth. Fabaceae Seeds extract Demonstrated activity against DENV-1 in vitro and in vivo level (doi:
10.1016/
S0166-3542(03)00175-X
)
Myrtopsis corymbosa (Labill.)
Guillaumin
Rutaceae Myrsellinol, ramosin, and
myrsellin
Inhibited about 87% against DENV polymerase (doi:
10.4103/phrev.
phrev_2_18
)
Ocimum sanctum L. Labiatae Leaves extract Inhibited the DENV-1 serotype in cell lines (doi:
10.4103/phrev.phrev_2_18)
Phyllanthus
urinaria L.
Phyllanthaceae Aqueous and
methanolic extract
Showed strongest inhibitory activity
against DENV-2 with more than 90% of
virus reduction (doi:
10.1186/1472-6882-
13-192
)
Psidium guajava L. Mrytaceae Bark extract Demonstrated good activity on DENV-2 serotype (doi:
10.1186/s12906-019-
2695-1
)
Tephrosia
madrensis Seem.
Fabaceae Flavonoids isolated
glabranine and 7-
O-methyl-
glabranine
Strongly inhibited dengue virus
replication in Rhesus monkey epithelial
cells (doi:
10.4103/phrev.phrev_2_18)
Uncaria tomentosa (Willd. ex
Schult.)DC.
Rutaceae Alkaloidal fraction Reduced monocyte infection rates and cytokine levels (doi:
10.1016/j.
intimp.2007.11.010
)
Zostera marina L. Zosteraceae p-sulfoxycinnamic acid Antidengue activity against DENV-2 serotype in LLC-MK2 cell lines
(doi:
10.1016/j.antiviral.2008.05.007)
The ethanolic leaf extract of guava was reported to have a good activity against the
DENV-2 with an IC
50
of 7.2 μg/mL (Saptawati et al., 2017). Two years later, Correa et al.
reported that the ethanolic extracts and fractions of guava bark demonstrated a good activ-
ity on DENV-2, along with five identified compounds (gallic acid, naringin, quercetin, cat-
echin, and hesperidin) from the most active fractions with EC
50
5 17.7 μg/mL and
SI 5 35.4. Catechin was the most active with more than 90% inhibition. From the in silico
molecular docking with virus protein NS5 and E protein, naringin and hesperidin had a
better interaction score than the theoretical threshold that showed higher affinity to NS5
protein (
Trujillo-Correa et al., 2019).
A metabolomic approach in antiviral compound identification
Metabolomics is an emerging “omics” that identifies and quantifies metabolites from cells,
biofluids, tissues, and organisms (
Schrimpe-Rutledge, Codreanu, Sherrod, & McLean, 2016).
Furthermore, metabolomic analysis is a powerful method to identify active compounds.
Metabolomics was previously described to decipher the most active redox compounds in
crude extracts of Viola alba subsp. dehnhardii, Violaceae (
Chervin et al., 2017) and to identify
antihepatocarcinogenic compounds from plants used to treat liver cancer in Cambodia
(
Chassagne et al., 2018). Metabolomic approaches can help to identify compounds having
potential antiviral activity, as in the case of herpes virus simplex (
Haggag et al., 2019; Prinsloo
& Vervoort, 2018
).
Objectives
The aim of this study is to find new antidengue compounds from leaf extracts of
P. guajava L. using a metabolomic approach and correlation analysis. Our approach aims
to link the chemical profile variability using a fast crude fractionation method to bioassay
results by means of multivariate data analysis (MVA). This holistic method has been
implemented to rapidly understand the diversity and role of chemical components
involved in bioactivity and is in contrast to the reductionist approach that involves succes-
sive fractionation steps for the purification of compounds responsible for the activity
(
Ayouni et al., 2016). The use of partial least square (PLS) regression models will provide
a ranking of putative active features detected from liquid chromatographymass spec-
trometry (LCMS) profiles. The annotation of peaks of interests was based on high-
resolution mass spectrometry (HRMS) and tandem mass spectrometry (MS/MS) patterns
mirrored to in silico fragmentation and confirmed by purification or commercial authentic
standards (
Tsugawa et al., 2016).
