Introduction
In Asia, medicinal plants are abundantly found to play a
key role by providing valuable products for medicine. Approximately 7580% of
people depend upon drugs prepared from these plants to cure various viral
diseases related to human and animals globally (Amber
et al. 2017). Medicinal plants
are relevant source to bring out innovative ideas in the research field regarding
pharmacology with minimum side effects. Exploring new herbs having antiviral activity becomes
limited because identifying antiviral ingredients from plants needs separation
techniques that were lacked. Plants with broad-spectrum antiviral activity
against emerging viruses of infectious diseases were screened (Mukhtar et al.
2008). In the past few years, synthetic drugs replaced by numerous
herbal products resulted in harmful effects. This led to reviving the
scientists interest to medicinal products which could not be even replaced by
modern chemistry. These plants are a rich source of bioactive ingredients with
characteristics of antiviral potential and strong efficacy (Akram et al.
2018).
Newcastle
disease is considered an economically major threat to the
poultry industry. This virus belongs to the Paramyxoviridae family cause hemorrhagic intestinal lesions,
respiratory distress, and impairs birds growth. To control its infection lot
of efforts have been put including vaccination but mutation within strain
develop resistance among pathogen (Harazem et al. 2019). If chickens are
infected with NDV it may results in extremely mild respiratory or enteric
disease to severe systemic infection, cause high mortality, thus characterized
by the rapid spread. Because of resistance development in the pathogen, there
should be another source to control NDV. However, to prevent the replication of
NDV or to decrease its severe effects on infected flocks
different strategies are required (Dortmans et al. 2012; Miller et al. 2013).
Similarly,
Influenza viruses outbreaks remained a major threat
worldwide with high mortality and morbidity. Influenza viruses are enveloped
viruses having a genome in the form of segments. It is a negative sense,
single-stranded RNA virus which belongs to Orthomyxoviridae family causes frequent
epidemics almost every year (Tripathi et al. 2020). A highly pathogenic
avian influenza virus can infect and kill humans directly. It causes various
infections such as respiratory tract problems or fatal systematic diseases in
poultry. Different scientists have reported the antiviral potential of natural
components against different viral infections in poultry birds and humans ( Chang et al.
2013; Ahmed et al. 2017).
According to WHO, natural medicines are being used about
three-quarters of the world population mainly different civilizations.
Different countries have standardized their formulas for pharmacological
products against various viral infections such as influenza virus and NDV.
Although, synthetic chemical components are widely used in the modern era and
the effectiveness of herbal products with the least side effects has been
proved by scientists (Lee et al. 2020). However, previous studies remain limited about
the antiviral activity of flowering parts of medicinal plants and their
bioactive components. So, the present study was conducted on various medicinal
flowers represents the strong antiviral activity of these against economically
threatening viruses NDV and Avian Influenza virus.
Materials and
Methods
Sample collection
100 g of dried flowers named Achillea millefolium L.,
Bombax ceiba L., Chrysanthemum cinerafolium (Trevis.) Vis., Hyssopus officinalis L., Rosa damascena Miller, Taraxacum officinale Weber and Woodfordia fruticosa Kurtz were purchased from local herbs market
and transferred in a pre-labeled and clean zip-lock plastic bag to the
Institute of Microbiology research lab. University of Veterinary and Animal
Sciences, Lahore to determine the activity of the herbal extracts.
Preparation of ethanolic herbal plant extracts
Firstly, the
Ethanolic extracts of medicinal flowers were prepared by grinding the flowers
into fine amorphous powder. Briefly, 10 g of powder of each flower was soaked
into 90 mL of 80 percent ethanol (1:10 w/v). After that, it was incubated for 2
days in a shaking incubator at 150 rpm at 37°C for the extraction of active
compounds. Following incubation, the extracts were filtered through filter
paper No.1 (Whatman, USA). The extracts were poured into glass Petri plates to
dry at 40°C. Dried extract in crystal form was
scratched and collected with the help of a spatula and further shifted in a
clean microfuge tube (Eppendorf, Germany). The stock solution of each extract
was prepared by using 10% dimethyl sulfoxide (DMSO) (Shaheen et al. 2015).
