Indian Journal of Natural Products and Resources
Vol. 3(2), June 2012, pp. 208-214
Screening and characterization of antiviral compounds from
Psidium guajava Linn. root bark against white spot syndrome virus
S Velmurugan, M Michael Babu, S M J Punitha, V Thanga Viji and T Citarasu*
Centre for Marine Science and Technology, Manonmaniam Sundaranar University, Rajakkamangalam-629 502,
Kanyakumari District, Tamil Nadu, India
Received 11 August 2011; 20 April 2012
White Spot Syndrome Virus (WSSV) has been reported to cause severe mortality and economic loss in shrimp culture
industry especially black tiger shrimp in worldwide. In present study, Psidium guajava Linn. root bark was serially extracted
with hexane, ethyl acetate and methanol then screened antiviral activity against WSSV by incubating the extracts with
WSSV infected haemolymph of Penaeus monodon which was propagated earlier. The incubated haemolymph was
intramuscularly injected to the second abdominal segment of Indian white shrimp Fennerropenaeus indicus and monitored
the survival up to 10 days. Two step PCR detection was performed from the genomic DNA of treated shrimps using VP 28WSSV diagnostic PCR primer. The survival and two step PCR detections revealed that, the ethyl acetate extract effectively
suppressed the WSSV followed by methanol extracts and no antiviral activity was observed in the hexane extracts.
Phytochemical analysis was performed with the active extracts and further this was purified through silica column
chromatography and Thin Layer Chromatography. The elution was screened again for anti-WSSV activity and the antiviral
active elution of P. guajava were analysed by GC-MS which revealed that the active elution contains active compounds
such as phenol, 2,5-bis(1,1-dimethylethyl), diethyl phthalate, asarone, phthalic acid, butyldodecyl ester, phytol and
1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester. The present study revealed that there is a possibility for developing
new antiviral drugs from P. guajava against WSSV infection.
Keywords: Psidium guajava, Common guava, Antiviral drugs, White Spot Syndrome Virus.
IPC code; Int. cl. (2011.01)—A61K 36/00, A61P 31/12
Introduction
Psidium guajava Linn. (Family-Myrtaceae), the
common guava tree, is widely distributed as a native
plant in Latin America, from Mexico to Brazil. The
leaves have been used in folk medicine for many
years to treat diarrhoea, stomachache and hepatic
problems1. Phytochemical studies undertaken by
researchers on different parts of the plant have
resulted in the isolation and identification of various
terpenoids, flavonoids and tannins2. However, among
the triterpenoids, only two pentacyclic triterpenoids of
the ursane series, namely ursolic acid3 and
2α-hydroxyursolic acid4 and three of the oleanane
series, oleanolic acid3, maslinic acid4 and arjunolic
acid5 were reported earlier.
Many herbs including the decoction of P. guajava
leaves have been used in folk medicine for the control
_________
*Correspondent author:
E-mail: [email protected]; [email protected];
Telefax: +91-4652-253078
of bacterial diseases. World Health Organization
indicated that studies on medicinal plants should
include both identification of chemical constituents
and determination of the biological activities of such
plants6.
White Spot Syndrome Virus (WSSV) of penaeid
shrimp, described by the appearance of white spots in
the exoskeleton with high mortality, was first reported
in early 1994 in Thailand as an accidental infection in
laboratory reared shrimp7. The causative organism of
WSSV is an enveloped, non-occluded and rod shaped
baculovirus. This virus is responsible for severe losses
to global shrimp aquaculture8 and the losses in India
alone have been estimated at several million dollars
per year9. WSSV has been isolated and characterized
from Indian shrimp, Penaeus indicus10.
To develop alternative practices aganist WSSV
control, attention should be diverted to find novel
drugs, especially from plant sources. Citarasu et al11
developed a herbal products having antibacterial,
VELMURUGAN et al: ANTIVIRAL COMPOUNDS FROM PSIDIUM GUAJAVA ROOT BARK
antiviral, immunostimulant and anti-stress effects on
fin and shellfish species and achieved successful
results. Antiviral research using plant extracts has
gained momentum since 195012 and many herbal
compounds have been found to have non-specific
immune stimulating properties in shrimp species13.