Results
UHPLC-HRMS-based metabolomics approach
We followed the same steps to validate our approach as previously described (
Chervin
et al., 2017
). Briefly, nine extracts collected in Lao PDR (three crude extracts from the
Champasak province, three crude extracts from the Savannakhet province, and three crude
extracts from the Vientiane province) were pooled according to their collection area and
445Case study: metabolomics reveal antidengue compounds isolated from Psidium guajava
Medicinal Plants as Anti-infectives
submitted to a rapid solid-phase extraction (SPE) step. This fractionation provided eight
fractions of increasing polarity for each pooled extract (a total of 24 fractions). The
ultra-high-performance liquid chromatographyhigh resolution mass spectrometry
(UHPLC-HRMS) profiles displayed 448 features (m/z-RT pairs) in negative ionization (NI)
mode and 163 features (m/z-RT pairs) in positive ionization (PI) mode after data proces-
sing using MS-CleanR workflow (
Fraisier-Vannier et al., 2020).
As an initial step, principal component analysis (PCA) was applied to obtain an unsuper-
vised overview of the LC-MS fingerprints of 24 SPE fractions. PCA gathers all independent
biological replicates from the same fraction. The corresponding results are presented in
Fig. 13.3. Crude extracts [denoted quality control (QC)] were plotted near the center of the
PCA score plot while each independent fraction clustered together according to their respec-
tive polarity. The first principal component axis separated polar and apolar fractions. These
results indicated that SPE fractionation and LC-MS workflows highlighted variability in the
data set and were reproducible. Moreover, it demonstrated a stable chemical composition
within extracts of guava collected in the three distinct areas.
FIGURE 13.3 PCA score plot of ESI-NI/PI dataset grouped between polar and apolar fractions.
PCA, Principal component analysis; NI, negative ionization; PI, positive ionization.
446 13. Antiviral potential of medicinal plants: a case study with guava tree against dengue virus using a metabolomic approach
Medicinal Plants as Anti-infectives
Antidengue activity
The c rude extracts and fractions were evaluated for their antiviral activity against
DENV-2 serotyp e in a cell-b ased infection assay as pre viously described
(
Cannalire et al., 2020; Watanabe et al., 2016). The inhibition percentages were evaluated
at three concentrations (200, 100, and 10 μg/mL) by both cotreatment and posttreatment.
Under the cotreatment condition, Huh-7 cells were supplemented by crude extracts or frac-
tions and DENV2 at the same time. All crude extracts of guava showed 100% DENV2 inhibi-
tion at 100 and 200 μg/mL and over 70% DENV-2 inhibition at 10 μg/mL. Under
posttreatment condition, Huh-7 cells were infected by DENV-2 and then treated with the
crude extracts or fractions. For the 24 SPE fractions tested in cotreatment or posttreatment, the
most active were the apolar fractions (fractions 7 and 8) that showed more than 90% DENV-2
inhibition activity at 100 and 200 μg/mL and over 70% DENV-2 inhibition at 10 μg/mL.
Guava crude extracts and fractions displayed low cytotoxicity at 10 μg/mL (
Fig. 13.4).
An orthogonal projection to latent structures (OPLS) regression model was built to get a
ranking of the loadings (m/z-RT pairs) toward the antidengue inhibition percentage
(input Y). This supervised technique provided a classification of potentially antidengue
compounds according to their regression coefficient values. The classification is presented
in
Fig. 13.5A: positive coefficients were related to the high correlation with antidengue
activity, whereas negative coefficients were relate d to low correlation with antidengue
activity. The coefficient plots of the four top-ranked features are presented in
Fig. 13.5B.
From the list of first features (Fig. 13.5C), we implemented the annotation procedure based
on their respective accurate masse and Ms/MS spectra.
Identification of putative antidengue compounds
The first four ranked compounds from the OPLS regression model were putatively
annotated by matching experimental mass spectra to several databases [Universal Natural
FIGURE 13.4 DENV-2 inhibition per-
centage of each fraction (Fr) obtained by
C18-SPE at 10 μg.
447Case study: metabolomics reveal antidengue compounds isolated from Psidium guajava
Medicinal Plants as Anti-infectives
Products Database (UNPD), KNApSAc, PlantCyc, Dictionary of Natural Products (DNP,
CRC Press, v25.2) and CheBI]. Compounds belonging to the Myrtaceae family and the
Psidium genus were prioritized to narrow down the possibilities. For each compound,
some candidates were ranked and proposed based on their similarity score according to
the comparison between experimental Ms/MS fragmentation and the in silico spectra of
candidates using MS-FINDER (
Tsugawa et al., 2016). This process resulted in the annota-
tion of four candidates belonging to the Psidium genus, and all were classified as triterpe-
noid compounds. Annotation results were supported by UV spectra of each peak
(
Table 13.2; column “UV”). The UV spectra of two triterpenoids (compounds 1 and 3)
showed an absorption band at 310 nm highlighting the presence of a conjugated radical.