Virus samples
LaSota strain of NDV lyophilized tablet (1000 doses
ampoule) was purchased from the market and Avian influenza virus (H9N2)
strain was taken from Quality operation laboratory UVAS. For further
processing, samples were transferred to Microbiology research Lab. UVAS, Lahore,
Pakistan.
Virus cultivation
Initially, reference strain of NDV (Lasota) and Avian
influenza virus (H9N2) was propagated in 9 days old chick
embryonated eggs that were purchased from the hatchery. To check the viability
of eggs, candling was performed. The inoculum was prepared by mixing 1 mL viral
suspension at room temperature (25°C) with antibiotic and antifungal agents.
After that, 0.1 mL of prepared inoculum was injected into each of 10 eggs
through the chorioallantoic sac route by using a sterile disposable syringe
followed by incubation in an egg incubator at 37°C and 6070
percent relative humidity. Out of all, two eggs served as a negative control
contains only normal saline and added antibiotic. After incubation, the eggs
were placed in refrigerator for 24 h and fluid was harvested (Grimes 2002).
Harvesting of
fluid
Firstly, the eggs shells were disinfected by using 70%
ethanol and the shells were removed using sterilized scissors. After that,
chorioallantoic fluid was harvested and the spot agglutination test was
performed by using 25 percent washed chicken RBCs to detect the presence of NDV
and H9N2 (Grimes 2002). For further confirmation,
hemagglutination test was performed and virus titers were found according to (Young et al.
2002) protocol. Harvested fluid was stored at -4°C for further testing.
Embryo
Infectivity (EID50)
The embryo
infective dose was calculated for each virus before evaluation of antiviral
activity. Ten-fold dilutions of viruses were inoculated in groups of
embryonated eggs as mentioned in Table 1 and 2. Each group contained four eggs.
The embryos infectivity was observed up to 3 days according to Chollom et
al. (2012). After that, the percentage index was calculated and the embryo
infective dose was determined which is the titer of virus particles in a single
dose causing 50% of embryos infection.
Antiviral activity of herbal extracts
Preparation of extracts dilutions: Dilutions of
all the extracts were prepared in normal saline according to three different
concentrations (200, 100 and 50 mg/mL) in microfuge tubes under sterile
conditions (Raza et al. 2015).
Dilution of extracts and suspension of extracts and
virus
Table 1: Embryo Infectious Dose 50 of NDV
|
Dilutions used |
Infected (I) |
Non-infected (NI) |
I (A) |
NI (B) |
Total |
Percentage |
|
10^-1 |
4 |
0 |
9 |
0 |
9 |
9/9 x 100 = 100% |
|
10^-2 |
4 |
0 |
5 |
0 |
5 |
5/5 x 100 = 100% |
|
10^-3 |
1 |
3 |
1 |
3 |
4 |
1/4 x 100 = 25% |
|
10^-4 |
0 |
4 |
0 |
7 |
7 |
0/7 x 100 = 0% |
|
10^-5 |
0 |
4 |
0 |
11 |
11 |
0/11 x 100 = 0% |
|
10^-6 |
0 |
4 |
0 |
15 |
15 |
0/15 x 100 = 0% |
|
10^-7 |
0 |
4 |
0 |
19 |
19 |
0/19 x 100 = 0% |
|
10^-8 |
0 |
4 |
0 |
23 |
23 |
0/23 x 100 = 0% |
Calculation
of the index:
Index= Percentage infected immediately above 50% - 50 ÷ Percentage infected at dilutions
immediately above 50%-%infected at dilutions immediately below 50%
= (100% - 50%) ÷ (100%-25%)
= 50 ÷ 75
= 0.6
= 10^2.61 EID50
/0.1mL
= 10^3.6 EID50 /mL
Table 2: Embryo Infectious Dose 50 of H9N2
|
Dilutions used |
Infected (I) |
Non-infected (NI) |
I (A) |
NI (B) |
Total |
Percentage |
|
10^-1 |
4 |
0 |
17 |
0 |
17 |
17/17 x 100 = 100% |
|
10^-2 |
4 |
0 |
13 |
0 |
13 |
13/13 x 100 = 100% |
|
10^-3 |
4 |
0 |
9 |
0 |
9 |
9/9 x 100 = 100% |
|
10^-4 |
2 |
2 |
5 |
2 |
7 |
5/7 x 100 = 71% |
|
10^-5 |
1 |
3 |
3 |
5 |
8 |
3/8 x 100 = 37.5% |
|
10^-6 |
1 |
3 |
2 |
8 |
10 |
2/10 x 100 = 20% |
|
10^-7 |
1 |
3 |
1 |
11 |
12 |
1/12 x 100 = 8% |
|
10^-8 |
0 |
4 |
0 |
15 |
15 |
0/15 x 100 = 0% |
Calculation
of the index:
Index = Percentage infected immediately above 50% - 50 ÷ Percentage infected at dilutions
immediately above 50%-%infected at dilutions immediately below 50%
= (71% - 50%) ÷ (71%-37%)
= 21 ÷ 34
= 0.61
= 10^4.61 EID50
/0.1 mL
= 10^5.6 EID50 /mL
Antiviral
activity of flowers was determined by using three different concentrations of
all extracts as C1, C2 and C3 against both NDV and H9N2.