The antimicrobial activity of five Chinese herb
extracts, against 12 bacterial and 2 viral fish
pathogens14 have been reported. In Vietnam, the
Institute of Ecology and Bioresources has undertaken
applied research on some medicinal herbs for
prophylaxis and treatment of fish and shrimp diseases
such as ulcer, intestinal disease, white mouth, white
head, red skin, and red spot in fish, and luminescence
and brown spot disease in shrimp15. The present work
investigates screening and characterization of antiviral
compounds from P. guajava root bark against the
White Spot Syndrome Virus (WSSV) through in vitro
and in vivo delivery of Indian white shrimp F. indicus.
Materials and Methods
Plant material and extraction
P. guajava roots were collected from Kanyakumari
district, Tamil Nadu, India during February 2011. The
root barks were separated, shade dried (Plate 1),
coarsely powdered and passed through fine sieve. One
hundred grams of fibre free powders were serially
extracted with 100 ml of the non polar solvent
hexane, mid polar extract ethyl acetate and polar
solvent methanol by percolation extraction method.
The extracts were filtered by Whatman no.1 filter
paper and the filtrate was condensed by rotary
evaporator under reduced pressure at a temperature of
50oC. Aqueous extracts were concentrated using
lyophilizer and stored at 4oC for further study.
209
Phytochemical screening for P. guajava extracts
Phytochemical screening was carried out for
P. guajava extracted by hexane, ethyl acetate and
methanol using standard methods to screen for the
presence of various chemical constituents16. The
active extracts were tested by different tests to
determine the presence of various phytochemicals,
viz. alkaloids (wakama test), terpenoids (salkowski
test), flavonoids, saponins, tannins, coumarins,
cardiac glycosides (keller-killiani test), steroids,
phenols, resins and carboxylic acids.
Primary antiviral screening against WSSV
WSSV Inoculums
WSSV infected shrimps with prominent white
spots were collected from shrimp farm located near
Nellore, Andhra Pradesh, India. The haemolymph
samples were drawn from the infected shrimps using
sterile syringes followed by centrifugation (3000 X g
for 20 min at 4°C). The supernatant fluid was then recentrifuged (8000 X g for 30 min at 4°C) and the final
supernatant fluid was filtered through a 0.4 µm filter.
The filtrate was then stored at -20°C for further
studies.
Incubation of P. guajava extracts with WSSV
Five hundred milligram of condensed plant extracts
was dissolved in 100 ml of NTE buffer (0.2 M NaCl,
0.02 M Tris–HCl and 0.02 M EDTA, pH 7.4) as stock
for further bioassay studies. Five micro litre of WSSV
suspension (300 µg of total protein) was mixed with
10 µl of individual extracts and incubated at 29°C for
3 h. After incubation period, the mixture was injected
intramuscularly to F. indicus had the average weight
of 10 ± 1 g. Three replicates were (n= 10 × 3= 30)
maintained in all treatments. Mortalities were
Plate 1—Psidium guajava plant and root bark
210
INDIAN J NAT PROD RESOUR, JUNE 2012
recorded daily and the experiment was carried out up
to 10 days. Control shrimps were injected with a
mixture of 10 µl NTE buffer and 5 µl viral
suspensions. Haemolymph was drawn directly from
the experimental and control shrimp groups using
prewashed syringe with anticoagulant (sodium citrate)
to avoid clotting. They were centrifuged at 3000 × g
at 4°C, after which, the supernatant was placed in
another centrifuge tube together with an
appropriate amount of digestion buffer (100 mM
NaCl, 10 mM Tris HCl, 50 mM EDTA, 0.5%
sodium dodecyl sulfate, 0.1 mg/ml proteinase K,
pH 8.0). After incubation at 65°C for 2h, the
digest
was
deproteinized
by
successive
phenol/chloroform/isoamyl
alcohol
extraction,
recovered by ethanol precipitation and dried; the dried
DNA pellet was resuspended in TE buffer17. Two step
PCR was performed by WSSV diagnostic PCR using
VP 28 primer (FP: 5’ – GCG CGC GGA TCC AAT
CAT GGA TCT TTC TTT CAC-3’; RP: 5’- GCG
CGC GAA TTC TTA CTC GGT CTC AGT GCC-3’)
designed by Namita et al18. Haemolymph samples of
experimental and control shrimps were tested by the
first step PCR. The negative samples detected in the
first step were further subjected for second step PCR
analysis. PCR products were analyzed by
electrophoresis in 1% agarose gels stained with
ethidium bromide, and visualized by ultraviolet
transillumination.