Four compounds were putatively identified, jacoumaric acid (JA), guavacoumaric acid, and
guajadial B which were already discovered in the Psidium genus, and terminolic acid first
isolated from Terminalia genus (
King, King, & Ross, 1955)(Fig. 13.6). Interestingly, three of
them have a ursane triterpenoid backbone, two of them (guavacoumaric acid and JA) are
position isomer of coumaric acid moiety. For three compounds, JA, terminolic acid, and gua-
jadial B, commercial authentic standards were used to confirm feature annotation and bio-
logical activity against DENV.
Antidengue assay of pure authentic standards
Antidengue assays on DENV serotypes DENV-1, DENV-2, DENV-3, and D ENV-4
were carried out for the first three compounds dereplicated by using chemical stan-
dards. The results, presented in
Table 13.3, showed that the first ranked compound, JA,
is more active than the other two aut hentic standards (terminolic acid and guajadial B)
with an EC
50
of 9.62, 4.62, 4.31, and 2.52 μg/mL, respectively. Furthermore, JA also
FIGURE 13.5 OPLS regression analysis and classification of putative antidengue. (A) Coefficient plot obtained
by OPLS regression; (B) emphasis on the first loadings; (C) First ranked feature details. OPLS, Orthogonal projec-
tion to latent structures.
448 13. Antiviral potential of medicinal plants: a case study with guava tree against dengue virus using a metabolomic approach
Medicinal Plants as Anti-infectives
TABLE 13.2 Summary of all the compounds identified or dereplicated for antidengue activity.
OPLS
rank
a
m/z
values RT
Adduct
type MF
Δ m/z
(mDa)
Main MS/Ms
fragments
UV
(nm)
Putative
annotation Score
Chemical
class
Coefficient
value
Biology
source
1 617.3825 10.31 [M-H]- C
39
H
54
O
6
1.563 601.1249 310 Jacoumaric
acid
b,c
6.2645 Ursane
triterpenoid
2.37962 Jacaranda
cauranda
497.3494
373.2319
2 549.342 7.1 [M1 FA-
H]-
C
30
H
48
O
6
1.2918 529.1945 ND Terminolic
acid
b,c
6.1864 Oleanane
triterpenoid
1.76963 Terminalia
glaucescens
435.2639
282.9619
161.1879
3 633.3774 8.94 [M-H]- C
39
H
54
O
7
1.0776 612.5383 310 Guavacoumaric
acid
a
5.24 Ursane
triterpenoid
1.66983 Plumeria
obtusa
513.4674
380.8951
4 473.2319 12.46 [M-H]- C
30
H
34
O
5
1.0477 445.3014 ND Guajadial B
b,c
5.6465 Meroterpenoid 1.53657 Psidium
guajava
356.8398
270.1221
a
Ranking based on OPLS regression coefficients, served as compound number.
b
Determined by in silico Ms/MS fragmentation with Ms-FINDER.
c
Confirmed by commercial authentic standard compounds.
ND, Not detected; MF, molecular formula; Ms/MS, tandem mass spectrometry; OPLS, orthogonal partial least squares/projections to latent structures; UV, ultraviolet.
FIGURE 13.6 Chemical structure of annotated compounds (see Table 13.3 for detail).
TABLE 13.3 Confirmation of the antidengue activity of top-ranked compounds by the OPLS regression model.
Compound Virus EC
50
(μg/mL) CC
50
(μg/mL) SI
Jacoumaric acid DENV-1 9.62 30 3.12
DENV-2 4.62 6.49
DENV-3 4.31 6.96
DENV-4 2.52 11.9
Terminolic acid DENV-1 . 50
DENV-2
DENV-3
DENV-4
Guajadial B DENV-1 . 50
DENV-2
DENV-3
DENV-4
OPLS, Orthogonal projection to latent structures.
450 13. Antiviral potential of medicinal plants: a case study with guava tree against dengue virus using a metabolomic approach
Medicinal Plants as Anti-infectives
showed a good selectivity towards the DENV serotypes 24 with a selective index value
over 5 for DENV 2 to 4.