Antibiotics and antifungal agents were added in all suspensions and 4HA virus
concentration was used for inoculum. The prepared virus/extract suspensions in
ratio of 1:1 were kept at 37°C for 1 h (Suriani et al.
2015).
Evaluation of in
ovo antiviral activity
To perform in ovo antiviral
activity, total seven embryonated chicken eggs (ECE) groups were made having
four eggs in each. Three groups were made according to concentrations C1, C2,
C3. Three groups served as a negative control, contained 10% DMSO, pure
extracts and normal saline while one group as positive control contained 4HA
virus for each. Firstly, the viability of 9 days old ECE was observed and a hole
was made above the air sac for inoculation. 0.1 mL of each concentration of
inoculum was injected through the allantoic sac route into eggs with the help
of a sterile syringe (0.1 cc) and a hole was sealed by sterile molten wax. The
eggs were then incubated at 37°C.
After 24 h, the embryonated eggs that died because of mechanical injury or
microbial contamination discarded for NDV but for H9N2,
the eggs died were placed in the refrigerator for chilling because it might be
due to H9N2 pathogenicity. After 48 h of post-inoculation
the eggs were placed in the refrigerator overnight for chilling. The
chorioallantoic fluid was harvested to perform spot agglutination and
hemagglutination test (Murakawa et al. 2003).
Viability and antiviral efficacy
Viability of
harvested fluid and antiviral efficacy of different concentrations of extracts
against NDV and H9N2 was checked by spot agglutination
test and further confirmed by hemagglutination test. The harvested fluid was
stored at -20°C in sterile
Eppendorf (1.5 mL).
Gas chromatography mass spectrometry (GC-MS)
The ethanolic
extract of all flowers in liquid form was sent to the Central laboratory
complex (CLC) laboratory UVAS, Ravi campus for analysis of all the chemical and
organic components in them. GCMS equipment used was of Agilant technologies,
7890B GC system, 5977B MSD MS system. The concentration and ratio of extracts
were determined through this system. Molecules were separated based on
volatility and polarity. Gas molecules used as mobile phase in it while column
act as stationary phase and retention time of molecules was found by the
detector. The results were shown in the form of peaks on the graph and
abundance of components indicated by percentage area as mentioned in Table 5.