Purification of ethyl acetate active extract of P. guajava
Column purification
Based on the primary screening, the active extract,
ethyl acetate were purified through preparative silica
column chromatography (mesh size 50-80 µm, 30 cm
length, 0.5 ml flow rate, 3 bed volume elution) with
hexane/ethyl acetate and ethyl acetate/methanol at
various proportions as mobile phase, fractions were
collected, condensed in a rotary evaporator and
stored.
Thin Layer Chromatography
Fractions were spotted on silica gel plates GF254
(Merck), 20 × 20 cm, 1 mm thick and the
chromatogram developed using, hexane: ethyl acetate
(7:3) and n-butanol: acetic acid: water (5:1:4) as
mobile phase. The plates were visualized under short
UV light and also the spots were developed using two
different spray reagents such as 10% Ammonium
molybdic acid containing 1 g of ceric sulphate and
10% of H2SO4 in MeOH according to Yrjönen19.
Secondary antiviral screening against WSSV
All elution were screened against WSSV following
the method mentioned previously and the survival and
PCR detections were carried out.
GCMS analysis for ethyl acetate active fraction
GC-MS analysis of active fraction of P. guajava
root bark were analysed individually using Agilent
GC-MS 5975 Inert XL MSD (United States) gas
chromatography equipped with J&W 122–5532G DB5 ms 30 × 0.25 mm × 0.25 µm and mass detector
(EM with replaceable horn) was operated in EMV
mode. Helium was used as carrier gas with the flow
rate of 1.0 ml/ min. The injection port temperature
was operated at 250°C. The column oven temperature
was held at 80°C for 2 min then programmed at
10°C/min to 250°C, which was held for 0 min, and
then at 5°C/min to 280°C which was held for 9 min.
Electron impact spectra in positive ionization mode
were acquired between m/z 40 and 450.
Data analysis
One way and two way Analysis of Variance
(ANOVA) were carried out using the software PASW
statistics data editor and Ky plot, respectively. Means
were compared at 0.05% for one way ANOVA and
0.001% level.
Results and Discussion
The phytochemical analysis revealed that the
hexane extracts enriched with steroids, saponins,
flavonoids, coumarin, carboxylic acids, phenols,
quinone, resins, terpenoids, and tannins, etc. The ethyl
acetate extract was highly enriched with flavonoids,
resins and quinines and other active compounds such
as alkaloids. The methanol extracts had steroid-II,
saponins, coumarin, carboxylic glycosides, phenols,
quinone, resins and alkaloids, etc (Table 1). Herbal
Table 1—Phytochemical analysis of the root bark extract of Psidium
guajava
Psidium guajava
Hexane Ethyl
acetate
Methanol
Saponins
Flavonoids
Coumarin
Carboxylic acids
Cardiac glycosides
Phenols
Quinone
Resins
Terpenoids
Alkaloids
+
+
+
+
+
+
+
+
+
+
-
+
++
+
+
++
++
++
+
+
+
++
+
+
+
+
+
+
Tannins
+
++
+
S. No.
Tests
1
Steroids
2
3
4
5
6
7
8
9
10
11
12
I
II
VELMURUGAN et al: ANTIVIRAL COMPOUNDS FROM PSIDIUM GUAJAVA ROOT BARK
Table 2—Primary antiviral screening of Psidium guajava root
bark extracts against WSSV
PCR Detection
Extractions
Hexane
Ethyl acetate
Methanol
Step - I
Step - II
++++
++
++++
++
Plate 2—Primary antiviral screening of Psidium guajava root bark
extracts against WSSV by PCR detection − M-Marker; 1-Positive
control; 2-Negetive control; 3 & 4-Ist & IInd step PCR for hexane
extract; 5 & 6- Ist & IInd step PCR for ethyl acetate extract;
7 & 8 - Ist & IInd step PCR for methanol extract.