Discussion
The aim of this study was to identify antidengue compounds from the ethanolic leaf
extract of P. guajava. According to the inhibition percentage at 10 μg/mL, crude extract of
P. guajava could inhibit more than 70% of DENV-2 serotype, which is more than the ethyl
acetate fraction of Euphorbia hirta L. (
Tayone, Tayone, & Hashimoto, 2014). Other studies
carried out on eight Indonesian plant extracts also confirmed that P. guajava and Carica
papaya leaf extracts were active against DENV-2 serotype at an EC
50
value of 7.2 and
6.57 μg/mL, respectively (
Saptawati et al., 2017), but no information about compounds
supporting this activity has been provided so far. Our metabolomics approach fills this
knowledge gap by using a fast fractionation step before hyphenation of LC-Ms profiles to
biological assays results. The apolar fractions obtained from SPE fractionation showed
over 60% inhibition against DENV-2. To rank statistically related compounds to the biolog-
ical activity against DENV, we applied an OPLS regression model. These analyses allowed
the identification of jacoumaric aid, terminolic acid, guavacoumaric acid, and guajadial B
as the putative most active compounds of apolar fractions.
Guava is widely used throughout the world for food and traditional medicine. The
Psidium guajava leaf has been used as an infusion or a decoction. Guava is mainly used as
an antidiarrheal. Also, guava was reported to be used as an antiinflammatory, for diabetes,
hypertension, wound, pain relief, and fever (
Gutie
´
rrez et al., 2008). All parts of guava are
used, especially its leaves (
Dakappa, Adhikari, Timilsina, & Sajjekhan, 2013; Elbert, 1964;
Gutie
´
rrez et al., 2008; Heinrich, Ankli, Frei, Weimann, & Sticher, 1998). Previous studies
reported various biological activities of guava including antioxidant, antiinflammatory,
antibacterial, lipid-lowering agent, anticough, antidiarrheal, antidiabetic, cardioprotective,
antimutagenic, hepatoprotective, and larvicidal effects (
Ngbolua et al., 2018). Various
types of phytochemicals were reported from the P. guajava including primary metabolites;
mineral, enzymes, proteins, and secondary metabolites; triterpenoids, sesquiterpenoids,
alkaloids, glycosides, flavonoids (guajaverin and quercetin), tannins, and saponins
(oleic acid, morin-3-O-α-
L-lyxopyranoside, and morin-3-O-α-L-arabopyranoside) (Arima &
Danno, 2002; Dakappa et al., 2013
). Moreover, the leaves of guava contain tannins, triter-
penes, flavonoids, alkaloids, phytosterols, and essential oil rich in cineol (
Manikandan,
Anand, & Muthumani, 2013
). Many compounds related to the triterpenoid class were iso-
lated from guava leaves, some of these compounds were already known and others were
newly identified (
Begum, Hassan, & Siddiqui, 2002; Begum, Hassan, Siddiqui, Shaheen,
et al., 2002; Shao et al., 2012
).
A previous study identified different antidengue compounds obtained from Psidium
guajava extract from the bark, belonging to phenolic acid and flavonoid, such as gallic
acid, quercetin, and catechin (
Trujillo-Correa et al., 2019). Catechin had the highest antivi-
ral activity. The difference observed with our study, where triterpenoids were identified
with high antiviral affinity from the leaf, could be explained by the part of the plant used.
Other triterpenoids purified from the fungi Ganoderma lucidum have been identified as a
451Case study: metabolomics reveal antidengue compounds isolated from Psidium guajava
Medicinal Plants as Anti-infectives
potential compound with antidengue activity, such as ganodermanontriol and celastrol
(
Bharadwaj et al., 2019; Yu et al., 2017). Furthermore, celastrol has the ability to inhibit rep-
lication of all four DENV serotypes, associated with innate immune stimulation by the
induction of the interferon expression and antiviral response in vitro. Another widespread
flavonoid, luteolin, which can be isolated from traditional Chinese medicinal plant, can
inhibit the replication of DENV1-4 serotypes by reducing infectiou s virus particle forma-
tion. Antiviral activity of luteolin was also demonstrated in vivo in DENV-infected mice
(
Peng et al., 2017). Luteolin acts on a host protease and counteracts the maturation process
of newly produced viruses (
Peng et al., 2018). This illustrates both the diversity of com-
pound classes, such as flavonoids and triterpenoids, that may have antidengue activity,
and the diverse activities on the viral infectious cycle of these compounds isolated from
medicinal plants.