Statistical
analysis
Table 3: Antiviral activity of flowers extracts
against NDV
|
Extracts |
Concentrations |
Spot
agglutination |
Haemagglutination |
||
|
C1 |
C2 |
C3 |
|||
|
R. damascena
|
1:8 |
1:4 |
1:2 |
-ve |
Bead
formation |
|
A. millefolium |
1:8 |
1:4 |
1:2 |
-ve |
Bead
formation |
|
B. ceiba |
1:8 |
1:4 |
1:2 |
-ve |
Bead
formation |
|
T. officianale |
1:8 |
1:4 |
1:2 |
+ve |
1:128 |
|
W. fruticosa |
1:8 |
1:4 |
1:2 |
-ve |
Bead
formation |
|
H. officianalis
|
1:8 |
1:4 |
1:2 |
+ve |
1:128 |
|
C. cinerafolium |
1:8 |
1:4 |
1:2 |
+ve |
1:128 |
Table 4: Antiviral activity of flowers extracts
against H9N2
|
Extracts |
Concentrations |
Spot agglutination |
Haemagglutination |
||
|
C1 |
C2 |
C3 |
|||
|
R. damascena
|
1:8 |
1:4 |
1:2 |
-ve |
Bead
formation |
|
A. millefolium |
1:8 |
1:4 |
1:2 |
-ve |
Bead
formation |
|
B. ceiba |
1:8 |
1:4 |
1:2 |
-ve |
Bead
formation |
|
T. officianale |
1:8 |
1:4 |
1:2 |
-ve |
Bead
formation |
|
W. fruticosa |
1:8 |
1:4 |
1:2 |
-ve |
Bead
formation |
|
H. officianalis
|
1:8 |
1:4 |
1:2 |
-ve |
Bead
formation |
|
C. cinerafolium |
1:8 |
1:4 |
1:2 |
-ve |
Bead
formation |
Table 5: Spectral analysis of ethanolic extracts of
Medicinal flowers
|
Flowers |
Compound |
Retention time (RT) |
Area (%) |
|
W. fruticosa |
3-heptanol,
2 methyl- |
6.165 |
21.87 |
|
T. officinale |
Cyclopentanone,
2-methyl- |
8.345 |
12.47 |
|
H. officinalis |
3-butene-1-o1,
2-methyl |
6.164 |
20.42 |
|
C. cinerarifolium |
cyclopentanol |
6.169 |
25.72 |
|
R. damascene |
4-cyclopentene-1,3-diol,
trans- |
6.171 |
25.52 |
|
B. ceiba |
Docosanoic acid |
15.854 |
90.04 |
|
A. millefolium |
5-Acetoxymethyl-2,6,10-trimethyl-2,9-undecadiene-6-ol |
6.164 |
19.22 |
The data was examined by using statistic package for
social sciences (SPSS) version 22.0. One way analysis of variance was applied
and P < 0.05 value was taken as significant.
Results
According to
the designed study, flower extracts were used to determine their antiviral
activity by spot agglutination and hemaggltination test. Spot agglutination
test for antiviral activity of extracts Ro.
damascena, A. millefolium, W. fruticosa
and B. ceiba against NDV was
found negative as no agglutination occurs. For further confirmation
hemagglutination test was performed against these extracts showed bead
formation (tear-shaped on tilt) confirmed the inhibition of NDV. While the remaining extracts Taxacum officianale Weber, H. officianalis,
C. cinerafolium showed positive results for both
spot agglutination and hemagglutination assay indicated that these extracts did
not effect on virus inhibition (Table 3). Multiple
comparisons of statistical analysis, for NDV showed mean significant difference
with P < 0.05 value of W. fruticosa extracts
A. millefolium, B. melabaricum,
H. officianalis, C. cinerafolium.
For the antiviral activity of all the extracts R. damascena, A. millefolium, W. fruticosa and B. ceiba , T. officianale, H. officianalis, C. cinerafolium against
H9N2 showed
negative results for both spot agglutination and hemagglutination test
indicates that these extracts strongly inhibited virus (Table 4). Multiple
comparisons of statistical analysis, for H9N2 showed mean
significant difference with P <
0.05 value of W. fruticosa
extracts A. millefolium, B. melabaricum, H. officianalis, C. cinerafolium, T. officianale and R.
damascena. The ethanolic extracts of all the
flowers were analyzed through GC-MS and their active chemical constituents were
determined. Spectral analysis of the ethanolic extract of B. ceiba (Gul-e-Simbal) contained seven
components and among them, Docosanoic acid was the most abundant component with
90.04 percentage while Octane, 4, 5-dimethyl found to had highest retention
time (Fig. 1). Spectral analysis of ethanolic extract of Taraxacum officinale Weber (Zar-e-Gul) examined eighteen components
and the most abundant was 1-heptene, 5-methyl with a percentage area of 17.02
among them (Table 5). The Retention time of Butanoic acid, 3-hydroxy- was 5.402
with first peak. Spectral analysis of the ethanolic extract of R. damascene (Gul-e-Surkh)
contained ten components among them Cyclopentanone, 2-methyl- was a major
component and its percentage area was 17.21 (Fig. 2). Hexane, 3-methyl with
retention time 5.399 with the first peak. C.
cinerarifolium (Gul-e-Dawoodi)
analysis through GC-MS examined ten active components and Cyclopentanone,
2-methyl was most abundant among them with 17.38 percentage area and Butyl
isopentyl with minimum retention time 5.401. Ethanolic extract of H. officinalis (Gul-e-Zoofa) examined ten chemical constituents and
3-butene-1-o1, 2-methyl was major among them 20.42 percentage area.