Plate 3—Secondary antiviral Screening of ethyl acetate fractions
of P. guajava root bark extracts against WSSV by PCR detection.
M-Marker; 1-Positive control; 2-Negetive control; 3, 5, 7, 9, 11,
13, 15: Ist step PCR for the fractions 1 to 7; 4, 6, 8, 10, 12, 14, 16:
IInd step PCR for the fractions 1 to 7.
compounds such as volatile oils, tannins, phenolics,
saponins, alkaloids, polysaccharides and polypeptides
were shown effective alternatives to that of
antibiotics. The screening of plant extracts and natural
products for antimicrobial activity has shown that
higher plants represent a potential source of new antiinfective agents20 as well as serve in drug discovery
from natural products for primary lead compounds.
The present study revealed that, presence of
coumarin, phenols, quinone, alkaloids and tannins in
the ethyl acetate extracts responsible for the antiviral
activity and successfully control the WSSV
replication.
Primary antiviral screening of the three extracts of
P. guajava is given in the Table 2 and Plate 2. The
ethyl acetate extract effectively suppressed the WSSV
during the in vitro incubation. The first and second
211
step PCR detection revealed that, the hexane extract
was failed to suppress the WSSV. Surprisingly, there
is no PCR positive signals received from the ethyl
acetate extract and the weak signals received from the
methanol extracts incubated with WSSV injected
shrimps. Twenty species of Indian traditional
medicinal plants such as Aegle marmelos Correa ex
Roxb., Allium sativum Linn., Aristolochia indica
Linn., Azadirachta indica A. Juss., Cassia fistula
Linn. , Catharanthus roseus G. Don, Curcuma longa
Linn., Cynodon dactylon Pers., Lantana camara
Linn., Melia azedarach Linn., Mimosa pudica Linn.,
Momordica charantia Linn., Morus alba Linn.,
Ocimum americanum Linn., Phyllanthus amarus
Schum. & Thonn., Emblica officinalis Gaertn.,
Psidium guajava Linn., Solanum nigrum Linn.,
Tridax procumbens Linn. and Tylophora indica
(Burm. f.) Merrill were tested for their antiviral
activity against WSSV21. Direkbusarakom et al22
tested guava (Psidium guajava) extract for antiviral
activity against the fish pathogenic viruses, infectious
haematopoietic necrosis virus (IHNV) infectious
pancreatic necrosis virus (IPNV) and Oncorhynchus
masou virus (OMV) using plaque reduction in CHSE214 cell lines. Pharmacological investigations
indicated that its bark, fruit, and leaves possess
antimicrobial, antidiabetic, hypoglycaemic and
hypotensive,
anticancer,
anti-inflammatory,
antiplaque, antiallergic, cardioprotective, antioxidant
and antimutagenic activities23-34.
Seven fractions were screened against WSSV after
purifying the P. guajava ethyl acetate extract through
column chromatography. Different fractions (F1 to
F7) were incubated with WSSV and injected to
F. indicus. Among the different P. guajava fractions
injected shrimps, the F1 fraction was effectively
suppressed the WSSV during incubation. There is
100 % mortality within 3 days was observed in
F. indicus of without P. guajava treatment (control).
The F1 treated F. indicus survived significantly
(P<0.05) at 80 % and the other groups had the
survival of 20, 30,40,35,35 and 25, respectively in the
F2 to F7 fractions treated groups, respectively. Also
two way ANOVA revealed that the survival was
varied significantly among the different groups
(F= 40.48868; 24. 58463; P<=0.001) (Figure 1). PCR
detections also prove the antiviral activity of the
fraction F1 (Plate 3). Citarasu et al11 fed with the
antiviral and immunostimulant herbal extracts
incorporated diets to the WSSV infected Shrimp,
212
INDIAN J NAT PROD RESOUR, JUNE 2012
Fig. 1—Survival of F. indicus after injection with ethyl acetate fractions of P. guajava incubated WSSV
Plate 4—Thin Layer Chromatogram analysis for the active
fraction of ethyl acetate extract of P. guajava
P. monodon juvenile and the impact of the herbals
were accessed with PCR diagnosis. The plants like
C. dactylon, A. marmelos, Tinospora cordifolia
(Willd.) Miers. ex Hook.f. & Thoms., Picrorhiza
kurroa Royle ex Benth. and Eclipta alba (Linn.)