We reported here the a ntidengue activity of three of the annotated compounds
(JA, terminolic acid, and guajadial B), confirmed by measuring the activity of their
authentic standards. The tests showed thatJAwasthemostactiveagainstDENV14with
EC
50
values of 9.62, 4.62, 4.31, and 2.52 μg/mL, respectively. Based on the EC
50
value,
jacoumaric acid is more active than previously reported compounds from P. guajava
bark, that is, gallic acid (25.8 μg/mL), naringin (47.9 μg/mL), quercetin (19.2 μg/mL), cat-
echin (33.7 μg/mL), and hesperidin (225.8 μg/mL) against DENV-2 serotype (
Trujillo-
Correa et al., 2019
). One of the major advantages of JA is its activity against all four serotypes
of DENV. JA was identified as potential histone deacetylase inhibitor by in silico methods,
which could play a role in epigenetic modifications and cell proliferation (
Adewole & Ishola,
2020
). JA could have antimicrobial activity, as we previously described against leishmania
using the same metabolomic approach (
Phakeovilay et al., 2019). Although JA was identified
as potential HIV-1 protease inhibitor by in silico virtual screening (
Yanuar, Suhartanto,
Mun’im, Anugraha, & Syahdi, 2014), to our knowledge, no further studies have shown any
activity against HIV.
Based on the results of our study, a metabolomic workflow has been successfully
applied to rapidly identify the antidengue compounds from the P. guajava leaf extract. Our
dereplication approach led to identify JA as the most active compounds against DENV14
serotypes in vitro. This is the first report of antidengue activity of JA. Further studies
would be required to understand the mechanism of action of JA against the DENV both
in vitro and in vivo.
Materials and methods
Plant collection
Nine samples of Psidium guajava L. (Pg) leaves were collected from middle and
south part of Lao PDR (Champasak: Pg1 to Pg 3,Savannakhet:Pg4toPg6andVientiane
province: Pg7 to Pg9,
Fig. 13.7). Samples were washed and dried before being
grounded into powder to obtain 1 kg of each. A voucher specimen of each sample was
collected and deposited at the herbarium of the Institute of traditional medicine of Lao,
Vientiane, Lao PDR. Permission fo r collection has been obtained from the competent
authorities.
452 13. Antiviral potential of medicinal plants: a case study with guava tree against dengue virus using a metabolomic approach
Medicinal Plants as Anti-infectives
Leaf extraction
For each accession area, one sample of P. guajava leaves (250 g) was extracted by 2.5 L
of 80% ethanol (EtOH) under agitation at room temperature for 24 h. The filtrated solution
was evaporated under reduced pressure (Buchi rotavapor R-114). Then, 100 mg of each
crude extract was dissolved in 1 mL of water and fractionated by SPE (1 g Sep-Pak C18
cartridge, Waters, Milford, MA, USA). Each extract was separated using eight aqueous
methanolic solutions of decreasing polarity (H
2
O/MeOH; 100/0, 90/10, 80/20, 70/30,
40/60, 30/70, 20/80, 0/100). Eight fractions were obtained: fraction 1 (25 mg), fraction
2 (5 mg), fraction 3 (10 mg), fraction 4 (8 mg), fraction 5 (6 mg), fraction 6 (8.5 mg), fraction
7 (4 mg), and fraction 8 (11 mg). All fractions were separated into two parts, first part was
used for UHPLC-HRMS profiling at 1 mg/mL in 80% of MeOH, and second part was sent
to Singapore laboratory for antidengue assay.
Cells and virus
Huh-7 [hepatocellular carcinoma cells, Aedes albopictus cell line (ATCC)] cells were cul-
tured in a DMEM medium containing 10% FBS, 1% penicillin/streptomycin (P/S) at 37
C
in 5% CO
2
. BHK-21 (baby hamster kidney fibroblast cells, ATCC) cells were cultured in
RPMI1640 medium containing 10% FBS, 1% P/S, at 37
Cin5%CO
2
. C6/36, an ATC C,
was maintained in RPMI1640 medium containing 25 mM HEPES, 10% FBS, and 1% P/S,
at 28
C in the absence of CO
2
.
DENV-2 EDEN 3295 (GenBank accession EU081177) was obtained from the Early
Dengue infection and outcome (EDEN) study in Singapore (
Low et al., 2006). Virus was
grown in C6/36 cells and the supernatants were stored at 2 80
C. Virus titer was deter-
mined by plaque assay on BHK-21 cells.
FIGURE 13.7 Collection areas of Psidium guajava L.
453Case study: metabolomics reveal antidengue compounds isolated from Psidium guajava
Medicinal Plants as Anti-infectives