2-bromopropionic acid, 2 ethylhexy1 ester among them had a minimum retention
time of 5.395. Spectral analysis of the ethanolic extract of W. fruticosa (gul-e-dhawa)
found 3-heptanol, 2 methyl as a most abundant component among sixteen
components with 21.87 percentage area and 3,7 dimethyloctylacetate with minimum
retention time of 5.183 (Fig. 3). Spectral analysis of ethanolic extract of A. millefolium (Gul-e-Birnajaisf) showed 5-Acetoxymethyl-2, 6,
10-trimethyl-2,9-undecadiene-6-ol as a major compound having 19.22 percentage
area among fifteen compounds while Bicyclo [1.1.0] butane-1-carboxylic acid had
minimum retention time of 5.175 (Fig. 4). GCMS analysis revealed the abundance
of alcohol and ethers in all the flowers. Besides these, terpenoids, alkanes,
and fatty acids were also found.
Discussion
%20185-192_files/image002.jpg)
Fig. 1: Spectral analysis of B. ceiba flowers ethanolic extracts
reveals different components in the form of peaks. Retention time of each
component is indicated on X-axis and their abundance is shown on Y-axis
%20185-192_files/image004.jpg)
Fig. 2: Spectral analysis of R. damascene flowers ethanolic extracts
reveals different components in the form of peaks. Retention time of each
component is indicated on X-axis and their abundance is shown on Y-axis
Medicinal plants have remarkable antiviral effects at
different stages of viral growth. Pharmacological products related to plants
are being ranked highly for viral infections at this time. W. fruticosa flowers
extract exhibit antiviral activity against enterovirus 71 but gallic acid
extracted from its flowers were found to show more strong activity against EV-71
with an IC50 of 0.76 µg/mL at a concentration of 100 µg/mL (Choi et al.
2010). A recent study evaluated the antiviral activity of Gallic acid from
W. fruticosa
flowers against herpes simplex virus type 1 and human immunodeficiency virus (Kratz et al.
2008) comparable to the present study which showed strong antiviral
activity of these flowers
against NDV and H9N2 in chicken embryonated eggs. GC-MS
examined sixteen chemical components of it and the most abundant among them was
3-heptanol, 2 methyl having 21.87 percentage areas with 6.165 RT. Retention
time (RT) indicates the time taken by components to elute from column shown on
chromatogram in the form of peaks.
A study conducted on the aqueous extract of T. officinale showed that it possesses
antiviral activity against the influenza virus. The analysis was done by mini
genome assay, real-time reverse transcription PCR found 0.6255 mg/mL of T. officinale extracts ability to
inhibit infections of PR8 or WSN viruses on human lung adenocarcinoma cell line
(He et al.
2011) comparable with present study where it showed antiviral activity
against H9N2 in contrast to NDV where it showed no
inhibitory effect. The ethanolic extract of Taraxacum
officinale Weber contained eighteen active components and 1-heptene, 5-methyl as most abundant
among them while Butanoic acid, 3-hydroxy had minimum retention time.
%20185-192_files/image006.jpg)
Fig. 3: Spectral analysis of W. fruticosa flowers
ethanolic extracts reveals different components in the form of peaks. Retention
time of each component is indicated on X-axis and their abundance is shown on
Y-axis
%20185-192_files/image008.jpg)
Fig. 4: Spectral analysis of A. millefolium flowers ethanolic
extracts reveals different components in the form of peaks. Retention time of
each component is indicated on X-axis and their abundance is shown on Y-axis
H. officinalis preparation are gaining much importance in food industries as well as
in herbal remedies (Dragland et al. 2003; Jung et al. 2004; Lugasi et al.
2006). The extracts of H.
officinalis contained tannins and some high molecular weight compounds
which are unidentified as well as caffeic acid having strong antiviral activity
against HIV. It might be used for the treatment of AIDS (Kreis et al. 1990).