Hassk. were effectively controlled the WSSV, in vivo
system. The present study, the F1 fraction effectively
suppressed the WSSV by presence of the active
compounds.
Thin layer chromatographic separation of the
P. guajava F1 fraction revealed that, around ten spots
were detected by low intensity UV wave detections.
Different spots were detected had the Rf value of
0.90, 0.85, 0.80, 0.75, 0.61, 0.59, 0.50, 0.41, 0.32,
respectively (Plate 4). GCMS analysis and the
database analysis revealed that, the F1 fraction of
P. guajava root bark extract had six identified
phytochemical compounds. The higher intensity
peaks and its quality revealed that the identified
compounds are phenol, 2, 5-bis (1, 1-dimethylethyl),
diethyl phthalate, asarone, phthalic acid-butyl dodecyl
ester, phytol and 1, 2-benzenedicarboxylic acid and
mono (2-ethylhexyl) ester (Table 3). The compounds
were deduced by matching with the NIST data bank
and also by their MS fragmentation pattern as
compared with the literature. The results obtained
from this analysis compare favourably well with
VELMURUGAN et al: ANTIVIRAL COMPOUNDS FROM PSIDIUM GUAJAVA ROOT BARK
213
Table 3—Major chemical compounds identified from the active fraction of ethyl acetate extract of P. guajava by GCMS analysis
S. No
Retention Time
1.
2.
3.
4.
5.
6.
10.262
11.618
12.022
18.220
20.873
27.812
Name of the compounds
Phenol, 2, 5-bis(1,1-dimethylethyl)
Diethyl phthalate
Asarone
Phthalic acid, butyl dodecyl ester
Phytol
1, 2-Benzenedicarboxylic acid,
mono(2-ethylhexyl) ester
compounds early identified in other parts of
P. guajava35,36. Guajadial, a phytochemical compound
isolated from the ethyl acetate extracts’ fraction of
P. guajava by NMR spectroscopic method37. It has
been reported that guava leaf contains some
polyphenols, such as peduncladgin, casuarinin and
isostrictinin38-40. However, high performance liquid
chromatography (HPLC) analysis demonstrated that
these elementary polyphenols were present in the
ethyl acetate extract of guava leaf41. Calotropis
procera (Ait.)W.T. Ait. extracts contains cardiac
glycosides, phenols, alkaloids, tannin and quinines
were effectively controlled WSSV at 90% level42.
2, 4-Bis (1, 1-dimethylethyl) phenol isolated and
characterized from terrestrial Streptomyces sp. TN272
had potent antimicrobial activity43. Different types of
asarone identified and characterized from several
plant oils including Acorus calamus Linn. had
antimicrobial activities44.
2
3
4
5
6
7
8
9
10
Conclusion
The present study revealed that the P. guajava root
bark extract had the antiviral activity against the
White spot Syndrome Virus (WSSV) with ethyl
acetate extraction. The purified active extracts contain
polyphenol derivatives. It concludes that, there is a
positive approach to develop antiviral drugs against
WSSV and save the huge economic loss and severe
damages in the shrimp aquaculture industry.
Acknowledgements
The authors gratefully acknowledges the University
Grants Commission (UGC), New Delhi, Government
of India, for its financial support, in the form of a
research grant [UGC-No 37/271 (SR) dated
01.02.2010].
11
12
13
14
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15
Molecular Formula
Molecular Weight
Quality %
C14H22O
C12H14O4
C12H16O3
C22H34O4
C20H40O
C16H22O4
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222
208
362
296
278
90
90
86
83
72
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