Essential oils and a lot of polyphenolic compounds are the main active
ingredients of this plant according to biological and chemical aspects studied
by the literature review (Benedec et al. 2003; Fathiazad and Hamedeyazdan
2011; Vlase et al. 2014). The
Current study revealed the flowers extracts of H. officinalis with strong antiviral activity against H9N2
inhibited viral growth in embryonated eggs but against NDV, did not show any
response. The Spectral analysis of H.
officinalis flowers were evaluated which contained ten chemical
constituents and 3-butene-1-o1, 2-methyl as abundant ingredient having 20.42
percentage areas. The study on R.
damascene plant found many active constituents including anthocyanins,
flavonoids, glycosides, and terpenes having beneficial properties for human
health. Petals of its flowers are rich in vitamin C and flavonoids. In
vitro study showed its inhibitory
effect on HIV infection with > 100 and 50 selective indices, respectively (Mahmood et al.
1996) relatable to the present study, it showed strong antiviral
activity of R. damascena
flowers against both NDV and H9N2. GCMS analysis of it found ten chemical constituents and
4-cyclopentene-1, 3-diol, trans was the most abundant.
The study of B. melabaricum flowers phenolic compounds showed its
strong activity against RSV in vitro
with a 50 µg/L IC50 value. All the compounds of ethanolic extracts were
evaluated and three among them were considered as having antiviral activity.
Flavonoid, glycoside, caffeoyl acid and kaempferol-3-O-β-D provide potent
antiviral activity against RSV. Many other compounds also possess anti-RSV
activity (Zhang et al. 2015) related to the present study showed antiviral
activity in chicken embryonated eggs against avian influenza virus but in
contrast to NDV having no inhibitory effect. The ethanolic extracts of A. kellalensis
flowers possess anti-rotavirus activity in vitro. The Dose of 100 µg/mL
extract of A. kellalensis
was observed as the effective concentration of extracts. Anti-bovine rotavirus
extracts of A. kellalensis
exhibit potent antiviral activity and it could be because of phenolic acids (Rustaiyan et al. 1999; Si et al. 2006; Kwon et al. 2010), flavonoids (Bae et al.
2000) that are RNA synthesis blockers (Kwon
et al. 2010). The study
analyzed that in veterinary medicine, the use of this herb for treatment
purposes will be effective (Taherkhani et al. 2013). In the present
study, A. millefolium flowers extract
also showed antiviral activity against avian influenza virus in contrast to NDV where it did not show any inhibitory
effect. The
ethanolic extract of c. cinerafolium found ten chemical components in
its flowers with cyclopentanol as abundant. It showed antiviral activity
against H9N2 but no response against NDV. ANOVA applied
for multiple comparisons of statistical analysis in the present study for AIV
showed mean significant difference with P-value < 0.05 of W. fruticosa extracts
A. millefolium, B. melabaricum,
H. officianalis and C. cinerafolium.
The components identified though GCMS in these extracts includes ethers, fatty
acids, flavonoids etc. However, further investigations are required about the
activity of these bioactive components. The in
vitro study on cell lines will be helpful to find out their particular mode
of action.
Conclusion
The study conducted on seven different medicinal flower
extracts showed their antiviral activity against H9N2 and
NDV. All the extracts have significant antiviral potential with a P < 0.05 value for both viruses except T. officianale Weber, H. officianalis and C. cinerafolium against NDV. Furthermore, GCMS analysis
examined several chemical ingredients in the form of different peaks. These
active constituents have a significant role in the flowers antiviral activity.
It is suggested to extracts these specific components for in vitro study so that can be used for therapeutic purpose and
prophylactic measures in future. Moreover, the active products of these flowers
should be isolated and commercialized for use in feed.
Author Contributions
MR, AAS, SR conceived and designed the
experiments. IN and MIR executed the experiments and analyzed the study
results. QA and AK helped in research work. AYS helped in writing the
manuscript. IN wrote and edited the paper. All authors critically revised the
manuscript for important intellectual contents and approved the final version.
Conflict of Interest
The authors declare that there is no conflict of
interest regarding the publication of this article.
Data Availability
Data presented in this study will be available on a fair
request to the corresponding author.
Ethics Approval
Not applicable in this paper.
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